Other articles by Hazel Sive on PubMed

• 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.
• 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.
• Vhnf1 and Fgf Signals Synergize to Specify Rhombomere Identity in the Zebrafish Hindbrain Development (Cambridge, England). Aug, 2003  |   Pubmed ID: 12835397 Vertebrate hindbrain segmentation is a highly conserved process but the mechanism of rhombomere determination is not well understood. Recent work in the zebrafish has shown a requirement for fibroblast growth factor (Fgf) signaling and for the transcription factor variant hepatocyte nuclear factor 1 (vhnf1) in specification of rhombomeres 5 and 6 (r5+r6). We show here that vhnf1 functions in two ways to subdivide the zebrafish caudal hindbrain domain (r4-r7) into individual rhombomeres. First, vhnf1 promotes r5+r6 identity through an obligate synergy with Fgf signals to activate valentino and krox20 expression. Second, vhnf1 functions independently of Fgf signals to repress hoxb1a expression. Although vhnf1 is expressed in a broad posterior domain during gastrulation, it promotes the specification of individual rhombomeres. This is achieved in part because vhnf1 gives cellular competence to respond to Fgf signals in a caudal hindbrain-specific manner.
• Early Requirement for Fgf8 Function During Hindbrain Pattern Formation in Zebrafish Developmental Dynamics : an Official Publication of the American Association of Anatomists. Feb, 2004  |   Pubmed ID: 14745965 Fibroblast growth factor (FGF) signaling is required for normal development of the vertebrate brain, including the isthmus and caudal regions of the hindbrain. Recent work in zebrafish has identified a requirement for the combination of fgf3 and fgf8 functions in specification of rhombomeres 5 and 6 (r5, r6), when evaluated at mid- and late somitogenesis stages. However, when examined earlier in development, during early somitogenesis stages, FGF8 alone is required to initiate r5 and r6 development. Both a mutation in fgf8 and injection of fgf8-targeted antisense morpholino-modified oligonucleotides result in suppression of genes normally expressed in r5 and r6 by the one- to two-somite stage. This expression recovers by the six-somite stage, and we propose that this recovery is a response to activation of fgf3 and to delayed accumulation of fgf8. These data demonstrate an early, nonredundant requirement for fgf8 function in hindbrain patterning.
• 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.
• The Meis3 Protein and Retinoid Signaling Interact to Pattern the Xenopus Hindbrain Developmental Biology. Jul, 2004  |   Pubmed ID: 15196951 In Xenopus embryos, proper hindbrain formation requires activities of both XMeis3 protein and retinoic acid (RA) signaling. In this study, we show that XMeis3 protein and RA signaling differentially interact to regulate hindbrain patterning. The knockdown of XMeis3 protein prevented RA-caudalizing activity from inducing hindbrain marker expression in both explants and embryos. In contrast, inhibition of RA signaling differentially modulated XMeis3 activity. Target genes that are jointly activated by either RA or XMeis3 activities could not be efficiently induced by XMeis3 when RA signaling was inhibited. However, transcription of an XMeis3 target gene that is not an RA target gene was hyper-induced in the absence of retinoid signaling. Target genes jointly induced by RA or XMeis3 protein were synergistically activated in the presence of both activities, while RA treatment inhibits the ability of XMeis3 to activate transcription of neural genes that are not RA targets. HoxD1, an RA direct-target gene was also identified as an XMeis3 direct-target gene. HoxD1 protein acts downstream of XMeis3 to induce hindbrain marker gene transcription. To pattern the hindbrain, RA requires functional XMeis3 protein activity. XMeis3 protein appears crucial for initial hindbrain induction, whereas RA signaling defines the spatial limits of hindbrain gene expression by modifying XMeis3 protein activity.
• 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.
• Identification and Characterisation of the Posteriorly-expressed Xenopus Neurotrophin Receptor Homolog Genes Fullback and Fullback-like Gene Expression Patterns : GEP. Nov, 2004  |   Pubmed ID: 15533829 We have identified fullback and fullback-like, two Xenopus laevis neurotrophin receptor homolog (NRH1) genes. The sequences of Fullback and Fullback-like are very similar to that of the neurotrophin receptor p75NTR, in both their extracellular and their intracellular domains. As their names imply, fullback and fullback-like are expressed in essentially identical patterns in the posterior of the embryo from the early gastrula stage onward. At tailbud and tadpole stages transcripts are also present in dorsal somites and the head, in addition to the growing tailbud. This expression pattern differs from that of p75NTR, suggesting that fullback and fullback-like have different functions from p75NTR during early development.
