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Fishman Lab
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Fishman Lab Publications
Preprints
Aspiras AC, Harpaz R, Chambule S, Tseng S, Engert F, Fishman MC, Bahl A. Collective behavior emerges from genetically controlled simple behavioral motifs in zebrafish. bioRxiv, published online 3 March 2021. doi: 10.1101/2021.03.03.433803. View preprint
All Publications
2024
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2024. Experience-dependent modulation of collective behavior in larval zebrafish. bioRxiv : the preprint server for biology. Pubmed: 39149341 DOI:10.1101/2024.08.02.606403 Harpaz R, Phillips M, Goel R, Fishman MC, Engert F. 2024. Experience-dependent modulation of collective behavior in larval zebrafish. bioRxiv : the preprint server for biology. Pubmed: 39149341 DOI:10.1101/2024.08.02.606403 Complex group behavior can emerge from simple inter-individual interactions. Commonly, these interactions are considered static and hardwired and little is known about how experience and learning affect collective group behavior. Young larvae use well described visuomotor transformations to guide interindividual interactions and collective group structure. Here, we use naturalistic and virtual-reality (VR) experiments to impose persistent changes in population density and measure their effects on future visually evoked turning behavior and the resulting changes in group structure. We find that neighbor distances decrease after exposure to higher population densities, and increase after the experience of lower densities. These adaptations develop slowly and gradually, over tens of minutes and remain stable over many hours. Mechanistically, we find that larvae estimate their current group density by tracking the frequency of neighbor-evoked looming events on the retina and couple the strength of their future interactions to that estimate. A time-varying state-space model that modulates agents' social interactions based on their previous visual-social experiences, accurately describes our behavioral observations and predicts novel aspects of behavior. These findings provide concrete evidence that inter-individual interactions are not static, but rather continuously evolve based on past experience and current environmental demands. The underlying neurobiological mechanisms of experience dependent modulation can now be explored in this small and transparent model organism. 2021
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Harpaz R, Aspiras AC, Chambule S, Tseng S, Bind MA, Engert F, Fishman MC, Bahl A. 2021. Collective behavior emerges from genetically controlled simple behavioral motifs in zebrafish. Science advances. 7(41):eabi7460. Pubmed: 34613782 DOI:10.1126/sciadv.abi7460 Harpaz R, Aspiras AC, Chambule S, Tseng S, Bind MA, Engert F, Fishman MC, Bahl A. 2021. Collective behavior emerges from genetically controlled simple behavioral motifs in zebrafish. Science advances. 7(41):eabi7460. Pubmed: 34613782 DOI:10.1126/sciadv.abi7460 It is not understood how changes in the genetic makeup of individuals alter the behavior of groups of animals. Here, we find that, even at early larval stages, zebrafish regulate their proximity and alignment with each other. Two simple visual responses, one that measures relative visual field occupancy and one that accounts for global visual motion, suffice to account for the group behavior that emerges. Mutations in genes known to affect social behavior in humans perturb these simple reflexes in individual larval zebrafish and change their emergent collective behaviors in the predicted fashion. Model simulations show that changes in these two responses in individual mutant animals predict well the distinctive collective patterns that emerge in a group. Hence, group behaviors reflect in part genetically defined primitive sensorimotor “motifs,” which are evident even in young larvae. 2020
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Tang W, Davidson JD, Zhang G, Conen KE, Fang J, Serluca F, Li J, Xiong X, Coble M, Tsai T, Molind G, Fawcett CH, Sanchez E, Zhu P, Couzin ID, Fishman MC. 2020. Genetic Control of Collective Behavior in Zebrafish. iScience. 23(3):100942. Pubmed: 32179471 DOI:10.1016/j.isci.2020.100942 Tang W, Davidson JD, Zhang G, Conen KE, Fang J, Serluca F, Li J, Xiong X, Coble M, Tsai T, Molind G, Fawcett CH, Sanchez E, Zhu P, Couzin ID, Fishman MC. 2020. Genetic Control of Collective Behavior in Zebrafish. iScience. 23(3):100942. Pubmed: 32179471 DOI:10.1016/j.isci.2020.100942 Many animals, including humans, have evolved to live and move in groups. In humans, disrupted social interactions are a fundamental feature of many psychiatric disorders. However, we know little about how genes regulate social behavior. Zebrafish may serve as a powerful model to explore this question. By comparing the behavior of wild-type fish with 90 mutant lines, we show that mutations of genes associated with human psychiatric disorders can alter the collective behavior of adult zebrafish. We identify three categories of behavioral variation across mutants: "scattered," in which fish show reduced cohesion; "coordinated," in which fish swim more in aligned schools; and "huddled," in which fish form dense but disordered groups. Changes in individual interaction rules can explain these differences. This work demonstrates how emergent patterns in animal groups can be altered by genetic changes in individuals and establishes a framework for understanding the fundamentals of social information processing.Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved. 2019
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Singh AP, Sosa MX, Fang J, Shanmukhappa SK, Hubaud A, Fawcett CH, Molind GJ, Tsai T, Capodieci P, Wetzel K, Sanchez E, Wang G, Coble M, Tang W, Cadena SM, Fishman MC, Glass DJ. 2019. αKlotho Regulates Age-Associated Vascular Calcification and Lifespan in Zebrafish. Cell reports. 28(11):2767-2776.e5. Pubmed: 31509740 DOI:S2211-1247(19)31046-0 Singh AP, Sosa MX, Fang J, Shanmukhappa SK, Hubaud A, Fawcett CH, Molind GJ, Tsai T, Capodieci P, Wetzel K, Sanchez E, Wang G, Coble M, Tang W, Cadena SM, Fishman MC, Glass DJ. 2019. αKlotho Regulates Age-Associated Vascular Calcification and Lifespan in Zebrafish. Cell reports. 28(11):2767-2776.e5. Pubmed: 31509740 DOI:S2211-1247(19)31046-0 The hormone αKlotho regulates lifespan in mice, as knockouts die early of what appears to be accelerated aging due to hyperphosphatemia and soft tissue calcification. In contrast, the overexpression of αKlotho increases lifespan. Given the severe mouse phenotype, we generated zebrafish mutants for αklotho as well as its binding partner fibroblast growth factor-23 (fgf23). Both mutations cause shortened lifespan in zebrafish, with abrupt onset of behavioral and degenerative physical changes at around 5 months of age. There is a calcification of vessels throughout the body, most dramatically in the outflow tract of the heart, the bulbus arteriosus (BA). This calcification is associated with an ectopic activation of osteoclast differentiation pathways. These findings suggest that the gradual loss of αKlotho found in normal aging might give rise to ectopic calcification.Copyright © 2019 Novartis Institutes for Biomedical Research. Published by Elsevier Inc. All rights reserved. -
Singh AP, Sosa MX, Fang J, Shanmukhappa S, Hubaud A, Fawcett C, Molind G, Tsai T, Capodieci P, Wetzel K, Sanchez E, Wang G, Coble M, Tang W, Cadena SM, Fishman MC, Glass DJ. 2019. αKlotho regulates age-associated vascular calcification and lifespan in zebrafish. Cell Reports. Singh AP, Sosa MX, Fang J, Shanmukhappa S, Hubaud A, Fawcett C, Molind G, Tsai T, Capodieci P, Wetzel K, Sanchez E, Wang G, Coble M, Tang W, Cadena SM, Fishman MC, Glass DJ. 2019. αKlotho regulates age-associated vascular calcification and lifespan in zebrafish. Cell Reports. -
Wang G, Zhang G, Li Z, Fawcett CH, Coble M, Sosa MX, Tsai T, Malesky K, Thibodeaux SJ, Zhu P, Glass DJ, Fishman MC. 2019. Abnormal Behavior of Zebrafish Mutant in Dopamine Transporter Is Rescued by Clozapine. iScience. 17:325-333. Pubmed: 31325771 DOI:S2589-0042(19)30222-6 Wang G, Zhang G, Li Z, Fawcett CH, Coble M, Sosa MX, Tsai T, Malesky K, Thibodeaux SJ, Zhu P, Glass DJ, Fishman MC. 2019. Abnormal Behavior of Zebrafish Mutant in Dopamine Transporter Is Rescued by Clozapine. iScience. 17:325-333. Pubmed: 31325771 DOI:S2589-0042(19)30222-6 Dopamine transporter (SLC6A3) deficiency causes infantile Parkinson disease, for which there is no effective therapy. We have explored the effects of genetically deleting SLC6A3 in zebrafish. Unlike the wild-type, slc6a3-/- fish hover near the tank bottom, with a repetitive digging-like behavior. slc6a3-/- fish manifest pruning and cellular loss of particular tyrosine hydroxylase-immunoreactive neurons in the midbrain. Clozapine, an effective therapeutic for treatment-resistant schizophrenia, rescues the abnormal behavior of slc6a3-/- fish. Clozapine also reverses the abnormalities in the A8 region of the mutant midbrain. By RNA sequencing analysis, clozapine increases the expression of erythropoietin pathway genes. Transgenic over-expression of erythropoietin in neurons of slc6a3-/- fish partially rescues the mutant behavior, suggesting a potential mechanistic basis for clozapine's efficacy.Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved. -
Diaz-Verdugo C, Sun GJ, Fawcett CH, Zhu P, Fishman MC. 2019. Mating Suppresses Alarm Response in Zebrafish. Current biology : CB. 29(15):2541-2546.e3. Pubmed: 31327717 DOI:S0960-9822(19)30774-2 Diaz-Verdugo C, Sun GJ, Fawcett CH, Zhu P, Fishman MC. 2019. Mating Suppresses Alarm Response in Zebrafish. Current biology : CB. 29(15):2541-2546.e3. Pubmed: 31327717 DOI:S0960-9822(19)30774-2 Mating and flight from threats are innate behaviors that enhance species survival [1, 2]. Stimuli to these behaviors often are contemporaneous and conflicting [3, 4]. Both how such conflicts are resolved and where in the brain such decisions are made are poorly understood. For teleosts, olfactory stimuli are key elements of mating and threat responses [5-7]. For example, zebrafish manifest a stereotypical escape response when exposed to an alarm substance released from injured conspecific skin ("skin extract") [8, 9]. We find that when mating, fish ignore this threatening stimulus. Water conditioned by the mating fish ("mating water") suffices to suppress much of the alarm-response behavior. By 2-photon imaging of calcium transients [10], we mapped the regions of the brain responding to skin extract and to mating water. In the telencephalon, we found regions where the responses overlap, one region (medial Dp) to be predominantly activated by skin extract, and another, Vs, to be predominantly activated by mating water. When mating water and skin extract were applied simultaneously, the alarm-specific response was suppressed, while the mating-water-specific response was retained, corresponding to the dominance of mating over flight behavior. The choice made, for reproduction over escape, is opposite to that of mammals, presumably reflecting how the balance affects species survival.Published by Elsevier Ltd. 2018
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Armstrong K, Ranganathan R, Fishman M. 2018. Toward a Culture of Scientific Inquiry - The Role of Medical Teaching Services. The New England journal of medicine. 378(1):1-3. Pubmed: 29298148 Armstrong K, Ranganathan R, Fishman M. 2018. Toward a Culture of Scientific Inquiry - The Role of Medical Teaching Services. The New England journal of medicine. 378(1):1-3. Pubmed: 29298148 -
Spector JM, Harrison RS, Fishman MC. 2018. Fundamental science behind today's important medicines. Science translational medicine. 10(438). Pubmed: 29695453 DOI:10.1126/scitranslmed.aaq1787 Spector JM, Harrison RS, Fishman MC. 2018. Fundamental science behind today's important medicines. Science translational medicine. 10(438). Pubmed: 29695453 DOI:10.1126/scitranslmed.aaq1787 Today's most transformative medicines exist because of fundamental discoveries that were made without regard to practical outcome and with their relevance to therapeutics only appearing decades later.Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. -
Musuku J, Engel ME, Musonda P, Lungu JC, Machila E, Schwaninger S, Mtaja A, Mulendele E, Kavindele D, Spector J, Tadmor B, Gutierrez MM, Van Dam J, Colin L, Long A, Fishman MC, Mayosi BM, Zühlke LJ. 2018. Prevalence of rheumatic heart disease in Zambian school children. BMC cardiovascular disorders. 18(1):135. Pubmed: 29969998 DOI:10.1186/s12872-018-0871-8 Musuku J, Engel ME, Musonda P, Lungu JC, Machila E, Schwaninger S, Mtaja A, Mulendele E, Kavindele D, Spector J, Tadmor B, Gutierrez MM, Van Dam J, Colin L, Long A, Fishman MC, Mayosi BM, Zühlke LJ. 2018. Prevalence of rheumatic heart disease in Zambian school children. BMC cardiovascular disorders. 18(1):135. Pubmed: 29969998 DOI:10.1186/s12872-018-0871-8 Array -
Vyas JM, Rajagopal J, Sokol CL, Wein MN, Mansour MK, Corey KE, Fishman MC, Armstrong KA. 2018. The Great Opportunity: Cultivating Scientific Inquiry in Medical Residency. The Journal of infectious diseases. 218(suppl_1):S44-S48. Pubmed: 29878132 DOI:10.1093/infdis/jiy200 Vyas JM, Rajagopal J, Sokol CL, Wein MN, Mansour MK, Corey KE, Fishman MC, Armstrong KA. 2018. The Great Opportunity: Cultivating Scientific Inquiry in Medical Residency. The Journal of infectious diseases. 218(suppl_1):S44-S48. Pubmed: 29878132 DOI:10.1093/infdis/jiy200 Residency training is a profound experience that greatly influences the career trajectory of every trainee. Currently, residency programs focus heavily (or almost exclusively) on the acquisition of medical knowledge and fail to foster intellectual curiosity and introduce residents to careers in investigation. We share 3 programs embedded in residency training where this focus is shifted with an emphasis on prompting intellectual curiosity and exciting residents about careers in investigation to revitalize the physician-scientist workforce. -
Tang W, Zhang G, Serluca F, Li J, Xiong X, Coble M, Tsai T, Li Z, Molind G, Zhu P, Fishman MC. 2018. Genetic architecture of collective behaviors in zebrafish. bioRxiv. DOI:https://doi.org/10.1101/350314 Tang W, Zhang G, Serluca F, Li J, Xiong X, Coble M, Tsai T, Li Z, Molind G, Zhu P, Fishman MC. 2018. Genetic architecture of collective behaviors in zebrafish. bioRxiv. DOI:https://doi.org/10.1101/350314 Collective behaviors of groups of animals, such as schooling and shoaling of fish, are central to species survival, but genes that regulate these activities are not known. Here we parsed collective behavior of groups of adult zebrafish using computer vision and unsupervised machine learning into a set of highly reproducible, unitary, several hundred millisecond states and transitions, which together can account for the entirety of relative positions and postures of groups of fish. Using CRISPR-Cas9 we then targeted for knockout 35 genes associated with autism and schizophrenia. We found mutations in three genes had distinctive effects on the amount of time spent in the specific states or transitions between states. Mutation in immp2l (inner mitochondrial membrane peptidase 2-like gene) enhances states of cohesion, so increases shoaling; mutation in in the Nav1.1 sodium channel, scn1lab+/− causes the fish to remain scattered without evident social interaction; and mutation in the adrenergic receptor, adra1aa−/−, keeps fish close together and retards transitions between states, leaving fish motionless for long periods. Motor and visual functions seemed relatively well-preserved. This work shows that the behaviors of fish engaged in collective activities are built from a set of stereotypical states. Single gene mutations can alter propensities to collective actions by changing the proportion of time spent in these states or the tendency to transition between states. This provides an approach to begin dissection of the molecular pathways used to generate and guide collective actions of groups of animals. 2017
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Long A, Lungu JC, Machila E, Schwaninger S, Spector J, Tadmor B, Fishman M, Mayosi BM, Musuku J. 2017. A programme to increase appropriate usage of benzathine penicillin for management of streptococcal pharyngitis and rheumatic heart disease in Zambia. Cardiovascular journal of Africa. 28(4):242-247. Pubmed: 28906539 DOI:10.5830/CVJA-2017-002 Long A, Lungu JC, Machila E, Schwaninger S, Spector J, Tadmor B, Fishman M, Mayosi BM, Musuku J. 2017. A programme to increase appropriate usage of benzathine penicillin for management of streptococcal pharyngitis and rheumatic heart disease in Zambia. Cardiovascular journal of Africa. 28(4):242-247. Pubmed: 28906539 DOI:10.5830/CVJA-2017-002 Rheumatic heart disease is highly prevalent and associated with substantial morbidity and mortality in many resource-poor areas of the world, including sub-Saharan Africa. Primary and secondary prophylaxis with penicillin has been shown to significantly improve outcomes and is recognised to be the standard of care, with intra-muscular benzathine penicillin G recommended as the preferred agent by many technical experts. However, ensuring compliance with therapy has proven to be challenging. As part of a public-private partnership initiative in Zambia, we conducted an educational and access-to-medicine programme aimed at increasing appropriate use of benzathine penicillin for the prevention and management of rheumatic heart disease, according to national guidelines. The programme was informed early on by identification of potential barriers to the administration of injectable penicillin, which included concern by health workers about allergic events. We describe this programme and report initial signs of success, as indicated by increased use of benzathine penicillin. We propose that a similar approach may have benefits in rheumatic heart disease programmes in other endemic regions. 2016
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Palacino J, Swalley SE, Song C, Cheung AK, Shu L, Zhang X, Van Hoosear M, Shin Y, Chin DN, Keller CG, Beibel M, Renaud NA, Smith TM, Salcius M, Shi X, Hild M, Servais R, Jain M, Deng L, Bullock C, McLellan M, Schuierer S, Murphy L, Blommers MJ, Blaustein C, Berenshteyn F, Lacoste A, Thomas JR, Roma G, Michaud GA, Tseng BS, Porter JA, Myer VE, Tallarico JA, Hamann LG, Curtis D, Fishman MC, Dietrich WF, Dales NA, Sivasankaran R. 2016. Corrigendum: SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice. Nature chemical biology. 12(4):304. Pubmed: 26991088 DOI:10.1038/nchembio0416-304c Palacino J, Swalley SE, Song C, Cheung AK, Shu L, Zhang X, Van Hoosear M, Shin Y, Chin DN, Keller CG, Beibel M, Renaud NA, Smith TM, Salcius M, Shi X, Hild M, Servais R, Jain M, Deng L, Bullock C, McLellan M, Schuierer S, Murphy L, Blommers MJ, Blaustein C, Berenshteyn F, Lacoste A, Thomas JR, Roma G, Michaud GA, Tseng BS, Porter JA, Myer VE, Tallarico JA, Hamann LG, Curtis D, Fishman MC, Dietrich WF, Dales NA, Sivasankaran R. 2016. Corrigendum: SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice. Nature chemical biology. 12(4):304. Pubmed: 26991088 DOI:10.1038/nchembio0416-304c -
Just S, Hirth S, Berger IM, Fishman MC, Rottbauer W. 2016. The mediator complex subunit Med10 regulates heart valve formation in zebrafish by controlling Tbx2b-mediated Has2 expression and cardiac jelly formation. Biochemical and biophysical research communications. 477(4):581-588. Pubmed: 27343557 DOI:S0006-291X(16)31006-3 Just S, Hirth S, Berger IM, Fishman MC, Rottbauer W. 2016. The mediator complex subunit Med10 regulates heart valve formation in zebrafish by controlling Tbx2b-mediated Has2 expression and cardiac jelly formation. Biochemical and biophysical research communications. 477(4):581-588. Pubmed: 27343557 DOI:S0006-291X(16)31006-3 In search for novel key regulators of cardiac valve formation, we isolated the zebrafish cardiac valve mutant ping pong (png). We find that an insertional promoter mutation within the zebrafish mediator complex subunit 10 (med10) gene is leading to impaired heart valve formation. Expression of the T-box transcription factor 2b (Tbx2b), known to be essential in cardiac valve development, is severely reduced in png mutant hearts. We demonstrate here that transient reconstitution of Tbx2b expression rescues AV canal development in png mutant zebrafish. By contrast, overexpression of Forkhead box N4 (Foxn4), a known upstream regulator of Tbx2b, is not capable to reconstitute tbx2b expression and heart valve formation in Med10-deficient png mutant hearts. Interestingly, hyaluronan synthase 2 (has2), a known downstream target of Tbx2 and producer of hyaluronan (HA) - a major ECM component of the cardiac jelly and critical for proper heart valve development - is completely absent in ping pong mutant hearts. We propose here a rather unique role of Med10 in orchestrating cardiac valve formation by mediating Foxn4 dependent tbx2b transcription, expression of Has2 and subsequently proper development of the cardiac jelly.Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved. 2015
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Palacino J, Swalley SE, Song C, Cheung AK, Shu L, Zhang X, Van Hoosear M, Shin Y, Chin DN, Keller CG, Beibel M, Renaud NA, Smith TM, Salcius M, Shi X, Hild M, Servais R, Jain M, Deng L, Bullock C, McLellan M, Schuierer S, Murphy L, Blommers MJ, Blaustein C, Berenshteyn F, Lacoste A, Thomas JR, Roma G, Michaud GA, Tseng BS, Porter JA, Myer VE, Tallarico JA, Hamann LG, Curtis D, Fishman MC, Dietrich WF, Dales NA, Sivasankaran R. 2015. SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice. Nature chemical biology. 11(7):511-7. Pubmed: 26030728 DOI:10.1038/nchembio.1837 Palacino J, Swalley SE, Song C, Cheung AK, Shu L, Zhang X, Van Hoosear M, Shin Y, Chin DN, Keller CG, Beibel M, Renaud NA, Smith TM, Salcius M, Shi X, Hild M, Servais R, Jain M, Deng L, Bullock C, McLellan M, Schuierer S, Murphy L, Blommers MJ, Blaustein C, Berenshteyn F, Lacoste A, Thomas JR, Roma G, Michaud GA, Tseng BS, Porter JA, Myer VE, Tallarico JA, Hamann LG, Curtis D, Fishman MC, Dietrich WF, Dales NA, Sivasankaran R. 2015. SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice. Nature chemical biology. 11(7):511-7. Pubmed: 26030728 DOI:10.1038/nchembio.1837 Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5' splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule-mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases. -
Wa Somwe S, Jumbe-Marsden E, Mateyo K, Senkwe MN, Sotomayor-Ruiz M, Musuku J, Soriano JB, Ancochea J, Fishman MC. 2015. Improving paediatric asthma care in Zambia. Bulletin of the World Health Organization. 93(10):732-736. Pubmed: 26600616 Wa Somwe S, Jumbe-Marsden E, Mateyo K, Senkwe MN, Sotomayor-Ruiz M, Musuku J, Soriano JB, Ancochea J, Fishman MC. 2015. Improving paediatric asthma care in Zambia. Bulletin of the World Health Organization. 93(10):732-736. Pubmed: 26600616 Array -
Palacino J, Swalley SE, Song C, Cheung AK, Shu L, Zhang X, Van Hoosear M, Shin Y, Chin DN, Keller CG, Beibel M, Renaud NA, Smith TM, Salcius M, Shi X, Hild M, Servais R, Jain M, Deng L, Bullock C, McLellan M, Schuierer S, Murphy L, Blommers MJ, Blaustein C, Berenshteyn F, Lacoste A, Thomas JR, Roma G, Michaud GA, Tseng BS, Porter JA, Myer VE, Tallarico JA, Hamann LG, Curtis D, Fishman MC, Dietrich WF, Dales NA, Sivasankaran R. 2015. Corrigendum: SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice. Nature chemical biology. 11(9):741. Pubmed: 26284678 DOI:10.1038/nchembio0915-741a Palacino J, Swalley SE, Song C, Cheung AK, Shu L, Zhang X, Van Hoosear M, Shin Y, Chin DN, Keller CG, Beibel M, Renaud NA, Smith TM, Salcius M, Shi X, Hild M, Servais R, Jain M, Deng L, Bullock C, McLellan M, Schuierer S, Murphy L, Blommers MJ, Blaustein C, Berenshteyn F, Lacoste A, Thomas JR, Roma G, Michaud GA, Tseng BS, Porter JA, Myer VE, Tallarico JA, Hamann LG, Curtis D, Fishman MC, Dietrich WF, Dales NA, Sivasankaran R. 2015. Corrigendum: SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice. Nature chemical biology. 11(9):741. Pubmed: 26284678 DOI:10.1038/nchembio0915-741a 2014
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Fishman MC. 2014. Author response to comment on "Power of rare diseases: found in translation". Science translational medicine. 6(228):228lr1. Pubmed: 24648339 DOI:10.1126/scitranslmed.3008809 Fishman MC. 2014. Author response to comment on "Power of rare diseases: found in translation". Science translational medicine. 6(228):228lr1. Pubmed: 24648339 DOI:10.1126/scitranslmed.3008809 The study of rare diseases in both academic and industry settings yields new knowledge about human pathophysiology and ultimately helps patients. -
Hoepfner D, Helliwell SB, Sadlish H, Schuierer S, Filipuzzi I, Brachat S, Bhullar B, Plikat U, Abraham Y, Altorfer M, Aust T, Baeriswyl L, Cerino R, Chang L, Estoppey D, Eichenberger J, Frederiksen M, Hartmann N, Hohendahl A, Knapp B, Krastel P, Melin N, Nigsch F, Oakeley EJ, Petitjean V, Petersen F, Riedl R, Schmitt EK, Staedtler F, Studer C, Tallarico JA, Wetzel S, Fishman MC, Porter JA, Movva NR. 2014. High-resolution chemical dissection of a model eukaryote reveals targets, pathways and gene functions. Microbiological research. 169(2-3):107-20. Pubmed: 24360837 DOI:S0944-5013(13)00193-6 Hoepfner D, Helliwell SB, Sadlish H, Schuierer S, Filipuzzi I, Brachat S, Bhullar B, Plikat U, Abraham Y, Altorfer M, Aust T, Baeriswyl L, Cerino R, Chang L, Estoppey D, Eichenberger J, Frederiksen M, Hartmann N, Hohendahl A, Knapp B, Krastel P, Melin N, Nigsch F, Oakeley EJ, Petitjean V, Petersen F, Riedl R, Schmitt EK, Staedtler F, Studer C, Tallarico JA, Wetzel S, Fishman MC, Porter JA, Movva NR. 2014. High-resolution chemical dissection of a model eukaryote reveals targets, pathways and gene functions. Microbiological research. 169(2-3):107-20. Pubmed: 24360837 DOI:S0944-5013(13)00193-6 Due to evolutionary conservation of biology, experimental knowledge captured from genetic studies in eukaryotic model organisms provides insight into human cellular pathways and ultimately physiology. Yeast chemogenomic profiling is a powerful approach for annotating cellular responses to small molecules. Using an optimized platform, we provide the relative sensitivities of the heterozygous and homozygous deletion collections for nearly 1800 biologically active compounds. The data quality enables unique insights into pathways that are sensitive and resistant to a given perturbation, as demonstrated with both known and novel compounds. We present examples of novel compounds that inhibit the therapeutically relevant fatty acid synthase and desaturase (Fas1p and Ole1p), and demonstrate how the individual profiles facilitate hypothesis-driven experiments to delineate compound mechanism of action. Importantly, the scale and diversity of tested compounds yields a dataset where the number of modulated pathways approaches saturation. This resource can be used to map novel biological connections, and also identify functions for unannotated genes. We validated hypotheses generated by global two-way hierarchical clustering of profiles for (i) novel compounds with a similar mechanism of action acting upon microtubules or vacuolar ATPases, and (ii) an un-annotated ORF, YIL060w, that plays a role in respiration in the mitochondria. Finally, we identify and characterize background mutations in the widely used yeast deletion collection which should improve the interpretation of past and future screens throughout the community. This comprehensive resource of cellular responses enables the expansion of our understanding of eukaryotic pathway biology.Copyright © 2013 Elsevier GmbH. All rights reserved. 2013
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Fishman MC. 2013. Power of rare diseases: found in translation. Science translational medicine. 5(201):201ps11. Pubmed: 24005157 DOI:10.1126/scitranslmed.3006800 Fishman MC. 2013. Power of rare diseases: found in translation. Science translational medicine. 5(201):201ps11. Pubmed: 24005157 DOI:10.1126/scitranslmed.3006800 Aside from established genetic evidence, the best proof of a model for disease pathogenesis rests on predicted perturbation via targeted medicines in clinical trials. Here, I discuss the strategy of performing exploratory first-in-human clinical studies on mechanistically homogeneous populations (often small groups of patients with rare diseases) as a routine entrance to full-registration clinical trials. Over the past decade, this approach has proved some pathogenic theories, disproved others, and guided investigators in new scientific directions. The immediate advantages have been smaller trials and provision of new treatments for rare diseases. Later, indications often can be expanded to subsets of more common diseases. 2012
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Hoepfner D, McNamara CW, Lim CS, Studer C, Riedl R, Aust T, McCormack SL, Plouffe DM, Meister S, Schuierer S, Plikat U, Hartmann N, Staedtler F, Cotesta S, Schmitt EK, Petersen F, Supek F, Glynne RJ, Tallarico JA, Porter JA, Fishman MC, Bodenreider C, Diagana TT, Movva NR, Winzeler EA. 2012. Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin. Cell host & microbe. 11(6):654-63. Pubmed: 22704625 DOI:10.1016/j.chom.2012.04.015 Hoepfner D, McNamara CW, Lim CS, Studer C, Riedl R, Aust T, McCormack SL, Plouffe DM, Meister S, Schuierer S, Plikat U, Hartmann N, Staedtler F, Cotesta S, Schmitt EK, Petersen F, Supek F, Glynne RJ, Tallarico JA, Porter JA, Fishman MC, Bodenreider C, Diagana TT, Movva NR, Winzeler EA. 2012. Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin. Cell host & microbe. 11(6):654-63. Pubmed: 22704625 DOI:10.1016/j.chom.2012.04.015 With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited.Copyright © 2012 Elsevier Inc. All rights reserved. -
Fishman M. 2012. Changing the practice of medicine: an interview with Mark Fishman. Interview by Sarah Allan. Disease models & mechanisms. 5(3):296-8. Pubmed: 22566557 DOI:10.1242/dmm.009936 Fishman M. 2012. Changing the practice of medicine: an interview with Mark Fishman. Interview by Sarah Allan. Disease models & mechanisms. 5(3):296-8. Pubmed: 22566557 DOI:10.1242/dmm.009936 Mark Fishman has served as President of the Novartis Institutes for BioMedical Research (NIBR) since 2002, and in a former academic role was among the first to develop the zebrafish system for studying development and disease. Here, he recalls what it was like to ride the first real wave of zebrafish research, discusses industry-academia collaborations and provides a current perspective on drug development. -
Fishman M. 2012. Mark Fishman. Interview by Asher Mullard. Nature reviews. Drug discovery. 11(1):14-5. Pubmed: 22212670 DOI:10.1038/nrd3640 Fishman M. 2012. Mark Fishman. Interview by Asher Mullard. Nature reviews. Drug discovery. 11(1):14-5. Pubmed: 22212670 DOI:10.1038/nrd3640 2011
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Just S, Meder B, Berger IM, Etard C, Trano N, Patzel E, Hassel D, Marquart S, Dahme T, Vogel B, Fishman MC, Katus HA, Strähle U, Rottbauer W. 2011. The myosin-interacting protein SMYD1 is essential for sarcomere organization. Journal of cell science. 124(Pt 18):3127-36. Pubmed: 21852424 DOI:10.1242/jcs.084772 Just S, Meder B, Berger IM, Etard C, Trano N, Patzel E, Hassel D, Marquart S, Dahme T, Vogel B, Fishman MC, Katus HA, Strähle U, Rottbauer W. 2011. The myosin-interacting protein SMYD1 is essential for sarcomere organization. Journal of cell science. 124(Pt 18):3127-36. Pubmed: 21852424 DOI:10.1242/jcs.084772 Assembly, maintenance and renewal of sarcomeres require highly organized and balanced folding, transport, modification and degradation of sarcomeric proteins. However, the molecules that mediate these processes are largely unknown. Here, we isolated the zebrafish mutant flatline (fla), which shows disturbed sarcomere assembly exclusively in heart and fast-twitch skeletal muscle. By positional cloning we identified a nonsense mutation within the SET- and MYND-domain-containing protein 1 gene (smyd1) to be responsible for the fla phenotype. We found SMYD1 expression to be restricted to the heart and fast-twitch skeletal muscle cells. Within these cell types, SMYD1 localizes to both the sarcomeric M-line, where it physically associates with myosin, and the nucleus, where it supposedly represses transcription through its SET and MYND domains. However, although we found transcript levels of thick filament chaperones, such as Hsp90a1 and UNC-45b, to be severely upregulated in fla, its histone methyltransferase activity - mainly responsible for the nuclear function of SMYD1 - is dispensable for sarcomerogenesis. Accordingly, sarcomere assembly in fla mutant embryos can be reconstituted by ectopically expressing histone methyltransferase-deficient SMYD1. By contrast, ectopic expression of myosin-binding-deficient SMYD1 does not rescue fla mutants, implicating an essential role for the SMYD1-myosin interaction in cardiac and fast-twitch skeletal muscle thick filament assembly. -
