Howard I Sirotkin

Stony Brook University, Stony Brook, NY, United States

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Publications (19)139.02 Total impact

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    ABSTRACT: Background: Regulation of developmental signaling pathways is essential for embryogenesis. The small putative zinc finger protein, Churchill (ChCh) has been implicated in modulation of both TGF-β and FGF signaling. Results: We employed zinc finger nuclease (ZFN) mediated gene targeting to disrupt the zebrafish chch locus and generate the first chch mutations. Three induced lesions produce frameshift mutations that truncate the protein in the third of five β-strands that comprise the protein. Surprisingly, zygotic and maternal zygotic chch mutants are viable. Mutants have elevated expression of mesodermal markers but progress normally through early development. chch mutants are sensitive to exogenous Nodal. However, neither misregulation of FGF targets nor sensitivity to exogenous FGF was detected. Finally, chch mutant cells were found to undergo inappropriate migration in cell transplant assays. Conclusions: Together, these results suggest that chch is not essential for survival, but functions to modulate early mesendodermal gene expression and limit cell migration. Developmental Dynamics, 2013. © 2013 Wiley Periodicals,Inc.
    Developmental Dynamics 02/2013; · 2.59 Impact Factor
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    ABSTRACT: Histone chaperones affect chromatin structure and gene expression through interaction with histones and RNA polymerase II (PolII). Here, we report that the histone chaperone Spt6 counteracts H3K27me3, an epigenetic mark deposited by the Polycomb Repressive Complex 2 (PRC2) and associated with transcriptional repression. By regulating proper engagement and function of the H3K27 demethylase KDM6A (UTX), Spt6 effectively promotes H3K27 demethylation, muscle gene expression, and cell differentiation. ChIP-Seq experiments reveal an extensive genome-wide overlap of Spt6, PolII, and KDM6A at transcribed regions that are devoid of H3K27me3. Mammalian cells and zebrafish embryos with reduced Spt6 display increased H3K27me3 and diminished expression of the master regulator MyoD, resulting in myogenic differentiation defects. As a confirmation for an antagonistic relationship between Spt6 and H3K27me3, inhibition of PRC2 permits MyoD re-expression in myogenic cells with reduced Spt6. Our data indicate that, through cooperation with PolII and KDM6A, Spt6 orchestrates removal of H3K27me3, thus controlling developmental gene expression and cell differentiation.
    The EMBO Journal 01/2013; · 9.82 Impact Factor
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    ABSTRACT: BACKGROUND: We have investigated a simple strategy for enhancing transgene expression specificity by leveraging genetic silencer elements. The approach serves to restrict transgene expression to a tissue of interest - the nervous system in the example provided here - thereby promoting specific/exclusive targeting of discrete cellular subtypes. Recent innovations are bringing us closer to understanding how the brain is organized, how neural circuits function, and how neurons can be regenerated. Fluorescent proteins enable mapping of the 'connectome', optogenetic tools allow excitable cells to be short-circuited or hyperactivated, and targeted ablation of neuronal subtypes facilitates investigations of circuit function and neuronal regeneration. Optimally, such toolsets need to be expressed solely within the cell types of interest as off-site expression makes establishing causal relationships difficult. To address this, we have exploited a gene 'silencing' system that promotes neuronal specificity by repressing expression in non-neural tissues. This methodology solves non-specific background issues that plague large-scale enhancer trap efforts and may provide a means of leveraging promoters/enhancers that otherwise express too broadly to be of value for in vivo manipulations. RESULTS: We show that a conserved neuron-restrictive silencer element (NRSE) can function to restrict transgene expression to the nervous system. The neuron-restrictive silencing factor/ repressor element 1 silencing transcription factor (NRSF/REST) transcriptional repressor binds NRSE/ repressor element 1 (RE1) sites and silences gene expression in non-neuronal cells. Inserting NRSE sites into transgenes strongly biased expression to neural tissues. NRSE sequences were effective in restricting expression of bipartite Gal4-based 'driver' transgenes within the context of an enhancer trap and when associated with a defined promoter and enhancer. However, NRSE sequences did not serve to restrict expression of an upstream activating sequence (UAS)-based reporter/effector transgene when associated solely with the UAS element. Morpholino knockdown assays showed that NRSF/REST expression is required for NRSE-based transgene silencing. CONCLUSIONS: Our findings demonstrate that the addition of NRSE sequences to transgenes can provide useful new tools for functional studies of the nervous system. However, the general approach may be more broadly applicable; tissue-specific silencer elements are operable in tissues other than the nervous system, suggesting this approach can be similarly applied to other paradigms. Thus, creating synthetic associations between endogenous regulatory elements and tissue-specific silencers may facilitate targeting of cellular subtypes for which defined promoters/enhancers are lacking.