• Combined Haploid and Insertional Mutation Screen in the Zebrafish Genesis (New York, N.Y. : 2000). Dec, 2004  |   Pubmed ID: 15593329 To identify genes required for development of the brain and somites, we performed a pilot screen of gynogenetic haploid zebrafish embryos produced from mothers mutagenized by viral insertion. We describe an efficient method to identify new mutations and the affected gene. In addition, we report the results of a small-scale screen that identified five genes required for brain development, including novel alleles of nagie oko, pou5f1, ribosomal protein L36, and n-cadherin, as well as a novel allele of the laminin g1 gene that is required for normal skeletal muscle fiber organization and somite patterning.
• 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.
• 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.
• Identification of a BMP Inhibitor-responsive Promoter Module Required for Expression of the Early Neural Gene Zic1 Developmental Biology. Jan, 2006  |   Pubmed ID: 16307736 Expression of the transcription factor zic1 at the onset of gastrulation is one of the earliest molecular indicators of neural fate determination in Xenopus. Inhibition of bone morphogenetic protein (BMP) signaling is critical for activation of zic1 expression and fundamental for establishing neural identity in both vertebrates and invertebrates. The mechanism by which interruption of BMP signaling activates neural-specific gene expression is not understood. Here, we report identification of a 215 bp genomic module that is both necessary and sufficient to activate Xenopus zic1 transcription upon interruption of BMP signaling. Transgenic analyses demonstrate that this BMP inhibitory response module (BIRM) is required for expression in the whole embryo. Multiple consensus binding sites for specific transcription factor families within the BIRM are required for its activity and some of these regions are phylogenetically conserved between orthologous vertebrate zic1 genes. These data suggest that interruption of BMP signaling facilitates neural determination via a complex mechanism, involving multiple regulatory factors that cooperate to control zic1 expression.
• 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.
• Zebrafish Promoter Microarrays Identify Actively Transcribed Embryonic Genes Genome Biology. 2006  |   Pubmed ID: 16889661 We have designed a zebrafish genomic microarray to identify DNA-protein interactions in the proximal promoter regions of over 11,000 zebrafish genes. Using these microarrays, together with chromatin immunoprecipitation with an antibody directed against tri-methylated lysine 4 of Histone H3, we demonstrate the feasibility of this method in zebrafish. This approach will allow investigators to determine the genomic binding locations of DNA interacting proteins during development and expedite the assembly of the genetic networks that regulate embryogenesis.
• 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.
• Disease Connections: a New Peer-reviewed Dialog Developmental Dynamics : an Official Publication of the American Association of Anatomists. Oct, 2006  |   Pubmed ID: 16972277
• 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.
• 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.
• Dejellying Xenopus Laevis Embryos CSH Protocols. 2007  |   Pubmed ID: 21357076 INTRODUCTIONThis protocol presents a method for preparing Xenopus embryos for manipulation. Embryos are surrounded by a series of thick, protective jelly membranes. Removal of these membranes is the first step in most micromanipulation procedures. These membranes must be completely removed for embryo dissection. For microinjection, membranes can be completely or partially removed.
• Removing the Vitelline Membrane from Xenopus Laevis Embryos CSH Protocols. 2007  |   Pubmed ID: 21357077 INTRODUCTIONThis protocol presents a method for the removal of the vitelline membrane from Xenopus embryos, which is essential for embryo dissection experiments. Removal of the vitelline membrane is a technique that becomes easier with practice. The easiest embryos on which to learn this technique are those with a large space between the membrane and the embryo, that is, late neurula stages.
• Handling Xenopus Laevis Adults CSH Protocols. 2007  |   Pubmed ID: 21357078 INTRODUCTIONThis protocol describes the proper technique for handling adult Xenopus animals.
• Inducing Ovulation in Xenopus Laevis CSH Protocols. 2007  |   Pubmed ID: 21357079 INTRODUCTIONThis protocol describes a method for inducing ovulation in Xenopus laevis for the purpose of in vitro fertilization. Ovulation is induced by injection of human chorionic gonadotropin (hCG) into the dorsal lymph sac of a female frog. Females can lay many hundreds of eggs. However, because the actual number of eggs laid and the efficiency of fertilization are unpredictable, ovulation should be induced in more than one female, even when only a few eggs are required.
