RESUMO
Generating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.
Assuntos
Padronização Corporal/genética , Corpos Embrioides/metabolismo , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias Murinas/metabolismo , Transdução de Sinais/genética , Animais , Ectoderma/citologia , Ectoderma/crescimento & desenvolvimento , Ectoderma/metabolismo , Embrião de Mamíferos , Corpos Embrioides/citologia , Endoderma/citologia , Endoderma/crescimento & desenvolvimento , Endoderma/metabolismo , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Gástrula/citologia , Gástrula/crescimento & desenvolvimento , Gástrula/metabolismo , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Tubo Neural/citologia , Tubo Neural/crescimento & desenvolvimento , Tubo Neural/metabolismo , Notocorda/citologia , Notocorda/crescimento & desenvolvimento , Notocorda/metabolismo , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismoRESUMO
CaMKII is a Ca2+/CaM-dependent protein kinase encoded by a family of conserved genes found throughout all metazoan species and expressed from fertilization into adulthood. One of these genes, camk2g1, is particularly important during early development as determined by pharmacologic, dominant negative and antisense morpholino approaches in zebrafish. Four other teleost fish species (cavefish, medaka, stickleback, and tilapia), exhibit sequence conservation of camk2g1 and duplication of the same CaMKII genes. A homozygous mutant of camk2g1 was generated in zebrafish using TALEN technology but yielded none of the phenotypic alterations seen using all other approaches and was reproductively viable. However, these camk2g1 mutant embryos showed a 4-fold over-expression of its paralog camk2g2. None of the other camk2 genes showed such transcriptional elevation, in fact, some of these genes were suppressed to 10% of wild type levels. In contrast, G0 camk2g1 CRISPR/Cas9 embryos recapitulated nearly all of the altered phenotypes observed in camk2g1 morphants, including renal, aural and ciliary defects. These findings validate the importance of this gene family during early zebrafish development and provide evidence for gene-specific transcriptional cross-talk consistent with genetic compensation.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Evolução Biológica , Sistemas CRISPR-Cas/genética , Embrião não Mamífero , Mutação com Perda de Função , Mutagênese , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
In many vertebrates, establishment of Left-Right (LR) asymmetry results from the activity of a ciliated organ functioning as the LR Organizer (LRO). While regulation of the formation of this structure by major signaling pathways has been described, the transcriptional control of LRO formation is poorly understood. Using the zebrafish model, we show that the transcription factors and cofactors mediating or regulating the transcriptional outcome of the Hippo signaling pathway play a pivotal role in controlling the expression of genes essential to the formation of the LRO including ligands and receptors of signaling pathways involved in this process and most genes required for motile ciliogenesis. Moreover, the transcription cofactor, Vgll4l regulates epigenetic programming in LRO progenitors by controlling the expression of writers and readers of DNA methylation marks. Altogether, our study uncovers a novel and essential role for the transcriptional effectors and regulators of the Hippo pathway in establishing LR asymmetry.
Assuntos
Padronização Corporal , Regulação da Expressão Gênica no Desenvolvimento , Transdução de Sinais , Fatores de Transcrição/metabolismo , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/embriologia , AnimaisRESUMO
In the zebrafish embryo, cells of the early blastula animal pole are all equivalent and are fully pluripotent until the midblastula transition that occurs at the tenth cell cycle (512 to 1K cells). This naive territory of the embryo is therefore perfectly suited to assay for morphogen activity. Here we describe different methods to generate ectopic morphogen gradients, either in vivo at the animal pole of the embryo, or in vitro in animal pole explants or in aggregates of animal pole blastomeres (also named embryoid bodies). These methods include injection of mRNA coding for growth factor(s) into animal pole blastomere(s), transplantation of growth factor(s) secreting cells, implantation of beads coated with purified protein(s), and various combinations of these different approaches. Our comparative study reveals that all these methods allow to generate morphogen gradient(s) that are able to induce, both in vivo and in vitro, the formation of a well-patterned embryonic axis.
Assuntos
Blástula/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Peixe-Zebra/embriologia , Animais , Blástula/citologia , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário , Morfogênese , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Despite the fundamental importance of patterning along the dorsal-ventral (DV) and anterior-posterior (AP) axes during embryogenesis, uncertainty exists in the orientation of these axes for the mesoderm. Here we examine the origin and formation of the zebrafish kidney, a ventrolateral mesoderm derivative, and show that AP patterning of the non-axial mesoderm occurs across the classic gastrula stage DV axis while DV patterning aligns along the animal-vegetal pole. We find that BMP signalling acts early to establish broad anterior and posterior territories in the non-axial mesoderm while retinoic acid (RA) functions later, but also across the classic DV axis. Our data support a model in which RA on the dorsal side of the embryo induces anterior kidney fates while posterior kidney progenitors are protected ventrally by the RA-catabolizing enzyme Cyp26a1. This work clarifies our understanding of vertebrate axis orientation and establishes a new paradigm for how the kidney and other mesodermal derivatives arise during embryogenesis.