    BMC Biology 11/2012; 10(1):93. · 7.43 Impact Factor
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    ABSTRACT: The transcriptional repressor Rest (Nrsf) recruits chromatin-modifying complexes to RE1 'silencer elements', which are associated with hundreds of neural genes. However, the requirement for Rest-mediated transcriptional regulation of embryonic development and cell fate is poorly understood. Conflicting views of the role of Rest in controlling cell fate have emerged from recent studies. To address these controversies, we examined the developmental requirement for Rest in zebrafish using zinc-finger nuclease-mediated gene targeting. We discovered that germ layer specification progresses normally in rest mutants despite derepression of target genes during embryogenesis. This analysis provides the first evidence that maternal rest is essential for repression of target genes during blastula stages. Surprisingly, neurogenesis proceeds largely normally in rest mutants, although abnormalities are observed within the nervous system, including defects in oligodendrocyte precursor cell development and a partial loss of facial branchiomotor neuron migration. Mutants progress normally through embryogenesis but many die as larvae (after 12 days). However, some homozygotes reach adulthood and are viable. We utilized an RE1/NRSE transgenic reporter system to dynamically monitor Rest activity. This analysis revealed that Rest is required to repress gene expression in mesodermal derivatives including muscle and notochord, as well as within the nervous system. Finally, we demonstrated that Rest is required for long-term repression of target genes in non-neural tissues in adult zebrafish. Our results point to a broad role for Rest in fine-tuning neural gene expression, rather than as a widespread regulator of neurogenesis or cell fate.
    Development 09/2012; 139(20):3838-48. · 6.60 Impact Factor
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    ABSTRACT: The spatial and temporal control of gene expression is key to generation of specific cellular fates during development. Studies of the transcriptional repressor REST/NRSF (RE1 Silencing Transcription Factor or Neural Restrictive Silencing Factor) have provided important insight into the role that epigenetic modifications play in differential gene expression. However, the precise function of REST during embryonic development is not well understood. We have discovered a novel interaction between zebrafish Rest and the Hedgehog (Hh) signaling pathway. We observed that Rest knockdown enhances or represses Hh signaling in a context-dependant manner. In wild-type embryos and embryos with elevated Hh signaling, Rest knockdown augments transcription of Hh target genes. Conversely, in contexts where Hh signaling is diminished, Rest knockdown has the opposite effect and Hh target gene expression is further attenuated. Epistatic analysis revealed that Rest interacts with the Hh pathway at a step downstream of Smo. Furthermore, we present evidence implicating the bifunctional, Hh signaling component Gli2a as key to the Rest modulation of the Hh response. The role of Rest as a regulator of Hh signaling has broad implications for many developmental contexts where REST and Hh signaling act.
    Developmental Biology 04/2010; 340(2):293-305. · 3.87 Impact Factor
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    Fatma O Kok, Iain T Shepherd, Howard I Sirotkin
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    ABSTRACT: Cell-type specific regulation of a small number of growth factor signal transduction pathways generates diverse developmental outcomes. The zinc finger protein Churchill (ChCh) is a key effector of fibroblast growth factor (FGF) signaling during gastrulation. ChCh is largely thought to act by inducing expression of the multifunctional Sip1 (Smad Interacting Protein 1). We investigated the function of ChCh and Sip1a during zebrafish somitogenesis. Knockdown of ChCh or Sip1a results in misshapen somites that are short and narrow. As in wild-type embryos, cycling gene expression occurs in the developing somites in ChCh and Sip1a compromised embryos, but expression of her1 and her7 is maintained in formed somites. In addition, tail bud fgf8 expression is expanded anteriorly in these embryos. Finally, we found that blocking FGF8 restores somite morphology in ChCh and Sip1a compromised embryos. These results demonstrate a novel role for ChCh and Sip1a in repression of FGF activity.