Peterson RT, Fishman MC. 2011. Designing zebrafish chemical screens. Methods in cell biology. 105:525-41. Pubmed: 21951546 DOI:10.1016/B978-0-12-381320-6.00023-0 Peterson RT, Fishman MC. 2011. Designing zebrafish chemical screens. Methods in cell biology. 105:525-41. Pubmed: 21951546 DOI:10.1016/B978-0-12-381320-6.00023-0 The zebrafish is proving to be highly amenable to in vivo small molecule screening. With a growing number of screens successfully completed, a rich interface is being created between disciplines that have historically used zebrafish (e.g., embryology and genetics) and disciplines focused on small molecules (e.g., chemistry and pharmacology). Navigating this interface requires consideration of the unique demands of conducting high-throughput screening in vivo. In this chapter, we discuss design elements of successful zebrafish screens, established screening methods, and approaches for mechanism of action studies following discovery of novel small molecules. These methods are enabling the zebrafish to have an increasingly positive impact on biomedical research and drug development.Copyright © 2011 Elsevier Inc. All rights reserved. 2010
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Sogah VM, Serluca FC, Fishman MC, Yelon DL, Macrae CA, Mably JD. 2010. Distinct troponin C isoform requirements in cardiac and skeletal muscle. Developmental dynamics : an official publication of the American Association of Anatomists. 239(11):3115-23. Pubmed: 20925115 DOI:10.1002/dvdy.22445 Sogah VM, Serluca FC, Fishman MC, Yelon DL, Macrae CA, Mably JD. 2010. Distinct troponin C isoform requirements in cardiac and skeletal muscle. Developmental dynamics : an official publication of the American Association of Anatomists. 239(11):3115-23. Pubmed: 20925115 DOI:10.1002/dvdy.22445 The zebrafish mutant silent partner is characterized by a dysmorphic, non-contractile ventricle resulting in an inability to generate normal blood flow. We have identified the genetic lesion in the zebrafish homolog of the slow twitch skeletal/cardiac troponin C gene. Although human troponin C1 (TNNC1) is expressed in both cardiac and skeletal muscle, duplication of this gene in zebrafish has resulted in tissue-specific partitioning of troponin C expression and function. Mutation of the zebrafish paralog tnnc1a, which is expressed predominantly in the heart, results in a loss of contractility and myofibrillar organization within ventricular cardiomyocytes, while skeletal muscle remains functional and intact. We further show that defective contractility in the developing heart results in abnormal atrial and ventricular chamber morphology. Together, our results suggest that tnnc1a is required both for the function and structural integrity of the contractile machinery in cardiomyocytes, helping to clarify potential mechanisms of troponin C-mediated cardiomyopathy.© 2010 Wiley-Liss, Inc. 2009
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Meder B, Laufer C, Hassel D, Just S, Marquart S, Vogel B, Hess A, Fishman MC, Katus HA, Rottbauer W. 2009. A single serine in the carboxyl terminus of cardiac essential myosin light chain-1 controls cardiomyocyte contractility in vivo. Circulation research. 104(5):650-9. Pubmed: 19168438 DOI:10.1161/CIRCRESAHA.108.186676 Meder B, Laufer C, Hassel D, Just S, Marquart S, Vogel B, Hess A, Fishman MC, Katus HA, Rottbauer W. 2009. A single serine in the carboxyl terminus of cardiac essential myosin light chain-1 controls cardiomyocyte contractility in vivo. Circulation research. 104(5):650-9. Pubmed: 19168438 DOI:10.1161/CIRCRESAHA.108.186676 Although it is well known that mutations in the cardiac essential myosin light chain-1 (cmlc-1) gene can cause hypertrophic cardiomyopathy, the precise in vivo structural and functional roles of cMLC-1 in the heart are only poorly understood. We have isolated the zebrafish mutant lazy susan (laz), which displays severely reduced contractility of both heart chambers. By positional cloning, we identified a nonsense mutation within the zebrafish cmlc-1 gene to be responsible for the laz phenotype, leading to expression of a carboxyl-terminally truncated cMLC-1. Whereas complete loss of cMLC-1 leads to cardiac acontractility attributable to impaired cardiac sarcomerogenesis, expression of a carboxyl-terminally truncated cMLC-1 in laz mutant hearts is sufficient for normal cardiac sarcomerogenesis but severely impairs cardiac contractility in a cell-autonomous fashion. Whereas overexpression of wild-type cMLC-1 restores contractility of laz mutant cardiomyocytes, overexpression of phosphorylation site serine 195-deficient cMLC-1 (cMLC-1(S195A)) does not reconstitute cardiac contractility in laz mutant cardiomyocytes. By contrast, introduction of a phosphomimetic amino acid on position 195 (cMLC-1(S195D)) rescues cardiomyocyte contractility, demonstrating for the first time an essential role of the carboxyl terminus and especially of serine 195 of cMLC-1 in the regulation of cardiac contractility. -
Serluca FC, Xu B, Okabe N, Baker K, Lin SY, Sullivan-Brown J, Konieczkowski DJ, Jaffe KM, Bradner JM, Fishman MC, Burdine RD. 2009. Mutations in zebrafish leucine-rich repeat-containing six-like affect cilia motility and result in pronephric cysts, but have variable effects on left-right patterning. Development (Cambridge, England). 136(10):1621-31. Pubmed: 19395640 DOI:10.1242/dev.020735 Serluca FC, Xu B, Okabe N, Baker K, Lin SY, Sullivan-Brown J, Konieczkowski DJ, Jaffe KM, Bradner JM, Fishman MC, Burdine RD. 2009. Mutations in zebrafish leucine-rich repeat-containing six-like affect cilia motility and result in pronephric cysts, but have variable effects on left-right patterning. Development (Cambridge, England). 136(10):1621-31. Pubmed: 19395640 DOI:10.1242/dev.020735 Cilia defects have been implicated in a variety of human diseases and genetic disorders, but how cilia motility contributes to these phenotypes is still unknown. To further our understanding of how cilia function in development, we have cloned and characterized two alleles of seahorse, a zebrafish mutation that results in pronephric cysts. seahorse encodes Lrrc6l, a leucine-rich repeat-containing protein that is highly conserved in organisms that have motile cilia. seahorse is expressed in zebrafish tissues known to contain motile cilia. Although mutants do not affect cilia structure and retain the ability to interact with Disheveled, both alleles of seahorse strongly affect cilia motility in the zebrafish pronephros and neural tube. Intriguingly, although seahorse mutations variably affect fluid flow in Kupffer's vesicle, they can have very weak effects on left-right patterning. Combined with recently published results, our alleles suggest that the function of seahorse in cilia motility is separable from its function in other cilia-related phenotypes. 2008
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Hassel D, Scholz EP, Trano N, Friedrich O, Just S, Meder B, Weiss DL, Zitron E, Marquart S, Vogel B, Karle CA, Seemann G, Fishman MC, Katus HA, Rottbauer W. 2008. Deficient zebrafish ether-à-go-go-related gene channel gating causes short-QT syndrome in zebrafish reggae mutants. Circulation. 117(7):866-75. Pubmed: 18250272 DOI:10.1161/CIRCULATIONAHA.107.752220 Hassel D, Scholz EP, Trano N, Friedrich O, Just S, Meder B, Weiss DL, Zitron E, Marquart S, Vogel B, Karle CA, Seemann G, Fishman MC, Katus HA, Rottbauer W. 2008. Deficient zebrafish ether-à-go-go-related gene channel gating causes short-QT syndrome in zebrafish reggae mutants. Circulation. 117(7):866-75. Pubmed: 18250272 DOI:10.1161/CIRCULATIONAHA.107.752220 Array 2006
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Bendig G, Grimmler M, Huttner IG, Wessels G, Dahme T, Just S, Trano N, Katus HA, Fishman MC, Rottbauer W. 2006. Integrin-linked kinase, a novel component of the cardiac mechanical stretch sensor, controls contractility in the zebrafish heart. Genes & development. 20(17):2361-72. Pubmed: 16921028 Bendig G, Grimmler M, Huttner IG, Wessels G, Dahme T, Just S, Trano N, Katus HA, Fishman MC, Rottbauer W. 2006. Integrin-linked kinase, a novel component of the cardiac mechanical stretch sensor, controls contractility in the zebrafish heart. Genes & development. 20(17):2361-72. Pubmed: 16921028 The vertebrate heart possesses autoregulatory mechanisms enabling it first to sense and then to adapt its force of contraction to continually changing demands. The molecular components of the cardiac mechanical stretch sensor are mostly unknown but of immense medical importance, since dysfunction of this sensing machinery is suspected to be responsible for a significant proportion of human heart failure. In the hearts of the ethylnitros-urea (ENU)-induced, recessive embryonic lethal zebrafish heart failure mutant main squeeze (msq), we find stretch-responsive genes such as atrial natriuretic factor (anf) and vascular endothelial growth factor (vegf) severely down-regulated. We demonstrate through positional cloning that heart failure in msq mutants is due to a mutation in the integrin-linked kinase (ilk) gene. ILK specifically localizes to costameres and sarcomeric Z-discs. The msq mutation (L308P) reduces ILK kinase activity and disrupts binding of ILK to the Z-disc adaptor protein beta-parvin (Affixin). Accordingly, in msq mutant embryos, heart failure can be suppressed by expression of ILK, and also of a constitutively active form of Protein Kinase B (PKB), and VEGF. Furthermore, antisense-mediated abrogation of zebrafish beta-parvin phenocopies the msq phenotype. Thus, we provide evidence that the heart uses the Integrin-ILK-beta-parvin network to sense mechanical stretch and respond with increased expression of ANF and VEGF, the latter of which was recently shown to augment cardiac force by increasing the heart's calcium transients. -
Mably JD, Chuang LP, Serluca FC, Mohideen MA, Chen JN, Fishman MC. 2006. santa and valentine pattern concentric growth of cardiac myocardium in the zebrafish. Development (Cambridge, England). 133(16):3139-46. Pubmed: 16873582 Mably JD, Chuang LP, Serluca FC, Mohideen MA, Chen JN, Fishman MC. 2006. santa and valentine pattern concentric growth of cardiac myocardium in the zebrafish. Development (Cambridge, England). 133(16):3139-46. Pubmed: 16873582 During embryogenesis, the myocardial layer of the primitive heart tube grows outward from the endocardial-lined lumen, with new cells added to generate concentric thickness to the wall. This is a key evolutionary step, demarcating vertebrates from more primitive chordates, and is essential for normal cardiac function. Zebrafish embryos with the recessive lethal mutations santa (san) and valentine (vtn) do not thicken, but do add the proper number of cells to the myocardium. Consequently, the heart chambers are huge, constituted of a monolayered myocardium lined by endocardium. This phenotype is similar to that of the heart of glass (heg) mutation, which we described previously as a novel endocardial expressed gene. By positional cloning, we here identify san as the zebrafish homolog of human CCM1, and vtn as the homolog of human CCM2. Dominant mutations of either in humans cause vascular anomalies in the brain, known as cerebral cavernous malformations. The synergistic effects of morpholino pairs indicate that san, vtn and heg are in a genetic pathway, and san and vtn contain protein motifs, NPxY and PTB domain, respectively, known to interact. This suggests that concentric growth of the myocardium, crucial for blood pressure generation, is dictated by a heg-san-vtn signaling pathway. -
Rottbauer W, Wessels G, Dahme T, Just S, Trano N, Hassel D, Burns CG, Katus HA, Fishman MC. 2006. Cardiac myosin light chain-2: a novel essential component of thick-myofilament assembly and contractility of the heart. Circulation research. 99(3):323-31. Pubmed: 16809551 Rottbauer W, Wessels G, Dahme T, Just S, Trano N, Hassel D, Burns CG, Katus HA, Fishman MC. 2006. Cardiac myosin light chain-2: a novel essential component of thick-myofilament assembly and contractility of the heart. Circulation research. 99(3):323-31. Pubmed: 16809551 Although it is well known that mutations in the cardiac regulatory myosin light chain-2 (mlc-2) gene cause hypertrophic cardiomyopathy, the precise in vivo structural and functional roles of MLC-2 in the heart are only poorly understood. We have isolated a mutation in zebrafish, tell tale heart (tel(m225)), which selectively perturbs contractility of the embryonic heart. By positional cloning, we identified tel to encode the zebrafish mlc-2 gene. In contrast to mammals, zebrafish have only 1 cardiac-specific mlc-2 gene, which we find to be expressed in atrial and ventricular cardiomyocytes during early embryonic development, but also in the adult heart. Accordingly, loss of zMLC-2 function cannot be compensated for by upregulation of another mlc-2 gene. Surprisingly, ultrastructural analysis of tel cardiomyocytes reveals complete absence of organized thick myofilaments. Thus, our findings provide the first in vivo evidence that cardiac MLC-2 is required for thick-filament stabilization and contractility in the vertebrate heart. -
Serluca FC, Fishman MC. 2006. Big, bad hearts: from flies to man. Proceedings of the National Academy of Sciences of the United States of America. 103(11):3947-8. Pubmed: 16537463 Serluca FC, Fishman MC. 2006. Big, bad hearts: from flies to man. Proceedings of the National Academy of Sciences of the United States of America. 103(11):3947-8. Pubmed: 16537463 2005
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Ebert AM, Hume GL, Warren KS, Cook NP, Burns CG, Mohideen MA, Siegal G, Yelon D, Fishman MC, Garrity DM. 2005. Calcium extrusion is critical for cardiac morphogenesis and rhythm in embryonic zebrafish hearts. Proceedings of the National Academy of Sciences of the United States of America. 102(49):17705-10. Pubmed: 16314582 Ebert AM, Hume GL, Warren KS, Cook NP, Burns CG, Mohideen MA, Siegal G, Yelon D, Fishman MC, Garrity DM. 2005. Calcium extrusion is critical for cardiac morphogenesis and rhythm in embryonic zebrafish hearts. Proceedings of the National Academy of Sciences of the United States of America. 102(49):17705-10. Pubmed: 16314582 Calcium entry into myocytes drives contraction of the embryonic heart. To prepare for the next contraction, myocytes must extrude calcium from intracellular space via the Na+/Ca2+ exchanger (NCX1) or sequester it into the sarcoplasmic reticulum, via the sarcoplasmic reticulum Ca2+-ATPase2 (SERCA2). In mammals, defective calcium extrusion correlates with increased intracellular calcium levels and may be relevant to heart failure and sarcoplasmic dysfunction in adults. We report here that mutation of the cardiac-specific NCX1 (NCX1h) gene causes embryonic lethal cardiac arrhythmia in zebrafish tremblor (tre) embryos. The tre ventricle is nearly silent, whereas the atrium manifests a variety of arrhythmias including fibrillation. Calcium extrusion defects in tre mutants correlate with severe disruptions in sarcomere assembly, whereas mutations in the L-type calcium channel that abort calcium entry do not produce this phenotype. Knockdown of SERCA2 activity by morpholino-mediated translational inhibition or pharmacological inhibition causes embryonic lethality due to defects in cardiac contractility and morphology but, in contrast to tre mutation, does not produce arrhythmia. Analysis of intracellular calcium levels indicates that homozygous tre embryos develop calcium overload, which may contribute to the degeneration of cardiac function in this mutant. Thus, the inhibition of NCX1h versus SERCA2 activity differentially affects the pathophysiology of rhythm in the developing heart and suggests that relative levels of NCX1 and SERCA2 function are essential for normal development. -
Fishman MC, Porter JA. 2005. Pharmaceuticals: a new grammar for drug discovery. Nature. 437(7058):491-3. Pubmed: 16177777 Fishman MC, Porter JA. 2005. Pharmaceuticals: a new grammar for drug discovery. Nature. 437(7058):491-3. Pubmed: 16177777 -
Fishman M. 2005. Mark Fishman on changing the grammar of drug discovery. Interviewed by Christopher Watson. Drug discovery today. 10(9):609-11. Pubmed: 15894223 Fishman M. 2005. Mark Fishman on changing the grammar of drug discovery. Interviewed by Christopher Watson. Drug discovery today. 10(9):609-11. Pubmed: 15894223 -
Rottbauer W, Just S, Wessels G, Trano N, Most P, Katus HA, Fishman MC. 2005. VEGF-PLCgamma1 pathway controls cardiac contractility in the embryonic heart. Genes & development. 19(13):1624-34. Pubmed: 15998812 Rottbauer W, Just S, Wessels G, Trano N, Most P, Katus HA, Fishman MC. 2005. VEGF-PLCgamma1 pathway controls cardiac contractility in the embryonic heart. Genes & development. 19(13):1624-34. Pubmed: 15998812 The strength of the heart beat can accommodate in seconds to changes in blood pressure or flow. The mechanism for such homeostatic adaptation is unknown. We sought the cause of poor contractility in the heart of the embryonic zebrafish with the mutation dead beat. We find through cloning that this is due to a mutation in the phospholipase C gamma1 (plcgamma1) gene. In mutant embryos, contractile function can be restored by PLCgamma1 expression directed selectively to cardiac myocytes. In other situations, PLCgamma1 is known to transduce the signal from vascular endothelial growth factor (VEGF), and we show here that abrogation of VEGF also interferes with cardiac contractility. Somewhat unexpectedly, FLT-1 is the responsible VEGF receptor. We show that the same system functions in the rat. Blockage of VEGF-PLCgamma1 signaling decreases calcium transients in rat ventricular cardiomyocytes, whereas VEGF imposes a positive inotropic effect on cardiomyocytes by increasing calcium transients. Thus, the muscle of the heart uses the VEGF-PLCgamma1 cascade to control the strength of the heart beat. We speculate that this paracrine system may contribute to normal and pathological regulation of cardiac contractility. -
Burns CG, Milan DJ, Grande EJ, Rottbauer W, MacRae CA, Fishman MC. 2005. High-throughput assay for small molecules that modulate zebrafish embryonic heart rate. Nature chemical biology. 1(5):263-4. Pubmed: 16408054 Burns CG, Milan DJ, Grande EJ, Rottbauer W, MacRae CA, Fishman MC. 2005. High-throughput assay for small molecules that modulate zebrafish embryonic heart rate. Nature chemical biology. 1(5):263-4. Pubmed: 16408054 To increase the facility and throughput of scoring phenotypic traits in embryonic zebrafish, we developed an automated micro-well assay for heart rate using automated fluorescence microscopy of transgenic embryos expressing green fluorescent protein in myocardium. The assay measures heart rates efficiently and accurately over a large linear dynamic range, and it rapidly characterizes dose dependence and kinetics of small molecule-induced changes in heart rate. This is the first high-throughput micro-well assay for organ function in an intact vertebrate. 2004
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Torres-Vázquez J, Gitler AD, Fraser SD, Berk JD, Van N Pham, Fishman MC, Childs S, Epstein JA, Weinstein BM. 2004. Semaphorin-plexin signaling guides patterning of the developing vasculature. Developmental cell. 7(1):117-23. Pubmed: 15239959 Torres-Vázquez J, Gitler AD, Fraser SD, Berk JD, Van N Pham, Fishman MC, Childs S, Epstein JA, Weinstein BM. 2004. Semaphorin-plexin signaling guides patterning of the developing vasculature. Developmental cell. 7(1):117-23. Pubmed: 15239959 Major vessels of the vertebrate circulatory system display evolutionarily conserved and reproducible anatomy, but the cues guiding this stereotypic patterning remain obscure. In the nervous system, axonal pathways are shaped by repulsive cues provided by ligands of the semaphorin family that are sensed by migrating neuronal growth cones through plexin receptors. We show that proper blood vessel pathfinding requires the endothelial receptor PlexinD1 and semaphorin signals, and we identify mutations in plexinD1 in the zebrafish vascular patterning mutant out of bounds. These results reveal the fundamental conservation of repulsive patterning mechanisms between axonal migration in the central nervous system and vascular endothelium during angiogenesis. -
Peterson RT, Shaw SY, Peterson TA, Milan DJ, Zhong TP, Schreiber SL, MacRae CA, Fishman MC. 2004. Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation. Nature biotechnology. 22(5):595-9. Pubmed: 15097998 Peterson RT, Shaw SY, Peterson TA, Milan DJ, Zhong TP, Schreiber SL, MacRae CA, Fishman MC. 2004. Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation. Nature biotechnology. 22(5):595-9. Pubmed: 15097998 Conventional drug discovery approaches require a priori selection of an appropriate molecular target, but it is often not obvious which biological pathways must be targeted to reverse a disease phenotype. Phenotype-based screens offer the potential to identify pathways and potential therapies that influence disease processes. The zebrafish mutation gridlock (grl, affecting the gene hey2) disrupts aortic blood flow in a region and physiological manner akin to aortic coarctation in humans. Here we use a whole-organism, phenotype-based, small-molecule screen to discover a class of compounds that suppress the coarctation phenotype and permit survival to adulthood. These compounds function during the specification and migration of angioblasts. They act to upregulate expression of vascular endothelial growth factor (VEGF), and the activation of the VEGF pathway is sufficient to suppress the gridlock phenotype. Thus, organism-based screens allow the discovery of small molecules that ameliorate complex dysmorphic syndromes even without targeting the affected gene directly. -
Fishman MC. 2004. Mark C. Fishman. Nature reviews. Drug discovery. 3(4):292. Pubmed: 15124621 Fishman MC. 2004. Mark C. Fishman. Nature reviews. Drug discovery. 3(4):292. Pubmed: 15124621 -
Peterson RT, Fishman MC. 2004. Discovery and use of small molecules for probing biological processes in zebrafish. Methods in cell biology. 76:569-91. Pubmed: 15602893 Peterson RT, Fishman MC. 2004. Discovery and use of small molecules for probing biological processes in zebrafish. Methods in cell biology. 76:569-91. Pubmed: 15602893 2003
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Mably JD, Mohideen MA, Burns CG, Chen JN, Fishman MC. 2003. heart of glass regulates the concentric growth of the heart in zebrafish. Current biology : CB. 13(24):2138-47. Pubmed: 14680629 Mably JD, Mohideen MA, Burns CG, Chen JN, Fishman MC. 2003. heart of glass regulates the concentric growth of the heart in zebrafish. Current biology : CB. 13(24):2138-47. Pubmed: 14680629 Array -
Mayer AN, Fishman MC. 2003. Nil per os encodes a conserved RNA recognition motif protein required for morphogenesis and cytodifferentiation of digestive organs in zebrafish. Development (Cambridge, England). 130(17):3917-28. Pubmed: 12874115 Mayer AN, Fishman MC. 2003. Nil per os encodes a conserved RNA recognition motif protein required for morphogenesis and cytodifferentiation of digestive organs in zebrafish. Development (Cambridge, England). 130(17):3917-28. Pubmed: 12874115 Digestive organ development occurs through a sequence of morphologically distinct stages, from overtly featureless endoderm, through organ primordia to, ultimately, adult form. The developmental controls that govern progression from one stage to the next are not well understood. To identify genes required for the formation of vertebrate digestive organs we performed a genetic screen in zebrafish. We isolated the nil per os (npo) mutation, which arrests morphogenesis and cytodifferentiation of the gut and exocrine pancreas in a primordial state. We identified the npo gene by positional cloning. It encodes a conserved protein, with multiple RNA recognition motifs, that is related to the yeast protein Mrd1p. During development npo is expressed in a dynamic fashion, functioning cell autonomously to promote organ cytodifferentiation. Antisense-mediated knockdown of npo results in organ hypoplasia, and overexpression of npo causes an overgrowth of gastrointestinal organs. Thus, npo is a gene essential for a key step in the gut morphogenetic sequence. 2002
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MacRae CA, Fishman MC. 2002. Zebrafish: the complete cardiovascular compendium. Cold Spring Harbor symposia on quantitative biology. 67:301-7. Pubmed: 12858553 MacRae CA, Fishman MC. 2002. Zebrafish: the complete cardiovascular compendium. Cold Spring Harbor symposia on quantitative biology. 67:301-7. Pubmed: 12858553 -
Serluca FC, Drummond IA, Fishman MC. 2002. Endothelial signaling in kidney morphogenesis: a role for hemodynamic forces. Current biology : CB. 12(6):492-7. Pubmed: 11909536 Serluca FC, Drummond IA, Fishman MC. 2002. Endothelial signaling in kidney morphogenesis: a role for hemodynamic forces. Current biology : CB. 12(6):492-7. Pubmed: 11909536 The local presence of endothelial cells seems necessary for proper embryonic development of several organs. However, the signals involved are unknown. The glomerulus is generated by the coalescence of podocytes around an ingrowing capillary and is the site of blood ultrafiltration. In the absence of vessels, glomerular assembly does not occur. We describe mutations in the zebrafish that prevent glomerulogenesis. All mutants display cardiac dysfunction. Pharmacological interference with cardiac output and focal laser occlusion of the vessel similarly prevent glomerular formation. The unifying feature of all these perturbations is absence of blood flow. We find that expression of matrix metalloproteinase-2 (MMP-2), known in other systems to be regulated in a stretch-responsive manner, is in renal endothelial cells and is regulated by flow, suggesting that an MMP-2-sensitive event may be downstream of the flow-related signal. In support of this, blockade of MMP-2 activity by injection of TIMP-2 does not perturb circulation but does prevent glomerular assembly. Thus, vascular flow is required for glomerular assembly, most probably acting via a stretch-responsive signaling system in the vessel wall. -
Childs S, Chen JN, Garrity DM, Fishman MC. 2002. Patterning of angiogenesis in the zebrafish embryo. Development (Cambridge, England). 129(4):973-82. Pubmed: 11861480 Childs S, Chen JN, Garrity DM, Fishman MC. 2002. Patterning of angiogenesis in the zebrafish embryo. Development (Cambridge, England). 129(4):973-82. Pubmed: 11861480 Little is known about how vascular patterns are generated in the embryo. The vasculature of the zebrafish trunk has an extremely regular pattern. One intersegmental vessel (ISV) sprouts from the aorta, runs between each pair of somites, and connects to the dorsal longitudinal anastomotic vessel (DLAV). We now define the cellular origins, migratory paths and cell fates that generate these metameric vessels of the trunk. Additionally, by a genetic screen we define one gene, out of bounds (obd), that constrains this angiogenic growth to a specific path. We have performed lineage analysis, using laser activation of a caged dye and mosaic construction to determine the origin of cells that constitute the ISV. Individual angioblasts destined for the ISVs arise from the lateral posterior mesoderm (LPM), and migrate to the dorsal aorta, from where they migrate between somites to their final position in the ISVs and dorsal longitudinal anastomotic vessel (DLAV). Cells of each ISV leave the aorta only between the ventral regions of two adjacent somites, and migrate dorsally to assume one of three ISV cell fates. Most dorsal is a T-shaped cell, based in the DLAV and branching ventrally; the second constitutes a connecting cell; and the third an inverted T-shaped cell, based in the aorta and branching dorsally. The ISV remains between somites during its ventral course, but changes to run mid-somite dorsally. This suggests that the pattern of ISV growth ventrally and dorsally is guided by different cues. We have also performed an ENU mutagenesis screen of 750 mutagenized genomes and identified one mutation, obd that disrupts this pattern. In obd mutant embryos, ISVs sprout precociously at abnormal sites and migrate anomalously in the vicinity of ventral somite. The dorsal extent of the ISV is less perturbed. Precocious sprouting can be inhibited in a VEGF morphant, but the anomalous site of origin of obd ISVs remains. In mosaic embryos, obd somite causes adjacent wild-type endothelial cells to assume the anomalous ISV pattern of obd embryos. Thus, the launching position of the new sprout and its initial trajectory are directed by inhibitory signals from ventral somites. Zebrafish ISVs are a tractable system for defining the origins and fates of vessels, and for dissecting elements that govern patterns of vessel growth. -
Yelon D, Weinstein BM, Fishman MC. 2002. Cardiovascular system. Results and problems in cell differentiation. 40:298-321. Pubmed: 12353483 Yelon D, Weinstein BM, Fishman MC. 2002. Cardiovascular system. Results and problems in cell differentiation. 40:298-321. Pubmed: 12353483 -
Xu X, Meiler SE, Zhong TP, Mohideen M, Crossley DA, Burggren WW, Fishman MC. 2002. Cardiomyopathy in zebrafish due to mutation in an alternatively spliced exon of titin. Nature genetics. 30(2):205-9. Pubmed: 11788825 Xu X, Meiler SE, Zhong TP, Mohideen M, Crossley DA, Burggren WW, Fishman MC. 2002. Cardiomyopathy in zebrafish due to mutation in an alternatively spliced exon of titin. Nature genetics. 30(2):205-9. Pubmed: 11788825 The zebrafish embryo is transparent and can tolerate absence of blood flow because its oxygen is delivered by diffusion rather than by the cardiovascular system. It is therefore possible to attribute cardiac failure directly to particular genes by ruling out the possibility that it is due to a secondary effect of hypoxia. We focus here on pickwickm171 (pikm171), a recessive lethal mutation discovered in a large-scale genetic screen. There are three other alleles in the pik complementation group with this phenotype (pikm242, pikm740, pikm186; ref. 3) and one allele (pikmVO62H) with additional skeletal paralysis. The pik heart develops normally but is poorly contractile from the first beat. Aside from the edema that inevitably accompanies cardiac dysfunction, development is normal during the first three days. We show by positional cloning that the 'causative' mutation is in an alternatively-spliced exon of the gene (ttn) encoding Titin. Titin is the biggest known protein and spans the half-sarcomere from Z-disc to M-line in heart and skeletal muscle. It has been proposed to provide a scaffold for the assembly of thick and thin filaments and to provide elastic recoil engendered by stretch during diastole. We found that nascent myofibrils form in pik mutants, but normal sarcomeres are absent. Mutant cells transplanted to wildtype hearts remain thin and bulge outwards as individual cell aneurysms without affecting nearby wildtype cardiomyocytes, indicating that the contractile deficiency is cell-autonomous. Absence of Titin function thus results in blockage of sarcomere assembly and causes a functional disorder resembling human dilated cardiomyopathies, one form of which is described in another paper in this issue. -
