RESUMO
Cardiac valve formation is a complex process that involves cell signaling events between the myocardial and endocardial layers of the heart across an elaborate extracellular matrix. These signals lead to marked morphogenetic movements and transdifferentiation of the endocardial cells at chamber boundaries. Here we identify the genetic defect in zebrafish jekyll mutants, which are deficient in the initiation of heart valve formation. The jekyll mutation disrupts a homolog of Drosophila Sugarless, a uridine 5'-diphosphate (UDP)-glucose dehydrogenase required for heparan sulfate, chondroitin sulfate, and hyaluronic acid production. The atrioventricular border cells do not differentiate from their neighbors in jekyll mutants, suggesting that Jekyll is required in a cell signaling event that establishes a boundary between the atrium and ventricle.
Assuntos
Valvas Cardíacas/embriologia , Coração/embriologia , Uridina Difosfato Glucose Desidrogenase/genética , Uridina Difosfato Glucose Desidrogenase/metabolismo , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Elementos Antissenso (Genética) , Padronização Corporal , Proteínas Morfogenéticas Ósseas/genética , Endocárdio/embriologia , Endocárdio/metabolismo , Feminino , Expressão Gênica , Glicosaminoglicanos/metabolismo , Valvas Cardíacas/citologia , Valvas Cardíacas/enzimologia , Valvas Cardíacas/metabolismo , Masculino , Dados de Sequência Molecular , Morfogênese , Mutação , Miocárdio/citologia , Miocárdio/metabolismo , Fenótipo , Mapeamento Físico do Cromossomo , Transdução de Sinais , Peixe-Zebra/genéticaRESUMO
BACKGROUND: Several potentially important regulators of vertebrate endoderm development have been identified, including Activin-related growth factors and their receptors; transcriptional regulators encoded by the genes Mixer, Xsox17, and HNF3beta; zebrafish One-eyed pinhead (Oep), a member of the Cripto/FRL-1/Cryptic family of epidermal growth factor related proteins (EGF-CFC); and the product of the zebrafish locus casanova, which plays an essential cell-autonomous role in endoderm formation. RESULTS: Using overexpression studies and the analysis of different zebrafish mutants, we have assembled a molecular pathway that leads to endoderm formation. We report that a zebrafish Sox17 homologue is expressed during gastrulation exclusively in the endoderm and that casanova mutants lack all sox17 expression. Overexpression of mixer induces ectopic sox17-expressing cells in wild-type embryos and promotes endoderm formation in oep mutants, but does not rescue sox17 expression or endoderm formation in casanova mutants. Overexpression of a constitutively active form of the type I transforming growth factor beta (TGF-beta) receptor TARAM-A also promotes sox17 expression in wild-type and oep mutant embryos, but not in casanova mutants. We also show that the Nodal-related molecules Cyclops and Squint and the transmembrane protein Oep are essential for normal mixer expression. CONCLUSIONS: The data indicate that the following pathway leads to zebrafish endoderm formation: Cyclops and Squint activate receptors such as TARAM-A; Oep also appears to act upstream of such receptors; signals transduced by these receptors lead to the expression of mixer, Mixer then acts through casanova to promote the expression of sox17 and differentiation of the endoderm.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Grupo de Alta Mobilidade , Proteínas Serina-Treonina Quinases , Receptores de Fatores de Crescimento Transformadores beta , Fatores de Transcrição , Proteínas de Peixe-Zebra , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Endoderma/citologia , Endoderma/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Dados de Sequência Molecular , Mutação , Ligantes da Sinalização Nodal , Proteínas/genética , Receptores de Fatores de Crescimento/genética , Fatores de Transcrição SOXF , Transdução de Sinais , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Peixe-Zebra/metabolismoRESUMO
Recent studies in mouse suggest that the extraembryonic endoderm has an important role in early embryonic patterning [1]. To analyze whether similar mechanisms operate in other vertebrates, we cloned the zebrafish homologue of Hex, a homeobox gene that is expressed asymmetrically in the mouse visceral endoderm [2]. Early expression of zebrafish hex is restricted to the dorsal portion of the yolk syncytial layer (YSL), an extraembryonic tissue. By the onset of gastrulation, hex is expressed in the entire dorsal half of the YSL, which directly underlies the cells fated to form the neural plate. We show that hex expression is initially regulated by the maternal Wnt pathway and later by a Bmp-mediated pathway. Overexpression experiments of wild-type and chimeric Hex constructs indicate that Hex functions as a transcriptional repressor and its overexpression led to the downregulation of bmp2b and wnt8 expression and the expansion of chordin expression. These findings provide further evidence that the zebrafish YSL is the functional equivalent of the mouse visceral endoderm and that extraembryonic structures may regulate early embryonic patterning in many vertebrates.