    Developmental Dynamics 02/2010; 239(2):548-58. · 2.59 Impact Factor
  • Keith P. Gates, Laura Mentzer, Howard I. Sirotkin
    Developmental Biology - DEVELOP BIOL. 01/2008; 319(2):570-570.
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    ABSTRACT: Somitogenesis is a highly controlled process that results in segmentation of the paraxial mesoderm. Notch pathway activity in the presomitic mesoderm is fundamental for management of synchronized gene expression which is necessary for regulation of somitogenesis. We have isolated an embryonic lethal mutation, SBU2, that causes somite formation defects very similar to Notch pathway mutants. SBU2 mutants generate only 6-7 asymmetrically arranged somites. However, in contrast to Notch pathway mutants, these mutants do not maintain previously formed somite boundaries and by 24 hpf, almost no somite boundaries remain. Other developmental processes disrupted in SBU2 mutants include tail morphogenesis, muscle fiber elongation, pigmentation, circulatory system development and neural differentiation. We demonstrated that these defects are the result of a nonsense mutation within the spt6 gene. spt6 encodes a transcription elongation factor that genetically interacts with the Paf-1 chromatin remodeling complex. SBU2 mutant phenotypes could be rescued by microinjection of spt6 mRNA and microinjection of spt6 morpholinos phenocopied the mutation. Our real-time PCR analysis revealed that Spt6 is essential for the transcriptional response to activation of the Notch pathway. Analysis of sbu2;mib double mutants indicates that Spt6 deficiency suppresses the neurogenic effects of the mib. Altogether, these results demonstrate that Spt6 is critical for somite formation in zebrafish and suggest that some defects observed in spt6 mutants result from alterations in Notch signaling. However, additional Spt6 mutant phenotypes are likely caused by vital functions of Spt6 in other pathways.
    Developmental Biology 08/2007; 307(2):214-26. · 3.87 Impact Factor
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    ABSTRACT: During gastrulation dynamic cell movements establish the germ layers and shape the body axis of the vertebrate embryo. The zinc finger protein Churchill (chch) has been proposed to be a key regulator of these movements. We examined the expression pattern of chch in zebrafish and studied the regulation of chch by FGF signaling. We observed zygotic expression of chch during early cleavage stages. Two lines of evidence demonstrate that chch is zygotically expressed prior to the mid-blastula transition. First, blocking transcription during early cleavage stages represses chch expression. Second, endogenous levels of chch transcripts increase between 1-cell and 16-cell embryos. chch remains widely expressed during blastula and gastrula stages but scattered cells express higher levels of chch. By somitogenesis, chch is expressed in the ventral-most cells of the embryo adjacent to the yolk. In addition, transcripts are also observed in superficial cells on the surface of the yolk, in presumptive mucous cells and keratinocytes. By 30 hpf transcripts are observed in anterior neural tissue and ventral cells adjacent to the yolk. Over the next three days chch expression is indistinct until 4 dpf when we observe expression in the pharynx and gut. We show that activation of FGF signaling during gastrulation is sufficient to induce chch expression. In addition, we demonstrate that blocking FGF signaling between the 4-cell and shield stages represses chch expression.