Assuntos
Padronização Corporal/genética , Proteína Morfogenética Óssea 2/genética , Rim/embriologia , Mesoderma/embriologia , Ácido Retinoico 4 Hidroxilase/genética , Tretinoína/metabolismo , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Animais , Padronização Corporal/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/genética , Embrião não Mamífero , Tretinoína/farmacologiaRESUMO
IQGAPs are scaffolding proteins that regulate actin assembly, exocyst function, cell motility, morphogenesis, adhesion and division. Vertebrates express 3 family members: IQGAP1, IQGAP2, and IQGAP3. IQGAP1 is known to stimulate nucleation of branched actin filaments through N-WASP and the Arp2/3 complex following direct binding to cytoplasmic tails of ligand-activated growth factor receptors, including EGFR, VEGFR2 and FGFR1. By contrast, little is known about functions of IQGAP2 or IQGAP3. Using in situ hybridization on whole mount zebrafish (Danio rerio) embryos, we show that IQGAP1 and IQGAP2 are associated with discrete tissues and organs, while IQGAP3 is mainly expressed in proliferative cells throughout embryonic and larval development. Morpholino knockdowns of IQGAP1 and IQGAP2 have little effect on embryo morphology while loss of function of IQGAP3 affects both cell proliferation and cell motility. IQGAP3 morphant phenotypes are similar to those resulting from overexpression of dominant negative forms of Ras or of Fibroblast Growth Factor Receptor 1 (FGFR1), suggesting that IQGAP3 plays a role in FGFR1-Ras-ERK signaling. In support of this hypothesis, dominant negative forms of FGFR1 or Ras could be rescued by co-injection of zebrafish IQGAP3 mRNA, strongly suggesting that IQGAP3 acts as a downstream regulator of the FGFR1-Ras signaling pathway.
Assuntos
Morfogênese , Receptores de Fatores de Crescimento/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Adesão Celular , Movimento Celular , Embrião não Mamífero/metabolismo , Peixe-Zebra/embriologiaRESUMO
During the course of their classic experiments, Hilde Mangold and Hans Spemann discovered that the dorsal blastopore lip of an amphibian gastrula was able to induce formation of a complete embryonic axis when transplanted into the ventral side of a host gastrula embryo. Since then, the inducing activity of the dorsal lip has been known as the Spemann or dorsal organizer. During the past 25 years, studies performed in a variety of species have led to the identification of molecular factors associated with the properties of this tissue. However, none of them is, by itself, able to induce formation of the main body axis from a population of naive pluripotent embryonic cells. Recently, experiments performed using the zebrafish (Danio rerio) revealed that the organizing activities present in the embryo are not restricted to the Spemann organizer but are distributed along the entire blastula/gastrula margin. These organizing activities result from the interaction between two opposing gradients of morphogens, BMP and Nodal, that are the primary signals that trigger the cascade of developmental events leading to the organization of the embryo. These studies mark the end of the era during which developmental biologists saw the Spemann organizer as the core element for the organization of the vertebrate embryonic axis and, instead, provides opportunities for the experimental control of morphogenesis starting with a population of embryonic pluripotent cells that will be instructed using those two morphogen gradients.
Assuntos
Organizadores Embrionários , Transdução de Sinais , Vertebrados/embriologia , Animais , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Humanos , Proteína Nodal/metabolismoRESUMO
Sialyltransferases are responsible for the synthesis of a diverse range of sialoglycoconjugates predicted to be pivotal to deuterostomes' evolution. In this work, we reconstructed the evolutionary history of the metazoan α2,3-sialyltransferases family (ST3Gal), a subset of sialyltransferases encompassing six subfamilies (ST3Gal I-ST3Gal VI) functionally characterized in mammals. Exploration of genomic and expressed sequence tag databases and search of conserved sialylmotifs led to the identification of a large data set of st3gal-related gene sequences. Molecular phylogeny and large scale sequence similarity network analysis identified four new vertebrate subfamilies called ST3Gal III-r, ST3Gal VII, ST3Gal VIII, and ST3Gal IX. To address the issue of the origin and evolutionary relationships of the st3gal-related genes, we performed comparative syntenic mapping of st3gal gene loci combined to ancestral genome reconstruction. The ten vertebrate ST3Gal subfamilies originated from genome duplication events at the base of vertebrates and are organized in three distinct and ancient groups of genes predating the early deuterostomes. Inferring st3gal gene family history identified also several lineage-specific gene losses, the significance of which was explored in a functional context. Toward this aim, spatiotemporal distribution of st3gal genes was analyzed in zebrafish and bovine tissues. In addition, molecular evolutionary analyses using specificity determining position and coevolved amino acid predictions led to the identification of amino acid residues with potential implication in functional divergence of vertebrate ST3Gal. We propose a detailed scenario of the evolutionary relationships of st3gal genes coupled to a conceptual framework of the evolution of ST3Gal functions.