• Isolating Xenopus Laevis Testes CSH Protocols. 2007  |   Pubmed ID: 21357080 INTRODUCTIONThis protocol describes a method of isolating Xenopus laevis testes for use in in vitro fertilization. The testes from one male Xenopus contain sufficient sperm to fertilize several thousand eggs.
• Xenopus Laevis Egg Collection CSH Protocols. 2007  |   Pubmed ID: 21357081 INTRODUCTIONThis protocol describes procedures for collecting Xenopus laevis eggs from females in which ovulation has been induced.
• Xenopus Laevis In Vitro Fertilization and Natural Mating Methods CSH Protocols. 2007  |   Pubmed ID: 21357082 INTRODUCTIONThis protocol describes in vitro fertilization and natural mating methods for Xenopus laevis.
• Embryo Dissection and Micromanipulation Tools CSH Protocols. 2007  |   Pubmed ID: 21357088 INTRODUCTIONThis article describes the creation and maintenance of tools for use in dissection and micromanipulation of embryos. All tools must be kept clean and rinsed with 70% ethanol to keep them sterile.
• Housing and Feeding of Xenopus Laevis CSH Protocols. 2007  |   Pubmed ID: 21357089 INTRODUCTIONGood animal husbandry is vital for maintaining a healthy frog population. This requires some effort but is generally rewarded by high-quality egg and embryo production. A healthy frog is placid, with moderately slimy skin and a nice pear shape. Jumpy frogs, frogs with dry or excessively slimy skin, bloated frogs, and frogs that look gray and thin or reddish are not healthy and should not be used for egg collection, as this would lead to further deterioration of the animals' condition, and the resulting eggs would be generally unsuitable for experimental purposes. This article describes proper husbandry techniques for Xenopus laevis in the laboratory.
• Animal Cap Isolation from Xenopus Laevis CSH Protocols. 2007  |   Pubmed ID: 21357092 INTRODUCTIONThis protocol presents a method for isolating Xenopus laevis animal cap cells, which are cells situated around the animal (pigmented) pole of a blastula or very early gastrula-stage embryo. This tissue is fated to become cement gland/neurectoderm on the dorsal side of the embryo and epidermis on the ventral side. Animal caps are composed of pluripotent cells that can be induced to form endodermal, mesodermal, or ectodermal cell types, and can therefore serve as a useful substrate to assess the activity of various inducing factors. Caps from embryos that have been injected with expression constructs can also be removed and analyzed to assess the activity of various genes. In addition, caps can be used in conjugation (induction) assays.
• Dissociation and Reaggregation of Xenopus Laevis Animal Caps CSH Protocols. 2007  |   Pubmed ID: 21357093 INTRODUCTIONPreparations of single cells from Xenopus laevis animal caps are useful for assaying the activity of inducing molecules. These dissociated cells can be exposed to more uniform concentrations of inducing factors than the multilayered intact cap. Single cells derived from caps are also used to analyze the role of cell-to-cell contact. This protocol describes the preparation of cells from Xenopus for use in such assays. Animal caps require divalent cations for their integrity and thus can be dissociated by exposure to medium lacking Ca(++) and Mg(++) ions. With this treatment, the cells comprising the inner layers of the cap dissociate within ~20 minutes. Cells can be reaggregated after this treatment and should survive well. The outer layer of cells is more recalcitrant to dissociation and requires a more severe treatment.
• Ectodermal (Animal Cap) Layer Separations in Xenopus Laevis CSH Protocols. 2007  |   Pubmed ID: 21357094 INTRODUCTIONIn Xenopus laevis, the blastula animal cap comprises two morphologically distinct cell layers, an outer monolayer termed the epithelial layer, which consists of tightly adherent pigmented cells, and an inner layer several cells thick termed the sensorial layer, which consists of loosely adherent cells. It is possible to isolate cell layers and to test their developmental potential and response to induction. This protocol describes how to separate the cell layers most easily, that is, by dissecting an intact Xenopus embryo.
• Xenopus Laevis Animal Cap/Vegetal Endoderm Conjugates CSH Protocols. 2007  |   Pubmed ID: 21357095 INTRODUCTIONIn Xenopus laevis, mesoderm can be induced in animal cap cells by contact with vegetal cells (presumptive endoderm). This juxtaposition of tissues was the first indication that the process of mesoderm induction could be separated from neural induction. This protocol describes how to set up a conjugate using Xenopus animal cap and vegetal tissue. In theory, a single embryo can provide both animal and vegetal cells; however, in practice, this is difficult to achieve.