    Gene Expression Patterns 07/2007; 7(6):645-50. · 1.64 Impact Factor
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    Eric R Londin, Laura Mentzer, Howard I Sirotkin
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    ABSTRACT: Cell movements are essential to the determination of cell fates during development. The zinc-finger transcription factor, Churchill (ChCh) has been proposed to regulate cell fate by regulating cell movements during gastrulation in the chick. However, the mechanism of action of ChCh is not understood. We demonstrate that ChCh acts to repress the response to Nodal-related signals in zebrafish. When ChCh function is abrogated the expression of mesodermal markers is enhanced while ectodermal markers are expressed at decreased levels. In cell transplant assays, we observed that ChCh-deficient cells are more motile than wild-type cells. When placed in wild-type hosts, ChCh-deficient cells often leave the epiblast, migrate to the germ ring and are later found in mesodermal structures. We demonstrate that both movement of ChCh-compromised cells to the germ ring and acquisition of mesodermal character depend on the ability of the donor cells to respond to Nodal signals. Blocking Nodal signaling in the donor cells at the levels of Oep, Alk receptors or Fast1 inhibited migration to the germ ring and mesodermal fate change in the donor cells. We also detect additional unusual movements of transplanted ChCh-deficient cells which suggests that movement and acquisition of mesodermal character can be uncoupled. Finally, we demonstrate that ChCh is required to limit the transcriptional response to Nodal. These data establish a broad role for ChCh in regulating both cell movement and Nodal signaling during early zebrafish development. We show that chch is required to limit mesodermal gene expression, inhibit Nodal-dependant movement of presumptive ectodermal cells and repress the transcriptional response to Nodal signaling. These findings reveal a dynamic role for chch in regulating cell movement and fate during early development.
    BMC Developmental Biology 02/2007; 7:120. · 2.73 Impact Factor
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    Michael A Bell, Kaitlyn E Ellis, Howard I Sirotkin
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    ABSTRACT: The pelvic skeleton of threespine stickleback fish contributes to defence against predatory vertebrates, but rare populations exhibit vestigial pelvic phenotypes. Low ionic strength water and absence of predatory fishes are associated with reduction of the pelvic skeleton, and lack of Pitx1 expression in the pelvic region is evidently the genetic basis for pelvic reduction in several populations. Pelvic vestiges in most populations are larger on the left (left-biased), apparently because Pitx2 is expressed only on that side. We used whole-mount in situ hybridization to study Pitx1 expression in 19 populations of Gasterosteus aculeatus from lakes around Cook Inlet, Alaska, USA. As expected, specimens from six populations with full pelvic structures usually expressed Pitx1 in the limb bud; those from eight populations with left-biased pelvic reduction usually did not express it. Specimens from one of three populations with right-biased or unbiased pelvic reduction sometimes expressed Pitx1. One of two populations in which the pelvic spines (but not the girdle) are usually absent often expressed Pitx1. In terms of Jacob's 1977 'tinkering' metaphor, Pitx1 was the spare part with which natural selection usually tinkered for stickleback pelvic reduction, but it also tinkered with other genes that have smaller effects.
    Novartis Foundation symposium 02/2007; 284:225-39; discussion 239-44.
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    Eric R Londin, Jack Niemiec, Howard I Sirotkin
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    ABSTRACT: The ectoderm gives rise to both neural tissue and epidermis. In vertebrates, specification of the neural plate requires repression of bone morphogenetic protein (BMP) signaling in the dorsal ectoderm. The extracellular BMP antagonist Chordin and other signals from the dorsal mesoderm play important roles in this process. We utilized zebrafish mutant combinations that disrupt Chordin and mesoderm formation to reveal additional signals that contribute to the establishment of the neural domain. We demonstrate that fibroblast growth factor (FGF) signaling accounts for the additional activity in neural specification. Impeding FGF signaling results in a shift of ectodermal markers from neural to epidermal. However, following inhibition of FGF signaling, expression of anterior neural markers recovers in a Nodal-dependent fashion. Simultaneously blocking, Chordin, mesoderm formation, and FGF signaling eliminates neural marker expression during gastrula stages. We observed that FGF signaling is required for chordin expression but that it also acts via other mechanisms to repress BMP transcription during late blastula stages. Activation of FGF signaling was also able to repress BMP transcription in the absence of protein synthesis. Our results support a model in which specification of anterior neural tissue requires early FGF-mediated repression of BMP transcript levels and later activities of Chordin and mesodermal factors.