Assuntos
Evolução Molecular , Sialiltransferases/genética , Vertebrados/genética , Sequência de Aminoácidos , Animais , Cordados/genética , Equinodermos/genética , Dados de Sequência Molecular , Filogenia , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Upon stimulation by Wnt ligands, the canonical Wnt/ß-catenin signalling pathway results in the stabilization of ß-catenin and its translocation into the nucleus to form transcriptionally active complexes with sequence-specific DNA-binding T-cell factor/lymphoid enhancer factor (TCF/LEF) family proteins. In the absence of nuclear ß-catenin, TCF proteins act as transcriptional repressors by binding to Groucho/Transducin-Like Enhancer of split (TLE) proteins that function as co-repressors by interacting with histone deacetylases whose activity leads to the generation of transcriptionally silent chromatin. Here we show that the transcription factor Ladybird homeobox 2 (Lbx2) positively controls the Wnt/ß-catenin signalling pathway in the posterior lateral and ventral mesoderm of the zebrafish embryo at the gastrula stage, by directly interfering with the binding of Groucho/TLE to TCF, thereby preventing formation of transcription repressor complexes. These findings reveal a novel level of regulation of the canonical Wnt/ß-catenin signalling pathway occurring in the nucleus and involving tissue-specific derepression of TCF by Lbx2.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Mesoderma/metabolismo , Proteínas Repressoras/fisiologia , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologia , beta Catenina/metabolismo , Animais , Proteínas Correpressoras/metabolismo , Gástrula/metabolismo , Transdução de Sinais , Peixe-ZebraAssuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Clonagem de Organismos/métodos , Morfogênese/efeitos dos fármacos , Proteína Nodal/farmacologia , Vertebrados/embriologia , Animais , Animais Geneticamente Modificados , Proteínas Morfogenéticas Ósseas/genética , Relação Dose-Resposta a Droga , Técnicas de Cultura Embrionária , Embrião não Mamífero , Humanos , Microinjeções , Morfogênese/genética , Proteína Nodal/genética , RNA Mensageiro/farmacologia , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
The in situ hybridization uses a labeled complementary RNA strand to localize a specific mRNA sequence in a tissue. This method is widely used to describe the spatial and temporal expression patterns of developmentally regulated genes. Here we describe a technique that employs in vitro synthesized RNA tagged with digoxigenin uridine-5'-triphosphate (UTP) to determine expression of genes on whole-mount zebrafish embryos and young larvae. Following hybridization, the localization of the specific transcript is visualized immunohistochemically using an anti-digoxigenin antibody conjugated to alkaline phosphatase that hydrolyzes the 5-bromo-4-chloro-3-indolyl phosphate (BCIP) to 5-bromo-4-chloro-3-indole and inorganic phosphate. 5-Bromo-4-chloro-3-indole can be oxidized by nitro blue tetrazolium (NBT), which forms an insoluble dark blue diformazan precipitate after reduction.This protocol has been used for performing large-scale analyses of the spatial and temporal expression of the zebrafish genome, resulting in the description of more than 8,400 expression patterns that are available at the zebrafish information network (ZFIN.org) in the gene expression section.
