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Other Publications (65)
- Development (Cambridge, England)
- BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology
- Development (Cambridge, England)
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Developmental Biology
- Mechanisms of Development
- Gene Expression Patterns : GEP
- Genesis (New York, N.Y. : 2000)
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Development (Cambridge, England)
- Developmental Biology
- Developmental Biology
- Genome Biology
- The International Journal of Developmental Biology
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Zebrafish
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- CSH Protocols
- Mechanisms of Development
- Anatomical Record (Hoboken, N.J. : 2007)
- Development (Cambridge, England)
- Genes & Development
- BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Development (Cambridge, England)
- Cold Spring Harbor Protocols
- Cold Spring Harbor Protocols
- Cold Spring Harbor Protocols
- Cold Spring Harbor Protocols
- Cold Spring Harbor Protocols
- Cold Spring Harbor Protocols
- Disease Models & Mechanisms
- Methods in Molecular Biology (Clifton, N.J.)
- FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology
- Neuron
- Cell
- Cold Spring Harbor Protocols
- Disease Models & Mechanisms
- Developmental Biology
- Genome Research
- Zebrafish
- Nature
- Nature
Articles by Hazel Sive in JoVE
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में चेहरे प्रत्यारोपण
Laura A. Jacox*1,3, Amanda J. Dickinson*4, Hazel Sive2,3
1Biological Sciences in Dental Medicine, Harvard University, 2Biology Department, Massachusetts Institute of Technology, 3Whitehead Institute, Massachusetts Institute of Technology, 4Biology Department, Virginia Commonwealth University
Xenopus के बीच चेहरे ऊतक "चरम पूर्वकाल डोमेन" रोपाई के लिए एक तकनीक भ्रूण विकसित किया गया है laevis. ऊतक craniofacial विकास के लिए और चेहरे क्षेत्रों के बीच बातचीत के संकेत के लिए स्थानीय आवश्यकताओं के अध्ययन की अनुमति, दूसरे में एक जीन की अभिव्यक्ति पृष्ठभूमि से ले जाया जा सकता है.
Other articles by Hazel Sive on PubMed
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Cement Gland-specific Activation of the Xag1 Promoter is Regulated by Co-operation of Putative Ets and ATF/CREB Transcription Factors
Development (Cambridge, England).
Oct, 2002 |
Pubmed ID: 12223398 The cement gland marks the extreme anterior ectoderm of the Xenopus embryo, and is determined through the overlap of several positional domains. In order to understand how these positional cues activate cement gland differentiation, the promoter of Xag1, a marker of cement gland differentiation, was analyzed. Previous studies have shown that Xag1 expression can be activated by the anterior-specific transcription factor Otx2, but that this activation is indirect. 102 bp of upstream genomic Xag1 sequence restricts reporter gene expression specifically to the cement gland. Within this region, putative binding sites for Ets and ATF/CREB transcription factors are both necessary and sufficient to drive cement gland-specific expression, and cooperate to do so. Furthermore, while the putative ATF/CREB factor is activated by Otx2, a factor acting through the putative Ets-binding site is not. These results suggest that Ets-like and ATF/CREB-like family members play a role in regulating Xag1 expression in the cement gland, through integration of Otx2 dependent and independent pathways.
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What's Your Position? the Xenopus Cement Gland As a Paradigm of Regional Specification
BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology.
Jul, 2003 |
Pubmed ID: 12815727 The correct positioning of organs during embryonic development requires multiple cues. The Xenopus cement gland is a mucus-secreting epithelium that is a simple model for organogenesis, allowing detailed analysis of this complex process. The cement gland forms at a conserved anterior position, where embryonic ectoderm and endoderm touch. In all deuterostomes, this region will form the stomodeum (primitive mouth) and, in some aquatic larva, will also form a cement gland. In recent years, a model has been put forward suggesting that an intermediate level of BMP signaling in the ectoderm leads to cement gland formation. We propose an alternative model whereby, during gastrulation, the cement gland (CG) is positioned by the overlap of three domains, corresponding to anterodorsal identity (AD), ventrolateral identity (VL), and ectodermal outer layer identity (EO), defining the equation (AD + VL + EO = CG). Anterodorsal identity requires a contribution by the transcription factor Otx2 while ventrolateral identity requires the BMP4 signaling pathway. These postional cues are integrated to activate cement gland differentiation. This integration appears to require intermediate steps, including expression of pitx genes, and members of the ATF/CREB and Ets transcription factor families.