Assuntos
Embrião não Mamífero/embriologia , Indução Embrionária , Endoderma/fisiologia , Proteínas de Homeodomínio/metabolismo , Saco Vitelino/fisiologia , Proteínas de Peixe-Zebra , Peixe-Zebra/embriologia , Animais , Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Camundongos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras , Fatores de Tempo , Fatores de Transcrição , Proteínas Wnt , Peixe-Zebra/genéticaRESUMO
BACKGROUND: The Par-3/Par-6/aPKC complex is a key regulator of cell polarity in a number of systems. In Drosophila, this complex acts at the zonula adherens (adherens junctions) to establish epithelial polarity and helps to orient the mitotic spindle during asymmetric neuroblast divisions. In MDCKII cells, this complex localizes to the zonula occludens (tight junctions) and appears to regulate epithelial polarity. However, the in vivo role of this complex during vertebrate embryogenesis is not known, due to the lack of relevant mutations. RESULTS: We have positionally cloned the zebrafish heart and soul (has) mutation, which affects the morphogenesis of several embryonic tissues, and show that it encodes atypical protein kinase C lambda (aPKC lambda). We find that loss of aPKC lambda affects the formation and maintenance of the zonula adherens in the polarized epithelia of the retina, neural tube, and digestive tract, leading to novel phenotypes, such as the formation of multiple lumens in the developing intestine. In addition, has mutants display defects in gut looping and endodermal organ morphogenesis that appear to be independent of the defects in epithelial polarity. Finally, we show that loss of aPKC lambda leads to defects in spindle orientation during progenitor cell divisions in the neural retina. CONCLUSIONS: Our results show that aPKC lambda is required for the formation and maintenance of the zonula adherens during early epithelial development in vertebrates and demonstrate a previously undescribed yet critical role for this protein in organ morphogenesis. Furthermore, our studies identify the first genetic locus regulating the orientation of cell division in vertebrates.
Assuntos
Junções Aderentes/fisiologia , Sistema Digestório/embriologia , Proteína Quinase C/fisiologia , Retina/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Divisão Celular , Clonagem Molecular , DNA Complementar , Sistema Digestório/metabolismo , Feminino , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Masculino , Camundongos , Dados de Sequência Molecular , Morfogênese , Mutagênese , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Retina/fisiologia , Peixe-Zebra/embriologiaRESUMO
The endoderm is the innermost embryonic germ layer, and in zebrafish, it gives rise to the lining of the gut, the gills, liver, pancreas, gallbladder, and derivatives of the pharyngeal pouch. These organs form the gastrointestinal tract and are involved with the absorption, delivery, and metabolism of nutrients. The liver has a central role in regulating these processes because it controls carbohydrate and lipid metabolism, protein synthesis, and breakdown of endogenous and xenobiotic products. Liver dysfunction frequently leads to significant morbidity and mortality; however, in most settings of organ injury, the liver exhibits remarkable regenerative capacity. In this chapter, we review the principal mechanisms of endoderm and liver formation and provide protocols to assess liver formation and liver regeneration.
Assuntos
Padronização Corporal/genética , Endoderma/crescimento & desenvolvimento , Fígado/crescimento & desenvolvimento , Peixe-Zebra/crescimento & desenvolvimento , Animais , Sistema Digestório/crescimento & desenvolvimento , Morfogênese/genética , Pâncreas/crescimento & desenvolvimento , Peixe-Zebra/genéticaRESUMO
The zebrafish is an important experimental system for vertebrate embryology, and is well suited to the molecular analysis of muscle development. Transcription factors, such as the MEF2s, regulate skeletal and cardiac muscle-specific genes during development. We report the identification of three zebrafish MEF2 genes which, like their mammalian counterparts, encode factors that function as DNA-binding transcriptional activators of muscle specific promoters. The pattern of MEF2 expression in zebrafish defines discrete cell populations in the developing somites and heart and has mechanistic implications for developmental regulation of the MEF2 genes, when compared with other species. Alteration of MEF2 expression in two mutants affecting somitogenesis provides insight into the control of muscle formation in the embryo.