    Developmental Biology 04/2005; 279(1):1-19. · 3.87 Impact Factor
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    ABSTRACT: Zebrafish acquire the ability for fast swimming early in development. The motility mutant accordion (acc) undergoes exaggerated and prolonged contractions on both sides of the body, interfering with the acquisition of patterned swimming responses. Our whole cell recordings from muscle indicate that the defect is not manifested in neuromuscular transmission. However, imaging of skeletal muscle of larval acc reveals greatly prolonged calcium transients and associated contractions in response to depolarization. Positional cloning of acc identified a serca mutation as the cause of the acc phenotype. SERCA is a sarcoplasmic reticulum transmembrane protein in skeletal muscle that mediates calcium re-uptake from the myoplasm. The mutation in SERCA, a serine to phenylalanine substitution, is likely to result in compromised protein function that accounts for the observed phenotype. Indeed, direct evidence that mutant SERCA causes the motility dysfunction was provided by the finding that wild type fish injected with an antisense morpholino directed against serca, exhibited accordion-like contractions and impaired swimming. We conclude that the motility dysfunction in embryonic and larval accordion zebrafish stems directly from defective calcium transport in skeletal muscle rather than defective CNS drive.
    Developmental Biology 01/2005; 276(2):441-51. · 3.87 Impact Factor
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    ABSTRACT: The mechanism controlling the development of dopaminergic (DA) and serotonergic (5HT) neurons in vertebrates is not well understood. Here we characterized a zebrafish mutant--too few (tof)--that develops hindbrain 5HT and noradrenergic neurons, but does not develop hypothalamic DA and 5HT neurons. tof encodes a forebrain-specific zinc finger transcription repressor that is homologous to the mammalian Fezl (forebrain embryonic zinc finger-like protein). Mosaic and co-staining analyses showed that fezl was not expressed in DA or 5HT neurons and instead controlled development of these neurons non-cell-autonomously. Both the eh1-related repressor motif and the second zinc finger domain were necessary for tof function. Our results indicate that tof/fezl is a key component in regulating the development of monoaminergic neurons in the vertebrate brain.
    Nature Neuroscience 02/2003; 6(1):28-33. · 15.25 Impact Factor
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    ABSTRACT: Nodal-related signals comprise a subclass of the transforming growth factor-beta (TGF-beta) superfamily and regulate key events in vertebrate embryogenesis, including mesoderm formation, establishment of left-right asymmetry and neural patterning [1-8]. Nodal ligands are thought to act with EGF-CFC protein co-factors to activate activin type I and II or related receptors, which phosphorylate Smad2 and trigger nuclear translocation of a Smad2/4 complex [8-12]. The winged-helix transcription factor forkhead activin signal transducer-1 (Fast-1) acts as a co-factor for Smad2 [12-20]. Xenopus Fast-1 is thought to function as a transcriptional effector of Nodal signals during mesoderm formation [17], but no mutations in the Fast-1 gene have been identified. We report the identification of the zebrafish fast1 gene and show that it is disrupted in schmalspur (sur) mutants, which have defects in the development of dorsal midline cell types and establishment of left-right asymmetry [21-25]. We find that prechordal plate and notochord are strongly reduced in maternal-zygotic sur mutants, whereas other mesendodermal structures are present - a less severe phenotype than that caused by complete loss of Nodal signaling. These results show that fast1 is required for development of dorsal axial structures and left-right asymmetry, and suggest that Nodal signals act through Fast1-dependent and independent pathways.