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Nlz Gene Family is Required for Hindbrain Patterning in the Zebrafish
Developmental Dynamics : an Official Publication of the American Association of Anatomists.
Apr, 2004 |
Pubmed ID: 15042707 This study describes the conserved nlz gene family whose members encode unusual zinc finger proteins. In the zebrafish neurectoderm, both nlz1 and the newly isolated nlz2 are expressed in the presumptive hindbrain and midbrain/hindbrain boundary, where expression of nlz1 is dependent on pax2a. In addition, nlz2 is uniquely expressed more anteriorly, in the presumptive midbrain and diencephalon. Overexpression of Nlz proteins during gastrula stages inhibits hindbrain development. In particular, ectopically expressed Nlz1 inhibits formation of future rhombomeres 2 and 3 (r2, r3), whereas neighboring r1 and r4 are not affected. Conversely, simultaneous reduction of Nlz1 and Nlz2 protein function by expression of antisense morpholino-modified oligomers leads to expansion of future r3 and r5, with associated loss of r4. These data indicate that one function of the nlz gene family is to specify or maintain r4 identity, and to limit r3 and r5 during hindbrain formation.
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Strategies of Vertebrate Neurulation and a Re-evaluation of Teleost Neural Tube Formation
Mechanisms of Development.
Oct, 2004 |
Pubmed ID: 15327780 The vertebrate neural tube develops by two distinct mechanisms. Anteriorly, in the brain and future trunk (cervicothoracic) region, 'primary neurulation' occurs, where an epithelial sheet rolls or bends into a tube. Posteriorly, in the future lumbar and tail region, the neural tube forms by 'secondary neurulation', where a mesenchymal cell population condenses to form a solid rod that undergoes transformation to an epithelial tube. Teleost neurulation has been described as different from that of other vertebrates. This is principally because the teleost trunk neural tube initially forms a solid rod (the neural keel) that later develops a lumen. This process has also been termed secondary neurulation. However, this description is not accurate since the teleost neural tube derives from an epithelial sheet that folds. This best fits the description of primary neurulation. It has also been suggested that teleost neurulation is primitive, however, both primary and secondary neurulation are found in groups with a more ancient origin than the teleosts. The similarity between neurulation in teleosts and other vertebrates indicates that this group includes viable models (such as the zebrafish) for understanding human neural tube development.
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Specification of the Enveloping Layer and Lack of Autoneuralization in Zebrafish Embryonic Explants
Developmental Dynamics : an Official Publication of the American Association of Anatomists.
Jan, 2005 |
Pubmed ID: 15543604 We have analyzed the roles of cell contact during determination of the outermost enveloping layer (EVL) and deeper neurectoderm in zebrafish embryos. Outer cells, but not deeper cells, are specified to express the EVL-specific marker, cyt1 by late blastula. EVL specification requires cell contact or close cell proximity, because cyt1 is not expressed after explant dissociation. The EVL may be homologous to the Xenopus epithelial layer, including the ventral larval epidermis. While Xenopus epidermal cytokeratin gene expression is activated by bone morphogenetic protein (BMP) signaling, zebrafish cyt1 is not responsive to BMPs. Zebrafish early gastrula ectodermal explants are specified to express the neural markers opl (zic1) and otx2, and this expression is prevented by BMP4. Dissociation of zebrafish explants prevents otx2 and opl expression, suggesting that neural specification in zebrafish requires cell contact or close cell proximity. This finding is in contrast to the case in Xenopus, where ectodermal dissociation leads to activation of neural gene expression, or autoneuralization. Our data suggest that distinct mechanisms direct development of homologous lineages in different vertebrates.
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Initial Formation of Zebrafish Brain Ventricles Occurs Independently of Circulation and Requires the Nagie Oko and Snakehead/atp1a1a.1 Gene Products
Development (Cambridge, England).