Assuntos
Proteínas de Ligação a DNA/genética , Coração/embriologia , Somitos/química , Fatores de Transcrição/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , DNA/metabolismo , Humanos , Fatores de Transcrição MEF2 , Camundongos , Dados de Sequência Molecular , Mutagênese , Fatores de Regulação Miogênica , Fatores de Transcrição/metabolismoRESUMO
The zebrafish, Brachydanio rerio, is rapidly becoming a system of choice for vertebrate developmental biologists. It presents unique embryological attributes and is amenable to saturation style mutagenesis, a powerful approach that, in invertebrates, has already led to the identification of a large number of key developmental genes. Since fertilization is external, the zebrafish embryo develops in the dish and is thus accessible for continued observation and manipulation at all stages of development. Furthermore, because the embryo is transparent, the developing heart and vessels can be resolved at the single-cell level. A large number of mutations that affect the development of cardiovascular form and function have recently been isolated from large-scale genetic screens for zygotic embryonic lethals. Our further understanding of the development of the cardiovascular system is important not only because of the high incidence, and familial inheritance, of congenital abnormalities, but also because it should lead to novel, differentiation-based strategies for the analysis and therapy of the diseased state.
RESUMO
The Huntington's disease (HD) gene contains a trinucleotide repeat that is expanded and unstable in patients with the disease (HDCRG, 1993). As the first step toward investigating a potential role for this gene in early vertebrate development, we isolated the homolog of the Huntington's disease (ZHD) cDNA in zebrafish. This cDNA encodes a predicted protein product of 3121 amino acids with 70% identity to human huntingtin. The first exon is predicted to encode four glutamines, followed by only one proline, demonstrating that the polymorphic polyproline stretch found in mammalian HD sequences is absent in the fish. We sequenced approximately 900bp upstream from the predicted start codon and found that it lacks a TATA box, CCAAT box, or Sp1 binding sites. Western blot analysis revealed that the protein is expressed at a high level in late embryonic development and at moderate levels in the adult head.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Doença de Huntington/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Repetições de Trinucleotídeos , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , DNA Complementar , Embrião não Mamífero/fisiologia , Peixes , Biblioteca Gênica , Humanos , Proteína Huntingtina , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/química , Proteínas Nucleares/biossíntese , Proteínas Nucleares/química , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Peixe-Zebra/embriologiaRESUMO
We have isolated and characterized four monoclonal antibodies (mAbs B33, E1.9, B30, and B10) that recognize mouse trigeminal sensory neurons at specific times during development. These antibodies permit the study of neuronal differentiation, axon outgrowth, and neuronal maturation in the trigeminal sensory system. With B33, we can follow migrating neural crest and placode cells into the anlagen of the trigeminal ganglion. E1.9 immunoreactivity marks neuronal differentiation and appears in the central nervous system at embryonic day 8.5 (E8.5) and in the peripheral nervous system at E9, E1.9 and B30 show the axonal outgrowth of trigeminal sensory neurons and reveal the pioneering of the peripheral tracts by an early population of ganglionic neurons. At this stage, in the central nervous system, mesencephalic trigeminal neurons are also E1.9 and B30 positive as they migrate to their final location in the rostral metencephalon. B30 and B10 allow us to follow the maturation of these neurons. Also, in about 1% of the embryos, we identified mispositioned or misrouted trigeminal neurons. Furthermore, these biochemical markers facilitate the study of neuronal development in vitro. We find that, based on morphological and biochemical criteria, the maturation of trigeminal neurons in culture is target independent.