• Xenopus Laevis Animal Cap/Dorsal Mesoderm Conjugates CSH Protocols. 2007  |   Pubmed ID: 21357096 INTRODUCTIONIn Xenopus laevis, dorsal mesoderm is also called "organizer" tissue or Spemann's organizer. It is fated to form the prechordal plate and notochord, as well as some somitic tissue. This protocol presents a method for the preparation of dorsal mesoderm conjugates, which can be useful in assessing whether test genes or promoters are responsive to dorsal mesodermal signals and, if so, by what region of the mesoderm they are induced. The example presented here is an animal cap/dorsal mesoderm conjugate.
• 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.
• Xenopus Laevis Einstecks CSH Protocols. 2007  |   Pubmed ID: 21357098 INTRODUCTION"Einstecks" refers to a procedure for placing a piece of tissue into the blastocoel of an early gastrula, in order to assess the inductive potential of the introduced tissue. The foreign tissue adheres to surrounding tissue and becomes incorporated into the host embryo. This simple transplant procedure, which is described here, has been used to assess which types of axial tissue can be induced by different regions of the mesendoderm or ectoderm, and how forced expression of genes or treatment with various factors can alter this potential.
• Transplantation of Xenopus Laevis Lens Ectoderm CSH Protocols. 2007  |   Pubmed ID: 21357099 INTRODUCTIONIn Xenopus laevis, transplanting a piece of tissue from one site to another (other than into the blastocoel) is a useful way of examining the sequence of inductions required to produce a particular organ. For example, the young primordium of a particular organ can be transplanted from a young individual into an older embryo to determine whether the older embryo still has the capacity to induce the tissue to form the organ. Although this capacity can also be assayed using explants, the complex surroundings of the host embryo cannot be reproduced in an explant. This protocol describes how to transplant a piece of gastrula-stage animal cap ectoderm into the presumptive lens of a neural-plate-stage host embryo. Note that the technique can be adapted for use in other regions of the embryo. Care must be taken to inflict as little damage on the embryo as possible during this procedure. Well-treated embryos recover rapidly, whereas embryos with extensive injuries do not.
• Dissection of Tightly Adhering Xenopus Laevis Tissues by Trypsin Treatment CSH Protocols. 2007  |   Pubmed ID: 21357100 INTRODUCTIONIn older Xenopus laevis embryos (late gastrula and beyond), tissues begin to stick to one another and cannot be peeled apart. For assays requiring isolated tissues, it is necessary to separate such tissues enzymatically, which can be readily accomplished by mild trypsin treatment. Embryos are treated singly, or in small numbers, to avoid possible toxic effects of excessive trypsin digestion. This protocol describes a method for the separation of neural tissue from a neural-plate-stage embryo, although the technique can be adapted to many different tissue types. Enzymatically dissected tissue can be used in quantitative gene expression assays or in specification or induction assays.
• Cortical Isolation from Xenopus Laevis Oocytes and Eggs CSH Protocols. 2007  |   Pubmed ID: 21357101 INTRODUCTIONIn Xenopus laevis, the cortex is the layer of gelatinous cytoplasm that lies just below the plasma membrane of the egg. Rotation of the cortex relative to the deeper cytoplasm soon after fertilization is intimately linked to normal dorsal axis specification. The cortex can be dissected from the egg to analyze its composition and activity or to clone associated RNAs. This protocol describes a procedure for isolating the vegetal cortex of the fertilized egg.
• Microdissection: Explant and Transplant Assays in Xenopus Laevis CSH Protocols. 2007  |   Pubmed ID: 21357111 INTRODUCTIONWhen analyzing any developmental process, important questions are those relating to the formation of different cell types. What makes a cell decide to become a particular cell type? What cell interactions are involved in this decision? The "commitment" of cells to a particular lineage is also called "specification" or "determination." Analysis of the cell interactions, or inductions, required for tissue-type determination is a critical step in the identification of the genes that control the process. Lineage commitment and inductive interactions can be assessed by explant or transplant assays with embryos from Xenopus laevis.
• 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.