May, 2005 |
Pubmed ID: 15788456 The mechanisms by which the vertebrate brain develops its characteristic three-dimensional structure are poorly understood. The brain ventricles are a highly conserved system of cavities that form very early during brain morphogenesis and that are required for normal brain function. We have initiated a study of zebrafish brain ventricle development and show here that the neural tube expands into primary forebrain, midbrain and hindbrain ventricles rapidly, over a 4-hour window during mid-somitogenesis. Circulation is not required for initial ventricle formation, only for later expansion. Cell division rates in the neural tube surrounding the ventricles are higher than between ventricles and, consistently, cell division is required for normal ventricle development. Two zebrafish mutants that do not develop brain ventricles are snakehead and nagie oko. We show that snakehead is allelic to small heart, which has a mutation in the Na+K+ ATPase gene atp1a1a.1. The snakehead neural tube undergoes normal ventricle morphogenesis; however, the ventricles do not inflate, probably owing to impaired ion transport. By contrast, mutants in nagie oko, which was previously shown to encode a MAGUK family protein, fail to undergo ventricle morphogenesis. This correlates with an abnormal brain neuroepithelium, with no clear midline and disrupted junctional protein expression. This study defines three steps that are required for brain ventricle development and that occur independently of circulation: (1) morphogenesis of the neural tube, requiring nok function; (2) lumen inflation requiring atp1a1a.1 function; and (3) localized cell proliferation. We suggest that mechanisms of brain ventricle development are conserved throughout the vertebrates.
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Development of the Primary Mouth in Xenopus Laevis
Developmental Biology.
Jul, 2006 |
Pubmed ID: 16678148 The initial opening between the gut and the outside of the deuterostome embryo breaks through at the extreme anterior. This region is unique in that ectoderm and endoderm are directly juxtaposed, without intervening mesoderm. This opening has been called the stomodeum, buccopharyngeal membrane or oral cavity at various stages of its formation, however, in order to clarify its function, we have termed this the "primary mouth". In vertebrates, the neural crest grows around the primary mouth to form the face and a "secondary mouth" forms. The primary mouth then becomes the pharyngeal opening. In order to establish a molecular understanding of primary mouth formation, we have begun to examine this process during Xenopus laevis development. An early step during this process occurs at tailbud and involves dissolution of the basement membrane between the ectoderm and endoderm. This is followed by ectodermal invagination to create the stomodeum. A subsequent step involves localized cell death in the ectoderm, which may lead to ectodermal thinning. Subsequently, ectoderm and endoderm apparently intercalate to generate one to two cell layers. The final step is perforation, where (after hatching) the primary mouth opens. Fate mapping has defined the ectodermal and endodermal regions that will form the primary mouth. Extirpations and transplants of these and adjacent regions indicate that, at tailbud, the oral ectoderm is not specifically required for primary mouth formation. In contrast, underlying endoderm and surrounding regions are crucial, presumably sources of necessary signals. This study indicates the complexity of primary mouth formation, and lays the groundwork for future molecular analyses of this important structure.
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The Zic1 Gene is an Activator of Wnt Signaling
The International Journal of Developmental Biology.
2006 |
Pubmed ID: 16892174 The zic1 gene plays an important role in early patterning of the Xenopus neurectoderm. While Zic1 does not act as a neural inducer, it synergizes with the neural inducing factor Noggin to activate expression of posterior neural genes, including the midbrain/hindbrain boundary marker engrailed-2. Since the Drosophila homologue of zic1, odd-paired (opa), regulates expression of the wingless and engrailed genes and since Wnt proteins posteriorize neural tissue in Xenopus, we asked whether Xenopus Zic1 acted through the Wnt pathway. Using Wnt signaling inhibitors, we demonstrate that an active Wnt pathway is required for activation of en-2 expression by zic1. Consistent with this result, Zic1 induces expression of several wnt genes, including wnt1, wnt4 and wnt8b. wnt1 gene expression activates expression of engrailed in various organisms, including Xenopus, as demonstrated here. Together, our data suggest that zic1 is an upstream regulator of several wnt genes and that the regulatory relationships between opa, wingless and engrailed seen in Drosophila are also present in vertebrates.
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Investigator Profile. An Interview with Hazel Sive, Ph.D. Interview by Vicki Glaser
Zebrafish.
2006 |
Pubmed ID: 18377221 Hazel L. Sive, Ph.D., is a Professor in the Department of Biology at the Massachusetts Institute of Technology, and a member of the Whitehead Institute in Cambridge, Massachusetts. Dr. Sive received a Bachelor's of Science degree from the University of Witwatersrand, Johannesburg, South Africa, in chemistry and zoology. She completed her Ph.D. in molecular biology from Rockefeller University in New York City. She pursued a postdoctoral fellowship in embryology at the Fred Hutchinson Cancer Center, in Seattle, Washington.
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Whitesnake/sfpq is Required for Cell Survival and Neuronal Development in the Zebrafish
Developmental Dynamics : an Official Publication of the American Association of Anatomists.