Ando A, Yang A, Mori K, Yamada H, Yamada E, Takahashi K, Saikia J, Kim M, Melia M, Fishman M, Huang P, Campochiaro PA. 2002. Nitric oxide is proangiogenic in the retina and choroid. Journal of cellular physiology. 191(1):116-24. Pubmed: 11920687 Ando A, Yang A, Mori K, Yamada H, Yamada E, Takahashi K, Saikia J, Kim M, Melia M, Fishman M, Huang P, Campochiaro PA. 2002. Nitric oxide is proangiogenic in the retina and choroid. Journal of cellular physiology. 191(1):116-24. Pubmed: 11920687 Nitric oxide (NO) has been shown to have proangiogenic or antiangiogenic effects depending upon the setting. In this study, we used mice with targeted deletion of one of the three isoforms of nitric oxide synthase (NOS) to investigate the effects of NO in ocular neovascularization. In transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors, deficiency of any of the three isoforms caused a significant decrease in subretinal neovascularization, but no alteration of VEGF expression. In mice with laser-induced rupture of Bruch's membrane, deficiency of inducible NOS (iNOS) or neuronal NOS (nNOS), but not endothelial NOS (eNOS), caused a significant decrease in choroidal neovascularization. In mice with oxygen-induced ischemic retinopathy, deficiency of eNOS, but not iNOS or nNOS caused a significant decrease in retinal neovascularization and decreased expression of VEGF. These data suggest that NO contributes to both retinal and choroidal neovascularization and that different isoforms of NOS are involved in different settings and different disease processes. A broad spectrum NOS inhibitor may have therapeutic potential for treatment of both retinal and choroidal neovascularization.Copyright 2002 Wiley-Liss, Inc. -
Rottbauer W, Saurin AJ, Lickert H, Shen X, Burns CG, Wo ZG, Kemler R, Kingston R, Wu C, Fishman M. 2002. Reptin and pontin antagonistically regulate heart growth in zebrafish embryos. Cell. 111(5):661-72. Pubmed: 12464178 Rottbauer W, Saurin AJ, Lickert H, Shen X, Burns CG, Wo ZG, Kemler R, Kingston R, Wu C, Fishman M. 2002. Reptin and pontin antagonistically regulate heart growth in zebrafish embryos. Cell. 111(5):661-72. Pubmed: 12464178 Organ size is precisely regulated during development, but the control mechanisms remain obscure. We have isolated a mutation in zebrafish, liebeskummer (lik), which causes development of hyperplastic embryonic hearts. lik encodes Reptin, a component of a DNA-stimulated ATPase complex. The mutation activates ATPase activity of Reptin complexes and causes a cell-autonomous proliferation of cardiomyocytes to begin well after progenitors have fashioned the primitive heart tube. With regard to heart growth, beta-catenin and Pontin, a DNA-stimulated ATPase that is often part of complexes with Reptin, are in the same genetic pathways. Pontin reduction phenocopies the cardiac hyperplasia of the lik mutation. Thus, the Reptin/Pontin ratio serves to regulate heart growth during development, at least in part via the beta-catenin pathway. -
Shin JT, Fishman MC. 2002. From Zebrafish to human: modular medical models. Annual review of genomics and human genetics. 3:311-40. Pubmed: 12142362 Shin JT, Fishman MC. 2002. From Zebrafish to human: modular medical models. Annual review of genomics and human genetics. 3:311-40. Pubmed: 12142362 Genetic screens in Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio clarified the logic of metazoan development by revealing critical unitary steps and pathways to embryogenesis. Can genetic screens similarly organize medicine? We here examine human diseases that resemble mutations in Danio rerio, the zebrafish, the one vertebrate species for which large-scale genetic screens have been performed and extensively analyzed. Zebrafish mutations faithfully phenocopy many human disorders. Each mutation, once cloned, provides candidate genes and pathways for evaluation in the human. The collection of mutations in their entirety potentially provides a medical taxonomy, one based in developmental biology and genetics. -
Sehnert AJ, Huq A, Weinstein BM, Walker C, Fishman M, Stainier DY. 2002. Cardiac troponin T is essential in sarcomere assembly and cardiac contractility. Nature genetics. 31(1):106-10. Pubmed: 11967535 Sehnert AJ, Huq A, Weinstein BM, Walker C, Fishman M, Stainier DY. 2002. Cardiac troponin T is essential in sarcomere assembly and cardiac contractility. Nature genetics. 31(1):106-10. Pubmed: 11967535 Mutations of the gene (TNNT2) encoding the thin-filament contractile protein cardiac troponin T are responsible for 15% of all cases of familial hypertrophic cardiomyopathy, the leading cause of sudden death in young athletes. Mutant proteins are thought to act through a dominant-negative mode that impairs function of heart muscle. TNNT2 mutations can also lead to dilated cardiomyopathy, a leading cause of heart failure. Despite the importance of cardiac troponin T in human disease, its loss-of-function phenotype has not been described. We show that the zebrafish silent heart (sih) mutation affects the gene tnnt2. We characterize two mutated alleles of sih that severely reduce tnnt2 expression: one affects mRNA splicing, and the other affects gene transcription. Tnnt2, together with alpha-tropomyosin (Tpma) and cardiac troponins C and I (Tnni3), forms a calcium-sensitive regulatory complex within sarcomeres. Unexpectedly, in addition to loss of Tnnt2 expression in sih mutant hearts, we observed a significant reduction in Tpma and Tnni3, and consequently, severe sarcomere defects. This interdependence of thin-filament protein expression led us to postulate that some mutations in tnnt2 may trigger misregulation of thin-filament protein expression, resulting in sarcomere loss and myocyte disarray, the life-threatening hallmarks of TNNT2 mutations in mice and humans. -
Roman BL, Pham VN, Lawson ND, Kulik M, Childs S, Lekven AC, Garrity DM, Moon RT, Fishman MC, Lechleider RJ, Weinstein BM. 2002. Disruption of acvrl1 increases endothelial cell number in zebrafish cranial vessels. Development (Cambridge, England). 129(12):3009-19. Pubmed: 12050147 Roman BL, Pham VN, Lawson ND, Kulik M, Childs S, Lekven AC, Garrity DM, Moon RT, Fishman MC, Lechleider RJ, Weinstein BM. 2002. Disruption of acvrl1 increases endothelial cell number in zebrafish cranial vessels. Development (Cambridge, England). 129(12):3009-19. Pubmed: 12050147 The zebrafish mutant violet beauregarde (vbg) can be identified at two days post-fertilization by an abnormal circulation pattern in which most blood cells flow through a limited number of dilated cranial vessels and fail to perfuse the trunk and tail. This phenotype cannot be explained by caudal vessel abnormalities or by a defect in cranial vessel patterning, but instead stems from an increase in endothelial cell number in specific cranial vessels. We show that vbg encodes activin receptor-like kinase 1 (Acvrl1; also known as Alk1), a TGFbeta type I receptor that is expressed predominantly in the endothelium of the vessels that become dilated in vbg mutants. Thus, vbg provides a model for the human autosomal dominant disorder, hereditary hemorrhagic telangiectasia type 2, in which disruption of ACVRL1 causes vessel malformations that may result in hemorrhage or stroke. Movies available on-line -
Ng JK, Kawakami Y, Büscher D, Raya A, Itoh T, Koth CM, Rodríguez Esteban C, Rodríguez-León J, Garrity DM, Fishman MC, Izpisúa Belmonte JC. 2002. The limb identity gene Tbx5 promotes limb initiation by interacting with Wnt2b and Fgf10. Development (Cambridge, England). 129(22):5161-70. Pubmed: 12399308 Ng JK, Kawakami Y, Büscher D, Raya A, Itoh T, Koth CM, Rodríguez Esteban C, Rodríguez-León J, Garrity DM, Fishman MC, Izpisúa Belmonte JC. 2002. The limb identity gene Tbx5 promotes limb initiation by interacting with Wnt2b and Fgf10. Development (Cambridge, England). 129(22):5161-70. Pubmed: 12399308 A major gap in our knowledge of development is how the growth and identity of tissues and organs are linked during embryogenesis. The vertebrate limb is one of the best models to study these processes. Combining mutant analyses with gain- and loss-of-function approaches in zebrafish and chick embryos, we show that Tbx5, in addition to its role governing forelimb identity, is both necessary and sufficient for limb outgrowth. We find that Tbx5 functions downstream of WNT signaling to regulate Fgf10, which, in turn, maintains Tbx5 expression during limb outgrowth. Furthermore, our results indicate that Tbx5 and Wnt2b function together to initiate and specify forelimb outgrowth and identity. The molecular interactions governed by members of the T-box, Wnt and Fgf gene families uncovered in this study provide a framework for understanding not only limb development, but how outgrowth and identity of other tissues and organs of the embryo may be regulated. -
Garrity DM, Childs S, Fishman MC. 2002. The heartstrings mutation in zebrafish causes heart/fin Tbx5 deficiency syndrome. Development (Cambridge, England). 129(19):4635-45. Pubmed: 12223419 Garrity DM, Childs S, Fishman MC. 2002. The heartstrings mutation in zebrafish causes heart/fin Tbx5 deficiency syndrome. Development (Cambridge, England). 129(19):4635-45. Pubmed: 12223419 Holt-Oram syndrome is one of the autosomal dominant human "heart-hand" disorders, with a combination of upper limb malformations and cardiac defects. Holt-Oram syndrome is caused by mutations in the TBX5 gene, a member of a large family of T-box transcription factors that play important roles in cell-type specification and morphogenesis. In a screen for mutations affecting zebrafish cardiac function, we isolated the recessive lethal mutant heartstrings, which lacks pectoral fins and exhibits severe cardiac dysfunction, beginning with a slow heart rate and progressing to a stretched, non-functional heart. We mapped and cloned the heartstrings mutation and find it to encode the zebrafish ortholog of the TBX5 gene. The heartstrings mutation causes premature termination at amino acid 316. Homozygous mutant embryos never develop pectoral fin buds and do not express several markers of early fin differentiation. The total absence of any fin bud differentiation distinguishes heartstrings from most other mutations that affect zebrafish fin development, suggesting that Tbx5 functions very early in the pectoral fin induction pathway. Moderate reduction of Tbx5 by morpholino causes fin malformations, revealing an additional early requirement for Tbx5 in coordinating the axes of fin outgrowth. The heart of heartstrings mutant embryos appears to form and function normally through the early heart tube stage, manifesting only a slight bradycardia compared with wild-type siblings. However, the heart fails to loop and then progressively deteriorates, a process affecting the ventricle as well as the atrium. Relative to mammals, fish require lower levels of Tbx5 to produce malformed appendages and display whole-heart rather than atrial-predominant cardiac defects. However, the syndromic deficiencies of tbx5 mutation are remarkably well retained between fish and mammals. -
Chen J, Tung CH, Mahmood U, Ntziachristos V, Gyurko R, Fishman MC, Huang PL, Weissleder R. 2002. In vivo imaging of proteolytic activity in atherosclerosis. Circulation. 105(23):2766-71. Pubmed: 12057992 Chen J, Tung CH, Mahmood U, Ntziachristos V, Gyurko R, Fishman MC, Huang PL, Weissleder R. 2002. In vivo imaging of proteolytic activity in atherosclerosis. Circulation. 105(23):2766-71. Pubmed: 12057992 Array 2001
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Zhong TP, Childs S, Leu JP, Fishman MC. 2001. Gridlock signalling pathway fashions the first embryonic artery. Nature. 414(6860):216-20. Pubmed: 11700560 Zhong TP, Childs S, Leu JP, Fishman MC. 2001. Gridlock signalling pathway fashions the first embryonic artery. Nature. 414(6860):216-20. Pubmed: 11700560 Arteries and veins are morphologically, functionally and molecularly very different, but how this distinction is established during vasculogenesis is unknown. Here we show, by lineage tracking in zebrafish embryos, that angioblast precursors for the trunk artery and vein are spatially mixed in the lateral posterior mesoderm. Progeny of each angioblast, however, are restricted to one of the vessels. This arterial-venous decision is guided by gridlock (grl), an artery-restricted gene that is expressed in the lateral posterior mesoderm. Graded reduction of grl expression, by mutation or morpholino antisense, progressively ablates regions of the artery, and expands contiguous regions of the vein, preceded by an increase in expression of the venous marker EphB4 receptor (ephb4) and diminution of expression of the arterial marker ephrin-B2 (efnb2). grl is downstream of notch, and interference with notch signalling, by blocking Su(H), similarly reduces the artery and increases the vein. Thus, a notch-grl pathway controls assembly of the first embryonic artery, apparently by adjudicating an arterial versus venous cell fate decision. -
Fishman MC. 2001. Genomics. Zebrafish--the canonical vertebrate. Science (New York, N.Y.). 294(5545):1290-1. Pubmed: 11701913 Fishman MC. 2001. Genomics. Zebrafish--the canonical vertebrate. Science (New York, N.Y.). 294(5545):1290-1. Pubmed: 11701913 -
Serluca FC, Sidow A, Mably JD, Fishman MC. 2001. Partitioning of tissue expression accompanies multiple duplications of the Na+/K+ ATPase alpha subunit gene. Genome research. 11(10):1625-31. Pubmed: 11591639 Serluca FC, Sidow A, Mably JD, Fishman MC. 2001. Partitioning of tissue expression accompanies multiple duplications of the Na+/K+ ATPase alpha subunit gene. Genome research. 11(10):1625-31. Pubmed: 11591639 Vertebrate genomes contain multiple copies of related genes that arose through gene duplication. In the past it has been proposed that these duplicated genes were retained because of acquisition of novel beneficial functions. A more recent model, the duplication-degeneration-complementation hypothesis (DDC), posits that the functions of a single gene may become separately allocated among the duplicated genes, rendering both duplicates essential. Thus far, empirical evidence for this model has been limited to the engrailed and sox family of developmental regulators, and it has been unclear whether it may also apply to ubiquitously expressed genes with essential functions for cell survival. Here we describe the cloning of three zebrafish alpha subunits of the Na(+),K(+)-ATPase and a comprehensive evolutionary analysis of this gene family. The predicted amino acid sequences are extremely well conserved among vertebrates. The evolutionary relationships and the map positions of these genes and of other alpha-like sequences indicate that both tandem and ploidy duplications contributed to the expansion of this gene family in the teleost lineage. The duplications are accompanied by acquisition of clear functional specialization, consistent with the DDC model of genome evolution. -
Rottbauer W, Baker K, Wo ZG, Mohideen MA, Cantiello HF, Fishman MC. 2001. Growth and function of the embryonic heart depend upon the cardiac-specific L-type calcium channel alpha1 subunit. Developmental cell. 1(2):265-75. Pubmed: 11702785 Rottbauer W, Baker K, Wo ZG, Mohideen MA, Cantiello HF, Fishman MC. 2001. Growth and function of the embryonic heart depend upon the cardiac-specific L-type calcium channel alpha1 subunit. Developmental cell. 1(2):265-75. Pubmed: 11702785 The heart must function from the moment of its embryonic assembly, but the molecular underpinnings of the first heart beat are not known, nor whether function determines form at this early stage. Here, we find by positional cloning that the embryonic lethal island beat (isl) mutation in zebrafish disrupts the alpha1 C L-type calcium channel subunit (C-LTCC). The isl atrium is relatively normal in size, and individual cells contract chaotically, in a pattern resembling atrial fibrillation. The ventricle is completely silent. Unlike another mutation with a silent ventricle, isl fails to acquire the normal number of myocytes. Thus, calcium signaling via C-LTCC can regulate heart growth independently of contraction, and plays distinctive roles in fashioning both form and function of the two developing chambers. -
Serluca FC, Fishman MC. 2001. Pre-pattern in the pronephric kidney field of zebrafish. Development (Cambridge, England). 128(12):2233-41. Pubmed: 11493543 Serluca FC, Fishman MC. 2001. Pre-pattern in the pronephric kidney field of zebrafish. Development (Cambridge, England). 128(12):2233-41. Pubmed: 11493543 Vertebrate embryos use a series of transient kidneys to regulate fluid balance, osmolarity and metabolic waste during development. The first kidney to form in the embryo is the pronephros. This kidney is composed of several cell types with very different functions and is organized into discrete segments: glomerulus, tubules and nephric duct. The site of origin of these cells is poorly understood, as are their lineage relationships. We have defined regions of the intermediate mesoderm as candidates for the pronephric field by expression patterns of the Wilms' Tumor suppressor gene (wt1), single-minded 1 (sim1) and pax2.1. All of these potential kidney markers are expressed in a stripe of intermediate mesoderm, with distinct, overlapping antero-posterior borders. We labeled small groups of cells in this area by laser uncaging of a fluorescent dextran, and then tracked their fates. We found that there was a bounded contiguous region of the intermediate mesoderm that provides pronephric progenitors. As is true for other organ fields, the pronephric field regulates after focal destruction, such that a normal pronephros forms after laser-mediated removal of the wt1 domain. The progenitors for podocytes, tubular cells and duct are restricted to subdomains within the pronephric field. The most anterior cells in the pronephric field give rise to podocytes. This corresponds to the wt1-expressing region. The next more posterior cells contribute to the tubule, and express both wt1 and pax2.1. The most posterior cells contribute to the nephric duct, and these express pax2.1 and sim1, but not wt1. Thus, there is a field for the pronephric kidney with classical attributes of defined border, pre-pattern and regulation. The pattern of the fate map reflects particular combinations of transcription factors. -
Fishman MC. 2001. The genomic cosmos. Nature. 410(6832):1033. Pubmed: 11323648 Fishman MC. 2001. The genomic cosmos. Nature. 410(6832):1033. Pubmed: 11323648 -
Chen JN, van Bebber F, Goldstein AM, Serluca FC, Jackson D, Childs S, Serbedzija G, Warren KS, Mably JD, Lindahl P, Mayer A, Haffter P, Fishman MC. 2001. Genetic steps to organ laterality in zebrafish. Comparative and functional genomics. 2(2):60-8. Pubmed: 18628903 DOI:10.1002/cfg.74 Chen JN, van Bebber F, Goldstein AM, Serluca FC, Jackson D, Childs S, Serbedzija G, Warren KS, Mably JD, Lindahl P, Mayer A, Haffter P, Fishman MC. 2001. Genetic steps to organ laterality in zebrafish. Comparative and functional genomics. 2(2):60-8. Pubmed: 18628903 DOI:10.1002/cfg.74 All internal organs are asymmetric along the left-right axis. Here we report a genetic screen to discover mutations which perturb organ laterality. Our particular focus is upon whether, and how, organs are linked to each other as they achieve their laterally asymmetric positions. We generated mutations by ENU mutagenesis and examined F3 progeny using a cocktail of probes that reveal early primordia of heart, gut, liver and pancreas. From the 750 genomes examined, we isolated seven recessive mutations which affect the earliest left-right positioning of one or all of the organs. None of these mutations caused discernable defects elsewhere in the embryo at the stages examined. This is in contrast to those mutations we reported previously (Chen et al., 1997) which, along with left-right abnormalities, cause marked perturbation in gastrulation, body form or midline structures. We find that the mutations can be classified on the basis of whether they perturb relationships among organ laterality. In Class 1 mutations, none of the organs manifest any left-right asymmetry. The heart does not jog to the left and normally leftpredominant BMP4 in the early heart tube remains symmetric. The gut tends to remain midline. There frequently is a remarkable bilateral duplication of liver and pancreas. Embryos with Class 2 mutations have organotypic asymmetry but, in any given embryo, organ positions can be normal, reversed or randomized. Class 3 reveals a hitherto unsuspected gene that selectively affects laterality of heart. We find that visceral organ positions are predicted by the direction of the preceding cardiac jog. We interpret this as suggesting that normally there is linkage between cardiac and visceral organ laterality. Class 1 mutations, we suggest, effectively remove the global laterality signals, with the consequence that organ positions are effectively symmetrical. Embryos with Class 2 mutations do manifest linkage among organs, but it may be reversed, suggesting that the global signals may be present but incorrectly orientated in some of the embryos. That laterality decisions of organs may be independently perturbed, as in the Class 3 mutation, indicates that there are distinctive pathways for reception and organotypic interpretation of the global signals. -
Warren KS, Baker K, Fishman MC. 2001. The slow mo mutation reduces pacemaker current and heart rate in adult zebrafish. American journal of physiology. Heart and circulatory physiology. 281(4):H1711-9. Pubmed: 11557562 Warren KS, Baker K, Fishman MC. 2001. The slow mo mutation reduces pacemaker current and heart rate in adult zebrafish. American journal of physiology. Heart and circulatory physiology. 281(4):H1711-9. Pubmed: 11557562 Genetic studies in zebrafish have focused on embryonic mutations, but many physiological mechanisms continue to mature after embryogenesis. We report here that zebrafish homozygous for the mutation slow mo can be raised to adulthood. In the embryo, the slow mo gene is needed to regulate heart rate, and its mutation causes a reduction in pacemaker current (I(h)) and slowing of heart rate (bradycardia). The homozygous adult slow mo fish continues to manifest bradycardia, without other evident ill effects. Patch-clamp analysis of isolated adult cardiomyocytes reveals that I(h) has chamber-specific properties such that the atrial current density of I(h) is far greater than the ventricular current density of I(h). I(h) is markedly diminished in cardiomyocytes from both chambers of slow mo mutant fish. Thus I(h) continues to be a critical determinant of pacemaker rate even after adult neural and humoral influences have developed. It is clear that zebrafish may be used for genetic dissection of selected physiological mechanisms in the adult. -
Peterson RT, Mably JD, Chen JN, Fishman MC. 2001. Convergence of distinct pathways to heart patterning revealed by the small molecule concentramide and the mutation heart-and-soul. Current biology : CB. 11(19):1481-91. Pubmed: 11591315 Peterson RT, Mably JD, Chen JN, Fishman MC. 2001. Convergence of distinct pathways to heart patterning revealed by the small molecule concentramide and the mutation heart-and-soul. Current biology : CB. 11(19):1481-91. Pubmed: 11591315 Array -
Chan J, Mably JD, Serluca FC, Chen JN, Goldstein NB, Thomas MC, Cleary JA, Brennan C, Fishman MC, Roberts TM. 2001. Morphogenesis of prechordal plate and notochord requires intact Eph/ephrin B signaling. Developmental biology. 234(2):470-82. Pubmed: 11397014 Chan J, Mably JD, Serluca FC, Chen JN, Goldstein NB, Thomas MC, Cleary JA, Brennan C, Fishman MC, Roberts TM. 2001. Morphogenesis of prechordal plate and notochord requires intact Eph/ephrin B signaling. Developmental biology. 234(2):470-82. Pubmed: 11397014 Eph receptors and their ligands, the ephrins, mediate cell-to-cell signals implicated in the regulation of cell migration processes during development. We report the molecular cloning and tissue distribution of zebrafish transmembrane ephrins that represent all known members of the mammalian class B ephrin family. The degree of homology among predicted ephrin B sequences suggests that, similar to their mammalian counterparts, zebrafish B-ephrins can also bind promiscuously to several Eph receptors. The dynamic expression patterns for each zebrafish B-ephrin support the idea that these ligands are confined to interact with their receptors at the borders of their complementary expression domains. Zebrafish B-ephrins are expressed as early as 30% epiboly and during gastrula stages: in the germ ring, shield, prechordal plate, and notochord. Ectopic overexpression of dominant-negative soluble ephrin B constructs yields reproducible defects in the morphology of the notochord and prechordal plate by the end of gastrulation. Notably disruption of Eph/ephrin B signaling does not completely destroy structures examined, suggesting that cell fate specification is not altered. Thus abnormal morphogenesis of the prechordal plate and the notochord is likely a consequence of a cell movement defect. Our observations suggest Eph/ephrin B signaling plays an essential role in regulating cell movements during gastrulation.Copyright 2001 Academic Press. 2000
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Warren KS, Wu JC, Pinet F, Fishman MC. 2000. The genetic basis of cardiac function: dissection by zebrafish (Danio rerio) screens. Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 355(1399):939-44. Pubmed: 11128987 Warren KS, Wu JC, Pinet F, Fishman MC. 2000. The genetic basis of cardiac function: dissection by zebrafish (Danio rerio) screens. Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 355(1399):939-44. Pubmed: 11128987 The vertebrate heart differs from chordate ancestors both structurally and functionally. Genetic units of form, termed 'modules', are identifiable by mutation, both in zebrafish and mouse, and correspond to features recently acquired in evolution, such as the ventricular chamber or endothelial lining of the vessels and heart. Zebrafish (Danio rerio) genetic screens have provided a reasonably inclusive set of such genes. Normal cardiac function may also be disrupted by single-gene mutations in zebrafish. Individual mutations may perturb contractility or rhythm generation. The zebrafish mutations which principally disturb cardiac contractility fall into two broad phenotypic categories, 'dilated' and 'hypertrophic'. Interestingly, these correspond to the two primary types of heart failure in humans. These disorders of early cardiac function provide candidate genes to be examined in complex human heart diseases, including arrhythmias and heart failure. -
Childs S, Weinstein BM, Mohideen MA, Donohue S, Bonkovsky H, Fishman MC. 2000. Zebrafish dracula encodes ferrochelatase and its mutation provides a model for erythropoietic protoporphyria. Current biology : CB. 10(16):1001-4. Pubmed: 10985389 Childs S, Weinstein BM, Mohideen MA, Donohue S, Bonkovsky H, Fishman MC. 2000. Zebrafish dracula encodes ferrochelatase and its mutation provides a model for erythropoietic protoporphyria. Current biology : CB. 10(16):1001-4. Pubmed: 10985389 Exposure to light precipitates the symptoms of several genetic disorders that affect both skin and internal organs. It is presumed that damage to non-cutaneous organs is initiated indirectly by light, but this is difficult to study in mammals. Zebrafish have an essentially transparent periderm for the first days of development. In a previous large-scale genetic screen we isolated a mutation, dracula (drc), which manifested as a light-dependent lysis of red blood cells [1]. We report here that protoporphyrin IX accumulates in the mutant embryos, suggesting a deficiency in the activity of ferrochelatase, the terminal enzyme in the pathway for heme biosynthesis. We find that homozygous drc(m248) mutant embryos have a G-->T transversion at a splice donor site in the ferrochelatase gene, creating a premature stop codon. The mutant phenotype, which shows light-dependent hemolysis and liver disease, is similar to that seen in humans with erythropoietic protoporphyria, a disorder of ferrochelatase. -
Gyurko R, Kuhlencordt P, Fishman MC, Huang PL. 2000. Modulation of mouse cardiac function in vivo by eNOS and ANP. American journal of physiology. Heart and circulatory physiology. 278(3):H971-81. Pubmed: 10710367 Gyurko R, Kuhlencordt P, Fishman MC, Huang PL. 2000. Modulation of mouse cardiac function in vivo by eNOS and ANP. American journal of physiology. Heart and circulatory physiology. 278(3):H971-81. Pubmed: 10710367 To study the role of endothelial nitric oxide synthase (eNOS) in cardiac function, we compared eNOS expression, contractility, and relaxation in the left ventricles of wild-type and eNOS-deficient mice. eNOS immunostaining is localized to the macro- and microvascular endothelium throughout the myocardium in wild-type mice and is absent in eNOS-/- mice. Whereas blood pressure is elevated in eNOS-/- mice, baseline cardiac contractility (dP/dt(max)) is similar in wild-type and eNOS-/- mice (9,673 +/- 2, 447 and 9,928 +/- 1,566 mmHg/s, respectively). The beta-adrenergic agonist isoproterenol (Iso) at doses of >/=1 ng causes enhanced increases in dP/dt(max) in eNOS-/- mice compared with wild-type controls in vivo (P < 0.01) as well as in Langendorff isolated heart preparations (P < 0.02). beta-Adrenergic receptor binding (B(max)) is not significantly different in the two groups of animals (B(max) = 41.4 +/- 9.4 and 36.1 +/- 5.1 fmol/mg for wild-type and eNOS-/-). Iso-stimulated ventricular relaxation is also enhanced in the eNOS-/- mice, as measured by dP/dt(min) in the isolated heart. However, baseline ventricular relaxation is normal in eNOS-/- mice (tau = 5.2 +/- 1.0 and 5.6 +/- 1.5 ms for wild-type and eNOS-/-, respectively), whereas it is impaired in wild-type mice after NOS inhibition (tau = 8.3 +/- 2.4 ms). cGMP levels in the left ventricle are unaffected by eNOS gene deletion (wild-type: 3.1 +/- 0.8 pmol/mg, eNOS-/-: 3.1 +/- 0.6 pmol/mg), leading us to examine the level of another physiological regulator of cGMP. Atrial natriuretic peptide (ANP) expression is markedly upregulated in the eNOS-/- mice, and exogenous ANP restores ventricular relaxation in wild-type mice treated with NOS inhibitors. These results suggest that eNOS attenuates both inotropic and lusitropic responses to beta-adrenergic stimulation, and it also appears to regulate baseline ventricular relaxation in conjunction with ANP. -
Zhong TP, Rosenberg M, Mohideen MA, Weinstein B, Fishman MC. 2000. gridlock, an HLH gene required for assembly of the aorta in zebrafish. Science (New York, N.Y.). 287(5459):1820-4. Pubmed: 10710309 Zhong TP, Rosenberg M, Mohideen MA, Weinstein B, Fishman MC. 2000. gridlock, an HLH gene required for assembly of the aorta in zebrafish. Science (New York, N.Y.). 287(5459):1820-4. Pubmed: 10710309 The first artery and vein of the vertebrate embryo assemble in the trunk by migration and coalescence of angioblasts to form endothelial tubes. The gridlock (grl) mutation in zebrafish selectively perturbs assembly of the artery (the aorta). Here it is shown that grl encodes a basic helix-loop-helix (bHLH) protein belonging to the Hairy/Enhancer of the split family of bHLH proteins. The grl gene is expressed in lateral plate mesoderm before vessel formation, and thereafter in the aorta and not in the vein. These results suggest that the arterial endothelial identity is established even before the onset of blood flow and implicate the grl gene in assignment of vessel-specific cell fate. -
Chen JN, Fishman MC. 2000. Genetic dissection of heart development. Ernst Schering Research Foundation workshop. Pubmed: 10943307 Chen JN, Fishman MC. 2000. Genetic dissection of heart development. Ernst Schering Research Foundation workshop. Pubmed: 10943307 -
Ton C, Hwang DM, Dempsey AA, Tang HC, Yoon J, Lim M, Mably JD, Fishman MC, Liew CC. 2000. Identification, characterization, and mapping of expressed sequence tags from an embryonic zebrafish heart cDNA library. Genome research. 10(12):1915-27. Pubmed: 11116087 Ton C, Hwang DM, Dempsey AA, Tang HC, Yoon J, Lim M, Mably JD, Fishman MC, Liew CC. 2000. Identification, characterization, and mapping of expressed sequence tags from an embryonic zebrafish heart cDNA library. Genome research. 10(12):1915-27. Pubmed: 11116087 The generation of expressed sequence tags (ESTs) has proven to be a rapid and economical approach by which to identify and characterize expressed genes. We generated 5102 ESTs from a 3-d-old embryonic zebrafish heart cDNA library. Of these, 57.6% matched to known genes, 14.2% matched only to other ESTs, and 27.8% showed no match to any ESTs or known genes. Clustering of all ESTs identified 359 unique clusters comprising 1771 ESTs, whereas the remaining 3331 ESTs did not cluster. This estimates the number of unique genes identified in the data set to be approximately 3690. A total of 1242 unique known genes were used to analyze the gene expression patterns in the zebrafish embryonic heart. These were categorized into seven categories on the basis of gene function. The largest class of genes represented those involved in gene/protein expression (25.9% of known transcripts). This class was followed by genes involved in metabolism (18.7%), cell structure/motility (16.4%), cell signaling and communication (9.6%), cell/organism defense (7.1%), and cell division (4.4%). Unclassified genes constituted the remaining 17.91%. Radiation hybrid mapping was performed for 102 ESTs and comparison of map positions between zebrafish and human identified new synteny groups. Continued comparative analysis will be useful in defining the boundaries of conserved chromosome segments between zebrafish and humans, which will facilitate the transfer of genetic information between the two organisms and improve our understanding of vertebrate evolution. -
Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Maller B, Hayward DC, Ball EE, Degnan B, Müller P, Spring J, Srinivasan A, Fishman M, Finnerty J, Corbo J, Levine M, Leahy P, Davidson E, Ruvkun G. 2000. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature. 408(6808):86-9. Pubmed: 11081512 Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Maller B, Hayward DC, Ball EE, Degnan B, Müller P, Spring J, Srinivasan A, Fishman M, Finnerty J, Corbo J, Levine M, Leahy P, Davidson E, Ruvkun G. 2000. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature. 408(6808):86-9. Pubmed: 11081512 Two small RNAs regulate the timing of Caenorhabditis elegans development. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA, and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs. Here we have detected let-7 RNAs of approximately 21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila, at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny. -
Chen JN, Fishman MC. 2000. Genetics of heart development. Trends in genetics : TIG. 16(9):383-8. Pubmed: 10973066 Chen JN, Fishman MC. 2000. Genetics of heart development. Trends in genetics : TIG. 16(9):383-8. Pubmed: 10973066 The genes that drive heart-cell differentiation in vertebrates and Drosophila are similar, even though the Drosophila 'heart' is a simple tube and the vertebrate heart is a multichambered physiologically complex organ. Mutational analysis in mice and, as particular focus of this review, in zebrafish, reveals the additional genes brought into play to fashion these evolutionarily 'new' organotypic components. -
Kubota I, Han X, Opel DJ, Zhao YY, Baliga R, Huang P, Fishman MC, Shannon RP, Michel T, Kelly RA. 2000. Increased susceptibility to development of triggered activity in myocytes from mice with targeted disruption of endothelial nitric oxide synthase. Journal of molecular and cellular cardiology. 32(7):1239-48. Pubmed: 10860766 Kubota I, Han X, Opel DJ, Zhao YY, Baliga R, Huang P, Fishman MC, Shannon RP, Michel T, Kelly RA. 2000. Increased susceptibility to development of triggered activity in myocytes from mice with targeted disruption of endothelial nitric oxide synthase. Journal of molecular and cellular cardiology. 32(7):1239-48. Pubmed: 10860766 Nitric oxide generated by cardiac myocytes or delivered by drugs has been shown to regulate cardiac contractile function and has been implicated in suppressing some cardiac arrhythmias, although this remains controversial. We examined the ability of the soluble cardiac glycoside, ouabain, to trigger arrhythmic contractions in ventricular myocytes isolated from mice lacking a functional endothelial nitric oxide synthase gene (eNOS(null)). Arrhythmic activity, defined as aftercontractions, was induced with ouabain (50 micromol/L) and recorded using a video-motion detector in isolated, electrically driven single ventricular myocytes from adult eNOS(null)or from their wild-type (WT) littermates. The rate of ouabain-induced arrhythmic contractions was significantly higher in eNOS(null)myocytes than in WT myocytes. Application of the NO donor S-nitroso-acetylcysteine (SNAC) significantly diminished the frequency of arrhythmic contractions in eNOS(null)myocytes. The antiarrhythmic effect of NO, whether generated by eNOS in WT cells or by SNAC, could be partially reversed by 1H-[1,2,4]oxadiazolo-[4, 3-a]- quinoxalin-1-one (ODQ), a specific soluble guanylyl cyclase inhibitor. Ouabain significantly increased intracellular cGMP in WT but not eNOS(null)hearts, and this cGMP response was blocked by ODQ. Since cardiac glycoside- induced aftercontractions are activated by the transient inward current (I(ti)), the role of NO in ouabain (100 micromol/L)- induced I(ti)was examined using the nystatin-perforated patch-clamp technique. The frequency of ouabain-induced I(ti)was significantly higher in eNOS(null)myocytes than in WT myocytes, and this could be suppressed by SNAC. These data demonstrate that NO derived from myocyte eNOS activation suppresses ouabain-induced arrhythmic contractions by a mechanism that might involve activation of guanylyl cyclase and elevation of cGMP.Copyright 2000 Academic Press. 1999
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Fishman MC. 1999. Zebrafish genetics: the enigma of arrival. Proceedings of the National Academy of Sciences of the United States of America. 96(19):10554-6. Pubmed: 10485858 Fishman MC. 1999. Zebrafish genetics: the enigma of arrival. Proceedings of the National Academy of Sciences of the United States of America. 96(19):10554-6. Pubmed: 10485858 -
Zhang L, Fishman MC, Huang PL. 1999. Estrogen mediates the protective effects of pregnancy and chorionic gonadotropin in a mouse model of vascular injury. Arteriosclerosis, thrombosis, and vascular biology. 19(9):2059-65. Pubmed: 10479646 Zhang L, Fishman MC, Huang PL. 1999. Estrogen mediates the protective effects of pregnancy and chorionic gonadotropin in a mouse model of vascular injury. Arteriosclerosis, thrombosis, and vascular biology. 19(9):2059-65. Pubmed: 10479646 To determine why pregnancy protects against intimal proliferation in a mouse model of vessel injury, we administered chorionic gonadotropin to intact and ovariectomized female mice. Chorionic gonadotropin markedly suppressed intimal proliferation in intact but not in ovariectomized female mice, indicating that the protective effects of chorionic gonadotropin require ovarian function. To test whether estrogen or progesterone might mediate the protective effects of pregnancy and chorionic gonadotropin, we administered estrogen and progesterone to ovariectomized mice. Estrogen administration to ovariectomized mice to achieve the elevated levels seen in pregnancy was sufficient to reproduce the marked suppression of intimal proliferation in response to vessel injury. Progesterone administration reduced intimal proliferation to a lesser degree and was correlated with increases in estrogen to levels seen in nonpregnant female mice. Staining for proliferating cell nuclear antigen suggested that estrogen reduced medial and intimal cell proliferation. Both the classic estrogen receptor-alpha and the recently discovered estrogen receptor-beta are present in vascular tissue as assessed by immunohistochemistry, providing a possible mechanism for the effects of estrogen. These results suggest that the protective effects of estrogen do not plateau at levels seen in normal females but increase further with estrogen levels up through levels seen during pregnancy. -
Shimoda N, Knapik EW, Ziniti J, Sim C, Yamada E, Kaplan S, Jackson D, de Sauvage F, Jacob H, Fishman MC. 1999. Zebrafish genetic map with 2000 microsatellite markers. Genomics. 58(3):219-32. Pubmed: 10373319 Shimoda N, Knapik EW, Ziniti J, Sim C, Yamada E, Kaplan S, Jackson D, de Sauvage F, Jacob H, Fishman MC. 1999. Zebrafish genetic map with 2000 microsatellite markers. Genomics. 58(3):219-32. Pubmed: 10373319 The zebrafish is the first vertebrate organism used for large-scale genetic screens seeking genes critical to development. These screens have been quite successful, with more than 1800 recessive mutations discovered that speak to morphogenesis of the vertebrate embryo. The cloning of the mutant genes depends on a dense genetic map. The 2000 markers we present here, using microsatellite (CA) repeats, provides 1.2-cM average resolution. One centimorgan in zebrafish is about 0. 74 megabase, so, for many mutations, these markers are close enough to begin positional cloning by YAC walks.Copyright 1999 Academic Press. -
Chataigneau T, Félétou M, Huang PL, Fishman MC, Duhault J, Vanhoutte PM. 1999. Acetylcholine-induced relaxation in blood vessels from endothelial nitric oxide synthase knockout mice. British journal of pharmacology. 126(1):219-26. Pubmed: 10051139 Chataigneau T, Félétou M, Huang PL, Fishman MC, Duhault J, Vanhoutte PM. 1999. Acetylcholine-induced relaxation in blood vessels from endothelial nitric oxide synthase knockout mice. British journal of pharmacology. 126(1):219-26. Pubmed: 10051139 1. Isometric tension was recorded in isolated rings of aorta, carotid, coronary and mesenteric arteries taken from endothelial nitric oxide synthase knockout mice (eNOS(-/-) mice) and the corresponding wild-type strain (eNOS(+/+) mice). The membrane potential of smooth muscle cells was measured in coronary arteries with intracellular microelectrodes. 2. In the isolated aorta, carotid and coronary arteries from the eNOS(+/+) mice, acetylcholine induced an endothelium-dependent relaxation which was inhibited by N(omega)-L-nitro-arginine. In contrast, in the mesenteric arteries, the inhibition of the cholinergic relaxation required the combination of N(omega)-L-nitro-arginine and indomethacin. 3. The isolated aorta, carotid and coronary arteries from the eNOS(-/-) mice did not relax in response to acetylcholine. However, acetylcholine produced an indomethacin-sensitive relaxation in the mesenteric artery from eNOS(-/-) mice. 4. The resting membrane potential of smooth muscle cells from isolated coronary arteries was significantly less negative in the eNOS(-/-) mice (-64.8 +/- 1.8 mV, n = 20 and -58.4 +/- 1.9 mV, n = 17, for eNOS(+/+) and eNOS(-/-) mice, respectively). In both strains, acetylcholine, bradykinin and substance P did not induce endothelium-dependent hyperpolarizations whereas cromakalim consistently produced hyperpolarizations (- 7.9 +/- 1.1 mV, n = 8 and -13.8 +/- 2.6 mV, n = 4, for eNOS(+/+) and eNOS(-/-) mice, respectively). 5. These findings demonstrate that in the blood vessels studied: (1) in the eNOS(+/+) mice, the endothelium-dependent relaxations to acetylcholine involve either NO or the combination of NO plus a product of cyclo-oxygenase but not EDHF; (2) in the eNOS(-/-) mice, NO-dependent responses and EDHF-like responses were not observed. In the mesenteric arteries acetylcholine releases a cyclo-oxygenase derivative. -
Serluca FC, Fishman MC. 1999. Cell lineage tracing in heart development. Methods in cell biology. 59:359-65. Pubmed: 9891369 Serluca FC, Fishman MC. 1999. Cell lineage tracing in heart development. Methods in cell biology. 59:359-65. Pubmed: 9891369 Photolysis-based lineage determination is especially useful in addressing late-stage lineage definitions. Although we have focused on the heart lineage, this technique is readily applicable to any region of the embryo. Furthermore, the concomitant analysis of gene expression and lineage restriction within the same embryo will help define the signaling cascades that direct normal cell fate and the regulative changes that mark vertebrate organ development. -
Amemiya CT, Zhong TP, Silverman GA, Fishman MC, Zon LI. 1999. Zebrafish YAC, BAC, and PAC genomic libraries. Methods in cell biology. 60:235-58. Pubmed: 9891341 Amemiya CT, Zhong TP, Silverman GA, Fishman MC, Zon LI. 1999. Zebrafish YAC, BAC, and PAC genomic libraries. Methods in cell biology. 60:235-58. Pubmed: 9891341 Numerous positional cloning projects directed at isolating genes responsible for the myriads of observed developmental defects in the zebrafish are anticipated in the very near future. In this chapter, we have reviewed the YAC, BAC, and PAC large-insert genomic resources available to the zebrafish community. We have discussed how these resources are screened and used in a positional cloning scheme and have pointed out frequently formidable logistical considerations in the approach. Despite being extremely tedious, positional cloning projects in the zebrafish will be comparatively easier to accomplish than in human and mouse, because of unique biological advantages of the zebrafish system. Moreover, the ease and speed at which genes are identified and cloned should rapidly increase as more mapping reagents and information become available, thereby paving the way for meaningful biological studies. 1998
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Han X, Kubota I, Feron O, Opel DJ, Arstall MA, Zhao YY, Huang P, Fishman MC, Michel T, Kelly RA. 1998. Muscarinic cholinergic regulation of cardiac myocyte ICa-L is absent in mice with targeted disruption of endothelial nitric oxide synthase. Proceedings of the National Academy of Sciences of the United States of America. 95(11):6510-5. Pubmed: 9600997 Han X, Kubota I, Feron O, Opel DJ, Arstall MA, Zhao YY, Huang P, Fishman MC, Michel T, Kelly RA. 1998. Muscarinic cholinergic regulation of cardiac myocyte ICa-L is absent in mice with targeted disruption of endothelial nitric oxide synthase. Proceedings of the National Academy of Sciences of the United States of America. 95(11):6510-5. Pubmed: 9600997 Cardiac myocytes have been shown to express constitutively endothelial nitric oxide synthase (eNOS) (nitric oxide synthase 3), the activation of which has been implicated in the regulation of myocyte L-type voltage-sensitive calcium channel current (ICa-L) and myocyte contractile responsiveness to parasympathetic nervous system signaling, although this implication remains controversial. Therefore, we examined the effect of the muscarinic cholinergic agonist carbachol (CCh) on ICa-L and contractile amplitude in isoproterenol (ISO)-prestimulated ventricular myocytes isolated from adult mice, designated eNOSnull mice, with targeted disruption of the eNOS gene. Although both eNOSnull and wild-type (WT) ventricular myocytes exhibited similar increases in ICa-L in response to ISO, there was no measurable suppression of ICa-L by CCh in cells from eNOSnull mice, in contrast to cells from WT mice. These results were reflected in the absence of an effect of CCh on the positive inotropic effect of ISO in eNOSnull myocytes. Also, unlike myocytes from WT animals, eNOSnull myocytes failed to exhibit an increase in cGMP content in response to CCh. Nevertheless, the pharmacologic nitric oxide donors 3-morpholino-sydnonimine and S-nitroso-acetyl-cystein increased cGMP generation and suppressed ISO-augmented ICa-L in eNOSnull cells, suggesting that the signal transduction pathway(s) downstream of eNOS remained intact. Of importance, activation of the acetylcholine-activated K+ channel by CCh was unaffected in atrial and ventricular eNOSnull myocytes. These results confirm the obligatory role of eNOS in coupling muscarinic receptor activation to cGMP-dependent control of ICa-L in cardiac myocytes. -
Murohara T, Asahara T, Silver M, Bauters C, Masuda H, Kalka C, Kearney M, Chen D, Symes JF, Fishman MC, Huang PL, Isner JM. 1998. Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. The Journal of clinical investigation. 101(11):2567-78. Pubmed: 9616228 Murohara T, Asahara T, Silver M, Bauters C, Masuda H, Kalka C, Kearney M, Chen D, Symes JF, Fishman MC, Huang PL, Isner JM. 1998. Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. The Journal of clinical investigation. 101(11):2567-78. Pubmed: 9616228 We tested the hypothesis that endothelial nitric oxide synthase (eNOS) modulates angiogenesis in two animal models in which therapeutic angiogenesis has been characterized as a compensatory response to tissue ischemia. We first administered L-arginine, previously shown to augment endogenous production of NO, to normal rabbits with operatively induced hindlimb ischemia. Angiogenesis in the ischemic hindlimb was significantly improved by dietary supplementation with L-arginine, compared to placebo-treated controls; angiographically evident vascularity in the ischemic limb, hemodynamic indices of limb perfusion, capillary density, and vasomotor reactivity in the collateral vessel-dependent ischemic limb were all improved by oral L-arginine supplementation. A murine model of operatively induced hindlimb ischemia was used to investigate the impact of targeted disruption of the gene encoding for ENOS on angiogenesis. Angiogenesis in the ischemic hindlimb was significantly impaired in eNOS-/- mice versus wild-type controls evaluated by either laser Doppler flow analysis or capillary density measurement. Impaired angiogenesis in eNOS-/- mice was not improved by administration of vascular endothelial growth factor (VEGF), suggesting that eNOS acts downstream from VEGF. Thus, (a) eNOS is a downstream mediator for in vivo angiogenesis, and (b) promoting eNOS activity by L-arginine supplementation accelerates in vivo angiogenesis. These findings suggest that defective endothelial NO synthesis may limit angiogenesis in patients with endothelial dysfunction related to atherosclerosis, and that oral L-arginine supplementation constitutes a potential therapeutic strategy for accelerating angiogenesis in patients with advanced vascular obstruction. -
Knapik EW, Goodman A, Ekker M, Chevrette M, Delgado J, Neuhauss S, Shimoda N, Driever W, Fishman MC, Jacob HJ. 1998. A microsatellite genetic linkage map for zebrafish (Danio rerio). Nature genetics. 18(4):338-43. Pubmed: 9537415 Knapik EW, Goodman A, Ekker M, Chevrette M, Delgado J, Neuhauss S, Shimoda N, Driever W, Fishman MC, Jacob HJ. 1998. A microsatellite genetic linkage map for zebrafish (Danio rerio). Nature genetics. 18(4):338-43. Pubmed: 9537415 We have constructed a zebrafish genetic linkage map consisting of 705 simple sequence-length polymorphism markers (SSLPs). The map covers 2350 centimorgans (cM) of the zebrafish genome with an average resolution of 3.3 cM. It is a complete map in genetic mapping terms (there is one linkage group for each of the 25 chromosomes), and it has been confirmed by somatic-cell hybrids and centromere-mapping using half-tetrad analysis. The markers are highly polymorphic in the zebrafish strains used for genetic crosses and provide a means to compare genetic segregation of developmental mutations between laboratories. These markers will provide an initial infrastructure for the positional cloning of the nearly 600 zebrafish genes identified as crucial to vertebrate development,and will become the anchor for the physical map of the zebrafish genome. -
Thorin E, Huang PL, Fishman MC, Bevan JA. 1998. Nitric oxide inhibits alpha2-adrenoceptor-mediated endothelium-dependent vasodilation. Circulation research. 82(12):1323-9. Pubmed: 9648729 Thorin E, Huang PL, Fishman MC, Bevan JA. 1998. Nitric oxide inhibits alpha2-adrenoceptor-mediated endothelium-dependent vasodilation. Circulation research. 82(12):1323-9. Pubmed: 9648729 This study was designed to investigate the interaction between the NO/L-arginine pathway and the alpha2-adrenoceptor-mediated endothelium-dependent vasorelaxation. Reactivity of isolated resistance mesenteric arterial segments from mice lacking the gene for constitutive endothelial NO synthase (eNOS- mice, n=14) and from their wild-type controls (WT mice, n=46) was studied in isometric conditions in the presence of indomethacin (blocker of cyclooxygenase). Oxymetazoline (OXY, 0.01 to 30 micromol/L; a selective alpha2-adrenoceptor agonist) induced an endothelium-dependent relaxation of eNOS- but not WT arteries preconstricted either with phenylephrine or serotonin. In the presence of Nomega-nitro-L-arginine (l-NNA, 100 micromol/L), an inhibitor of NOS, OXY induced an endothelium-dependent relaxation of WT mesenteric arteries. l-NNA had no effect on the relaxation caused by OXY in eNOS- arterial rings. Therefore, the relaxation caused by OXY was independent of NO formation. To demonstrate the inhibitory role of NO on the alpha2-adrenoceptor-mediated relaxation, subthreshold (0.1 nmol/L) to threshold (1 nmol/L) concentrations of sodium nitroprusside (donor of NO) were added to l-NNA-treated arteries before OXY challenges: in these conditions, the alpha2-adrenoceptor-mediated relaxation of eNOS- and WT arteries was inhibited. OXY-induced relaxation was restored on readdition of methylene blue (1 micromol/L, inhibitor of guanylate cyclase), suggesting that cGMP may be the mechanism of inhibition of the alpha2-adrenergic pathway in the presence of NO. Finally, OXY-mediated relaxation was blocked by tetraethylammonium (1 mmol/L) but not glibenclamide (1 micromol/L), suggesting the involvement of an endothelium-derived hyperpolarizing factor that activates Ca2+-activated K+ channels. In conclusion, alpha2-adrenoceptor activation caused relaxation of isolated murine mesenteric arteries that was functionally blocked by NO through a mechanism that may involve activation of the soluble guanylate cyclase and cGMP formation. The endothelium-dependent alpha2-adrenoceptor-mediated relaxation is likely to be due to an endothelium-derived hyperpolarizing factor, whose release and/or production is reduced by concurrent NO formation. -
Serbedzija GN, Chen JN, Fishman MC. 1998. Regulation in the heart field of zebrafish. Development (Cambridge, England). 125(6):1095-101. Pubmed: 9463356 Serbedzija GN, Chen JN, Fishman MC. 1998. Regulation in the heart field of zebrafish. Development (Cambridge, England). 125(6):1095-101. Pubmed: 9463356 In many vertebrates, removal of early embryonic heart precursors can be repaired, leaving the heart and embryo without visible deficit. One possibility is that this 'regulation' involves a cell fate switch whereby cells, perhaps in regions surrounding normal progenitors, are redirected to the heart cell fate. However, the lineage and spatial relationships between cells that are normal heart progenitors and those that can assume that role after injury are not known, nor are their molecular distinctions. We have adapted a laser-activated technique to label single or small patches of cells in the lateral plate mesoderm of the zebrafish and to track their subsequent lineage. We find that the heart precursor cells are clustered in a region adjacent to the prechordal plate, just anterior to the notochord tip. Complete unilateral ablation of all heart precursors with a laser does not disrupt heart development, if performed before the 18-somite stage. By combining extirpation of the heart precursors with cell labeling, we find that cells anterior to the normal cardiogenic compartments constitute the source of regulatory cells that compensate for the loss of the progenitors. One of the earliest embryonic markers of the premyocardial cells is the divergent homeodomain gene, Nkx2.5. Interestingly, normal cardiogenic progenitors derive from only the anterior half of the Nkx2.5-expressing region in the lateral plate mesoderm. The posterior half, adjacent to the notochord, does not include cardiac progenitors and the posterior Nkx2.5-expressing cells do not contribute to the heart, even after ablation of the normal cardiogenic region. The cells that can acquire a cardiac cell fate after injury to the normal progenitors also reside near the prechordal plate, but anterior to the Nkx2.5-expressing domain. Normally they give rise to head mesenchyme. They share with cardiac progenitors early expression of GATA 4. The location of the different elements of the cardiac field, and their response to injury, suggests that the prechordal plate supports and/or the notochord suppresses the cardiac fate. -
Zhong TP, Kaphingst K, Akella U, Haldi M, Lander ES, Fishman MC. 1998. Zebrafish genomic library in yeast artificial chromosomes. Genomics. 48(1):136-8. Pubmed: 9503028 Zhong TP, Kaphingst K, Akella U, Haldi M, Lander ES, Fishman MC. 1998. Zebrafish genomic library in yeast artificial chromosomes. Genomics. 48(1):136-8. Pubmed: 9503028 We have constructed a zebrafish yeast artificial chromosome (YAC) library using genomic DNA isolated from the inbred AB zebrafish strain. The average insert size is 470 kb, estimated from analysis of 155 random selected YACs. The library consists of 17,000 clones, providing about a 4.7-fold coverage of zebrafish genome. The YAC clones have been arrayed in individual wells of 96-well microplates and also pooled to permit rapid polymerase chain reaction screening of the entire library. We have also found that the YAC ends can be easily rescued and sequenced from pRML1/pRML2-based mini-YAC clones. -
Tuffereau C, Benejean J, Alfonso AM, Flamand A, Fishman MC. 1998. Neuronal cell surface molecules mediate specific binding to rabies virus glycoprotein expressed by a recombinant baculovirus on the surfaces of lepidopteran cells. Journal of virology. 72(2):1085-91. Pubmed: 9445003 Tuffereau C, Benejean J, Alfonso AM, Flamand A, Fishman MC. 1998. Neuronal cell surface molecules mediate specific binding to rabies virus glycoprotein expressed by a recombinant baculovirus on the surfaces of lepidopteran cells. Journal of virology. 72(2):1085-91. Pubmed: 9445003 The existence of specific rabies virus (RV) glycoprotein (G) binding sites on the surfaces of neuroblastoma cells is demonstrated. Spodoptera frugiperda (Sf21) cells expressing G of the RV strain CVS (Gcvs-Sf21 cells) bind specifically to neuroblastoma cells of different species but not to any other cell type (fibroblast, myoblast, epithelial, or glioma). Attachment to mouse neuroblastoma NG108-15 cells is abolished by previous treatment of Gcvs-Sf2 cells with anti-G antibody. Substitutions for lysine at position 330 and for arginine at position 333 in RV G greatly reduce interaction between Gcvs-Sf21 cells and NG108-15 cells. These data are consistent with in vivo results: an avirulent RV mutant bearing the same double mutation is not able to infect sensory neurons or motoneurons (P. Coulon, J.-P. Ternaux, A. Flamand, and C. Tuffereau, J. Virol. 72:273-278, 1998) after intramuscular inoculation into a mouse. Furthermore, infection of NG108-15 cells by RV but not by vesicular stomatitis virus leads to a reduction of the number of binding sites at the neuronal-cell surface. Our data strongly suggest that these specific attachment sites on neuroblastoma cells represent a neuronal receptor(s) used by RV to infect certain types of neurons in vivo. -
Warren KS, Fishman MC. 1998. "Physiological genomics": mutant screens in zebrafish. The American journal of physiology. 275(1):H1-7. Pubmed: 9688889 DOI:10.1152/ajpheart.1998.275.1.H1 Warren KS, Fishman MC. 1998. "Physiological genomics": mutant screens in zebrafish. The American journal of physiology. 275(1):H1-7. Pubmed: 9688889 DOI:10.1152/ajpheart.1998.275.1.H1 Large-scale mutagenesis screens have proved essential in the search for genes that are important to development in the fly, worm, and yeast. Here we present the power of large-scale screening in a vertebrate, the zebrafish Danio rerio, and propose the use of this genetic system to address fundamental questions of vertebrate developmental physiology. As an example, we focus on zebrafish mutations that reveal single genes essential for normal development of the cardiovascular system. These single gene mutations disrupt specific aspects of rate, rhythm, conduction, or contractility of the developing heart. -
Park M, Lewis C, Turbay D, Chung A, Chen JN, Evans S, Breitbart RE, Fishman MC, Izumo S, Bodmer R. 1998. Differential rescue of visceral and cardiac defects in Drosophila by vertebrate tinman-related genes. Proceedings of the National Academy of Sciences of the United States of America. 95(16):9366-71. Pubmed: 9689086 Park M, Lewis C, Turbay D, Chung A, Chen JN, Evans S, Breitbart RE, Fishman MC, Izumo S, Bodmer R. 1998. Differential rescue of visceral and cardiac defects in Drosophila by vertebrate tinman-related genes. Proceedings of the National Academy of Sciences of the United States of America. 95(16):9366-71. Pubmed: 9689086 tinman, a mesodermal NK2-type homeobox gene, is absolutely required for the subdivision of the early Drosophila mesoderm and for the formation of the heart as well as the visceral muscle primordia. Several vertebrate relatives of tinman, many of which are predominately expressed in the very early cardiac progenitors (and pharyngeal endoderm), also seem to promote heart development. Here, we show that most of these vertebrate tinman-related genes can readily substitute for Drosophila tinman function in promoting visceral mesoderm-specific marker gene expression, but much less in promoting cardiac-specific gene expression indicative of heart development. In addition, another mesodermal NK2-type gene from Drosophila, bagpipe, which is normally only needed for visceral mesoderm but not heart development, cannot substitute for tinman at all. These data indicate that the functional equivalence of the tinman-related subclass of NK2-type genes (in activating markers of visceral mesoderm development in Drosophila) is specific to this subclass and distinct from other homeobox genes. Despite the apparent overall conservation of heart development between vertebrates and invertebrates, the differential rescue of visceral mesoderm versus heart development suggests that some of the molecular mechanisms of organ formation may have diverged during evolution. -
Goldstein AM, Fishman MC. 1998. Notochord regulates cardiac lineage in zebrafish embryos. Developmental biology. 201(2):247-52. Pubmed: 9740662 Goldstein AM, Fishman MC. 1998. Notochord regulates cardiac lineage in zebrafish embryos. Developmental biology. 201(2):247-52. Pubmed: 9740662 We focus here upon regulation by the notochord of myocardial cell fate in zebrafish. Myocardial precursors, defined by lineage tracing in the living embryo, are in the lateral plate mesoderm adjacent to the notochord-prechordal plate junction. Interestingly, the anterior end of the notochord corresponds to the posterior extent of the heart progenitor field, defined by this lineage analysis. This suggested that the notochord might suppress, or the prechordal plate might enhance, the cardiogenic fate. Nkx2.5 expression is, in the zebrafish embryo, closely correlated with the position of myocardial precursors, which reside adjacent to the notochord-prechordal plate junction. This expression, however, is extinguished in the region posterior to this junction, a region normally not contributing cells to the heart. Laser ablation of the notochord tip between the 4-somite and 12-somite stage causes posterior expansion of the Nkx2. 5-expressing region. The ntl mutation of the notochord is associated with posterior extension of Nkx2.5 expression. Lineage tracking, by laser activation of caged fluoresceinated dextran, confirms that, normally, lateral plate cells next to the notochord do not contribute progeny to the heart. After anterior notochord ablation, these cells are redirected to a heart cell fate. These data suggest that the anterior notochord delimits the posterior extent of the heart field by suppressing the heart cell fate.Copyright 1998 Academic Press. -
Mende U, Zagrovic B, Cohen A, Li Y, Valenzuela D, Fishman MC, Neer EJ. 1998. Effect of deletion of the major brain G-protein alpha subunit (alpha(o)) on coordination of G-protein subunits and on adenylyl cyclase activity. Journal of neuroscience research. 54(2):263-72. Pubmed: 9788285 Mende U, Zagrovic B, Cohen A, Li Y, Valenzuela D, Fishman MC, Neer EJ. 1998. Effect of deletion of the major brain G-protein alpha subunit (alpha(o)) on coordination of G-protein subunits and on adenylyl cyclase activity. Journal of neuroscience research. 54(2):263-72. Pubmed: 9788285 Heterotrimeric G-proteins, composed of alpha and betagamma subunits, transmit signals from cell-surface receptors to cellular effectors and ion channels. Cellular responses to receptor agonists depend on not only the type and amount of G-protein subunits expressed but also the ratio of alpha and betagamma subunits. Thus far, little is known about how the amounts of alpha and betagamma subunits are coordinated. Targeted disruption of the alpha(o) gene leads to loss of both isoforms of alpha(o), the most abundant alpha subunit in the brain. We demonstrate that loss of alpha(o) protein in the brain is accompanied by a reduction of beta protein to 32+/-2% (n = 4) of wild type. Sucrose density gradient experiments show that all of the betagamma remaining in the brains of alpha(o)-/- mice sediments as a heterotrimer (s20,w = 4.4 S, n = 2), with no detectable free alpha or betagamma subunits. Thus, the level of the remaining betagamma subunits matches that of the remaining alpha subunits. Protein levels of alpha subunits other than alpha(o) are unchanged, suggesting that they are controlled independently. Coordination of betagamma to alpha occurs posttranscriptionally because the mRNA level of the predominant beta1 subtype in the brains of alpha(o)-/- mice was unchanged. Adenylyl cyclase can be positively or negatively regulated by betagamma. Because the level of other alpha subunits is unchanged and alpha(o) itself has little or no effect on adenylyl cyclase, we could examine how a large change in the level of betagamma affects this enzyme. Surprisingly, we could not detect any difference in the adenylyl cyclase activity between brain membranes from wild-type and alpha(o)-/- mice. We propose that alpha(o) and its associated betagamma are sequestered in a distinct pool of membranes that does not contribute to the regulation of adenylyl cyclase. -