Assuntos
Neurônios Aferentes/fisiologia , Nervo Trigêmeo/citologia , Animais , Anticorpos Monoclonais , Axônios/fisiologia , Diferenciação Celular , Células Cultivadas , Feminino , Gangliosídeos/análise , Imuno-Histoquímica , Mesencéfalo/citologia , Mesencéfalo/crescimento & desenvolvimento , Camundongos , Gravidez , Gânglio Trigeminal/citologia , Gânglio Trigeminal/crescimento & desenvolvimento , Nervo Trigêmeo/crescimento & desenvolvimento , Núcleos do Trigêmeo/citologia , Núcleos do Trigêmeo/crescimento & desenvolvimentoRESUMO
Forward-genetic analyses in Drosophila and Caenorhabditis elegans have given us unprecedented insights into many developmental mechanisms. To study the formation of organs that contain cell types and structures not present in invertebrates, a vertebrate model system amenable to forward genetics would be very useful. Recent work shows that a newly initiated genetic approach in zebrafish is already making significant contributions to understanding the development of the vertebrate heart, an organ that contains several vertebrate-specific features. These and other studies point to the utility of the zebrafish system for studying a wide range of vertebrate-specific processes.
Assuntos
Coração/embriologia , Vertebrados/embriologia , Vertebrados/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Caenorhabditis/genética , Diferenciação Celular , Drosophila/genética , Morfogênese , Mutação , Miocárdio/citologiaRESUMO
Pioneer neurons establish preliminary nerve pathways that are followed by later-growing axons. The existence of pioneers and their importance is well documented in invertebrate systems. In mammals, early neuronal development has generally been difficult to study because of the size and complexity of the embryos, and the lack of adequate markers. Here we look at the time of earliest axonal outgrowth in the mouse embryo by using specific monoclonal antibodies to stain wholemount preparations. During the period of formation and closure of the neuropore beginning at embryonic day 8.5, we can follow the earliest trigeminal sensory neurons extending axons along stereotyped pathways. In the trigeminal ganglion, an early wave of neurogenesis gives rise to a small number of neurons whose axons pioneer the different trigeminal tracts in the periphery. After a brief pause (12 hr), these primary axons branch out to innervate individual targets. Emerging a day later, secondary fibers extend along the pioneers. By contrast, in the central nervous system, neurons of the mesencephalic trigeminal nucleus extend toward the rhombencephalon independently, ignoring preexisting fibers. These results show the existence of an early set of axonal tracts in the mouse peripheral nervous system that may be used for the guidance of later-differentiating neurons.
Assuntos
Neurônios Aferentes/fisiologia , Gânglio Trigeminal/embriologia , Núcleos do Trigêmeo/embriologia , Animais , Anticorpos Monoclonais , Axônios/fisiologia , Axônios/ultraestrutura , Células Cultivadas , Embrião de Mamíferos , Feminino , Camundongos , Neurônios Aferentes/citologia , Gravidez , Gânglio Trigeminal/citologia , Núcleos do Trigêmeo/citologiaRESUMO
The patterning of an internal organ, like the heart, is little understood. Central to this patterning is the formation, or the acquisition, of an anteroposterior (A-P) axis. We have approached the question of how the heart tube acquires polarity in the zebrafish, Brachydanio rerio, which offers numerous advantages for studying cardiac morphogenesis. During the early stages of organogenesis in the fish, the heart tube lies in an A-P orientation with the venous end lying anteriorly and the arterial end lying posteriorly. High doses (10(-6)-10(-5)M) of retinoic acid (RA) cause truncation of the body axis, as they do in Xenopus. Low doses of retinoic acid (10(-8)-10(-7) M), which do not appear to affect the rest of the embryo, have pronounced effects upon heart tube morphogenesis, causing it to shrink progressively along the A-P axis. To investigate this further, we identified monoclonal antibodies that distinguish between the zebrafish cardiac chambers and used them to show that the RA-induced cardiac truncation always begins at the arterial end of the heart tube. There is a continuous gradient of sensitivity from the arterial to the venous end, such that increasing RA exposure causes the progressive and sequential deletion first of the bulbus arteriosus and then, in order, of the ventricle, the atrium, and the sinus venosus. As exposure increases, parts of chambers are deleted before entire chambers; thus, the sensitivity to RA appears to be independent of chamber boundaries. The analysis of the heart tube's sensitivity to RA and its timing suggest that polarity is established during or shortly after initial commitment to the cardiac lineage.