May, 2007 |
Pubmed ID: 17393485 Organogenesis involves both the development of specific cell types and their organization into a functional three-dimensional structure. We are using the zebrafish to assess the genetic basis for brain organogenesis. We show that the whitesnake mutant corresponds to the sfpq (splicing factor, proline/glutamine rich) gene, encoding the PSF protein (polypyrimidine tract-binding protein-associated splicing factor). In vitro studies have shown that PSF is important for RNA splicing and transcription and is a candidate brain-specific splicing factor, however, the in vivo function of this gene is unclear. sfpq is expressed throughout development and in the adult zebrafish, with strong expression in the developing brain, particularly in regions enriched for neuronal progenitors. In the whitesnake mutant, a brain phenotype is visible by 28 hr after fertilization, when it becomes apparent that the midbrain and hindbrain are abnormally shaped. Neural crest, heart, and muscle development or function is also abnormal. sfpq function appears to be required in two distinct phases during development. First, loss of sfpq gene function leads to increased cell death throughout the early embryo, suggesting that cell survival requires functional PSF protein. Second, sfpq function is required for differentiation, but not for determination, of specific classes of brain neurons. These data indicate that, in vertebrates, sfpq plays a key role in neuronal development and is essential for normal brain development.
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Xenopus Laevis Keller Explants
CSH Protocols.
2007 |
Pubmed ID: 21357097 INTRODUCTIONThe basic Keller explant is a rectangle of dorsal mesendoderm and ectoderm from an early-gastrula-stage Xenopus laevis embryo. It is ~60° to 90° wide, extending from the bottle cells to the animal pole. This protocol describes how to dissect, assemble, and cultivate Keller explants. The purpose of Keller explants was initially to allow observation of gastrulation movements, particularly convergent extension, in culture. This is difficult to do when explants curl up, but in Keller sandwiches, the explants are cultured flat, either as a single sheet (open-face explant) or more frequently as two sheets sandwiched together with their inner surfaces apposed (closed sandwich). Explants are cultured beneath a coverslip fragment or a glass bridge resting on silicone vacuum grease until the desired stage, usually during or after neurulation. Instead of involuting beneath the ectoderm, mesoderm elongates in a plane with adjacent ectoderm. Explants are made at the onset of gastrulation before significant vertical juxtaposition of ectoderm and mesoderm has occurred.
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Baskets for in Situ Hybridization and Immunohistochemistry
CSH Protocols.
2007 |
Pubmed ID: 21357142 INTRODUCTIONFor large in situ hybridization and immunohistochemistry experiments, changing solutions in vials becomes tedious, and baskets should be used. Commercially available baskets, such as 15-mm Netwell baskets (Corning), fit into 12-well tissue culture plates. Although they are expensive, they can, with extensive washing, be reused. However, these baskets are not readily adaptable to large-scale use, and their relatively shallow wells make cross-contamination between wells a real danger. This protocol describes a procedure for making homemade baskets with plastic microcentrifuge tubes and nylon mesh that are more adaptable to large-scale experiments. The baskets are narrow and deep and thus can be placed in a rack at high density. To change solutions, individual tubes or whole racks of tubes can be moved from one bath of solution to another. Because many tubes can be manipulated simultaneously, there is an enormous saving in work. It is also quite difficult to lose embryos in baskets; embryos in vials stand a good chance of being sucked into the aspirator during solution changes and lost.
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Formation of the Zebrafish Midbrain-hindbrain Boundary Constriction Requires Laminin-dependent Basal Constriction
Mechanisms of Development.
Nov-Dec, 2008 |
Pubmed ID: 18682291 The midbrain-hindbrain boundary (MHB) is a highly conserved fold in the vertebrate embryonic brain. We have termed the deepest point of this fold the MHB constriction (MHBC) and have begun to define the mechanisms by which it develops. In the zebrafish, the MHBC is formed soon after neural tube closure, concomitant with inflation of the brain ventricles. The MHBC is unusual, as it forms by bending the basal side of the neuroepithelium. At single cell resolution, we show that zebrafish MHBC formation involves two steps. The first is a shortening of MHB cells to approximately 75% of the length of surrounding cells. The second is basal constriction, and apical expansion, of a small group of cells that contribute to the MHBC. In the absence of inflated brain ventricles, basal constriction still occurs, indicating that the MHBC is not formed as a passive consequence of ventricle inflation. In laminin mutants, basal constriction does not occur, indicating an active role for the basement membrane in this process. Apical expansion also fails to occur in laminin mutants, suggesting that apical expansion may be dependent on basal constriction. This study demonstrates laminin-dependent basal constriction as a previously undescribed molecular mechanism for brain morphogenesis.