Bernstein HG, Keilhoff G, Seidel B, Stanarius A, Huang PL, Fishman MC, Reiser M, Bogerts B, Wolf G. 1998. Expression of hypothalamic peptides in mice lacking neuronal nitric oxide synthase: reduced beta-END immunoreactivity in the arcuate nucleus. Neuroendocrinology. 68(6):403-11. Pubmed: 9873204 Bernstein HG, Keilhoff G, Seidel B, Stanarius A, Huang PL, Fishman MC, Reiser M, Bogerts B, Wolf G. 1998. Expression of hypothalamic peptides in mice lacking neuronal nitric oxide synthase: reduced beta-END immunoreactivity in the arcuate nucleus. Neuroendocrinology. 68(6):403-11. Pubmed: 9873204 The gas nitric oxide (NO) is an important messenger in brain signaling. Along with many other functions, NO is thought to influence the expression and/or release of various hypothalamic hormones (corticotropin-releasing hormone (CRH), gonadotropin-releasing hormone (GnRH) and vasopressin). To learn more about the role of NO in neuroendocrine mechanisms, we studied in mutant mice lacking neuronal isoform of NO synthase (nNOS) the cellular expression of CRH, neurophysin (the carrier protein of vasopressin/oxytocin) and pro-opiomelanocortin (POMC), as well as of the POMC-derived peptides beta-endorphin (beta-END), alpha-melanocyte-stimulating hormone (alpha-MSH) and corticotropin (ACTH) by use of immunohistochemistry and in situ hybridization. Additionally, the remaining NO-generating capacities of the nNOS minus mice were investigated by NADPH-diaphorase histochemistry and citrulline immunohistochemistry as well as by immunohistochemical localization and Western blot analysis of endothelial NOS (eNOS) and nNOS isoforms. Amongst all hypothalamic peptides under investigation, only beta-END was found to be altered in mutant mice. A morphometric analysis of beta-END producing neurons of the arcuate nucleus revealed that significantly less cells were immunoreactive in mutant mice, whereas the expression of the precursor POMC as well as of other POMC-derived peptides was found to be unchanged. In addition to that, fewer beta-END-immunoreactive fibers were found in the paraventricular nucleus of nNOS minus mice in comparison to wild-type animals. Hence, the reduction of hypothalamic beta-END is probably a posttranslational event that might reflect a disturbed endorphinergic innervation of those hypothalamic neurons which normally express nNOS. -
Drummond IA, Majumdar A, Hentschel H, Elger M, Solnica-Krezel L, Schier AF, Neuhauss SC, Stemple DL, Zwartkruis F, Rangini Z, Driever W, Fishman MC. 1998. Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function. Development (Cambridge, England). 125(23):4655-67. Pubmed: 9806915 Drummond IA, Majumdar A, Hentschel H, Elger M, Solnica-Krezel L, Schier AF, Neuhauss SC, Stemple DL, Zwartkruis F, Rangini Z, Driever W, Fishman MC. 1998. Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function. Development (Cambridge, England). 125(23):4655-67. Pubmed: 9806915 The zebrafish pronephric kidney provides a simplified model of nephron development and epithelial cell differentiation which is amenable to genetic analysis. The pronephros consists of two nephrons with fused glomeruli and paired pronephric tubules and ducts. Nephron formation occurs after the differentiation of the pronephric duct with both the glomeruli and tubules being derived from a nephron primordium. Fluorescent dextran injection experiments demonstrate that vascularization of the zebrafish pronephros and the onset of glomerular filtration occurs between 40 and 48 hpf. We isolated fifteen recessive mutations that affect development of the pronephros. All have visible cysts in place of the pronephric tubule at 2-2.5 days of development. Mutants were grouped in three classes: (1) a group of twelve mutants with defects in body axis curvature and manifesting the most rapid and severe cyst formation involving the glomerulus, tubule and duct, (2) the fleer mutation with distended glomerular capillary loops and cystic tubules, and (3) the mutation pao pao tang with a normal glomerulus and cysts limited to the pronephric tubules. double bubble was analyzed as a representative of mutations that perturb the entire length of the pronephros and body axis curvature. Cyst formation begins in the glomerulus at 40 hpf at the time when glomerular filtration is established suggesting a defect associated with the onset of pronephric function. Basolateral membrane protein targeting in the pronephric duct epithelial cells is also severely affected, suggesting a failure in terminal epithelial cell differentiation and alterations in electrolyte transport. These studies reveal the similarity of normal pronephric development to kidney organogenesis in all vertebrates and allow for a genetic dissection of genes needed to establish the earliest renal function. -
Moroi M, Zhang L, Yasuda T, Virmani R, Gold HK, Fishman MC, Huang PL. 1998. Interaction of genetic deficiency of endothelial nitric oxide, gender, and pregnancy in vascular response to injury in mice. The Journal of clinical investigation. 101(6):1225-32. Pubmed: 9502763 Moroi M, Zhang L, Yasuda T, Virmani R, Gold HK, Fishman MC, Huang PL. 1998. Interaction of genetic deficiency of endothelial nitric oxide, gender, and pregnancy in vascular response to injury in mice. The Journal of clinical investigation. 101(6):1225-32. Pubmed: 9502763 To begin to dissect atherogenesis as a complex genetic disorder affected by genetic makeup and environment, we have (a) generated a reproducible mouse model of neointimal growth; (b) evaluated the effect of disruption of a single gene, endothelial nitric oxide synthase, believed to be central to intimal growth, and (c) examined the modifying effects of gender and pregnancy upon the vascular response. Cuff placement around the femoral artery causes reproducible intimal growth. We assessed the response to injury by quantitative morphometry, measuring the intimal to medial (I/M) volume ratio. In wild-type mice, cuff placement causes pronounced intimal proliferation without affecting the media, resulting in I/M ratios of 31% (SV129 males) and 27% (C57BL/6 males). eNOS mutant male mice have a much greater degree of intimal growth (I/M ratio of 70%). Female mice show less intimal response than do males, although eNOS mutant female mice still have more response than do wild-type females. Most dramatic, however, is the effect of pregnancy, which essentially abolishes the intimal response to injury, even overriding the effect of eNOS mutation. We conclude that eNOS deficiency is a genetic predisposition to intimal proliferation that is enhanced by male gender, and that may be overridden by pregnancy. -
Goldstein AM, Ticho BS, Fishman MC. 1998. Patterning the heart's left-right axis: from zebrafish to man. Developmental genetics. 22(3):278-87. Pubmed: 9621434 Goldstein AM, Ticho BS, Fishman MC. 1998. Patterning the heart's left-right axis: from zebrafish to man. Developmental genetics. 22(3):278-87. Pubmed: 9621434 Normal left-right asymmetry is highly conserved among vertebrates. Errors in the proper patterning of this axis are believed to lead to congenital anomalies of the heart and abdominal viscera, often with profound clinical consequences. We review briefly the nature of potential signals and signaling sources that lead to the break in left-right symmetry. The evidence suggests that left-right reversal, or homogenization, of these signals may lead to different consequences, and we explain some malpositions and malalignments of the atria, ventricles, and/or outflow tract that are seen in a variety of congenital cardiac diseases. We speculate that there are units of organ assembly responsive to laterality signals, and these units may be driven independently. One crucial source of signals appears to be the notochord and floorplate. In order to examine the clinical relationship of these midline structures to putative disorders of laterality, we review all patients with disturbances of normal laterality seen at the Massachusetts General Hospital over the past 20 years. We find a significant association between laterality defects and anomalies of the spine and other midline structures. 1997
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Zaharchuk G, Hara H, Huang PL, Fishman MC, Moskowitz MA, Jenkins BG, Rosen BR. 1997. Neuronal nitric oxide synthase mutant mice show smaller infarcts and attenuated apparent diffusion coefficient changes in the peri-infarct zone during focal cerebral ischemia. Magnetic resonance in medicine. 37(2):170-5. Pubmed: 9001139 Zaharchuk G, Hara H, Huang PL, Fishman MC, Moskowitz MA, Jenkins BG, Rosen BR. 1997. Neuronal nitric oxide synthase mutant mice show smaller infarcts and attenuated apparent diffusion coefficient changes in the peri-infarct zone during focal cerebral ischemia. Magnetic resonance in medicine. 37(2):170-5. Pubmed: 9001139 Diffusion-weighted MRI at 2 T was used to monitor and assess tissue damage after permanent middle cerebral artery occlusion (MCAO) in wild-type (WT) and mice deficient in nitric oxide synthase gene expression (nNOS-). The ischemic lesion was evaluated 3 h after occlusion and subdivided into the lesion core and peri-infarct zone based on the magnitude of the apparent diffusion coefficient (ADC) change. Infarct volume, measured by using histochemical staining 24 h after MCA occlusion, correlated best with MRI infarct volume as assessed by an ADC threshold of 25% decrease from baseline at 3 h. For ADC thresholds of greater than 25% decrease, lesion size was not significantly different in nNOS- and WT mice. However, brain tissue showing ADC decreases of 10-25% was significantly smaller in the ipsilateral hemisphere of mutants (27 +/- 2% and 21 +/- 2% in WT and nNOS-, respectively; P < 0.05). These findings occurred independently of infarct volume and are consistent with a smaller peri-infarct zone in nNOS- mice. We postulate that the smaller peri-infarct zone is a reflection of less severe metabolic disturbance after ischemia in nNOS- mice, possibly related to diminished production of nitric oxide (NO) or a related product. We conclude that magnetic resonance techniques previously used to assess ischemic damage in larger animals can be extended to the mouse, raising the possibility that the molecular mechanisms leading to ischemic damage can be examined by using genetically engineered mice. -
Harding P, Sigmon DH, Alfie ME, Huang PL, Fishman MC, Beierwaltes WH, Carretero OA. 1997. Cyclooxygenase-2 mediates increased renal renin content induced by low-sodium diet. Hypertension (Dallas, Tex. : 1979). 29(1 Pt 2):297-302. Pubmed: 9039118 Harding P, Sigmon DH, Alfie ME, Huang PL, Fishman MC, Beierwaltes WH, Carretero OA. 1997. Cyclooxygenase-2 mediates increased renal renin content induced by low-sodium diet. Hypertension (Dallas, Tex. : 1979). 29(1 Pt 2):297-302. Pubmed: 9039118 We hypothesized that neuronal nitric oxide synthase and cyclooxygenase-2, which both exist in the renal cortex, predominantly in the macula densa, play a role in the control of renal renin tissue content. We studied the possible role of neuronal nitric oxide synthase in regulating renal renin content by using mice in which the neuronal nitric oxide synthase gene has been disrupted (nNOS-/-) compared with its two progenitor strains, the 129/SvEv and the C57BL/6, to determine if the absence of neuronal nitric oxide synthase would result in decreased renal renin content or blunt the increase observed during low sodium intake. Renal renin content from cortical slices was determined in adult mice from all three strains maintained on a normal sodium diet. Renal renin content was significantly reduced in the nNOS-/- mice compared with the 129/SvEv and the C57BL/6 mice (3.11 +/- 0.23 versus 5.66 +/- 0.50 and 7.55 +/- 1.17 micrograms angiotensin l/mg dry weight, respectively; P < .005), suggesting that neuronal nitric oxide synthase may stimulate renal renin content under basal conditions. Neither selective pharmacological inhibition of neuronal nitric oxide synthase using 7-nitroindazole or disruption of the neuronal nitric oxide synthase gene affected the increase in renal content observed during dietary sodium restriction. The influence of cyclooxygenase-2 on renal renin content through a macula densa-mediated pathway was studied using a selective cyclooxygenase-2 inhibitor, NS398, in 129/SvEv mice. A low-sodium diet increased renal renin content from 6.97 +/- 0.52 to 11.59 +/- 0.79 micrograms angiotensin l/mg dry weight (P < .005); but this increase was blocked by NS398. In addition, treatment with NS398 reduced renin mRNA in response to a low-sodium diet. Thus, increased renal renin content in response to dietary sodium restriction appears to require the induction of cyclooxygenase-2, while neuronal nitric oxide synthase appears to affect basal but not stimulated renal renin content. -
Fishman MC, Stainier DY, Breitbart RE, Westerfield M. 1997. Zebrafish: genetic and embryological methods in a transparent vertebrate embryo. Methods in cell biology. 52:67-82. Pubmed: 9379966 Fishman MC, Stainier DY, Breitbart RE, Westerfield M. 1997. Zebrafish: genetic and embryological methods in a transparent vertebrate embryo. Methods in cell biology. 52:67-82. Pubmed: 9379966 -
Matthews RT, Beal MF, Fallon J, Fedorchak K, Huang PL, Fishman MC, Hyman BT. 1997. MPP+ induced substantia nigra degeneration is attenuated in nNOS knockout mice. Neurobiology of disease. 4(2):114-21. Pubmed: 9331901 Matthews RT, Beal MF, Fallon J, Fedorchak K, Huang PL, Fishman MC, Hyman BT. 1997. MPP+ induced substantia nigra degeneration is attenuated in nNOS knockout mice. Neurobiology of disease. 4(2):114-21. Pubmed: 9331901 Recent studies showed that neuronal nitric oxide synthase (nNOS) plays a role in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity. In the present study we examined the effects of striatal injection of 1-methyl-4-phenylpyridinium (MPP+) on substantia nigra degeneration in mutant mice lacking the nNOS gene or the endothelial nitric oxide synthase (eNOS) gene. Both striatal lesion volume and substantia nigra degeneration were significantly attenuated in the nNOS mutant mice but not in the eNOS mutant mice. The mice lacking nNOS showed a significant attenuation of MPP+(-) induced increases of 3-nitrotyrosine concentrations in the striatum. In a separate experiment administration of 7-nitroindazole for 48 h after MPP+ injections significantly attenuated substantia nigra degeneration in rats. Immunohistochemical studies showed apposition of nNOS-positive neuronal processes on tyrosine hydroxylase-positive neurons. These results provide further evidence that neuronally derived NO and peroxynitrite play a role in MPP+ neurotoxicity. -
Eckhardt F, Behar O, Calautti E, Yonezawa K, Nishimoto I, Fishman MC. 1997. A novel transmembrane semaphorin can bind c-src. Molecular and cellular neurosciences. 9(5-6):409-19. Pubmed: 9361278 Eckhardt F, Behar O, Calautti E, Yonezawa K, Nishimoto I, Fishman MC. 1997. A novel transmembrane semaphorin can bind c-src. Molecular and cellular neurosciences. 9(5-6):409-19. Pubmed: 9361278 The semaphorins/collapsins constitute a family of genes unified by the presence of a "semaphorin domain" which has been conserved through metazoan evolution. The semaphorin family comprises both secreted and transmembrane molecules and is thought to be made up of ligands for as yet unidentified receptors. The functions are not known, with the exception of those of sema III (also referred as sem D and collapsin 1), D-sema I, and D-sema II, which have been shown to be involved in axonal pathfinding. Here report the identification of a mouse semaphorin cDNA, termed Sema VIb. Although Sema VIb contains the extracellular semaphorin domain, it lacks the immunoglobulin domain or thrombospondin repeats which are present in other described vertebrate (but not invertebrate) transmembrane semaphorins. During development Sema VIb mRNA is expressed in subregions of the nervous system and is particularly prominent in muscle. In adulthood, Sema VIb mRNA is expressed ubiquitously. The cytoplasmic domain of Sema VIb contains several proline-rich potential SH3 domain binding sites. Using an in vitro binding assay, we show that Sema VIb binds specifically the SH3 domain of the protooncogene c-src. In transfected COS cells Sema VIb coimmunoprecipitates with c-src. These results, along with our evidence that Sema VIb can form dimers, suggests that the semaphorin family not only serves as ligands but may include members, especially those which are transmembrane, which serve as receptors, triggering intracellular signaling via an src-related cascade. -
Fishman MC, Olson EN. 1997. Parsing the heart: genetic modules for organ assembly. Cell. 91(2):153-6. Pubmed: 9346232 Fishman MC, Olson EN. 1997. Parsing the heart: genetic modules for organ assembly. Cell. 91(2):153-6. Pubmed: 9346232 -
Fouquet B, Weinstein BM, Serluca FC, Fishman MC. 1997. Vessel patterning in the embryo of the zebrafish: guidance by notochord. Developmental biology. 183(1):37-48. Pubmed: 9119113 Fouquet B, Weinstein BM, Serluca FC, Fishman MC. 1997. Vessel patterning in the embryo of the zebrafish: guidance by notochord. Developmental biology. 183(1):37-48. Pubmed: 9119113 We have cloned the zebrafish homolog of the receptor tyrosine kinase flk-1 to provide us with a tool to study normal vascular pattern formation in the developing zebrafish embryo and to compare it to mutants in which vascular pattern is perturbed. We find that during normal development the first angioblasts arise laterally in the mesoderm and then migrate medially to form the primordia of the large axial vessels, the dorsal aorta (axial artery) and the axial vein. Lumen formation occurs shortly before onset of circulation at 24 hr postfertilization. We examined the specification of vascular progenitors in the mutant cloche, which fails to form both vessels and blood. cloche lacks all flk-expressing cells and therefore appears to lack angioblasts. The axial vessels of the trunk form in close proximity to notochord and endoderm, which may provide cues for their formation. The dorsal aorta is normally just ventral to the notochord; the axial vein is just below the dorsal aorta and above the endoderm. floating head (flh) and no tail (ntl) mutants both have defects in the formation of notochord. Both are cell-autonomous lesions, flh abolishing notochord and ntl preventing its differentiation. In both mutants the dorsal aorta fails to form, while formation of the axial vein is less affected. Mosaic analysis of mutant embryos shows that transplanted wild-type cells can become notochord in mutant flh embryos. In these mosaic embryos flh cells expressing flk assemble at the midline, beneath the wild-type notochord, and form an aortic primordium. This suggests that signals from the notochord may guide angioblasts in the fashioning of the dorsal aorta. The notochord seems to be less important for the formation of the vein. -
Friedlander RM, Gagliardini V, Hara H, Fink KB, Li W, MacDonald G, Fishman MC, Greenberg AH, Moskowitz MA, Yuan J. 1997. Expression of a dominant negative mutant of interleukin-1 beta converting enzyme in transgenic mice prevents neuronal cell death induced by trophic factor withdrawal and ischemic brain injury. The Journal of experimental medicine. 185(5):933-40. Pubmed: 9120399 Friedlander RM, Gagliardini V, Hara H, Fink KB, Li W, MacDonald G, Fishman MC, Greenberg AH, Moskowitz MA, Yuan J. 1997. Expression of a dominant negative mutant of interleukin-1 beta converting enzyme in transgenic mice prevents neuronal cell death induced by trophic factor withdrawal and ischemic brain injury. The Journal of experimental medicine. 185(5):933-40. Pubmed: 9120399 To explore the role of the interleukin (IL)-1 beta converting enzyme (ICE) in neuronal apoptosis, we designed a mutant ICE gene (C285G) that acts as a dominant negative ICE inhibitor. Microinjection of the mutant ICE gene into embryonal chicken dorsal root ganglial neurons inhibits trophic factor withdrawal-induced apoptosis. Transgenic mice expressing the fused mutant ICE-lacZ gene under the control of the neuron specific enolase promoter appeared neurologically normal. These mice are deficient in processing pro-IL-1 beta, indicating that mutant ICEC285G blocks ICE function. Dorsal root ganglial neurons isolated from transgenic mice were resistant to trophic factor withdrawal-induced apoptosis. In addition, the neurons isolated from newborn ICE knockout mice are similarly resistant to trophic factor withdrawal-induced apoptosis. After permanent focal ischemia by middle cerebral artery occlusion, the mutant ICEC285G transgenic mice show significantly reduced brain injury as well as less behavioral deficits when compared to the wild-type controls. Since ICE is the only enzyme with IL-1 beta convertase activity in mice, our data indicates that the mutant ICEC285G inhibits ICE, and hence mature IL-1 beta production, and through this mechanism, at least in part, inhibits apoptosis. Our data suggest that genetic manipulation using ICE family dominant negative inhibitors can ameliorate the extent of ischemia-induced brain injury and preserve neurological function. -
Chen JN, van Eeden FJ, Warren KS, Chin A, Nüsslein-Volhard C, Haffter P, Fishman MC. 1997. Left-right pattern of cardiac BMP4 may drive asymmetry of the heart in zebrafish. Development (Cambridge, England). 124(21):4373-82. Pubmed: 9334285 Chen JN, van Eeden FJ, Warren KS, Chin A, Nüsslein-Volhard C, Haffter P, Fishman MC. 1997. Left-right pattern of cardiac BMP4 may drive asymmetry of the heart in zebrafish. Development (Cambridge, England). 124(21):4373-82. Pubmed: 9334285 The first evident break in left-right symmetry of the primitive zebrafish heart tube is the shift in pattern of BMP4 expression from radially symmetric to left-predominant. The midline heart tube then 'jogs' to the left and subsequently loops to the right. We examined 279 mutations, affecting more than 200 genes, and found 21 mutations that perturb this process. Some cause BMP4 to remain radially symmetric. Others randomize the asymmetric BMP4 pattern. Retention of BMP4 symmetry is associated with failure to jog: right-predominance of the BMP4 pattern is associated with reversal of the direction of jogging and looping. Raising BMP4 diffusely throughout the heart, via sonic hedgehog injection, or the blocking of its action by injection of a dominant negative BMP4 receptor, prevent directional jogging or looping. The genes crucial to directing cardiac asymmetry include a subset of those needed for patterning the dorsoventral axis and for notochord and ventral spinal cord development. Thus, the pattern of cardiac BMP4 appears to be in the pathway by which the heart interprets lateralizing signals from the midline. -
Valenzuela D, Han X, Mende U, Fankhauser C, Mashimo H, Huang P, Pfeffer J, Neer EJ, Fishman MC. 1997. G alpha(o) is necessary for muscarinic regulation of Ca2+ channels in mouse heart. Proceedings of the National Academy of Sciences of the United States of America. 94(5):1727-32. Pubmed: 9050846 Valenzuela D, Han X, Mende U, Fankhauser C, Mashimo H, Huang P, Pfeffer J, Neer EJ, Fishman MC. 1997. G alpha(o) is necessary for muscarinic regulation of Ca2+ channels in mouse heart. Proceedings of the National Academy of Sciences of the United States of America. 94(5):1727-32. Pubmed: 9050846 Heterotrimeric G proteins, composed of G alpha and G betagamma subunits, transmit signals from cell surface receptors to cellular effector enzymes and ion channels. The G alpha(o) protein is the most abundant G alpha subtype in the nervous system, but it is also found in the heart. Its function is not completely known, although it is required for regulation of N-type Ca2+ channels in GH3 cells and also interacts with GAP43, a major protein in growth cones, suggesting a role in neuronal pathfinding. To analyze the function of G alpha(o), we have generated mice lacking both isoforms of G alpha(o) by homologous recombination. Surprisingly, the nervous system is grossly intact, despite the fact that G alpha(o) makes up 0.2-0.5% of brain particulate protein and 10% of the growth cone membrane. The G alpha(o)-/- mice do suffer tremors and occasional seizures, but there is no obvious histologic abnormality in the nervous system. In contrast, G alpha(o)-/- mice have a clear and specific defect in ion channel regulation in the heart. Normal muscarinic regulation of L-type calcium channels in ventricular myocytes is absent in the mutant mice. The L-type calcium channel responds normally to isoproterenol, but there is no evident muscarinic inhibition. Muscarinic regulation of atrial K+ channels is normal, as is the electrocardiogram. The levels of other G alpha subunits (G alpha(s), G alpha(q), and G alpha(i)) are unchanged in the hearts of G alpha(o)-/- mice, but the amount of G betagamma is decreased. Whichever subunit, G alpha(o) or G betagamma, carries the signal forward, these studies show that muscarinic inhibition of L-type Ca2+ channels requires coupling of the muscarinic receptor to G alpha(o). Other cardiac G alpha subunits cannot substitute. -
Baker K, Warren KS, Yellen G, Fishman MC. 1997. Defective "pacemaker" current (Ih) in a zebrafish mutant with a slow heart rate. Proceedings of the National Academy of Sciences of the United States of America. 94(9):4554-9. Pubmed: 9114028 Baker K, Warren KS, Yellen G, Fishman MC. 1997. Defective "pacemaker" current (Ih) in a zebrafish mutant with a slow heart rate. Proceedings of the National Academy of Sciences of the United States of America. 94(9):4554-9. Pubmed: 9114028 At a cellular level, cardiac pacemaking, which sets the rate and rhythm of the heartbeat, is produced by the slow membrane depolarization that occurs between action potentials. Several ionic currents could account for this pacemaker potential, but their relative prominence is controversial, and it is not known which ones actually play a pacemaking role in vivo. To correlate currents in individual heart cells with the rhythmic properties of the intact heart, we have examined slow mo (smo), a recessive mutation we discovered in the zebrafish Danio rerio. This mutation causes a reduced heart rate in the embryo, a property we can quantitate because the embryo is transparent. We developed methods for culture of cardiocytes from zebrafish embryos and found that, even in culture, cells from smo continue to beat relatively slowly. By patch-clamp analysis, we discovered that a large repertoire of cardiac currents noted in other species are present in these cultured cells, including sodium, T-type, and L-type calcium and several potassium currents, all of which appear normal in the mutant. The only abnormality appears to be in a hyperpolarization-activated inward current with the properties of Ih, a current described previously in the nervous system, pacemaker, and other cardiac tissue. smo cardiomyocytes have a reduction in Ih that appears to result from severe diminution of one kinetic component of the Ih current. This provides strong evidence that Ih is an important contributor to the pacemaking behavior of the intact heart. -
Fishman MC, Chien KR. 1997. Fashioning the vertebrate heart: earliest embryonic decisions. Development (Cambridge, England). 124(11):2099-117. Pubmed: 9187138 Fishman MC, Chien KR. 1997. Fashioning the vertebrate heart: earliest embryonic decisions. Development (Cambridge, England). 124(11):2099-117. Pubmed: 9187138 Our goal here is to set out the types of unitary decisions made by heart progenitor cells, from their appearance in the heart field until they form the simple heart tube. This provides a context to evaluate cell fate, lineage and, finally, morphogenetic decisions that configure global heart form and function. Some paradigms for cellular differentiation and for pattern generation may be borrowed from invertebrates, but neither Drosophila nor Caenorhabditis elegans suffice to unravel higher order decisions. Genetic analyses in mouse and zebrafish may provide one entrance to these pathways. -
Steudel W, Ichinose F, Huang PL, Hurford WE, Jones RC, Bevan JA, Fishman MC, Zapol WM. 1997. Pulmonary vasoconstriction and hypertension in mice with targeted disruption of the endothelial nitric oxide synthase (NOS 3) gene. Circulation research. 81(1):34-41. Pubmed: 9201025 Steudel W, Ichinose F, Huang PL, Hurford WE, Jones RC, Bevan JA, Fishman MC, Zapol WM. 1997. Pulmonary vasoconstriction and hypertension in mice with targeted disruption of the endothelial nitric oxide synthase (NOS 3) gene. Circulation research. 81(1):34-41. Pubmed: 9201025 NO, synthesized in endothelial cells by endothelial NO synthase (NOS 3), is believed to be an important endogenous pulmonary vasodilator substance that contributes to the normal low pulmonary vascular resistance. To selectively investigate the role of NOS 3 in the pulmonary circulation, mice with targeted disruption of the NOS 3 gene were studied. Pulmonary hemodynamics were studied by measuring pulmonary artery pressure, left ventricular end-diastolic pressure, and lower thoracic aortic flow by using a novel open-chest technique. Transient partial occlusion of the inferior vena cava was used to assess the pulmonary artery pressure-flow relationship. Tension developed by isolated pulmonary artery segments after acetylcholine stimulation was measured in vitro. The histological appearance of NOS 3-deficient and wild-type murine lungs was compared. NOS 3-deficient mice (n = 27), when compared with wild-type mice (n = 32), had pulmonary hypertension (pulmonary artery pressure, 19.0 +/- 0.8 versus 16.4 +/- 0.6 mm Hg [mean +/- SE]; P < .05) that was due to an increased total pulmonary resistance (62 +/- 6 versus 33 +/- 2 mm Hg.min.g.mL-1; P < .001). In vitro, acetylcholine induced vasodilation in the main pulmonary arteries of wild-type but not NOS 3-deficient mice. The morphology of the lungs of NOS 3-deficient mice did not differ from that of wild-type mice. We conclude that NOS 3 is a key enzyme responsible for providing basal pulmonary NO release. Congenital NOS 3 deficiency produces mild pulmonary hypertension in mice. -
Vorwerk CK, Hyman BT, Miller JW, Husain D, Zurakowski D, Huang PL, Fishman MC, Dreyer EB. 1997. The role of neuronal and endothelial nitric oxide synthase in retinal excitotoxicity. Investigative ophthalmology & visual science. 38(10):2038-44. Pubmed: 9331267 Vorwerk CK, Hyman BT, Miller JW, Husain D, Zurakowski D, Huang PL, Fishman MC, Dreyer EB. 1997. The role of neuronal and endothelial nitric oxide synthase in retinal excitotoxicity. Investigative ophthalmology & visual science. 38(10):2038-44. Pubmed: 9331267 Array 1996
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Knapik EW, Goodman A, Atkinson OS, Roberts CT, Shiozawa M, Sim CU, Weksler-Zangen S, Trolliet MR, Futrell C, Innes BA, Koike G, McLaughlin MG, Pierre L, Simon JS, Vilallonga E, Roy M, Chiang PW, Fishman MC, Driever W, Jacob HJ. 1996. A reference cross DNA panel for zebrafish (Danio rerio) anchored with simple sequence length polymorphisms. Development (Cambridge, England). 123:451-60. Pubmed: 9007262 Knapik EW, Goodman A, Atkinson OS, Roberts CT, Shiozawa M, Sim CU, Weksler-Zangen S, Trolliet MR, Futrell C, Innes BA, Koike G, McLaughlin MG, Pierre L, Simon JS, Vilallonga E, Roy M, Chiang PW, Fishman MC, Driever W, Jacob HJ. 1996. A reference cross DNA panel for zebrafish (Danio rerio) anchored with simple sequence length polymorphisms. Development (Cambridge, England). 123:451-60. Pubmed: 9007262 The ultimate informativeness of the zebrafish mutations described in this issue will rest in part on the ability to clone these genes. However, the genetic infrastructure required for the positional cloning in zebrafish is still in its infancy. Here we report a reference cross panel of DNA, consisting of 520 F2 progeny (1040 meioses) that has been anchored to a zebrafish genetic linkage map by 102 simple sequence length polymorphisms. This reference cross DNA provides: (1) a panel of DNA from the cross that was used to construct the genetic linkage map, upon which polymorphic gene(s) and genetic markers can be mapped; (2) a fine order mapping tool, with a maximum resolution of 0.1 cM; and (3) a foundation for the development of a physical map (an ordered array of clones each containing a known portion of the genome). This reference cross DNA will serve as a resource enabling investigators to relate genes or genetic markers directly to a single genetic linkage map and avoid the problem of integrating different maps with different genetic markers, as must be currently done when using randomly amplified polymorphic DNA markers, or as has occurred with human genetic linkage maps. -