Assuntos
Coração/embriologia , Peixe-Zebra/embriologia , Animais , Anticorpos Monoclonais , Relação Dose-Resposta a Droga , Gástrula/efeitos dos fármacos , Coração/efeitos dos fármacos , Imuno-Histoquímica , Morfogênese , Tretinoína/farmacologiaRESUMO
A monoclonal antibody, B30, obtained with whole cells from embryonic brain as an immunogen, recognizes a neuronal cell surface antigen that appears only in 2 distinct systems in the developing mouse brain: the trigeminal system and the cerebellum. In the trigeminal system, B30 labels the surface of neurons, including their axons and their transient dendrites, in 2 groups of cells: the centrally located mesencephalic trigeminal nucleus and the peripheral trigeminal ganglion. Immunoreactivity is detectable during axon outgrowth, peaks around the seventh postnatal day, and disappears around 2 weeks after birth. In the cerebellum, B30 labels 2 layers of cells during development. Perinatally, and for about a week after birth, the layer of premigratory granule cells stains. After their maturation, Purkinje cells start to stain and by 12 d postnatally all the Purkinje cell bodies, their axons, and their dendritic trees show strong immunoreactivity. Subsequently, and in the adult, this staining is lost from some cells to reveal bands of antigen positive and negative Purkinje cells. Initial biochemical characterization of the epitope shows that it is carried on 2 minor gangliosides.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Superfície/imunologia , Neurônios/imunologia , Núcleos do Trigêmeo/imunologia , Animais , Animais Recém-Nascidos , Cerebelo/embriologia , Cerebelo/imunologia , Feto/imunologia , Imuno-Histoquímica , Mesencéfalo , Camundongos , Células de Purkinje/imunologia , Células de Purkinje/ultraestrutura , Ratos , Coloração e Rotulagem , Gânglio Trigeminal/citologia , Gânglio Trigeminal/embriologia , Gânglio Trigeminal/imunologia , Núcleos do Trigêmeo/citologia , Núcleos do Trigêmeo/embriologiaRESUMO
Genetics is a powerful tool, especially when used in combination with embryology, in the seeking of genes necessary for assembly of the cardiovasculature. The first questions must address the types of cellular decisions that are made during development. As for simpler systems in C elegans and D melanogaster, the lineage and cell-fate decisions of the cardiovascular progenitors need to be assessed. In addition it is likely that new paradigms will emerge for multicellular assembly. The study of cardiovascular mutations will define individual genetic steps that define organotypic decisions. A genetic approach is a natural extension of embryology, physiology, and anatomy, fields of great sophistication with regard to the cardiovasculature, because, like them, it focuses on integrative biology and on the intact organism. The zebrafish is particularly well suited to a combination genetic-embryologic study of the fashioning of the cardiovasculature.
Assuntos
Coração/embriologia , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Sistema Cardiovascular/embriologia , Diferenciação Celular/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Peixes/embriologia , Peixes/genética , Humanos , Morfogênese , Mutação/genética , Xenopus/embriologia , Xenopus/genéticaRESUMO
Vertebrate embryonic hematopoiesis is a complex process that involves a number of cellular interactions, notably those occurring between endothelial and blood cells. The zebrafish cloche mutation affects both the hematopoietic and endothelial lineages from an early stage (Stainier, D. Y. R., Weinstein, B. M., Detrich, H. W. R., Zon, L. I. and Fishman, M. C. (1995) Development 121, 3141-3150). cloche mutants lack endocardium, as well as head and trunk endothelium, and nearly all blood cells. Cell transplantation studies have revealed that the endocardial defect in cloche is cell-autonomous: wild-type cells can form endocardium in mutant hosts, but mutant cells never contribute to the endocardium in wild-type or mutant hosts. In this paper, we analyze the cell-autonomy of the blood defect in cloche. The blood cell deficiency in cloche mutants could be an indirect effect of the endothelial defects. Alternatively, cloche could be required cell-autonomously in the blood cells themselves. To distinguish between these possibilities, we cotransplanted wild-type and mutant cells into a single wild-type host in order to compare their respective hematopoietic capacity. We found that transplanted wild-type cells were much more likely than mutant cells to contribute to circulating blood in a wild-type host. Furthermore, in the few cases where both wild-type and mutant donors contributed to blood in a wild-type host, the number of blood cells derived from the wild-type donor was always much greater than the number of blood cells derived from the mutant donor. These data indicate that cloche is required cell-autonomously in blood cells for their differentiation and/or proliferation. When we assessed early expression of the erythropoietic gene gata-1 in transplant recipients, we found that mutant blastomeres were as likely as wild-type blastomeres to give rise to gata-1-expressing cells in a wild-type host. Together, these two sets of data argue that cloche is not required cell-autonomously for the differentiation of red blood cells, as assayed by gata-1 expression, but rather for their proliferation and/or survival, as assayed by their contribution to circulating blood. In addition, we found that transplanted wild-type cells were less likely to express gata-1 in a mutant environment than in a wild-type one, suggesting that cloche also acts non-autonomously in red blood cell differentiation. This non-autonomous function of cloche in red blood cell differentiation may reflect its cell-autonomous requirement in the endothelial lineage. Thus, cloche appears to be required in erythropoiesis cell non-autonomously at a step prior to gata-1 expression, and cell-autonomously subsequently.