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Characterization and Classification of Zebrafish Brain Morphology Mutants
Anatomical Record (Hoboken, N.J. : 2007).
Jan, 2009 |
Pubmed ID: 19051268 The mechanisms by which the vertebrate brain achieves its three-dimensional structure are clearly complex, requiring the functions of many genes. Using the zebrafish as a model, we have begun to define genes required for brain morphogenesis, including brain ventricle formation, by studying 16 mutants previously identified as having embryonic brain morphology defects. We report the phenotypic characterization of these mutants at several timepoints, using brain ventricle dye injection, imaging, and immunohistochemistry with neuronal markers. Most of these mutants display early phenotypes, affecting initial brain shaping, whereas others show later phenotypes, affecting brain ventricle expansion. In the early phenotype group, we further define four phenotypic classes and corresponding functions required for brain morphogenesis. Although we did not use known genotypes for this classification, basing it solely on phenotypes, many mutants with defects in functionally related genes clustered in a single class. In particular, Class 1 mutants show midline separation defects, corresponding to epithelial junction defects; Class 2 mutants show reduced brain ventricle size; Class 3 mutants show midbrain-hindbrain abnormalities, corresponding to basement membrane defects; and Class 4 mutants show absence of ventricle lumen inflation, corresponding to defective ion pumping. Later brain ventricle expansion requires the extracellular matrix, cardiovascular circulation, and transcription/splicing-dependent events. We suggest that these mutants define processes likely to be used during brain morphogenesis throughout the vertebrates. Anat Rec, 2009. (c) 2008 Wiley-Liss, Inc.
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The Wnt Antagonists Frzb-1 and Crescent Locally Regulate Basement Membrane Dissolution in the Developing Primary Mouth
Development (Cambridge, England).
Apr, 2009 |
Pubmed ID: 19224982 The primary mouth forms from ectoderm and endoderm at the extreme anterior of the embryo, a conserved mesoderm-free region. In Xenopus, a very early step in primary mouth formation is loss of the basement membrane between the ectoderm and endoderm. In an unbiased microarray screen, we defined genes encoding the sFRPs Frzb-1 and Crescent as transiently and locally expressed in the primary mouth anlage. Using antisense oligonucleotides and ;face transplants', we show that frzb-1 and crescent expression is specifically required in the primary mouth region at the time this organ begins to form. Several assays indicate that Frzb-1 and Crescent modulate primary mouth formation by suppressing Wnt signaling, which is likely to be mediated by beta-catenin. First, a similar phenotype (no primary mouth) is seen after loss of Frzb-1/Crescent function to that seen after temporally and spatially restricted overexpression of Wnt-8. Second, overexpression of either Frzb-1 or Dkk-1 results in an enlarged primary mouth anlage. Third, overexpression of Dkk-1 can restore a primary mouth to embryos in which Frzb-1/Crescent expression has been inhibited. We show that Frzb-1/Crescent function locally promotes basement membrane dissolution in the primary mouth primordium. Consistently, Frzb-1 overexpression decreases RNA levels of the essential basement membrane genes fibronectin and laminin, whereas Wnt-8 overexpression increases the levels of these RNAs. These data are the first to connect Wnt signaling and basement membrane integrity during primary mouth development, and suggest a general paradigm for the regulation of basement membrane remodeling.
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Coherent but Overlapping Expression of MicroRNAs and Their Targets During Vertebrate Development
Genes & Development.
Feb, 2009 |
Pubmed ID: 19240133 MicroRNAs (miRNAs) are small noncoding RNAs that direct post-transcriptional repression of protein-coding genes. In vertebrates, each highly conserved miRNA typically regulates hundreds of target mRNAs. However, the precise relationship between expression of the miRNAs and that of their targets has remained unclear, in part because of the scarcity of quantitative expression data at cellular resolution. Here we report quantitative analyses of mRNA levels in miRNA-expressing cells of the zebrafish embryo, capturing entire miRNA expression domains, purified to cellular resolution using fluorescent-activated cell sorting (FACS). Focus was on regulation by miR-206 and miR-133 in the developing somites and miR-124 in the developing central nervous system. Comparison of wild-type embryos and those lacking miRNAs revealed predicted targets that responded to the miRNAs and distinguished miRNA-mediated mRNA destabilization from other regulatory effects. For all three miRNAs examined, expression of the miRNAs and that of their predicted targets usually overlapped. A few targets were expressed at higher levels in miRNA-expressing cells than in the rest of the embryo, demonstrating that miRNA-mediated repression can act in opposition to other regulatory processes. However, for most targets expression was lower in miRNA-expressing cells than in the rest of the embryo, indicating that miRNAs usually operate in concert with the other regulatory machinery of the cell.