    Current Biology 10/2000; 10(17):1051-4. · 9.49 Impact Factor
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    H I Sirotkin, S T Dougan, A F Schier, W S Talbot
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    ABSTRACT: In vertebrate embryos, maternal (beta)-catenin protein activates the expression of zygotic genes that establish the dorsal axial structures. Among the zygotically acting genes with key roles in the specification of dorsal axial structures are the homeobox gene bozozok (boz) and the nodal-related (TGF-(beta) family) gene squint (sqt). Both genes are expressed in the dorsal yolk syncytial layer, a source of dorsal mesoderm inducing signals, and mutational analysis has indicated that boz and sqt are required for dorsal mesoderm development. Here we examine the regulatory interactions among boz, sqt and a second nodal-related gene, cyclops (cyc). Three lines of evidence indicate that boz and sqt act in parallel to specify dorsal mesoderm and anterior neuroectoderm. First, boz requires sqt function to induce high levels of ectopic dorsal mesoderm, consistent with sqt acting either downstream or in parallel to boz. Second, sqt mRNA is expressed in blastula stage boz mutants, indicating that boz is not essential for activation of sqt transcription, and conversely, boz mRNA is expressed in blastula stage sqt mutants. Third, boz;sqt double mutants have a much more severe phenotype than boz and sqt single mutants. Double mutants consistently lack the anterior neural tube and axial mesoderm, and ventral fates are markedly expanded. Expression of chordin and noggin1 is greatly reduced in boz;sqt mutants, indicating that the boz and sqt pathways have overlapping roles in activating secreted BMP antagonists. In striking contrast to boz;sqt double mutants, anterior neural fates are specified in boz;sqt;cyc triple mutants. This indicates that cyc represses anterior neural development, and that boz and sqt counteract this repressive function. Our results support a model in which boz and sqt act in parallel to induce dorsalizing BMP-antagonists and to counteract the repressive function of cyc in neural patterning.
    Development 07/2000; 127(12):2583-92. · 6.21 Impact Factor
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    ABSTRACT: The dorsal gastrula organizer plays a fundamental role in establishment of the vertebrate axis. We demonstrate that the zebrafish bozozok (boz) locus is required at the blastula stages for formation of the embryonic shield, the equivalent of the gastrula organizer and expression of multiple organizer-specific genes. Furthermore, boz is essential for specification of dorsoanterior embryonic structures, including notochord, prechordal mesendoderm, floor plate and forebrain. We report that boz mutations disrupt the homeobox gene dharma. Overexpression of boz in the extraembryonic yolk syncytial layer of boz mutant embryos is sufficient for normal development of the overlying blastoderm, revealing an involvement of extraembryonic structures in anterior patterning in fish similarly to murine embryos. Epistatic analyses indicate that boz acts downstream of beta-catenin and upstream to TGF-beta signaling or in a parallel pathway. These studies provide genetic evidence for an essential function of a homeodomain protein in beta-catenin-mediated induction of the dorsal gastrula organizer and place boz at the top of a hierarchy of zygotic genes specifying the dorsal midline of a vertebrate embryo.
    Development 05/1999; 126(7):1427-38. · 6.21 Impact Factor
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    ABSTRACT: Genetic screens in zebrafish (Danio rerio) have isolated mutations in hundreds of genes with essential functions. To facilitate the identification of candidate genes for these mutations, we have genetically mapped 104 genes and expressed sequence tags by scoring single-strand conformational polymorphisms in a panel of haploid siblings. To integrate this map with existing genetic maps, we also scored 275 previously mapped genes, microsatellites, and sequence-tagged sites in the same haploid panel. Systematic phylogenetic analysis defined likely mammalian orthologs of mapped zebrafish genes, and comparison of map positions in zebrafish and mammals identified significant conservation of synteny. This comparative analysis also identified pairs of zebrafish genes that appear to be orthologous to single mammalian genes, suggesting that these genes arose in a genome duplication that occurred in the teleost lineage after the divergence of fish and mammal ancestors. This comparative map analysis will be useful in predicting the locations of zebrafish genes from mammalian gene maps and in understanding the evolution of the vertebrate genome.
    Genome Research 05/1999; 9(4):334-47. · 14.40 Impact Factor
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    ABSTRACT: The vertebrate body plan is established during gastrulation, when cells move inwards to form the mesodermal and endodermal germ layers. Signals from a region of dorsal mesoderm, which is termed the organizer, pattern the body axis by specifying the fates of neighbouring cells
    Nature 09/1998; 395(6698):181-185. · 38.60 Impact Factor

Publication Stats

843 Citations
139.02 Total Impact Points

Institutions

  • 2005–2013
    • Stony Brook University
      • • Department of Neurobiology & Behavior
      • • Department of Neurobiology and Behavior
      Stony Brook, NY, United States
  • 2003
    • Stanford Medicine
      • Department of Developmental Biology
      Stanford, California, United States
  • 2000
    • Stanford University
      • Department of Developmental Biology
      Stanford, CA, United States