Huang PL, Fishman MC. 1996. Genetic analysis of nitric oxide synthase isoforms: targeted mutation in mice. Journal of molecular medicine (Berlin, Germany). 74(8):415-21. Pubmed: 8872855 Huang PL, Fishman MC. 1996. Genetic analysis of nitric oxide synthase isoforms: targeted mutation in mice. Journal of molecular medicine (Berlin, Germany). 74(8):415-21. Pubmed: 8872855 Since the discovery that nitric oxide is an endogenous vasodilator responsible for endothelium-derived relaxing factor activity, nitric oxide has been found in many different cell types and implicated in many diverse biological processes. Because pharmacological blockade does not distinguish between the three major isoforms of nitric oxide synthase, the tissue and enzyme source of nitric oxide is unclear in many situations. Targeted disruption of the genes for the various isoforms of nitric oxide synthase offers a useful genetic approach to study the roles of each isoform and to examine the effects of their deletion on physiological processes in intact animals. Here we review the phenotypes of the various nitric oxide synthase mutant mice and examine what they reveal about the complexities of the nitric oxide signaling system and about molecular and physiological compensations brought into play in the absence of individual isoforms. -
Eder PS, Srinivasan A, Fishman MC, Altman S. 1996. The RNA subunit of ribonuclease P from the zebrafish, Danio rerio. The Journal of biological chemistry. 271(35):21031-6. Pubmed: 8702867 Eder PS, Srinivasan A, Fishman MC, Altman S. 1996. The RNA subunit of ribonuclease P from the zebrafish, Danio rerio. The Journal of biological chemistry. 271(35):21031-6. Pubmed: 8702867 A simple strategy has been devised to identify the gene encoding the RNA subunit of RNase P from the zebrafish, Danio rerio. The sequence obtained by amplification of genomic DNA with primers based on sequences common to two other vertebrates was confirmed by reverse transcription and amplification of RNA from a partially purified preparation of the holoenzyme. The 5' and 3' ends were determined by cyclizing the RNA, followed by reverse transcription and sequencing across the ligated RNA junction. The zebrafish sequence is 63% identical to that of Xenopus laevis nuclear RNase P RNA and 69% identical to the human RNase P RNA. A consensus secondary structure was constructed based on these nucleotide identities and on the many compensatory base changes in several regions among these three RNAs. The strategy used to obtain the zebrafish sequence should be useful in deriving analogous gene sequences from diverse classes of eukaryotes. -
Li W, Fishman MC, Yuan J. 1996. Prevention of apoptosis in CNTF-dependent neurons by a mutant ICE and by viral protein CrmA but not by proto-oncogene product Bcl-2. Cell death and differentiation. 3(1):105-12. Pubmed: 17180061 Li W, Fishman MC, Yuan J. 1996. Prevention of apoptosis in CNTF-dependent neurons by a mutant ICE and by viral protein CrmA but not by proto-oncogene product Bcl-2. Cell death and differentiation. 3(1):105-12. Pubmed: 17180061 The interleukin-1beta converting enzyme (ICE) gene family, (homologues of C. elegans cell death gene product Ced-3) plays an important role in controlling programmed cell death. Nerve growth factor (NGF) promotes survival of cultured embryonic chicken dorsal root ganglion neurons. Ciliary ganglion neurons depend exclusively on ciliary neurotrophic factor (CNTF) for survival. Complete depletion of NGF or CNTF from culture medium induces apoptosis in both types of neurons. We can prevent apoptosis, due either to NGF or CNTF withdrawal and in either type of neuron, by overexpression of a mutant inactive ICE and an ICE inhibitor, the product of cowpox virus gene crmA. Bcl-2 does not prevent apoptosis in CNTF-dependent ciliary neurons or DRG neurons as it does in NGF-dependent neurons. These results suggest that neuronal cell death is mediated through a common effector mechanism involving the Ice family of genes, whereas different suppression mechanisms are engaged depending upon the specific neurotrophic factors present. -
Son H, Hawkins RD, Martin K, Kiebler M, Huang PL, Fishman MC, Kandel ER. 1996. Long-term potentiation is reduced in mice that are doubly mutant in endothelial and neuronal nitric oxide synthase. Cell. 87(6):1015-23. Pubmed: 8978606 Son H, Hawkins RD, Martin K, Kiebler M, Huang PL, Fishman MC, Kandel ER. 1996. Long-term potentiation is reduced in mice that are doubly mutant in endothelial and neuronal nitric oxide synthase. Cell. 87(6):1015-23. Pubmed: 8978606 Nitric oxide (NO) has been implicated in hippocampal long-term potentiation (LTP), but LTP is normal in mice with a targeted mutation in the neuronal form of NO synthase (nNOS-). LTP was also normal in mice with a targeted mutation in endothelial NOS (eNOS-), but LTP in stratum radiatum of CA1 was significantly reduced in doubly mutant mice (nNOS-/eNOS-). By contrast, LTP in stratum oriens was normal in the doubly mutant mice. These results provide the first genetic evidence that NOS is involved in LTP in stratum radiatum and suggest that the neuronal and endothelial forms can compensate for each other in mice with a single mutation. They further suggest that there is also a NOS-independent component of LTP in stratum radiatum and that LTP in stratum oriens is largely NOS independent. -
Carpenter C, Honkanen AA, Mashimo H, Goss KA, Huang P, Fishman MC, Asaad M, Dorso CR, Cheung H. 1996. Renal abnormalities in mutant mice. Nature. 380(6572):292. Pubmed: 8598926 Carpenter C, Honkanen AA, Mashimo H, Goss KA, Huang P, Fishman MC, Asaad M, Dorso CR, Cheung H. 1996. Renal abnormalities in mutant mice. Nature. 380(6572):292. Pubmed: 8598926 -
Lo EH, Hara H, Rogowska J, Trocha M, Pierce AR, Huang PL, Fishman MC, Wolf GL, Moskowitz MA. 1996. Temporal correlation mapping analysis of the hemodynamic penumbra in mutant mice deficient in endothelial nitric oxide synthase gene expression. Stroke. 27(8):1381-5. Pubmed: 8711806 Lo EH, Hara H, Rogowska J, Trocha M, Pierce AR, Huang PL, Fishman MC, Wolf GL, Moskowitz MA. 1996. Temporal correlation mapping analysis of the hemodynamic penumbra in mutant mice deficient in endothelial nitric oxide synthase gene expression. Stroke. 27(8):1381-5. Pubmed: 8711806 Array -
Weinstein BM, Fishman MC. 1996. Cardiovascular morphogenesis in zebrafish. Cardiovascular research. 31 Spec No:E17-24. Pubmed: 8681341 Weinstein BM, Fishman MC. 1996. Cardiovascular morphogenesis in zebrafish. Cardiovascular research. 31 Spec No:E17-24. Pubmed: 8681341 -
Mashimo H, He XD, Huang PL, Fishman MC, Goyal RK. 1996. Neuronal constitutive nitric oxide synthase is involved in murine enteric inhibitory neurotransmission. The Journal of clinical investigation. 98(1):8-13. Pubmed: 8690808 Mashimo H, He XD, Huang PL, Fishman MC, Goyal RK. 1996. Neuronal constitutive nitric oxide synthase is involved in murine enteric inhibitory neurotransmission. The Journal of clinical investigation. 98(1):8-13. Pubmed: 8690808 Array -
Ma J, Ayata C, Huang PL, Fishman MC, Moskowitz MA. 1996. Regional cerebral blood flow response to vibrissal stimulation in mice lacking type I NOS gene expression. The American journal of physiology. 270(3 Pt 2):H1085-90. Pubmed: 8780207 Ma J, Ayata C, Huang PL, Fishman MC, Moskowitz MA. 1996. Regional cerebral blood flow response to vibrissal stimulation in mice lacking type I NOS gene expression. The American journal of physiology. 270(3 Pt 2):H1085-90. Pubmed: 8780207 The role of nitric oxide (NO) in cerebral blood flow-metabolism coupling was assessed in SV-129 wild-type (WT) and neuronal (type I) NO synthase (NOS) knockout mice (Kn). Regional cerebral blood flow (rCBF; laser-Doppler flowmetry) was measured over the contralateral cortical barrel field during unilateral mechanical vibrissal deflection (2-3 Hz, 60 s) under urethan anesthesia. The rCBF response was similar in WT and Kn and did not differ when recorded over the intact skull or closed cranial window preparations. Whisker stimulation increased rCBF by 41 +/- 8% (maximum) and 27 +/- 6% (mean) in WT (n = 6) and 41 +/- 7% (maximum) and 26 +/- 6% (mean) in Kn (n = 6) when recorded through a closed cranial window. After superfusion with topical N omega-nitro-L-arginine (L-NNA; 1 mM), the rCBF response was inhibited by approximately 45% in WT mice (P < 0.05), whereas there was no inhibition in Kn. Endothelium-dependent relaxation, assessed by pial vessel dilation in response to topical acetylcholine (100 microM) and inhibition by L-NNA (1 mM), was the same in both groups. Our results suggest that 1) endothelial NO production does not mediate the rCBF coupling to neuronal activity in Kn, 2) the inhibitory effect of L-NNA on the rCBF response to whisker stimulation in WT is a consequence of type I (neuronal) NOS inhibition, and 3) NO-independent mechanisms couple rCBF and metabolism during whisker stimulation in mice lacking expression of neuronal NOS. -
Schulz JB, Huang PL, Matthews RT, Passov D, Fishman MC, Beal MF. 1996. Striatal malonate lesions are attenuated in neuronal nitric oxide synthase knockout mice. Journal of neurochemistry. 67(1):430-3. Pubmed: 8667023 Schulz JB, Huang PL, Matthews RT, Passov D, Fishman MC, Beal MF. 1996. Striatal malonate lesions are attenuated in neuronal nitric oxide synthase knockout mice. Journal of neurochemistry. 67(1):430-3. Pubmed: 8667023 Intrastriatal administration of the reversible succinate dehydrogenase inhibitor malonate produces both energy depletion and striatal lesions by a secondary excitotoxic mechanism. To investigate the role of nitric oxide (NO.) in the pathogenesis of the lesions we examined malonate toxicity in mice in which the genes for neuronal nitric oxide synthase (nNOS) or endothelial nitric oxide synthase (eNOS) were disrupted. Malonate striatal lesions were significantly attenuated in the nNOS mutant mice, and they were significantly increased in the eNOS mutant mice. Malonate-induced increases in levels of 2,3- and 2,5-dihydroxybenzoic acid/salicylate, markers of hydroxyl radical generation, were significantly attenuated in the nNOS knockout mice. Malonate-induced increases in 3-nitrotyrosine, a marker for peroxynitrite-mediated damage, were blocked in the nNOS mice, whereas a significant increase occurred in the eNOS mice. These findings show that NO. produced by nNOS results in generation of peroxynitrite, which plays a role in malonate neurotoxicity. -
Behar O, Golden JA, Mashimo H, Schoen FJ, Fishman MC. 1996. Semaphorin III is needed for normal patterning and growth of nerves, bones and heart. Nature. 383(6600):525-8. Pubmed: 8849723 Behar O, Golden JA, Mashimo H, Schoen FJ, Fishman MC. 1996. Semaphorin III is needed for normal patterning and growth of nerves, bones and heart. Nature. 383(6600):525-8. Pubmed: 8849723 The expression patterns of the recently discovered family of semaphorin genes suggests that they have widespread roles in embryonic development. Some seem to guide neuronal growth cones, but otherwise their functions are unknown. Semaphorin III is a membrane-associated secreted protein with a developmentally dynamic pattern of expression, including particular domains of the nervous system, the borders of developing bones, and the heart. In vitro, semaphorin III causes growth-cone collapse, and repels cutaneous sensory axons from the ventral spinal cord. Mutants in the Drosophila gene semaII, which encodes a related semaphorin, die after eclosion, but no responsible abnormality is evident. We have generated mice mutant in the semaIII gene by homologous recombination. Here we show that in the mutants, some sensory axons project into inappropriate regions of the spinal cord where semaIII is normally expressed. The cerebral cortex of homozygous mutant mice shows a paucity of neuropil and abnormally oriented neuronal processes, especially of the large pyramidal neurons. Certain embryonic bones and cartilaginous structures develop abnormally, with vertebral fusions and partial rib duplications. The few mice that survive more than a few days postnatally manifest pronounced and selective hypertrophy of the right ventricle of the heart and dilation of the right atrium. Thus, semaphorin III might serve as a signal that restrains growth in several developing organs. -
Hara H, Huang PL, Panahian N, Fishman MC, Moskowitz MA. 1996. Reduced brain edema and infarction volume in mice lacking the neuronal isoform of nitric oxide synthase after transient MCA occlusion. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 16(4):605-11. Pubmed: 8964799 Hara H, Huang PL, Panahian N, Fishman MC, Moskowitz MA. 1996. Reduced brain edema and infarction volume in mice lacking the neuronal isoform of nitric oxide synthase after transient MCA occlusion. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 16(4):605-11. Pubmed: 8964799 Infarct volume and edema were assessed after transient focal ischemia in mice lacking neuronal nitric oxide synthase (NOS) gene expression. With use of an 8-0 coated monofilament, the middle cerebral artery (MCA) of mutant (n = 32) and wild-type mice [SV-129 (n = 31), C57Black/6 (n = 18)] were occluded for 3 h and reperfused for up to 24 h. Regional CBF (rCBF), neurological deficits, water content, and infarct volume were examined in all three strains. rCBF, blood pressure, and heart rate did not differ between groups when measured for 1 h after reperfusion. Neurological deficits were less severe in mutant mice after MCA occlusion. Brain water content at 3 h after reperfusion and infarct volume at 24 h after reperfusion were greater in wild-type mice. These data indicate that genetic deletion of neuronal NOS confers resistance to focal ischemic injury in a reperfusion model. The findings agree with previous studies showing that tissue injury is less extensive after both permanent MCA occlusion and global ischemia in mice lacking neuronal NOS gene expression. Hence, NO may play a pivotal role in the pathogenesis of ischemic brain damage. -
Ticho BS, Stainier DY, Fishman MC, Breitbart RE. 1996. Three zebrafish MEF2 genes delineate somitic and cardiac muscle development in wild-type and mutant embryos. Mechanisms of development. 59(2):205-18. Pubmed: 8951797 Ticho BS, Stainier DY, Fishman MC, Breitbart RE. 1996. Three zebrafish MEF2 genes delineate somitic and cardiac muscle development in wild-type and mutant embryos. Mechanisms of development. 59(2):205-18. Pubmed: 8951797 The zebrafish is an important experimental system for vertebrate embryology, and is well suited to the molecular analysis of muscle development. Transcription factors, such as the MEF2s, regulate skeletal and cardiac muscle-specific genes during development. We report the identification of three zebrafish MEF2 genes which, like their mammalian counterparts, encode factors that function as DNA-binding transcriptional activators of muscle specific promoters. The pattern of MEF2 expression in zebrafish defines discrete cell populations in the developing somites and heart and has mechanistic implications for developmental regulation of the MEF2 genes, when compared with other species. Alteration of MEF2 expression in two mutants affecting somitogenesis provides insight into the control of muscle formation in the embryo. -
Mashimo H, Wu DC, Podolsky DK, Fishman MC. 1996. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor. Science (New York, N.Y.). 274(5285):262-5. Pubmed: 8824194 Mashimo H, Wu DC, Podolsky DK, Fishman MC. 1996. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor. Science (New York, N.Y.). 274(5285):262-5. Pubmed: 8824194 The mechanisms that maintain the epithelial integrity of the gastrointestinal tract remain largely undefined. The gene encoding intestinal trefoil factor (ITF), a protein secreted throughout the small intestine and colon, was rendered nonfunctional in mice by targeted disruption. Mice lacking ITF had impaired mucosal healing and died from extensive colitis after oral administration of dextran sulfate sodium, an agent that causes mild epithelial injury in wild-type mice. ITF-deficient mice manifested poor epithelial regeneration after injury. These findings reveal a central role for ITF in the maintenance and repair of the intestinal mucosa. -
Pack M, Solnica-Krezel L, Malicki J, Neuhauss SC, Schier AF, Stemple DL, Driever W, Fishman MC. 1996. Mutations affecting development of zebrafish digestive organs. Development (Cambridge, England). 123:321-8. Pubmed: 9007252 Pack M, Solnica-Krezel L, Malicki J, Neuhauss SC, Schier AF, Stemple DL, Driever W, Fishman MC. 1996. Mutations affecting development of zebrafish digestive organs. Development (Cambridge, England). 123:321-8. Pubmed: 9007252 The zebrafish gastrointestinal system matures in a manner akin to higher vertebrates. We describe nine mutations that perturb development of these organs. Normally, by the fourth day postfertilization the digestive organs are formed, the epithelial cells of the intestine are polarized and express digestive enzymes, the hepatocytes secrete bile, and the pancreatic islets and acini generate immunoreactive insulin and carboxypeptidase A, respectively. Seven mutations cause arrest of intestinal epithelial development after formation of the tube but before cell polarization is completed. These perturb different regions of the intestine. Six preferentially affect foregut, and one the hindgut. In one of the foregut mutations the esophagus does not form. Two mutations cause hepatic degeneration. The pancreas is affected in four mutants, all of which also perturb anterior intestine. The pancreatic exocrine cells are selectively affected in these four mutations. Exocrine precursor cells appear, as identified by GATA-5 expression, but do not differentiate and acini do not form. The pancreatic islets are spared, and endocrine cells mature and synthesize insulin. These gastrointestinal mutations may be informative with regard to patterning and crucial lineage decisions during organogenesis, and may be relevant to diabetes, congenital dysmorphogenesis and disorders of cell proliferation. -
Weinstein BM, Schier AF, Abdelilah S, Malicki J, Solnica-Krezel L, Stemple DL, Stainier DY, Zwartkruis F, Driever W, Fishman MC. 1996. Hematopoietic mutations in the zebrafish. Development (Cambridge, England). 123:303-9. Pubmed: 9007250 Weinstein BM, Schier AF, Abdelilah S, Malicki J, Solnica-Krezel L, Stemple DL, Stainier DY, Zwartkruis F, Driever W, Fishman MC. 1996. Hematopoietic mutations in the zebrafish. Development (Cambridge, England). 123:303-9. Pubmed: 9007250 We have identified mutations that perturb the formation or differentiation of the first embryonic blood cells in the zebrafish embryo. These 'primitive' red blood cells originate in the intermediate cell mass of the trunk, a derivative of the dorsal lateral plate mesoderm. By transfusion of blood between embryos we demonstrate that this cohort of cells provides the embryo with all, or nearly all, of its blood cells until at least day 5 postfertilization. Larval lethal mutations generated by ENU mutagenesis affect different steps in the development of these cells. Some cause defects in precursor generation, others defects in differentiation, and others an increase in cellular photosensitivity. -
Ma J, Meng W, Ayata C, Huang PL, Fishman MC, Moskowitz MA. 1996. L-NNA-sensitive regional cerebral blood flow augmentation during hypercapnia in type III NOS mutant mice. The American journal of physiology. 271(4 Pt 2):H1717-9. Pubmed: 8897969 Ma J, Meng W, Ayata C, Huang PL, Fishman MC, Moskowitz MA. 1996. L-NNA-sensitive regional cerebral blood flow augmentation during hypercapnia in type III NOS mutant mice. The American journal of physiology. 271(4 Pt 2):H1717-9. Pubmed: 8897969 The effect of NG-nitro-L-arginine (L-NNA) on regional cerebral blood flow (rCBF) response to hypercapnia (5% CO2 inhalation) was studied in urethan-anesthetized wild-type (SV-129) and type III nitric oxide (NO) synthase (NOS)-deficient mice, using laser-Doppler flowmetry and the closed cranial window technique. Resting rCBF during normocapnia decreased by approximately 25% after L-NNA superfusion in wild-type mice only (n = 18), suggesting a role for type III NOS in baseline blood flow. Hypercapnia augmented rCBF approximately 50% in both wild-type and type III NOS mutant mice. L-NNA superfusion (1 mM) inhibited this increase by approximately 60% in both strains. Hence, synthesis of NO by the constitutively expressed type I NOS contributes to blood flow augmentation during hypercapnia. -
Stainier DY, Fouquet B, Chen JN, Warren KS, Weinstein BM, Meiler SE, Mohideen MA, Neuhauss SC, Solnica-Krezel L, Schier AF, Zwartkruis F, Stemple DL, Malicki J, Driever W, Fishman MC. 1996. Mutations affecting the formation and function of the cardiovascular system in the zebrafish embryo. Development (Cambridge, England). 123:285-92. Pubmed: 9007248 Stainier DY, Fouquet B, Chen JN, Warren KS, Weinstein BM, Meiler SE, Mohideen MA, Neuhauss SC, Solnica-Krezel L, Schier AF, Zwartkruis F, Stemple DL, Malicki J, Driever W, Fishman MC. 1996. Mutations affecting the formation and function of the cardiovascular system in the zebrafish embryo. Development (Cambridge, England). 123:285-92. Pubmed: 9007248 As part of a large-scale mutagenesis screen of the zebrafish genome, we have identified 58 mutations that affect the formation and function of the cardiovascular system. The cardiovascular system is particularly amenable for screening in the transparent zebrafish embryo because the heart and blood vessels are prominent and their function easily examined. We have classified the mutations affecting the heart into those that affect primarily either morphogenesis or function. Nine mutations clearly disrupt the formation of the heart. cloche deletes the endocardium. In cloche mutants, the myocardial layer forms in the absence of the endocardium but is dysmorphic and exhibits a weak contractility. Two loci, miles apart and bonnie and clyde, play a critical role in the fusion of the bilateral tubular primordia. Three mutations lead to an abnormally large heart and one to the formation of a diminutive, dysmorphic heart. We have found no mutation that deletes the myocardial cells altogether, but one, pandora, appears to eliminate the ventricle selectively. Seven mutations interfere with vascular integrity, as indicated by hemorrhage at particular sites. In terms of cardiac function, one large group exhibits a weak beat. In this group, five loci affect both chambers and seven a specific chamber (the atrium or ventricle). For example, the weak atrium mutation exhibits an atrium that becomes silent but has a normally beating ventricle. Seven mutations affect the rhythm of the heart causing, for example, a slow rate, a fibrillating pattern or an apparent block to conduction. In several other mutants, regurgitation of blood flow from ventricle to atrium is the most prominent abnormality, due either to the absence of valves or to poor coordination between the chambers with regard to the timing of contraction. The mutations identified in this screen point to discrete and critical steps in the formation and function of the heart and vasculature. -
Huang Z, Huang PL, Ma J, Meng W, Ayata C, Fishman MC, Moskowitz MA. 1996. Enlarged infarcts in endothelial nitric oxide synthase knockout mice are attenuated by nitro-L-arginine. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 16(5):981-7. Pubmed: 8784243 Huang Z, Huang PL, Ma J, Meng W, Ayata C, Fishman MC, Moskowitz MA. 1996. Enlarged infarcts in endothelial nitric oxide synthase knockout mice are attenuated by nitro-L-arginine. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 16(5):981-7. Pubmed: 8784243 Infarct size and vascular hemodynamics were measured 24 h after middle cerebral artery (MCA) occlusion in mice genetically deficient in the endothelial nitric oxide synthase (eNOS) isoform. eNOS mutant mice developed larger infarcts (21%) than the wild-type strain when assessed 24 h after intraluminal filament occlusion. Moreover, regional CBF values recorded in the MCA territory by laser-Doppler flowmetry were more severely reduced after occlusion and were disproportionately reduced during controlled hemorrhagic hypotension in autoregulation experiments. Unlike the situation in wild-type mice, nitro-L-arginine superfusion (1 mM) dilated pial arterioles of eNOS knockout mice in a closed cranial window preparation. As noted previously, eNOS mutant mice were hypertensive. However, infarct size remained increased despite lowering blood pressure to normotensive levels by hydralazine treatment. Systemic administration of nitro-L-arginine decreased infarct size in eNOS mutant mice (24%) but not in the wild-type strain. This finding complements published data showing that nitro-L-arginine increases infarct size in knockout mice expressing the eNOS but not the neuronal NOS isoform (i.e., neuronal NOS knockout mice). We conclude that NO production within endothelium may protect brain tissue, perhaps by hemodynamic mechanisms, whereas neuronal NO overproduction may lead to neurotoxicity. -
Meng W, Ma J, Ayata C, Hara H, Huang PL, Fishman MC, Moskowitz MA. 1996. ACh dilates pial arterioles in endothelial and neuronal NOS knockout mice by NO-dependent mechanisms. The American journal of physiology. 271(3 Pt 2):H1145-50. Pubmed: 8853353 Meng W, Ma J, Ayata C, Hara H, Huang PL, Fishman MC, Moskowitz MA. 1996. ACh dilates pial arterioles in endothelial and neuronal NOS knockout mice by NO-dependent mechanisms. The American journal of physiology. 271(3 Pt 2):H1145-50. Pubmed: 8853353 We used mice with deletions in either the endothelial nitric oxide synthase (eNOS) or neuronal NOS (nNOS) gene to investigate the role of eNOS and nNOS in acetylcholine (ACh)-induced relaxation of pial arterioles (20-30 microns). Pial arteriolar diameter was measured by intravital microscopy through a closed cranial window, and NOS activity was determined by the conversion of [3H]arginine to [3H]citrulline in subjacent cortex. ACh superfusion (1, 10 microM) caused atropine-sensitive dose-dependent arteriolar dilation in all three mouse strains. At 10 microM, increases of 20 +/- 2, 31 +/- 3, and 23 +/- 3% were recorded in wild-type (n = 25), nNOS mutant (n = 15), and eNOS mutant (n = 20) mice, respectively. NG-nitro-L-arginine (L-NNA, 1 mM) superfusion inhibited cortical NOS activity by > 70% and abrogated the response in wild-type mice while blocking the dilation by approximately 50% in eNOS mutant and nNOS mutant mice. Only in the eNOS mutant did tetrodotoxin (TTX) superfusion (1 microM) attenuate ACh-induced dilation (n = 6). The residual dilation after L-NNA in eNOS mutant mice could be blocked completely by TTX-plus L-NNA. Our findings indicate that 1) ACh dilates pial arterioles of wild-type mice by NOS-dependent mechanisms as reported in other species, 2) the response in nNOS mutant mice resembles the wild-type response except for enhanced dilation to ACh and reduced L-NNA sensitivity, and 3) surprisingly, the response in eNOS mutant mice is partially NOS dependent and attenuated by both TTX and L-NNA. Because nNOS is constitutively expressed in eNOS mutants, these findings coupled with the TTX results suggest that an nNOS-dependent mechanism may compensate for the chronic loss of eNOS activity after targeted gene disruption. -
Chen JN, Fishman MC. 1996. Zebrafish tinman homolog demarcates the heart field and initiates myocardial differentiation. Development (Cambridge, England). 122(12):3809-16. Pubmed: 9012502 Chen JN, Fishman MC. 1996. Zebrafish tinman homolog demarcates the heart field and initiates myocardial differentiation. Development (Cambridge, England). 122(12):3809-16. Pubmed: 9012502 The fashioning of a vertebrate organ requires integration of decisions of cell fate by individual cells with those that regulate organotypic form. Logical candidates for this role, in an organ such as the heart, are genes that initiate the differentiation process leading to heart muscle and those that define the earliest embryonic heart field, but for neither class are genes defined. We cloned zebrafish Nkx2.5, a homolog of the tinman homeodomain gene needed for visceral and cardiac mesoderm formation in Drosophila. In the zebrafish, its expression is associated with cardiac precursor cells throughout development, even in the early gastrula, where the level of zebrafish Nkx2.5 is in a gradient which spatially matches the regional propensity of ventral-marginal cells to become heart. Overexpression of Nkx2.5 causes formation of disproportionally larger hearts in otherwise apparently normal embryos. Transplanted cell expressing high levels of Nkx2.5 express cardiac genes even in ectopic locales. Fibroblasts transfected with myc-tagged Nkx2.5 express cardiac genes. These effects require the homeodomain. Thus, Nkx2.5 appears to mark the earliest embryonic heart field and to be capable of initiating the cardiogenic differentiation program. Because ectopic cells or transfected fibroblasts do not beat, Nkx2.5 is likely to be but one step in the determination of cardiac myocyte cell fate. Its overexpression increases heart size, perhaps by bringing cells on the edge of the field to a threshold level for initiation of cardiac differentiation. -
Hara H, Waeber C, Huang PL, Fujii M, Fishman MC, Moskowitz MA. 1996. Brain distribution of nitric oxide synthase in neuronal or endothelial nitric oxide synthase mutant mice using [3H]L-NG-nitro-arginine autoradiography. Neuroscience. 75(3):881-90. Pubmed: 8951881 Hara H, Waeber C, Huang PL, Fujii M, Fishman MC, Moskowitz MA. 1996. Brain distribution of nitric oxide synthase in neuronal or endothelial nitric oxide synthase mutant mice using [3H]L-NG-nitro-arginine autoradiography. Neuroscience. 75(3):881-90. Pubmed: 8951881 The regional distribution of nitric oxide synthase in the central nervous system was assessed by quantitative autoradiography using [3H]L-NG-nitro-arginine binding in wild-type mice (SV-129 and C57black/6) and in mice lacking expression of the neuronal (type 1) and endothelial (type 3) nitric oxide synthase gene. The distribution of nitric oxide synthase binding sites in wild-type mice was similar to that described for rat brain by nicotinamide adenine dinucleotide phosphate-diaphorase staining and immunohistochemistry, and as determined by quantitative autoradiography. In the wild-type mice, the densest labelling was observed in the granular layer of the olfactory bulb, tenia tecta, rhinal fissure, amygdaloid complex and molecular layer of cerebellum. The islands of Calleja, the hippocampal CA1 and CA3 subfields, dentate gyrus, cortical layers I-II, the superficial gray layer of superior colliculus and the granule layer of cerebellum displayed intermediate binding. Cortical layers III-VI, the striatum and the thalamus were only weakly labelled. Binding was saturable and of high affinity, and was displaced by 7-nitroindazole (100 microM), a potent and selective inhibitor of type 1 nitric oxide synthase, and by unlabelled L-NG-nitro-arginine (10 microM). The density of [3H]L-NG-nitro-arginine binding was dramatically reduced in all brain regions in type 1 mutant mice, whereas there were no detectable binding differences between wild-type and type 3 nitric oxide synthase mutant mice. Hence, type 1 nitric oxide synthase is the major source of [3H]L-NG-nitro-arginine binding in the mouse brain. [3H]L-NG-Nitro-arginine autoradiography may be a useful tool to quantify nitric oxide synthase in different brain areas after pharmacological or physiological manipulations. -
Driever W, Fishman MC. 1996. The zebrafish: heritable disorders in transparent embryos. The Journal of clinical investigation. 97(8):1788-94. Pubmed: 8621761 Driever W, Fishman MC. 1996. The zebrafish: heritable disorders in transparent embryos. The Journal of clinical investigation. 97(8):1788-94. Pubmed: 8621761 -