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Epithelial Relaxation Mediated by the Myosin Phosphatase Regulator Mypt1 is Required for Brain Ventricle Lumen Expansion and Hindbrain Morphogenesis
Development (Cambridge, England).
Mar, 2010 |
Pubmed ID: 20147380 We demonstrate that in the zebrafish hindbrain, cell shape, rhombomere morphogenesis and, unexpectedly, brain ventricle lumen expansion depend on the contractile state of the neuroepithelium. The hindbrain neural tube opens in a specific sequence, with initial separation along the midline at rhombomere boundaries, subsequent openings within rhombomeres and eventual coalescence of openings into the hindbrain ventricle lumen. A mutation in the myosin phosphatase regulator mypt1 results in a small ventricle due to impaired stretching of the surrounding neuroepithelium. Although initial hindbrain opening remains normal, mypt1 mutant rhombomeres do not undergo normal morphological progression. Three-dimensional reconstruction demonstrates cell shapes within rhombomeres and at rhombomere boundaries are abnormal in mypt1 mutants. Wild-type cell shape requires that surrounding cells are also wild type, whereas mutant cell shape is autonomously regulated. Supporting the requirement for regulation of myosin function during hindbrain morphogenesis, wild-type embryos show dynamic levels of phosphorylated myosin regulatory light chain (pMRLC). By contrast, mutants show continuously high pMRLC levels, with concentration of pMRLC and myosin II at the apical side of the epithelium, and myosin II and actin concentration at rhombomere boundaries. Brain ventricle lumen expansion, rhombomere morphology and cell shape are rescued by inhibition of myosin II function, indicating that each defect is a consequence of overactive myosin. We suggest that the epithelium must ;relax', via activity of myosin phosphatase, to allow for normal hindbrain morphogenesis and expansion of the brain ventricular lumen. Epithelial relaxation might be a widespread strategy to facilitate tube inflation in many organs.
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Keeping Two Animal Systems in One Lab - a Frog Plus Fish Case Study
Methods in Molecular Biology (Clifton, N.J.).
2011 |
Pubmed ID: 21805281 For two decades, my lab has been studying development using two vertebrate animals, the frog Xenopus and the zebrafish, Danio. This has been both productive and challenging. The initial rationale for the choice was to compare the same process in two species, as a means to find commonalities that may carry through all vertebrates. As time progressed, however, each species has become exploited for its specific attributes, more than for comparative studies. Maintaining two species simultaneously has been challenging, as has the division of research between the two and making sure that lab members know both systems well enough to communicate productively. Other significant issues concern funding for disparate research, figuring out how to make contributions to both fish and frog communities, and being accepted as a member of two communities. I discuss whether this dual allegiance has been a good idea.
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Disc1 Regulates Both β-catenin-mediated and Noncanonical Wnt Signaling During Vertebrate Embryogenesis
FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology.
Dec, 2011 |
Pubmed ID: 21859895 Disc1 is a schizophrenia risk gene that engages multiple signaling pathways during neurogenesis and brain development. Using the zebrafish as a tool, we analyze the function of zebrafish Disc1 (zDisc1) at the earliest stages of brain and body development. We define a "tool" as a biological system that gives insight into mechanisms underlying a human disorder, although the system does not phenocopy the disorder. A zDisc1 peptide binds to GSK3β, and zDisc1 directs early brain development and neurogenesis, by promoting β-catenin-mediated Wnt signaling and inhibiting GSK3β activity. zDisc1 loss-of-function embryos additionally display a convergence and extension phenotype, demonstrated by abnormal movement of dorsolateral cells during gastrulation, through changes in gene expression, and later through formation of abnormal, U-shaped muscle segments, and a truncated tail. These phenotypes are caused by alterations in the noncanonical Wnt pathway, via Daam and Rho signaling. The convergence and extension phenotype can be rescued by a dominant negative GSK3β construct, suggesting that zDisc1 inhibits GSK3β activity during noncanonical Wnt signaling. This is the first demonstration that Disc1 modulates the noncanonical Wnt pathway and suggests a previously unconsidered mechanism by which Disc1 may contribute to the etiology of neuropsychiatric disorders.