• Synthesis and Purification of Digoxigenin-labeled RNA Probes for in Situ Hybridization CSH Protocols. 2007  |   Pubmed ID: 21357143 INTRODUCTIONIn situ hybridization is the most versatile method for determining when and where embryonic transcripts are expressed. Although a detailed analysis of gene expression often requires analysis of sectioned material, the whole-mount approach is invariably the first method used to localize gene expression. Whole-mount in situ hybridization has also become an invaluable tool for analyzing experimentally manipulated embryos and explants. The information gained using this technique is similar to that obtained from immunohistochemistry and includes not only the location of expression but a semiquantitative estimate of the relative levels of gene expression in different parts of the embryo. Because most genes are first analyzed as genomic or cDNA clones, it is straightforward to derive specific probes from these sequences. In this protocol, standard RNA synthesis using a bacteriophage polymerase is carried out incorporating a digoxigenin-substituted ribonucleotide, dig-UTP. In vitro transcription in the direction opposite to in vivo transcription of the mRNA makes an antisense RNA that is complementary to the mRNA. The ratio of dig-UTP to UTP has been optimized to give efficient synthesis and efficient detection of the probe. The three commercially available bacteriophage polymerases (SP6, T7, and T3) all incorporate the substituted nucleotide with equivalent efficiencies, and thus there is a wide choice of plasmid vectors to prepare the template.
• 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.
• 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.
• 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.
• 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.
• Totally Tubular: the Mystery Behind Function and Origin of the Brain Ventricular System BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology. Apr, 2009  |   Pubmed ID: 19274662 A unique feature of the vertebrate brain is the ventricular system, a series of connected cavities which are filled with cerebrospinal fluid (CSF) and surrounded by neuroepithelium. While CSF is critical for both adult brain function and embryonic brain development, neither development nor function of the brain ventricular system is fully understood. In this review, we discuss the mystery of why vertebrate brains have ventricles, and whence they originate. The brain ventricular system develops from the lumen of the neural tube, as the neuroepithelium undergoes morphogenesis. The molecular mechanisms underlying this ontogeny are described. We discuss possible functions of both adult and embryonic brain ventricles, as well as major brain defects that are associated with CSF and brain ventricular abnormalities. We conclude that vertebrates have taken advantage of their neural tube to form the essential brain ventricular system.
• Understanding the Role of DISC1 in Psychiatric Disease and During Normal Development The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Oct, 2009  |   Pubmed ID: 19828788 The biology of schizophrenia is complex with multiple hypotheses (dopamine, glutamate, neurodevelopmental) well supported to underlie the disease. Pathways centered on the risk factor "disrupted in schizophrenia 1" (DISC1) may be able to explain and unite these disparate hypotheses and will be the topic of this mini-symposium preview. Nearly a decade after its original identification at the center of a translocation breakpoint in a large Scottish family that was associated with major psychiatric disease, we are starting to obtain credible insights into its function and role in disease etiology. This preview will highlight a number of exciting areas of current DISC1 research that are revealing roles for DISC1 during normal brain development and also in the disease state. Together these different threads will provide a timely and exciting overview of the DISC1 field and its potential in furthering our understanding of psychiatric diseases and in developing new therapies.
• 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.
• Microinjection of Xenopus Embryos Cold Spring Harbor Protocols. Dec, 2010  |   Pubmed ID: 21123415 This article describes the factors that should be considered when microinjecting Xenopus oocytes. Forward planning is particularly important for embryo injection because the frogs lay eggs only for a limited time, and once the eggs are fertilized there is a very short period during which the embryos are suitable for injection.
• Isolation of Xenopus Oocytes Cold Spring Harbor Protocols. Dec, 2010  |   Pubmed ID: 21123421 Xenopus oocytes are obtained from sexually mature females by surgically removing parts of the ovary. The operation is not fatal and can be performed on an anesthetized frog several times during its lifetime. However, a recovery period of 2 wk is recommended between operations. A careful record of all operations performed, including details of oocyte quality, should be kept. A frog that produces one good batch of oocytes; e.g., those that translate injected messenger RNAs (mRNAs) efficiently, should be recorded and used again, because oocyte quality is generally frog-dependent.
• Defolliculation of Xenopus Oocytes Cold Spring Harbor Protocols. Dec, 2010  |   Pubmed ID: 21123422 It is possible to microinject Xenopus oocytes that are still contained within their ovarian follicles, but most researchers find it more convenient to work with defolliculated oocytes. Defolliculation can be carried out enzymatically by treatment with collagenase, or it can be performed manually. Enzymatic treatment is recommended for preparation of large numbers of oocytes (more than 1000); however, this treatment causes complications because it often damages the quality of the oocytes. The manual procedure, which requires some practice, is recommended for experiments requiring only a few hundred oocytes. This protocol details both the enzymatic and manual procedures for defolliculation of Xenopus oocytes.