Panahian N, Yoshida T, Huang PL, Hedley-Whyte ET, Dalkara T, Fishman MC, Moskowitz MA. 1996. Attenuated hippocampal damage after global cerebral ischemia in mice mutant in neuronal nitric oxide synthase. Neuroscience. 72(2):343-54. Pubmed: 8737405 Panahian N, Yoshida T, Huang PL, Hedley-Whyte ET, Dalkara T, Fishman MC, Moskowitz MA. 1996. Attenuated hippocampal damage after global cerebral ischemia in mice mutant in neuronal nitric oxide synthase. Neuroscience. 72(2):343-54. Pubmed: 8737405 To address the importance of nitric oxide or its reaction products as mediators of neurotoxicity in brain, tissue injury was assessed after transient global ischemia in mice rendered mutant in the gene for neuronal nitric oxide synthase. Halothane-anesthetized wild type and mutant mice were subjected to temporary occlusion of the basilar plus both carotid arteries for 5 or 10 min followed by three days of reperfusion. Hippocampal injury, assessed both by qualitative grading and by cell counting in the CA1 subregion, was significantly less in the mutant mice group after 5 or 10 min of ischemia. Mutant mice exhibited a lower mortality (P < 0.01), less weight loss, more normal grooming and spontaneous motor activity and better grasping in the 10 min group. There were no obvious differences in cerebrovascular anatomy or hemodynamics between wild type and mutant mice. The data suggest that a deficiency of neuronal nitric oxide synthase confers increased resistance to transient global cerebral ischemia, and support the suggestion that selective neuronal nitric oxide synthase inhibitors might reduce tissue injury associated with global cerebral ischemia. 1995
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Igarashi M, Li WW, Sudo Y, Fishman MC. 1995. Ligand-induced growth cone collapse: amplification and blockade by variant GAP-43 peptides. The Journal of neuroscience : the official journal of the Society for Neuroscience. 15(8):5660-7. Pubmed: 7643208 Igarashi M, Li WW, Sudo Y, Fishman MC. 1995. Ligand-induced growth cone collapse: amplification and blockade by variant GAP-43 peptides. The Journal of neuroscience : the official journal of the Society for Neuroscience. 15(8):5660-7. Pubmed: 7643208 Growth cones are powerful amplifiers for signals from the microenvironment. Their collapse can be triggered by cell surface components of myelin and brain membranes, as well as by soluble ligands, including neurotransmitters. GAP-43 is a protein concentrated on the inner surface of the growth cone membrane. Assayed in isolation, it interacts with the heterotrimeric protein, G(o), and in oocytes it amplifies the effects of ligand-triggered G protein activation. We wished to examine whether GAP-43 serves to amplify signals at the growth cone. The G(o) stimulating region of GAP-43 is encoded in the 10 amino acids (MLCCMRRT-KQ) of the first exon. We examined the effect of this peptide upon chick dorsal root ganglion growth cone collapse and neurite retraction triggered by brain membranes or myelin, as well as by serotonin. We find that application of the GAP-43 1-10 peptide amplifies the effects of all three ligands. The amplification is greater when GAP-43 1-10 is injected intracellularly. Peptides with amino acid substitutions for the two cysteine residues manifest parallel changes in growth cone collapse and G(o) stimulation. In particular, tyrosine or methionine substitutions cause the peptide to inhibit G(o) and to block induced growth cone collapse. The GAP-43 peptides therefore regulate the sensitivity of growth cones to extrinsic signals. The modified peptides serve as a starting point for the design of reagents to enhance CNS regeneration. -
Mashimo H, Podolsky DK, Fishman MC. 1995. Structure and expression of murine intestinal trefoil factor: high evolutionary conservation and postnatal expression. Biochemical and biophysical research communications. 210(1):31-7. Pubmed: 7741746 Mashimo H, Podolsky DK, Fishman MC. 1995. Structure and expression of murine intestinal trefoil factor: high evolutionary conservation and postnatal expression. Biochemical and biophysical research communications. 210(1):31-7. Pubmed: 7741746 Intestinal Trefoil Factor (ITF) is a member of a family of gastrointestinal tract peptides with region-specific expression which are enhanced at sites of injury and repair. In the present study, the murine homologue gene of ITF was molecularly cloned in order to characterize the structure and expression of this peptide in mice. Murine ITF exhibited 78, 95 and 94% nucleotide homology respectively in exons I, II and III, with overall 90% predicted amino acid identity when compared to the rat ITF. Murine ITF exhibited 70% inferred amino acid identity compared with human ITF. Northern blot analysis of various adult mouse tissues demonstrated that ITF is expressed abundantly in the intestine and colon, and minimally in stomach, but not in brain, lung, spleen, kidney, uterus, pancreas, liver, heart or thymus tissues. Expression of ITF appeared to occur as a post-natal event: antibody specific for ITF stains intensely goblet cells in the intestine and colon of three-day old and older mice, but not in the gastrointestinal tract of younger mice or embryos. -
Strittmatter SM, Fankhauser C, Huang PL, Mashimo H, Fishman MC. 1995. Neuronal pathfinding is abnormal in mice lacking the neuronal growth cone protein GAP-43. Cell. 80(3):445-52. Pubmed: 7859286 Strittmatter SM, Fankhauser C, Huang PL, Mashimo H, Fishman MC. 1995. Neuronal pathfinding is abnormal in mice lacking the neuronal growth cone protein GAP-43. Cell. 80(3):445-52. Pubmed: 7859286 GAP-43 has been termed a "growth" or "plasticity" protein because it is expressed at high levels in neuronal growth cones during development and during axonal regeneration. By homologous recombination, we generated mice lacking GAP-43. The mice die in the early postnatal period. GAP-43-deficient retinal axons remain trapped in the chiasm for 6 days, unable to navigate past this midline decision point. Over the subsequent weeks of life, most GAP-43-deficient axons do enter the appropriate tracts, and the adult CNS is grossly normal. There is no evidence for interference with nerve growth rate, and cultured neurons extend neurites and growth cones in a fashion indistinguishable from controls. Thus, the GAP-43 protein is not essential for axonal outgrowth or growth cone formation per se, but is required at certain decision points, such as the optic chiasm. This is compatible with the hypothesis that GAP-43 serves to amplify pathfinding signals from the growth cone. -
Irikura K, Huang PL, Ma J, Lee WS, Dalkara T, Fishman MC, Dawson TM, Snyder SH, Moskowitz MA. 1995. Cerebrovascular alterations in mice lacking neuronal nitric oxide synthase gene expression. Proceedings of the National Academy of Sciences of the United States of America. 92(15):6823-7. Pubmed: 7542777 Irikura K, Huang PL, Ma J, Lee WS, Dalkara T, Fishman MC, Dawson TM, Snyder SH, Moskowitz MA. 1995. Cerebrovascular alterations in mice lacking neuronal nitric oxide synthase gene expression. Proceedings of the National Academy of Sciences of the United States of America. 92(15):6823-7. Pubmed: 7542777 Nitric oxide (NO) is known to mediate increases in regional cerebral blood flow elicited by CO2 inhalation. In mice with deletion of the gene for neuronal NO synthase (NOS), CO2 inhalation augments cerebral blood flow to the same extent as in wild-type mice. However, unlike wild-type mice, the increased flow in mutants is not blocked by the NOS inhibition, N omega-nitro-L-arginine, and CO2 exposure fails to increase brain levels of cGMP. Topical acetylcholine elicits vasodilation in the mutants which is blocked by N omega-nitro-L-arginine, indicating normal functioning of endothelial NOS. Moreover, immunohistochemical staining for endothelial NOS is normal in the mutants. Thus, following loss of neuronal NOS, the cerebral circulatory response is maintained by a compensatory system not involving NO. -
Darius S, Wolf G, Huang PL, Fishman MC. 1995. Localization of NADPH-diaphorase/nitric oxide synthase in the rat retina: an electron microscopic study. Brain research. 690(2):231-5. Pubmed: 8535841 Darius S, Wolf G, Huang PL, Fishman MC. 1995. Localization of NADPH-diaphorase/nitric oxide synthase in the rat retina: an electron microscopic study. Brain research. 690(2):231-5. Pubmed: 8535841 The activity of NADPH-diaphorase (NADPH-d), a marker for nitric oxide synthase (NOS), was examined histochemically in the rat and mice retina. Mice in which the neuronal NOS gene has been disrupted (nNOS- mice) were used for specificity controls. Light microscopically a few amacrine cells were heavily stained. Other cells were stained weakly or not at all. Under the electron microscope, formazan precipitates were detectable on membranes of endoplasmic reticulum, nuclear envelope, mitochondria, and, in a few cases, the Golgi complex. Bipolar, horizontal, and Müller cells, were if at all, sparsely labeled with formazan. Labeled mitochondria were observed in rod endings and in inner segments of photoreceptors. Outer segments of photoreceptors and ganglion cells were completely free of reaction product. The NADPH-d reaction in wild-type mice displayed a similar distribution pattern to that in rats. Retinae of nNOS- mice showed a complete lack of prominent NADPH-d stained (amacrine) cells. None or a very few labeled membranes were seen. -
Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA, Fishman MC. 1995. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature. 377(6546):239-42. Pubmed: 7545787 Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA, Fishman MC. 1995. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature. 377(6546):239-42. Pubmed: 7545787 Nitric oxide (NO), a potent vasodilator produced by endothelial cells, is thought to be the endothelium-dependent relaxing factor (EDRF) which mediates vascular relaxation in response to acetylcholine, bradykinin and substance P in many vascular beds. NO has been implicated in the regulation of blood pressure and regional blood flow, and also affects vascular smooth-muscle proliferation and inhibits platelet aggregation and leukocyte adhesion. Abnormalities in endothelial production of NO occur in atherosclerosis, diabetes and hypertension. Pharmacological blockade of NO production with arginine analogues such as L-nitroarginine (L-NA) or L-N-arginine methyl ester affects multiple isoforms of nitric oxide synthase (NOS), and so cannot distinguish their physiological roles. To study the role of endothelial NOS (eNOS) in vascular function, we disrupted the gene encoding eNOS in mice. Endothelium-derived relaxing factor activity, as assayed by acetylcholine-induced relaxation, is absent, and the eNOS mutant mice are hypertensive. Thus eNOS mediates basal vasodilation. Responses to NOS blockade in the mutant mice suggest that non-endothelial isoforms of NOS may be involved in maintaining blood pressure. -
Stainier DY, Weinstein BM, Detrich HW, Zon LI, Fishman MC. 1995. Cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages. Development (Cambridge, England). 121(10):3141-50. Pubmed: 7588049 Stainier DY, Weinstein BM, Detrich HW, Zon LI, Fishman MC. 1995. Cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages. Development (Cambridge, England). 121(10):3141-50. Pubmed: 7588049 Endothelial and hematopoietic cells appear synchronously on the extra-embryonic membranes of amniotes in structures known as blood islands. This observation has led to the suggestion that these two ventral lineages share a common progenitor. Recently, we have shown in the zebrafish, Danio rerio, that a single cell in the ventral marginal zone of the early blastula can give rise to both endothelial and blood cells as well as to other mesodermal cells (Stainier, D. Y. R., Lee, R. K. and Fishman, M. C. (1993). Development 119, 31-40; Lee, R. K. K., Stainier, D. Y. R., Weinstein, B. M. and Fishman, M. C. (1994). Development 120, 3361-3366). Here we describe a zebrafish mutation, cloche, that affects both the endothelial and hematopoietic lineages at a very early stage. The endocardium, the endothelial lining of the heart, is missing in mutant embryos. This deletion is selective as evidenced by the presence of other endothelial cells, for example those lining the main vessels of the trunk. Early cardiac morphogenesis proceeds normally even in the absence of the endocardium. The myocardial cells form a tube that is demarcated into chambers, beats rhythmically, but exhibits a reduced contractility. This functional deficit is likely due to the absence of the endocardial cells, although it may be a direct effect of the mutation on the myocardial cells. Cell transplantation studies reveal that the endothelial defect, i.e. the endocardial deletion, is a cell-autonomous lesion, consistent with the possibility that cloche is part of a signal transduction pathway. In addition, the number of blood cells is greatly reduced in cloche mutants and the hematopoietic tissues show no expression of GATA-1 or GATA-2, two key hematopoietic transcription factors that are first expressed during early embryogenesis. These results show that cloche is involved in the genesis and early diversification of the endothelial and blood lineages, possibly by affecting a common progenitor cell population. -
Weinstein BM, Stemple DL, Driever W, Fishman MC. 1995. Gridlock, a localized heritable vascular patterning defect in the zebrafish. Nature medicine. 1(11):1143-7. Pubmed: 7584985 Weinstein BM, Stemple DL, Driever W, Fishman MC. 1995. Gridlock, a localized heritable vascular patterning defect in the zebrafish. Nature medicine. 1(11):1143-7. Pubmed: 7584985 We are using the zebrafish, Danio rerio, to identify genes that generate and pattern the vertebrate vasculature. We have isolated a recessive mutation, gridlockm145 (grlm145) in which blood flow to the tail is impeded by a localized vascular defect. Using a novel microangiographic method, we show that the blockade is in the anterior trunk, where the paired lateral dorsal aortae normally merge to form the single midline aorta. Arterial-venous shunts and collateral vessels develop in most mutant embryos, bypassing the lesion and reconstituting caudal blood flow. The grl defect resembles coarctation of the aorta, a human congenital cardiovascular malformation of unknown aetiology, in the location of the lesion and its consequences and in the mutants' dependence on collateral vessels for survival. -
Nelson RJ, Demas GE, Huang PL, Fishman MC, Dawson VL, Dawson TM, Snyder SH. 1995. Behavioural abnormalities in male mice lacking neuronal nitric oxide synthase. Nature. 378(6555):383-6. Pubmed: 7477374 Nelson RJ, Demas GE, Huang PL, Fishman MC, Dawson VL, Dawson TM, Snyder SH. 1995. Behavioural abnormalities in male mice lacking neuronal nitric oxide synthase. Nature. 378(6555):383-6. Pubmed: 7477374 In addition to its role in blood vessel and macrophage function, nitric oxide (NO) is a neurotransmitter found in high densities in emotion-regulating brain regions. Mice with targeted disruption of neuronal NO synthase (nNOS) display grossly normal appearance, locomotor activity, breeding, long-term potentiation and long-term depression. The nNOS- mice are resistant to neural stroke damage following middle cerebral artery ligation. Although CO2-induced cerebral vasodilatation in wild-type mice is NO-dependent, in nNOS- mice this vasodilation is unaffected by NOS inhibitors. Establishing a behavioural role for NO has, until now, not been feasible, as NOS inhibitor drugs can only be administered acutely and because their pronounced effects on blood pressure and other body functions obfuscate behavioural interpretations. We now report a large increase in aggressive behaviour and excess, inappropriate sexual behaviour in nNOS- mice. -
Grabczyk E, Fishman MC. 1995. A long purine-pyrimidine homopolymer acts as a transcriptional diode. The Journal of biological chemistry. 270(4):1791-7. Pubmed: 7829515 Grabczyk E, Fishman MC. 1995. A long purine-pyrimidine homopolymer acts as a transcriptional diode. The Journal of biological chemistry. 270(4):1791-7. Pubmed: 7829515 Polypurine-polypyrimidine (R.Y) sequences have the unusual ability to form DNA triple helices. Such tracts are overrepresented upstream of eukaryotic genes, although a function there has not been clear. We report that transcription in vitro into one such upstream R.Y tract in the direction that makes a predominantly purine RNA is effectively blocked by formation of an intramolecular triple helix. The triplex is triggered by transcription and stabilized by the binding of nascent purine RNA to the template. Transcription in the opposite direction is not restricted. Polypurine-polypyrimidine DNA may provide a dynamic and selective block to transcription without the aid of accessory proteins. 1994
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Lee RK, Stainier DY, Weinstein BM, Fishman MC. 1994. Cardiovascular development in the zebrafish. II. Endocardial progenitors are sequestered within the heart field. Development (Cambridge, England). 120(12):3361-6. Pubmed: 7821208 Lee RK, Stainier DY, Weinstein BM, Fishman MC. 1994. Cardiovascular development in the zebrafish. II. Endocardial progenitors are sequestered within the heart field. Development (Cambridge, England). 120(12):3361-6. Pubmed: 7821208 We have examined the zebrafish embryo to ascertain the location of endocardial and myocardial progenitors prior to gastrulation, in an attempt to define the earliest stages of cardiac patterning. Currently there is uncertainty as to the spatial and lineage relationship of the progenitors for these two phenotypically distinct cell types that form the two concentric layers of the primitive heart tube. By single-cell injection and tracking, we distinguish a region in the early and midblastula which has the properties of a heart field, in that it defines a zone of cardiac progenitors within which there is a spatial gradient of propensity to generate heart cells, and which regulates, in the sense of adapting to the transplantation of pluripotential cells. This zone extends from the future ventral axis dorsally along the margin, with cardiogenic propensity tapering off laterally and dorsally. Myocardial progenitors are spread throughout this region, but endocardial precursors are restricted to the ventral marginal region. The cardiovascular progeny of the ventral cells include, in addition to endocardium and myocardium, cells in the endothelium and blood. -
Strittmatter SM, Igarashi M, Fishman MC. 1994. GAP-43 amino terminal peptides modulate growth cone morphology and neurite outgrowth. The Journal of neuroscience : the official journal of the Society for Neuroscience. 14(9):5503-13. Pubmed: 8083750 Strittmatter SM, Igarashi M, Fishman MC. 1994. GAP-43 amino terminal peptides modulate growth cone morphology and neurite outgrowth. The Journal of neuroscience : the official journal of the Society for Neuroscience. 14(9):5503-13. Pubmed: 8083750 The neuronal growth-associated protein GAP-43 is expressed maximally during development and regeneration, and is enriched at the cytosolic surface of the growth cone membrane. GAP-43 can activate the GTP-binding protein G(o) which is also a major component of the growth cone membrane. These findings have led to the hypothesis that GAP-43 might modulate neurite outgrowth by altering G-protein activity. Here we define the sequence requirements for GAP-43 amino terminal peptide stimulation of G(o), and test these peptides as potential modulators of neurite outgrowth. The first 10 amino acids of GAP-43, Met-Leu-Cys-Cys-Met-Arg-Arg-Thr-Lys-Gln, stimulate G(o). Substitutions at particular residues reveal that cys3, cys4, arg6, and lys9 are critical, but arg7 is not. Both the GAP-43(1-10) peptide and the G-protein-activating peptide mastoparan induce growth cone collapse and inhibit neurite extension from embryonic chick dorsal root ganglion and retinal neurons. This is likely to be mediated by G-proteins: pertussis toxin blocks the inhibition, and mutant peptides that do not activate G(o) do not alter outgrowth. In contrast to the case with embryonic chick dorsal root ganglion cells, neurite outgrowth from N1E-115 neuroblastoma cells is stimulated by GAP-43(1-10). This is probably also a G-protein-mediated event because it is blocked by pertussis toxin, because the sequence requirements match those for G(o) stimulation, and because mastoparan stimulates outgrowth from these cells. The longer GAP-43(1-25) peptide does not alter neurite outgrowth unless the cells are permeabilized, suggesting an intracellular site of action. These data identify a novel set of compounds that modulate neurite outgrowth, and also support the notion that GAP-43 can alter neurite extension by modulating pertussis toxin-sensitive G-protein activity in the growth cone. -
Stainier DY, Fishman MC. 1994. The zebrafish as a model system to study cardiovascular development. Trends in cardiovascular medicine. 4(5):207-12. Pubmed: 21244869 DOI:10.1016/1050-1738(94)90036-1 Stainier DY, Fishman MC. 1994. The zebrafish as a model system to study cardiovascular development. Trends in cardiovascular medicine. 4(5):207-12. Pubmed: 21244869 DOI:10.1016/1050-1738(94)90036-1 The zebrafish, Brachydanio rerio, is rapidly becoming a system of choice for vertebrate developmental biologists. It presents unique embryological attributes and is amenable to saturation style mutagenesis, a powerful approach that, in invertebrates, has already led to the identification of a large number of key developmental genes. Since fertilization is external, the zebrafish embryo develops in the dish and is thus accessible for continued observation and manipulation at all stages of development. Furthermore, because the embryo is transparent, the developing heart and vessels can be resolved at the single-cell level. A large number of mutations that affect the development of cardiovascular form and function have recently been isolated from large-scale genetic screens for zygotic embryonic lethals. Our further understanding of the development of the cardiovascular system is important not only because of the high incidence, and familial inheritance, of congenital abnormalities, but also because it should lead to novel, differentiation-based strategies for the analysis and therapy of the diseased state.Copyright © 1994. Published by Elsevier Inc. -
Kass S, MacRae C, Graber HL, Sparks EA, McNamara D, Boudoulas H, Basson CT, Baker PB, Cody RJ, Fishman MC. 1994. A gene defect that causes conduction system disease and dilated cardiomyopathy maps to chromosome 1p1-1q1. Nature genetics. 7(4):546-51. Pubmed: 7951328 Kass S, MacRae C, Graber HL, Sparks EA, McNamara D, Boudoulas H, Basson CT, Baker PB, Cody RJ, Fishman MC. 1994. A gene defect that causes conduction system disease and dilated cardiomyopathy maps to chromosome 1p1-1q1. Nature genetics. 7(4):546-51. Pubmed: 7951328 Longitudinal evaluation of a seven generation kindred with an inherited conduction system defect and dilated cardiomyopathy demonstrated autosomal dominant transmission of a progressive disorder that both perturbs atrioventricular conduction and depresses cardiac contractility. To elucidate the molecular genetic basis for this disorder, a genome-wide linkage analysis was performed. Polymorphic loci near the centromere of chromosome 1 demonstrated linkage to the disease locus (maximum multipoint lod score = 13.2 in the interval between D1S305 and D1S176). Based on the disease phenotype and map location we speculate that gap junction protein connexin 40 is a candidate for mutations that result in conduction system disease and dilated cardiomyopathy. -
Huang Z, Huang PL, Panahian N, Dalkara T, Fishman MC, Moskowitz MA. 1994. Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science (New York, N.Y.). 265(5180):1883-5. Pubmed: 7522345 Huang Z, Huang PL, Panahian N, Dalkara T, Fishman MC, Moskowitz MA. 1994. Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science (New York, N.Y.). 265(5180):1883-5. Pubmed: 7522345 The proposal that nitric oxide (NO) or its reactant products mediate toxicity in brain remains controversial in part because of the use of nonselective agents that block NO formation in neuronal, glial, and vascular compartments. In mutant mice deficient in neuronal NO synthase (NOS) activity, infarct volumes decreased significantly 24 and 72 hours after middle cerebral artery occlusion, and the neurological deficits were less than those in normal mice. This result could not be accounted for by differences in blood flow or vascular anatomy. However, infarct size in the mutant became larger after endothelial NOS inhibition by nitro-L-arginine administration. Hence, neuronal NO production appears to exacerbate acute ischemic injury, whereas vascular NO protects after middle cerebral artery occlusion. The data emphasize the importance of developing selective inhibitors of the neuronal isoform. -
Fishman MC, Stainier DY. 1994. Cardiovascular development. Prospects for a genetic approach. Circulation research. 74(5):757-63. Pubmed: 8156624 Fishman MC, Stainier DY. 1994. Cardiovascular development. Prospects for a genetic approach. Circulation research. 74(5):757-63. Pubmed: 8156624 Genetics is a powerful tool, especially when used in combination with embryology, in the seeking of genes necessary for assembly of the cardiovasculature. The first questions must address the types of cellular decisions that are made during development. As for simpler systems in C elegans and D melanogaster, the lineage and cell-fate decisions of the cardiovascular progenitors need to be assessed. In addition it is likely that new paradigms will emerge for multicellular assembly. The study of cardiovascular mutations will define individual genetic steps that define organotypic decisions. A genetic approach is a natural extension of embryology, physiology, and anatomy, fields of great sophistication with regard to the cardiovasculature, because, like them, it focuses on integrative biology and on the intact organism. The zebrafish is particularly well suited to a combination genetic-embryologic study of the fashioning of the cardiovasculature. -
Strittmatter SM, Valenzuela D, Fishman MC. 1994. An amino-terminal domain of the growth-associated protein GAP-43 mediates its effects on filopodial formation and cell spreading. Journal of cell science. 107 ( Pt 1):195-204. Pubmed: 8175908 Strittmatter SM, Valenzuela D, Fishman MC. 1994. An amino-terminal domain of the growth-associated protein GAP-43 mediates its effects on filopodial formation and cell spreading. Journal of cell science. 107 ( Pt 1):195-204. Pubmed: 8175908 GAP-43 is a neuronal protein that is believed to be important to neuronal growth and nerve terminal plasticity. It is enriched on the inner surface of growth cone membranes, a localization that may depend upon palmitoylation of Cys3 and Cys4. It is a major substrate for protein kinase C, which phosphorylates Ser41. Isolated GAP-43 can bind to actin and to calmodulin, and can activate the heterotrimeric GTP-binding proteins, G(o) and Gi. A peptide consisting of the GAP-43 sequence 39-55 binds calmodulin, and an amino-terminal GAP-43 (1-10) peptide activates G(o), suggesting that these stretches may be functional domains of the intact protein. When expressed in non-neuronal cells, GAP-43 enhances filopodial extension and has effects upon cell spreading. We have examined the effects of various GAP-43 domains upon this assay, by expression of GAP-43, GAP-43 mutant proteins, and GAP-43-CAT fusion proteins in COS-7 cells. We find that the amino terminus (Met-Leu-Cys-Cys-Met-Arg-Arg-Thr-Lys-Gln) is an important contributor to these effects on cell shape. A GAP-43 protein mutant in Cys3 and Cys4 does not bind to the membrane, and is inactive. Mutants in Arg6 or Lys9 also are inactive, although they remain localized to particulate fractions; Arg7 mutants are active. A chimeric gene consisting of GAP-43 (1-10) fused to chloramphenicol acetyl transferase (CAT) also causes cell shape changes. As for GAP-43, the effects of this fusion protein are abolished by mutations of Cys3, Cys4, Arg6 or Lys9, but not by mutation of Arg7. Therefore, the cell surface activity of transfected GAP-43 depends upon its amino terminus, although other domains may regulate it in this regard. Since the amino-terminal domain includes the peptide stretch known to be capable of activating G(o) and Gi, we examined the effect of GAP-43 on a Gi-regulated second messenger system, the inhibition of cAMP production in A431 cells. A431 cells stably transfected with GAP-43 spread less well than do controls. In addition, they evidence decreased levels of forskolin-stimulated cAMP, consistent with chronic stimulation of Gi. Stimulation of adenylate cyclase by isoproterenol reverses the GAP-43-induced changes in cell shape. This suggests that G protein stimulation is involved in GAP-43 effects upon cell shape. -
Vanselow J, Grabczyk E, Ping J, Baetscher M, Teng S, Fishman MC. 1994. GAP-43 transgenic mice: dispersed genomic sequences confer a GAP-43-like expression pattern during development and regeneration. The Journal of neuroscience : the official journal of the Society for Neuroscience. 14(2):499-510. Pubmed: 8301350 Vanselow J, Grabczyk E, Ping J, Baetscher M, Teng S, Fishman MC. 1994. GAP-43 transgenic mice: dispersed genomic sequences confer a GAP-43-like expression pattern during development and regeneration. The Journal of neuroscience : the official journal of the Society for Neuroscience. 14(2):499-510. Pubmed: 8301350 Using transgenic mice, we have examined the expression pattern conferred by regions of genomic GAP-43 coupled to beta-galactosidase. We demonstrate that gene constructions that include the GAP-43 5'-flanking region along with sufficient sequences of the first intron drive beta-galactosidase (lacZ) expression to mimic in many regards the complex spatial and temporal pattern of endogenous GAP-43 expression. Transgene expression reaches peak levels during development, and persists at high levels in particular adult brain regions, such as the hippocampus and olfactory bulb. The inclusion of a stretch of the first intron in the construction is necessary to prevent expression outside of the nervous system, indicating that some of the cell specificity of GAP-43 expression is due to suppression of expression in inappropriate tissues. Injury caused by sciatic nerve crush causes reexpression of the transgene in adult sensory and motor neurons. This genomic region of GAP-43, therefore, includes elements responsive to neuronal growth signals that regulate both development and regeneration. -
Gagliardini V, Fernandez PA, Lee RK, Drexler HC, Rotello RJ, Fishman MC, Yuan J. 1994. Prevention of vertebrate neuronal death by the crmA gene. Science (New York, N.Y.). 263(5148):826-8. Pubmed: 8303301 Gagliardini V, Fernandez PA, Lee RK, Drexler HC, Rotello RJ, Fishman MC, Yuan J. 1994. Prevention of vertebrate neuronal death by the crmA gene. Science (New York, N.Y.). 263(5148):826-8. Pubmed: 8303301 Interleukin-1 beta converting enzyme (ICE) is a mammalian homolog of CED-3, a protein required for programmed cell death in the nematode Caenorhabditis elegans. The activity of ICE can be specifically inhibited by the product of crmA, a cytokine response modifier gene encoded by cowpox virus. Microinjection of the crmA gene into chicken dorsal root ganglion neurons was found to prevent cell death induced by deprivation of nerve growth factor. Thus, ICE is likely to participate in neuronal death in vertebrates. -
Strittmatter SM, Fishman MC, Zhu XP. 1994. Activated mutants of the alpha subunit of G(o) promote an increased number of neurites per cell. The Journal of neuroscience : the official journal of the Society for Neuroscience. 14(4):2327-38. Pubmed: 8158271 Strittmatter SM, Fishman MC, Zhu XP. 1994. Activated mutants of the alpha subunit of G(o) promote an increased number of neurites per cell. The Journal of neuroscience : the official journal of the Society for Neuroscience. 14(4):2327-38. Pubmed: 8158271 The high concentration of the GTP-binding protein G(o) in the neuronal growth cone suggests that G(o) activation state may after neurite outgrowth. We find that activation of pertussis toxin-sensitive G-proteins by mastoparan increases neurite outgrowth from neuroblastoma cells. To examine G(o) activation specifically, point mutations homologous to activating, oncogenic mutations in alpha i2 and alpha s were introduced into the alpha subunit of G(o). The stability of the alpha o mutants to tryptic digestion confirms that they are activated. When expressed in PC12 or N1E-115 cells, activated alpha o doubles total neurites length per cell, primarily by increasing the number of neurites per cell. The growth cones of cells expressing activated alpha o are narrower than control growth cones. Expression of wildtype alpha o or the activated alpha subunits of other G-proteins did not affect total neurite length per cell. Thus, factors that lead to activation of G(o) can modulate neurite number per cell. -