Assuntos
Hematopoese/genética , Mutação , Peixe-Zebra/sangue , Peixe-Zebra/genética , Animais , Células Sanguíneas/patologia , Diferenciação Celular/genética , Transplante de Células , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião não Mamífero/citologia , Indução Embrionária/genética , Endotélio Vascular/patologia , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Modelos Biológicos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Proteínas de Peixe-ZebraRESUMO
A classical genetic approach, in which mutagenized organisms are screened for phenotypes of interest, is appealing for the analysis of developmental processes. Here, we describe the advantages of zebrafish genetics for the study of heart development. As an example of the utility of this strategy, we discuss its potential to illuminate the molecular mechanisms of cardiac chamber formation. The signals that specify ventricular and atrial lineages and the differentiation pathways that produce distinct chambers are poorly understood. Recently identified zebrafish mutations that disrupt ventricular or atrial development promise to reveal genes essential for these processes.
Assuntos
Padronização Corporal/genética , Átrios do Coração/embriologia , Ventrículos do Coração/embriologia , Animais , Linhagem da Célula/genética , Genes , Testes Genéticos , Modelos Biológicos , Mutação , Transdução de Sinais/genética , Tretinoína/fisiologia , Peixe-Zebra/embriologiaRESUMO
We have analyzed the origin of cardiac progenitors in the zebrafish embryo by injection of single blastomeres with a lineage tracer dye, and examined the formation of the zebrafish heart tube by serial sectioning of immunostained embryos. At the 512-cell stage (early blastula), most cardiac progenitors lie in a marginal zone that extends from 90 degrees longitude (midway between the future dorsal and ventral axis) through 180 degrees longitude (the future ventral axis) to 270 degrees longitude. By focusing on myocardial progenitors located at 90 degrees (and 270 degrees) longitude, we found that a single cell injected in the early blastula can contribute progeny to both the atrium and ventricle. A cell injected in the midblastula contributes progeny to either the atrium or ventricle, but not both. This analysis suggests that, at least for these myocardial progenitors, the atrial and ventricular lineages separate in the midblastula. Precardiac cells involute early during gastrulation and turn towards the animal pole with other early involuting cells. These cardiogenic cells reach the embryonic axis around the 8-somite stage, and there they coalesce to form a pair of myocardial tubular primordia on either side of the midline. By the 21-somite stage, the tropomyosin-immunoreactive myocardial tubes have moved closer to each other, and a distinct group of cells, the endocardial progenitor cells, sits medially between them. The myocardial tubes then fuse to enclose the endocardial cells and form the definitive heart tube. By 22 hours postfertilization (26-somite stage), the heart tube is clearly beating. The regionalization of cardiac myosin heavy chain expression distinguishes the cardiac chambers at this stage, although they are not morphologically delineated until 36 hours. This work shows that cardiogenic regions can be identified in the early blastula, and that chamber restriction seems to arise in the midblastula. Additionally, it provides the basis for embryological perturbation at the single cell level, as well as for the genetic analysis of heart tube formation in the zebrafish.