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Common DISC1 Polymorphisms Disrupt Wnt/GSK3β Signaling and Brain Development
Neuron.
Nov, 2011 |
Pubmed ID: 22099458 Disrupted in Schizophrenia-1 (DISC1) is a candidate gene for psychiatric disorders and has many roles during brain development. Common DISC1 polymorphisms (variants) are associated with neuropsychiatric phenotypes including altered cognition, brain structure, and function; however, it is unknown how this occurs. Here, we demonstrate using mouse, zebrafish, and human model systems that DISC1 variants are loss of function in Wnt/GSK3β signaling and disrupt brain development. The DISC1 variants A83V, R264Q, and L607F, but not S704C, do not activate Wnt signaling compared with wild-type DISC1 resulting in decreased neural progenitor proliferation. In zebrafish, R264Q and L607F could not rescue DISC1 knockdown-mediated aberrant brain development. Furthermore, human lymphoblast cell lines endogenously expressing R264Q displayed impaired Wnt signaling. Interestingly, S704C inhibited the migration of neurons in the developing neocortex. Our data demonstrate DISC1 variants impair Wnt signaling and brain development and elucidate a possible mechanism for their role in neuropsychiatric phenotypes.
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Zebrafish Homologs of Genes Within 16p11.2, a Genomic Region Associated with Brain Disorders, Are Active During Brain Development, and Include Two Deletion Dosage Sensor Genes
Disease Models & Mechanisms.
Jul, 2012 |
Pubmed ID: 22566537 Deletion or duplication of one copy of the human 16p11.2 interval is tightly associated with impaired brain function, including autism spectrum disorders (ASDs), intellectual disability disorder (IDD) and other phenotypes, indicating the importance of gene dosage in this copy number variant region (CNV). The core of this CNV includes 25 genes; however, the number of genes that contribute to these phenotypes is not known. Furthermore, genes whose functional levels change with deletion or duplication (termed 'dosage sensors'), which can associate the CNV with pathologies, have not been identified in this region. Using the zebrafish as a tool, a set of 16p11.2 homologs was identified, primarily on chromosomes 3 and 12. Use of 11 phenotypic assays, spanning the first 5 days of development, demonstrated that this set of genes is highly active, such that 21 out of the 22 homologs tested showed loss-of-function phenotypes. Most genes in this region were required for nervous system development - impacting brain morphology, eye development, axonal density or organization, and motor response. In general, human genes were able to substitute for the fish homolog, demonstrating orthology and suggesting conserved molecular pathways. In a screen for 16p11.2 genes whose function is sensitive to hemizygosity, the aldolase a (aldoaa) and kinesin family member 22 (kif22) genes were identified as giving clear phenotypes when RNA levels were reduced by ~50%, suggesting that these genes are deletion dosage sensors. This study leads to two major findings. The first is that the 16p11.2 region comprises a highly active set of genes, which could present a large genetic target and might explain why multiple brain function, and other, phenotypes are associated with this interval. The second major finding is that there are (at least) two genes with deletion dosage sensor properties among the 16p11.2 set, and these could link this CNV to brain disorders such as ASD and IDD.
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Multiple Roles for the Na,K-ATPase Subunits, Atp1a1 and Fxyd1, During Brain Ventricle Development
Developmental Biology.
Aug, 2012 |
Pubmed ID: 22683378 Formation of the vertebrate brain ventricles requires both production of cerebrospinal fluid (CSF), and its retention in the ventricles. The Na,K-ATPase is required for brain ventricle development, and we show here that this protein complex impacts three associated processes. The first requires both the alpha subunit (Atp1a1) and the regulatory subunit, Fxyd1, and leads to formation of a cohesive neuroepithelium, with continuous apical junctions. The second process leads to modulation of neuroepithelial permeability, and requires Atp1a1, which increases permeability with partial loss of function and decreases it with overexpression. In contrast, fxyd1 overexpression does not alter neuroepithelial permeability, suggesting that its activity is limited to neuroepithelium formation. RhoA regulates both neuroepithelium formation and permeability, downstream of the Na,K-ATPase. A third process, likely to be CSF production, is RhoA-independent, requiring Atp1a1, but not Fxyd1. Consistent with a role for Na,K-ATPase pump function, the inhibitor ouabain prevents neuroepithelium formation, while intracellular Na(+) increases after Atp1a1 and Fxyd1 loss of function. These data include the first reported role for Fxyd1 in the developing brain, and indicate that the Na,K-ATPase regulates three aspects of brain ventricle development essential for normal function: formation of a cohesive neuroepithelium, restriction of neuroepithelial permeability, and production of CSF.