• Microinjection of Xenopus Oocytes Cold Spring Harbor Protocols. Dec, 2010  |   Pubmed ID: 21123423 Xenopus oocytes can be injected with messenger RNA (mRNA) or DNA constructs into the cytoplasm or nucleus, respectively. The cytoplasm can withstand the introduction of up to 50 nL of injected material, and the nucleus can tolerate up to 20 nL. This protocol describes microinjection of both defolliculated and folliculated Xenopus oocytes. Oocytes remain in good condition for a relatively long time, and injections can be continued, if necessary, over a period of days.
• Calibration of the Injection Volume for Microinjection of Xenopus Oocytes and Embryos Cold Spring Harbor Protocols. Dec, 2010  |   Pubmed ID: 21123424 Microinjection of Xenopus oocytes or embryos with messenger RNA (mRNA) or DNA is a powerful technique for studying development. Before microinjection can be performed, the injection volume must be calibrated carefully. This protocol describes the calibration procedure for a pressure injector.
• Microinjection of RNA and Preparation of Secreted Proteins from Xenopus Oocytes Cold Spring Harbor Protocols. Dec, 2010  |   Pubmed ID: 21123425 Microinjection of Xenopus oocytes or embryos with messenger RNA (mRNA) is a powerful technique for studying development, including protein expression during development. This protocol describes a method for the preparation of secreted (35)S-labeled proteins following mRNA microinjection into Xenopus oocytes.
• 'Model' or 'tool'? New Definitions for Translational Research Disease Models & Mechanisms. Mar, 2011  |   Pubmed ID: 21357758 The term 'model' often describes non-human biological systems that are used to obtain a better understanding of human disorders. According to the most stringent definition, an animal 'model' would display exactly the same phenotype as seen in the relevant human disorder; however, this precise correspondence is often not present. In this Editorial, I propose the alternative, broader term 'tool' to describe a biological system that does not obviously (or precisely) recapitulate a human disorder, but that nonetheless provides useful insight into the etiology or treatment of that disorder. Applying the term 'tool' to biological systems used in disease-related studies will help to identify those systems that can most effectively address mechanisms underlying human disease. Conversely, differentiating 'models' from 'tools' will help to define more clearly the limitations of biological systems used in preclinical analyses.
• 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.
• 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.
• 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.
• Conserved Function of LincRNAs in Vertebrate Embryonic Development Despite Rapid Sequence Evolution Cell. Dec, 2011  |   Pubmed ID: 22196729 Thousands of long intervening noncoding RNAs (lincRNAs) have been identified in mammals. To better understand the evolution and functions of these enigmatic RNAs, we used chromatin marks, poly(A)-site mapping and RNA-Seq data to identify more than 550 distinct lincRNAs in zebrafish. Although these shared many characteristics with mammalian lincRNAs, only 29 had detectable sequence similarity with putative mammalian orthologs, typically restricted to a single short region of high conservation. Other lincRNAs had conserved genomic locations without detectable sequence conservation. Antisense reagents targeting conserved regions of two zebrafish lincRNAs caused developmental defects. Reagents targeting splice sites caused the same defects and were rescued by adding either the mature lincRNA or its human or mouse ortholog. Our study provides a roadmap for identification and analysis of lincRNAs in model organisms and shows that lincRNAs play crucial biological roles during embryonic development with functionality conserved despite limited sequence conservation.
• Isolation of DNA from Red Blood Cells in Xenopus Cold Spring Harbor Protocols. Jan, 2012  |   Pubmed ID: 22194257 The amphibian Xenopus laevis is an important model organism that is particularly valuable for studies of early vertebrate development. Genomic DNA constitutes the total genetic information of an organism and it is used for Southern blotting, for determining gene structure, and for detecting the presence or absence of genes of interest. Genomic DNA can be extracted from Xenopus red blood cells, which are unlike the mammalian equivalent in that they contain nuclei. This article describes a protocol for the isolation of genomic DNA from frog red blood cells.
• 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.
• 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.
• 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.
• 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.
• Education: Online On-ramps Nature. Jul, 2013  |   Pubmed ID: 23868246
• 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.