Assuntos
Blastocisto/citologia , Sistema Cardiovascular/embriologia , Peixe-Zebra/embriologia , Animais , Diferenciação Celular/fisiologia , Gástrula/citologia , Coração/embriologia , Imuno-HistoquímicaRESUMO
Previous studies have indicated that gata5, a zinc-finger transcription factor gene, is required for the development of the zebrafish gut tube. Here, we show that gata5 mutants also display defects in the development of other endodermal organs such as the liver, pancreas, thyroid and thymus. gata5 is expressed in the endodermal progenitors from late blastula stages, suggesting that it functions early during endoderm development. We indeed find that during gastrulation stages, gata5 mutants form fewer endodermal cells than their wild-type siblings. In addition, the endodermal cells that form in gata5 mutants appear to express lower than wild-type levels of endodermal genes such as sox17 and axial/foxA2. Conversely, overexpression of gata5 leads to expanded endodermal gene expression. These data indicate that Gata5 is involved both in the generation of endodermal cells at late blastula stages and in the maintenance of endodermal sox17 expression during gastrulation. We have also analyzed the relationship of Gata5 to other factors involved in endoderm formation. Using complementary mutant and overexpression analyses, we show that Gata5 regulates endoderm formation in cooperation with the Mix-type transcription factor Bon, that Gata5 and Bon function downstream of Nodal signaling, and that cas function is usually required for the activity of Gata5 in endoderm formation. Finally, we show that fau/gata5, bon and cas exhibit dominant genetic interactions providing additional support that they function in the same pathway. Together, these data demonstrate that Gata5 plays multiple roles in endoderm development in zebrafish, and position Gata5 relative to other regulators of endoderm formation.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/fisiologia , Animais , Embrião não Mamífero/fisiologia , Endoderma/fisiologia , Fator de Transcrição GATA5 , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Transdução de Sinais , Proteínas de Peixe-ZebraRESUMO
We have isolated a monoclonal antibody, mAb 52G9, that recognizes a 55-kDa cell surface protein restricted to the early embryonic rat forebrain and to placode-derived structures. In the central nervous system (CNS), 52G9 immunoreactivity appears at Embryonic Day 11 (E11) in the rostral-most area of the telencephalon. It then spreads to the neuroepithelium of the telencephalon and basal diencephalon. Most strikingly, it appears at E14 in a distinct zone at the caudal end of the ventral diencephalic neuroepithelium. This area is sharply defined by strong 52G9 immunoreactivity bounded by unlabeled neuroepithelium. The pattern revealed by 52G9 is the first biochemical demonstration of spatial domains in the forebrain at a time prior to neuronal differentiation. By E18, 52G9 immunoreactivity has progressively disappeared from the forebrain; the glomerular layer of the olfactory bulb is the only 52G9-positive area in the CNS. The olfactory, otic, and hypophyseal placodes, which can be identified as early as E10, are also 52G9 positive as are their derivatives, the sensory epithelial of the nasal passage and inner ear, and also Rathke's pouch. The distribution and regulation of the 52G9 protein suggests that this novel cell surface molecule may be involved in the formation of spatial domains in the developing forebrain.
Assuntos
Encéfalo/embriologia , Proteínas de Membrana/análise , Proteínas do Tecido Nervoso/análise , Animais , Anticorpos Monoclonais , Encéfalo/anatomia & histologia , Encéfalo/citologia , Células Cultivadas , Feminino , Imunofluorescência , Idade Gestacional , Peso Molecular , Especificidade de Órgãos , Gravidez , Ratos , Medula Espinal/citologia , Medula Espinal/embriologiaRESUMO
We have previously reported the isolation of a monoclonal antibody, mAb B30, that recognizes two minor gangliosides specifically expressed in a small subset of neurons in the developing mouse central nervous system (Stainier and Gilbert, 1989). B30 labels mesencephalic trigeminal neurons shortly after differentiation until about 2 weeks after birth. Postnatally, it also labels two specific monolayers of cerebellar neurons. In this study, we have characterized the B30 immunoreactivity in the developing peripheral nervous system of the mouse. We report that B30 is a marker for neural crest-derived neurons and have used it to follow the neuronal differentiation of neural crest cells in a serum-free chemically defined culture system. Within hours after plating, neural crest cells migrate away from the neural tube explant on a fibronectin or laminin substrate and by 24 hr, up to 15% of them have differentiated into morphologically identifable neurons. In vitro as in vivo, undifferentiated mouse neural crest cells express the GD3 ganglioside which is recognized by mAb B33, and neural crest-derived neurons can be labeled by mAbs B33, B30, and also E1.9, a specific neuronal cytoskeletal marker. We also show the unique biochemical specificity of mAb B30 and provide experimental evidence for the role of the B30 ganglioside in the cellular adhesion process.