O'Dell TJ, Huang PL, Dawson TM, Dinerman JL, Snyder SH, Kandel ER, Fishman MC. 1994. Endothelial NOS and the blockade of LTP by NOS inhibitors in mice lacking neuronal NOS. Science (New York, N.Y.). 265(5171):542-6. Pubmed: 7518615 O'Dell TJ, Huang PL, Dawson TM, Dinerman JL, Snyder SH, Kandel ER, Fishman MC. 1994. Endothelial NOS and the blockade of LTP by NOS inhibitors in mice lacking neuronal NOS. Science (New York, N.Y.). 265(5171):542-6. Pubmed: 7518615 Long-term potentiation (LTP) is a persistent increase in synaptic strength implicated in certain forms of learning and memory. In the CA1 region of the hippocampus, LTP is thought to involve the release of one or more retrograde messengers from the postsynaptic cell that act on the presynaptic terminal to enhance transmitter release. One candidate retrograde messenger is the membrane-permeant gas nitric oxide (NO), which in the brain is released after activation of the neuronal-specific NO synthase isoform (nNOS). To assess the importance of NO in hippocampal synaptic plasticity, LTP was examined in mice where the gene encoding nNOS was disrupted by gene targeting. In nNOS- mice, LTP induced by weak intensity tetanic stimulation was normal except for a slight reduction in comparison to that in wild-type mice and was blocked by NOS inhibitors, just as it was in wild-type mice. Immunocytochemical studies indicate that in the nNOS- mice as in wild-type mice, the endothelial form of NOS (eNOS) is expressed in CA1 neurons. These findings suggest that eNOS, rather than nNOS, generates NO within the postsynaptic cell during LTP. 1993
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Stainier DY, Lee RK, Fishman MC. 1993. Cardiovascular development in the zebrafish. I. Myocardial fate map and heart tube formation. Development (Cambridge, England). 119(1):31-40. Pubmed: 8275863 Stainier DY, Lee RK, Fishman MC. 1993. Cardiovascular development in the zebrafish. I. Myocardial fate map and heart tube formation. Development (Cambridge, England). 119(1):31-40. Pubmed: 8275863 We have analyzed the origin of cardiac progenitors in the zebrafish embryo by injection of single blastomeres with a lineage tracer dye, and examined the formation of the zebrafish heart tube by serial sectioning of immunostained embryos. At the 512-cell stage (early blastula), most cardiac progenitors lie in a marginal zone that extends from 90 degrees longitude (midway between the future dorsal and ventral axis) through 180 degrees longitude (the future ventral axis) to 270 degrees longitude. By focusing on myocardial progenitors located at 90 degrees (and 270 degrees) longitude, we found that a single cell injected in the early blastula can contribute progeny to both the atrium and ventricle. A cell injected in the midblastula contributes progeny to either the atrium or ventricle, but not both. This analysis suggests that, at least for these myocardial progenitors, the atrial and ventricular lineages separate in the midblastula. Precardiac cells involute early during gastrulation and turn towards the animal pole with other early involuting cells. These cardiogenic cells reach the embryonic axis around the 8-somite stage, and there they coalesce to form a pair of myocardial tubular primordia on either side of the midline. By the 21-somite stage, the tropomyosin-immunoreactive myocardial tubes have moved closer to each other, and a distinct group of cells, the endocardial progenitor cells, sits medially between them. The myocardial tubes then fuse to enclose the endocardial cells and form the definitive heart tube. By 22 hours postfertilization (26-somite stage), the heart tube is clearly beating. The regionalization of cardiac myosin heavy chain expression distinguishes the cardiac chambers at this stage, although they are not morphologically delineated until 36 hours. This work shows that cardiogenic regions can be identified in the early blastula, and that chamber restriction seems to arise in the midblastula. Additionally, it provides the basis for embryological perturbation at the single cell level, as well as for the genetic analysis of heart tube formation in the zebrafish. -
Huang PL, Dawson TM, Bredt DS, Snyder SH, Fishman MC. 1993. Targeted disruption of the neuronal nitric oxide synthase gene. Cell. 75(7):1273-86. Pubmed: 7505721 Huang PL, Dawson TM, Bredt DS, Snyder SH, Fishman MC. 1993. Targeted disruption of the neuronal nitric oxide synthase gene. Cell. 75(7):1273-86. Pubmed: 7505721 By homologous recombination, we have generated mice that lack the neuronal nitric oxide synthase (NOS) gene. Neuronal NOS expression and NADPH-diaphorase (NDP) staining are absent in the mutant mice. Very low level residual catalytic activity suggests that other enzymes in the brain may generate nitric oxide. The neurons normally expressing NOS appear intact, and the mutant NOS mice are viable, fertile, and without evident histopathological abnormalities in the central nervous system. The most evident effect of disrupting the neuronal NOS gene is the development of grossly enlarged stomachs, with hypertrophy of the pyloric sphincter and the circular muscle layer. This phenotype resembles the human disorder infantile pyloric stenosis, in which gastric outlet obstruction is associated with the lack of NDP neurons in the pylorus. -
Igarashi M, Strittmatter SM, Vartanian T, Fishman MC. 1993. Mediation by G proteins of signals that cause collapse of growth cones. Science (New York, N.Y.). 259(5091):77-9. Pubmed: 8418498 Igarashi M, Strittmatter SM, Vartanian T, Fishman MC. 1993. Mediation by G proteins of signals that cause collapse of growth cones. Science (New York, N.Y.). 259(5091):77-9. Pubmed: 8418498 During development, motion of nerve growth cones ceases on contact with particular targets. The signaling mechanism is unknown. In culture, growth cone collapse can be caused by solubilized embryonic brain membranes, central nervous system myelin, a 35-kilodalton protein isolated from myelin, and mastoparan. Collapse induced by each of these is blocked by pertussis toxin. Thus, collapse of growth cones is mediated by G protein-coupled receptors, which may be activated by proteins associated with the cell surface as well as by soluble ligands. -
Strittmatter SM, Cannon SC, Ross EM, Higashijima T, Fishman MC. 1993. GAP-43 augments G protein-coupled receptor transduction in Xenopus laevis oocytes. Proceedings of the National Academy of Sciences of the United States of America. 90(11):5327-31. Pubmed: 7685122 Strittmatter SM, Cannon SC, Ross EM, Higashijima T, Fishman MC. 1993. GAP-43 augments G protein-coupled receptor transduction in Xenopus laevis oocytes. Proceedings of the National Academy of Sciences of the United States of America. 90(11):5327-31. Pubmed: 7685122 The neuronal protein GAP-43 is thought to play a role in determining growth-cone motility, perhaps as an intracellular regulator of signal transduction, but its molecular mechanism of action has remained unclear. We find that GAP-43, when microinjected into Xenopus laevis oocytes, increases the oocyte response to G protein-coupled receptor agonists by 10- to 100-fold. Higher levels of GAP-43 cause a transient current flow, even without receptor stimulation. The GAP-43-induced current, like receptor-stimulated currents, is mediated by a calcium-activated chloride channel and can be desensitized by injection of inositol 1,4,5-trisphosphate. This suggests that neuronal GAP-43 may serve as an intracellular signal to greatly enhance the sensitivity of G protein-coupled receptor transduction. 1992
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Sudo Y, Valenzuela D, Beck-Sickinger AG, Fishman MC, Strittmatter SM. 1992. Palmitoylation alters protein activity: blockade of G(o) stimulation by GAP-43. The EMBO journal. 11(6):2095-102. Pubmed: 1534749 Sudo Y, Valenzuela D, Beck-Sickinger AG, Fishman MC, Strittmatter SM. 1992. Palmitoylation alters protein activity: blockade of G(o) stimulation by GAP-43. The EMBO journal. 11(6):2095-102. Pubmed: 1534749 The addition of palmitate to cysteine residues enhances the hydrophobicity of proteins, and consequently their membrane association. Here we have investigated whether this type of fatty acylation also regulates protein-protein interactions. GAP-43 is a neuronal protein that increases guanine nucleotide exchange by heterotrimeric G proteins. Two cysteine residues near the N-terminus of GAP-43 are subject to palmitoylation, and are necessary for membrane binding as well as for G(o) activation. N-terminal peptides, which include these cysteines, stimulate G(o). Monopalmitoylation reduces, and dipalmitoylation abolishes the activity of the peptides. The activity of GAP-43 protein purified from brain also is reversibly blocked by palmitoylation. This suggests that palmitoylation controls a cycle of GAP-43 between an acylated, membrane-bound reservoir of inactive GAP-43, and a depalmitoylated, active pool of protein. -
Strittmatter SM, Vartanian T, Fishman MC. 1992. GAP-43 as a plasticity protein in neuronal form and repair. Journal of neurobiology. 23(5):507-20. Pubmed: 1431834 Strittmatter SM, Vartanian T, Fishman MC. 1992. GAP-43 as a plasticity protein in neuronal form and repair. Journal of neurobiology. 23(5):507-20. Pubmed: 1431834 Neurons exhibit a remarkable plasticity of form, both during neural development and during the subsequent remodelling of synaptic connectivity. Here we review work on GAP-43 and G0, and focus upon the thesis that their interaction may endow neurons with such plasticity. We also present new data on the role of G proteins in neurite growth, and on the interaction of GAP-43 and actin. GAP-43 is a protein induced during periods of axonal extension and highly enriched on the inner surface of the growth cone membrane. Its membrane localization is primarily due to a short amino terminal sequence which is subject to palmitoylation. Binding to actin filaments may also assist in restricting the protein to specific cellular domains. Consistent with its role as a "plasticity protein," there is evidence that GAP-43 can directly alter cell shape and neurite extension, and several theses have been advanced for how it might do so. Two other prominent components of the growth cone membrane are the alpha and beta subunits of G0. GAP-43 regulates their guanine nucleotide exchange, which is an unusual role for an intracellular protein. We speculate that GAP-43 may adjust the "set point" of responsiveness for G0 stimulation by receptors, thereby altering the neuronal propensity to growth, without actually causing growth. To begin to address how G protein activity affects axon growth, we have developed a means to introduce guanine nucleotide analogs into sympathetic neurons. Stimulation of G proteins with GTP-gamma-S retards axon growth, whereas GDP-beta-S enhances it. This is compatible with G protein registration of inhibitory signals. -
Stainier DY, Fishman MC. 1992. Patterning the zebrafish heart tube: acquisition of anteroposterior polarity. Developmental biology. 153(1):91-101. Pubmed: 1516755 Stainier DY, Fishman MC. 1992. Patterning the zebrafish heart tube: acquisition of anteroposterior polarity. Developmental biology. 153(1):91-101. Pubmed: 1516755 The patterning of an internal organ, like the heart, is little understood. Central to this patterning is the formation, or the acquisition, of an anteroposterior (A-P) axis. We have approached the question of how the heart tube acquires polarity in the zebrafish, Brachydanio rerio, which offers numerous advantages for studying cardiac morphogenesis. During the early stages of organogenesis in the fish, the heart tube lies in an A-P orientation with the venous end lying anteriorly and the arterial end lying posteriorly. High doses (10(-6)-10(-5)M) of retinoic acid (RA) cause truncation of the body axis, as they do in Xenopus. Low doses of retinoic acid (10(-8)-10(-7) M), which do not appear to affect the rest of the embryo, have pronounced effects upon heart tube morphogenesis, causing it to shrink progressively along the A-P axis. To investigate this further, we identified monoclonal antibodies that distinguish between the zebrafish cardiac chambers and used them to show that the RA-induced cardiac truncation always begins at the arterial end of the heart tube. There is a continuous gradient of sensitivity from the arterial to the venous end, such that increasing RA exposure causes the progressive and sequential deletion first of the bulbus arteriosus and then, in order, of the ventricle, the atrium, and the sinus venosus. As exposure increases, parts of chambers are deleted before entire chambers; thus, the sensitivity to RA appears to be independent of chamber boundaries. The analysis of the heart tube's sensitivity to RA and its timing suggest that polarity is established during or shortly after initial commitment to the cardiac lineage. 1991
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Strittmatter SM, Valenzuela D, Vartanian T, Sudo Y, Zuber MX, Fishman MC. 1991. Growth cone transduction: Go and GAP-43. Journal of cell science. Supplement. 15:27-33. Pubmed: 1840457 Strittmatter SM, Valenzuela D, Vartanian T, Sudo Y, Zuber MX, Fishman MC. 1991. Growth cone transduction: Go and GAP-43. Journal of cell science. Supplement. 15:27-33. Pubmed: 1840457 The neuronal growth cone plays a crucial role in forming the complex brain architecture achieved during development, and similar nerve terminal mechanisms may operate to modify synaptic structure during adulthood. The growth cone leads the elongating axon towards appropriate synaptic targets by altering motility in response to a variety of extracellular signals. Independently of extrinsic clues, neurons manifest intrinsic control of their growth and form (Banker and Cowan, 1979). Hence, there must be intracellular proteins which control nerve cell shape, so-called 'plasticity' or 'growth' genes. GAP-43 may be such a molecule (Skene and Willard, 1981; Benowitz and Lewis, 1983). For example, GAP-43 is localized to the growth cone membrane (Meiri et al. 1986; Skene et al. 1986) and can enhance filopodial formation even in non-neuronal cells (Zuber et al. 1989a). It includes a small region at the amino terminus for membrane association and perhaps growth cone targeting (Zuber et al. 1989b, Liu et al. 1991). We have found that Go, a member of the G protein family that links receptors and second messengers, is the major non-cytoskeletal protein in the growth cone membrane (Strittmatter et al. 1990). Double staining immunohistochemistry for GAP-43 and Go shows that the distributions of the two proteins are quite similar. Purified GAP-43 regulates the activity of purified Go (Strittmatter et al. 1990), a surprising observation since GAP-43 is an intracellular protein. We have compared the mechanism of GAP-43 activation of Go with that of G protein-linked receptors.2+ interactions between Go and GAP-43 suggest that Go plays a pivotal role in growth cone function, coordinating the effects of both extracellular signals and intracellular growth proteins. -
Fishman MC, Valenzuela D. 1991. GAP-43 and neuronal remodeling. Progress in brain research. 89:89-95. Pubmed: 1839073 Fishman MC, Valenzuela D. 1991. GAP-43 and neuronal remodeling. Progress in brain research. 89:89-95. Pubmed: 1839073 -
Strittmatter SM, Fishman MC. 1991. The neuronal growth cone as a specialized transduction system. BioEssays : news and reviews in molecular, cellular and developmental biology. 13(3):127-34. Pubmed: 1831353 Strittmatter SM, Fishman MC. 1991. The neuronal growth cone as a specialized transduction system. BioEssays : news and reviews in molecular, cellular and developmental biology. 13(3):127-34. Pubmed: 1831353 Neuronal growth and remodelling are guided by both intracellular gene programs and extracellular stimuli. The growth cone is one site where the effects of these extrinsic and intrinsic factors converge upon the mechanical determinants of cell shape. We review the growth cone as a transduction device, converting extracellular signals into mechanical forces. A variety of soluble, extracellular matrix and membrane bound molecules control growth cone behavior. In addition, GAP-43 is discussed as a possible component of the intraneuronal gene program which modulates growth cone activity. The GTP-binding protein, Go, is a major growth cone membrane protein that may transduce signals not only from outside the cell, but from within as well. This may provide a molecular site in the growth cone for the coordination of a genetic growth program with environmental signals. -
Strittmatter SM, Valenzuela D, Sudo Y, Linder ME, Fishman MC. 1991. An intracellular guanine nucleotide release protein for G0. GAP-43 stimulates isolated alpha subunits by a novel mechanism. The Journal of biological chemistry. 266(33):22465-71. Pubmed: 1834672 Strittmatter SM, Valenzuela D, Sudo Y, Linder ME, Fishman MC. 1991. An intracellular guanine nucleotide release protein for G0. GAP-43 stimulates isolated alpha subunits by a novel mechanism. The Journal of biological chemistry. 266(33):22465-71. Pubmed: 1834672 G protein-coupled membrane receptors activate G proteins by enhancing guanine nucleotide exchange. G0 is a major component of the growing regions (growth cones) of neurons. GAP-43 is a neuronal protein associated with the cytosolic face of the growth cone plasma membrane and stimulates binding of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) to Go (Strittmatter, S. M., Valenzuela, D., Kennedy, T. E., Neer, E. J., and Fishman, M. C. (1990) Nature 344, 836-841). Here we have examined the mechanism by which GAP-43 affects G0. Like G protein-coupled receptors, GAP-43 enhances GDP release from G0, increases the initial rate of GTP gamma S binding, and increases the GTPase activity of Go, all without altering the intrinsic kappa cat for the GTPase. Unlike the case for receptors, however, the GAP-43 effect is not blocked by pertussis toxin, nor affected by the presence or absence of beta gamma or of phospholipids. There is specificity to the interaction, in that GAP-43 increases GTP gamma S binding to recombinant alpha o and alpha i1, but not to recombinant alpha s. Thus, GAP-43 is a guanine nucleotide release protein with a novel mechanism of action, potentially controlling membrane-associated G proteins from within the cell. -
Baetscher M, Schmidt E, Shimizu A, Leder P, Fishman MC. 1991. SV40 T antigen transforms calcitonin cells of the thyroid but not CGRP-containing neurons in transgenic mice. Oncogene. 6(7):1133-8. Pubmed: 1650439 Baetscher M, Schmidt E, Shimizu A, Leder P, Fishman MC. 1991. SV40 T antigen transforms calcitonin cells of the thyroid but not CGRP-containing neurons in transgenic mice. Oncogene. 6(7):1133-8. Pubmed: 1650439 Neurons are postmitotic for the adult life of animals. Tumors rarely, if ever, arise from neurons in the adult, although they do from other cells from the same lineage, such as the neuroendocrine C cells of the thyroid. We have found that 2 kb of the calcitonin gene-related peptide (CGRP)/calcitonin gene suffices to target expression to CGRP-containing neurons, such as those in the dorsal root ganglia (DRG), and to the calcitonin-secreting C cells of the thyroid. Using this promoter we have examined the effect of two potentially transforming oncogenes in these two different populations. Overexpression of c-myc for periods of up to two years does not transform either cell type, whereas SV40 Tag causes early onset medullary thyroid carcinoma, but does not transform the dorsal root ganglia neurons. This suggests that as part of the terminal differentiation process of these neurons, the cessation of mitosis is accompanied by a relative refractoriness to oncogenes that may transform other cells of the same lineage. 1990
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Grabczyk E, Zuber MX, Federoff HJ, Ng SC, Pack A, Fishman MC. 1990. Cloning and Characterization of the Rat Gene Encoding GAP-43. The European journal of neuroscience. 2(10):822-827. Pubmed: 12106089 Grabczyk E, Zuber MX, Federoff HJ, Ng SC, Pack A, Fishman MC. 1990. Cloning and Characterization of the Rat Gene Encoding GAP-43. The European journal of neuroscience. 2(10):822-827. Pubmed: 12106089 GAP-43 is a gene expressed only in the nervous system. The protein product is believed to be important to neuronal growth and plasticity. Most, and likely all, neurons express high levels of GAP-43 during periods of neurite elongation. To initiate studies of GAP-43 gene regulation we have cloned the rat gene encoding GAP-43. The GAP-43 gene includes three exons. The first exon encodes only the amino terminal 10 amino acids, which corresponds to the membrane targeting domain of GAP-43. The second exon encodes a putative calmodulin binding domain and a protein kinase C phosphorylation site. The 5'-flanking sequence is unusual in that it lacks CAAT or TATA elements, and directs RNA transcription initiation from several sites. Some of the transcription start sites are used to a different degree in the central and peripheral nervous systems. -
Strittmatter SM, Valenzuela D, Kennedy TE, Neer EJ, Fishman MC. 1990. G0 is a major growth cone protein subject to regulation by GAP-43. Nature. 344(6269):836-41. Pubmed: 2158629 Strittmatter SM, Valenzuela D, Kennedy TE, Neer EJ, Fishman MC. 1990. G0 is a major growth cone protein subject to regulation by GAP-43. Nature. 344(6269):836-41. Pubmed: 2158629 G0, a GTP-binding protein that transduces information from transmembrane receptors, has been found to be a major component of the neuronal growth cone membrane. GAP-43, an intracellular growth cone protein closely associated with neuronal growth, stimulates GTP-gamma-S binding to G0. It does so through an amino-terminal domain homologous to G-linked transmembrane receptors. Thus, G0 in the growth cone may be regulated by intracellular as well as extracellular signals. 1989
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Zuber MX, Goodman DW, Karns LR, Fishman MC. 1989. The neuronal growth-associated protein GAP-43 induces filopodia in non-neuronal cells. Science (New York, N.Y.). 244(4909):1193-5. Pubmed: 2658062 Zuber MX, Goodman DW, Karns LR, Fishman MC. 1989. The neuronal growth-associated protein GAP-43 induces filopodia in non-neuronal cells. Science (New York, N.Y.). 244(4909):1193-5. Pubmed: 2658062 The neuron-specific protein GAP-43 is associated with the membrane of the nerve growth cone and thus may be important to the activity of this distinctive neuronal structure. Transient transfection of COS and NIH 3T3 cells with appropriate vectors resulted in expression of GAP-43 in these non-neuronal cells; as in neurons, transfected GAP-43 associated with the membrane. In addition, many long fine filopodial processes extended from the periphery of such transfected cells. Stable CHO cell lines expressing GAP-43 also exhibited processes that were more numerous, far longer, and more complex than those of CHO cell lines not transfected or transfected with control plasmids. Thus GAP-43 may directly contribute to growth cone activity by regulating cell membrane structure and enhancing extension of filopodial processes. -
De la Monte SM, Federoff HJ, Ng SC, Grabczyk E, Fishman MC. 1989. GAP-43 gene expression during development: persistence in a distinctive set of neurons in the mature central nervous system. Brain research. Developmental brain research. 46(2):161-8. Pubmed: 2720952 De la Monte SM, Federoff HJ, Ng SC, Grabczyk E, Fishman MC. 1989. GAP-43 gene expression during development: persistence in a distinctive set of neurons in the mature central nervous system. Brain research. Developmental brain research. 46(2):161-8. Pubmed: 2720952 GAP-43 is a rapidly transported axonal protein most prominently expressed in regenerating and developing nerves. However, the low level persistence of GAP-43 in the adult CNS where growth and regenerative capacity are minimal may additionally indicate a role for this molecule in neuronal remodeling. Previous studies have revealed GAP-43 immunoreactivity in neurites throughout many regions of the CNS. To identify the CNS neurons that express GAP-43 at different stages of development, we utilized in situ hybridization and immunocytochemistry; the latter was performed with an antibody that recognizes GAP-43 immunoreactivity in both perikarya and neurites. In the perinatal period GAP-43 is expressed in all neurons. Subsequently its expression becomes progressively restricted such that by maturity most neurons no longer express detectable levels, although GAP-43 expression is still moderately high in the adult entorhinal cortex, and strikingly high in the adult hippocampus and olfactory bulb. In light of current notions about the function of GAP-43, it is tempting to speculate that this anatomy denotes neurons engaged in structural remodeling and functional plasticity. -
Ng SC, Perkins LA, Conboy G, Perrimon N, Fishman MC. 1989. A Drosophila gene expressed in the embryonic CNS shares one conserved domain with the mammalian GAP-43. Development (Cambridge, England). 105(3):629-38. Pubmed: 2693037 Ng SC, Perkins LA, Conboy G, Perrimon N, Fishman MC. 1989. A Drosophila gene expressed in the embryonic CNS shares one conserved domain with the mammalian GAP-43. Development (Cambridge, England). 105(3):629-38. Pubmed: 2693037 By cross hybridization with the mammalian growth-related protein, GAP-43, we have isolated several Drosophila cDNAs and genomic sequences. These sequences correspond to a single copy gene that encodes two developmentally regulated transcripts 2.4 and 2.0 kb in length. The predicted protein sequence from the cDNAs contains a stretch of 20 amino acids closely related to the mammalian GAP-43 protein. These residues are also highly conserved in a cDNA isolated from the nematode C. elegans. Prior to dorsal closure, expression of the Drosophila gene is observed in non-neuronal tissues, especially in the mesectoderm and presumptive epidermis, both in a metameric pattern. After dorsal closure, expression becomes restricted to sets of cells that are segmentally reiterated along the periphery of the nervous system. These cells appear to include at least one specific set of glia that may establish scaffolding for the development of the longitudinal neuropile. -
Zuber MX, Strittmatter SM, Fishman MC. 1989. A membrane-targeting signal in the amino terminus of the neuronal protein GAP-43. Nature. 341(6240):345-8. Pubmed: 2797153 Zuber MX, Strittmatter SM, Fishman MC. 1989. A membrane-targeting signal in the amino terminus of the neuronal protein GAP-43. Nature. 341(6240):345-8. Pubmed: 2797153 Neurons and other cells, such as those of epithelia, accumulate particular proteins in spatially discrete domains of the plasma membrane. This enrichment is probably important for localization of function, but it is not clear how it is accomplished. One proposal for epithelial cells is that proteins contain targeting signals which guide preferential accumulation in basal or apical membranes. The growth-cone membrane of a neuron serves as a specialized transduction system, which helps to convert cues from its environment into regulated growth. Because it can be physically separated from the cell soma, it has been possible to show that the growth-cone membrane contains a restricted set of total cellular proteins, although, to our knowledge, no proteins are limited to that structure. One of the most prominent proteins in the growth-cone membrane is GAP-43. Basi et al. have suggested that the N-terminus of GAP-43 might be important for the binding of GAP-43 to the growth-cone membrane. Skene and Virag recently found that the cysteines in the N-terminus are fatty-acylated and that this post-translational modification correlates with membrane-binding ability. We investigated the binding of GAP-43 to the growth-cone membrane by mutational analysis and by laser-scanning confocal microscopy of fusion proteins that included regions of GAP-43 and chloramphenicol acetyltransferase (CAT). We found that a short stretch of the GAP-43 N-terminus suffices to direct accumulation in growth-cone membranes, especially in the filopodia. This supports a previous proposal for the importance of this region of GAP-43 in determining the membrane distribution of GAP-43. 1988
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Federoff HJ, Grabczyk E, Fishman MC. 1988. Dual regulation of GAP-43 gene expression by nerve growth factor and glucocorticoids. The Journal of biological chemistry. 263(36):19290-5. Pubmed: 2461937 Federoff HJ, Grabczyk E, Fishman MC. 1988. Dual regulation of GAP-43 gene expression by nerve growth factor and glucocorticoids. The Journal of biological chemistry. 263(36):19290-5. Pubmed: 2461937 GAP-43 is a neural-specific protein that is believed integral to neurite growth and to the plasticity of neuronal structure. Its gene expression is regulated in vivo and correlates with periods of axonal growth. We investigated the regulation of GAP-43 gene expression in PC12 cells, which are believed to resemble precursor cells of the adrenomedullary lineage. In these cells, nerve growth factor (NGF) increases GAP-43 expression, and corticosteroids decrease it. Corticosteroids diminish GAP-43 levels even in cells already differentiated by NGF, as well as in primary neurons of the superior cervical ganglion. Neither the NGF nor the steroid effect requires new protein synthesis. Nuclear run-on experiments show that the steroid repression is mediated at the level of gene transcription but that the NGF effect is likely to be posttranscriptional. NGF and corticosteroids are known to regulate bimodally the cell fate decision of sympathoadrenal precursors, with NGF promoting the neuronal phenotype and steroids promoting the chromaffin phenotype. The regulation of GAP-43 is consistent with the notion that this gene is bimodally regulated during these cell fate decisions. -
Ng SC, de la Monte SM, Conboy GL, Karns LR, Fishman MC. 1988. Cloning of human GAP-43: growth association and ischemic resurgence. Neuron. 1(2):133-9. Pubmed: 3272163 Ng SC, de la Monte SM, Conboy GL, Karns LR, Fishman MC. 1988. Cloning of human GAP-43: growth association and ischemic resurgence. Neuron. 1(2):133-9. Pubmed: 3272163 GAP-43 is a growth cone protein expressed in neurons especially during periods of axonal elongation. Poor repair in the adult mammalian CNS has been ascribed to restraints upon its expression. We have cloned human GAP-43 cDNA to investigate its potential involvement in neurological illness. Analysis of postmortem human brain tissue disclosed uniformly high expression of GAP-43 throughout the neonatal brain, whereas in the adult brain high levels of GAP-43 persist only in discrete regions. However, in the wake of ischemic injury in the adult brain, regions normally low in GAP-43 reexpress it at high levels, suggesting a role for GAP-43 in remodeling and repair of mature CNS neurons. 1987
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Karns LR, Ng SC, Freeman JA, Fishman MC. 1987. Cloning of complementary DNA for GAP-43, a neuronal growth-related protein. Science (New York, N.Y.). 236(4801):597-600. Pubmed: 2437653 Karns LR, Ng SC, Freeman JA, Fishman MC. 1987. Cloning of complementary DNA for GAP-43, a neuronal growth-related protein. Science (New York, N.Y.). 236(4801):597-600. Pubmed: 2437653 GAP-43 is one of a small subset of cellular proteins selectively transported by a neuron to its terminals. Its enrichment in growth cones and its increased levels in developing or regenerating neurons suggest that it has an important role in neurite growth. A complementary DNA (cDNA) that encodes rat GAP-43 has been isolated to study its structural characteristics and regulation. The predicted molecular size is 24 kilodaltons, although its migration in SDS-polyacrylamide gels is anomalously retarded. Expression of GAP-43 is limited to the nervous system, where its levels are highest during periods of neurite outgrowth. Nerve growth factor or adenosine 3',5'-monophosphate induction of neurites from PC12 cells is accompanied by increased GAP-43 expression. GAP-43 RNA is easily detectable, although at diminished levels, in the adult rat nervous system. This regulation of GAP-43 is concordant with a role in growth-related processes of the neuron, processes that may continue in the mature animal.