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Extensive Alternative Polyadenylation During Zebrafish Development
Genome Research.
Oct, 2012 |
Pubmed ID: 22722342 The post-transcriptional fate of messenger RNAs (mRNAs) is largely dictated by their 3' untranslated regions (3' UTRs), which are defined by cleavage and polyadenylation (CPA) of pre-mRNAs. We used poly(A)-position profiling by sequencing (3P-seq) to map poly(A) sites at eight developmental stages and tissues in the zebrafish. Analysis of over 60 million 3P-seq reads substantially increased and improved existing 3' UTR annotations, resulting in confidently identified 3' UTRs for >79% of the annotated protein-coding genes in zebrafish. mRNAs from most zebrafish genes undergo alternative CPA, with those from more than a thousand genes using different dominant 3' UTRs at different stages. These included one of the poly(A) polymerase genes, for which alternative CPA reinforces its repression in the ovary. 3' UTRs tend to be shortest in the ovaries and longest in the brain. Isoforms with some of the shortest 3' UTRs are highly expressed in the ovary, yet absent in the maternally contributed RNAs of the embryo, perhaps because their 3' UTRs are too short to accommodate a uridine-rich motif required for stability of the maternal mRNA. At 2 h post-fertilization, thousands of unique poly(A) sites appear at locations lacking a typical polyadenylation signal, which suggests a wave of widespread cytoplasmic polyadenylation of mRNA degradation intermediates. Our insights into the identities, formation, and evolution of zebrafish 3' UTRs provide a resource for studying gene regulation during vertebrate development.
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Efficient ShRNA-Mediated Inhibition of Gene Expression in Zebrafish
Zebrafish.
Sep, 2012 |
Pubmed ID: 22788660 Abstract Despite the broad repertoire of loss of function (LOF) tools available for use in the zebrafish, there remains a need for a simple and rapid method that can inhibit expression of genes at later stages. RNAi would fulfill that role, and a previous report (Dong et al. 2009) provided encouraging data. The goal of this study was to further address the ability of expressed shRNAs to inhibit gene expression. This included quantifying RNA knockdown, testing specificity of shRNA effects, and determining whether tissue-specific LOF could be achieved. Using an F0 transgenic approach, this report demonstrates that for two genes, wnt5b and zDisc1, each with described mutant and morphant phenotypes, shRNAs efficiently decrease endogenous RNA levels. Phenotypes elicited by shRNA resemble those of mutants and morphants, and are reversed by expression of cognate RNA, further demonstrating specificity. Tissue-specific expression of zDisc1 shRNAs in F0 transgenics demonstrates that conditional LOF can be readily obtained. These results suggest that shRNA expression presents a viable approach for rapid inhibition of zebrafish gene expression.
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Poly(A)-tail Profiling Reveals an Embryonic Switch in Translational Control
Nature.
Jan, 2014 |
Pubmed ID: 24476825 Poly(A) tails enhance the stability and translation of most eukaryotic messenger RNAs, but difficulties in globally measuring poly(A)-tail lengths have impeded greater understanding of poly(A)-tail function. Here we describe poly(A)-tail length profiling by sequencing (PAL-seq) and apply it to measure tail lengths of millions of individual RNAs isolated from yeasts, cell lines, Arabidopsis thaliana leaves, mouse liver, and zebrafish and frog embryos. Poly(A)-tail lengths were conserved between orthologous mRNAs, with mRNAs encoding ribosomal proteins and other 'housekeeping' proteins tending to have shorter tails. As expected, tail lengths were coupled to translational efficiencies in early zebrafish and frog embryos. However, this strong coupling diminished at gastrulation and was absent in non-embryonic samples, indicating a rapid developmental switch in the nature of translational control. This switch complements an earlier switch to zygotic transcriptional control and explains why the predominant effect of microRNA-mediated deadenylation concurrently shifts from translational repression to mRNA destabilization.
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