Assuntos
Embrião não Mamífero/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ/métodos , Larva/ultraestrutura , RNA/análise , Peixe-Zebra/embriologia , Animais , Digoxigenina/análogos & derivados , Embrião não Mamífero/metabolismo , Feminino , Imuno-Histoquímica/métodos , Indicadores e Reagentes , Indóis , Larva/genética , Reação em Cadeia da Polimerase/métodos , Sondas RNA/análise , Sondas RNA/genética , Fixação de Tecidos/métodos , Uridina Trifosfato/análogos & derivados , Peixe-Zebra/genéticaRESUMO
The role of the habenular nuclei in modulating fear and reward pathways has sparked a renewed interest in this conserved forebrain region. The bilaterally paired habenular nuclei, each consisting of a medial/dorsal and lateral/ventral nucleus, can be further divided into discrete subdomains whose neuronal populations, precise connectivity, and specific functions are not well understood. An added complexity is that the left and right habenulae show pronounced morphological differences in many non-mammalian species. Notably, the dorsal habenulae of larval zebrafish provide a vertebrate genetic model to probe the development and functional significance of brain asymmetry. Previous reports have described a number of genes that are expressed in the zebrafish habenulae, either in bilaterally symmetric patterns or more extensively on one side of the brain than the other. The goal of our study was to generate a comprehensive map of the zebrafish dorsal habenular nuclei, by delineating the relationship between gene expression domains, comparing the extent of left-right asymmetry at larval and adult stages, and identifying potentially functional subnuclear regions as defined by neurotransmitter phenotype. Although many aspects of habenular organization appear conserved with rodents, the zebrafish habenulae also possess unique properties that may underlie lateralization of their functions.
Assuntos
Habenula/embriologia , Neurotransmissores/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Habenula/metabolismo , Imuno-Histoquímica , Neurônios/metabolismo , Neurotransmissores/genética , Especificidade de Órgãos/genética , Fenótipo , Peixe-Zebra/genéticaRESUMO
Development of vertebrate embryos involves tightly regulated molecular and cellular processes that progressively instruct proliferating embryonic cells about their identity and behavior. Whereas numerous gene activities have been found to be essential during early embryogenesis, little is known about the minimal conditions and factors that would be sufficient to instruct pluripotent cells to organize the embryo. Here, we show that opposing gradients of bone morphogenetic protein (BMP) and Nodal, two transforming growth factor family members that act as morphogens, are sufficient to induce molecular and cellular mechanisms required to organize, in vivo or in vitro, uncommitted cells of the zebrafish blastula animal pole into a well-developed embryo.
Assuntos
Blástula/fisiologia , Proteínas Morfogenéticas Ósseas/fisiologia , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário , Proteína Nodal/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Padronização Corporal , Proteínas Morfogenéticas Ósseas/genética , Gástrula/fisiologia , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento , Morfogênese , Proteína Nodal/genética , RNA Mensageiro/genética , Transdução de Sinais , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The habenulo-interpeduncular pathway, a highly conserved cholinergic system, has emerged as a valuable model to study left-right asymmetry in the brain. In larval zebrafish, the bilaterally paired dorsal habenular nuclei (dHb) exhibit prominent left-right differences in their organization, gene expression, and connectivity, but their cholinergic nature was unclear. Through the discovery of a duplicated cholinergic gene locus, we now show that choline acetyltransferase and vesicular acetylcholine transporter homologs are preferentially expressed in the right dHb of larval zebrafish. Genes encoding the nicotinic acetylcholine receptor subunits α2 and ß4 are transcribed in the target interpeduncular nucleus (IPN), suggesting that the asymmetrical cholinergic pathway is functional. To confirm this, we activated channelrhodopsin-2 specifically in the larval dHb and performed whole-cell patch-clamp recording of IPN neurons. The response to optogenetic or electrical stimulation of the right dHb consisted of an initial fast glutamatergic excitatory postsynaptic current followed by a slow-rising cholinergic current. In adult zebrafish, the dHb are divided into discrete cholinergic and peptidergic subnuclei that differ in size between the left and right sides of the brain. After exposing adults to nicotine, fos expression was activated in subregions of the IPN enriched for specific nicotinic acetylcholine receptor subunits. Our studies of the newly identified cholinergic gene locus resolve the neurotransmitter identity of the zebrafish habenular nuclei and reveal functional asymmetry in a major cholinergic neuromodulatory pathway of the vertebrate brain.
Assuntos
Lateralidade Funcional/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Habenula/fisiologia , Modelos Animais , Tegmento Mesencefálico/fisiologia , Acetilcolina/metabolismo , Animais , Sequência de Bases , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Primers do DNA/genética , Estimulação Elétrica , Habenula/metabolismo , Hibridização In Situ , Larva/fisiologia , Dados de Sequência Molecular , Vias Neurais/fisiologia , Optogenética , Técnicas de Patch-Clamp , Receptores Nicotínicos/metabolismo , Análise de Sequência de RNA , Tegmento Mesencefálico/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Peixe-ZebraRESUMO
The dorsal habenular nuclei of the zebrafish epithalamus have become a valuable model for studying the development of left-right (L-R) asymmetry and its function in the vertebrate brain. The bilaterally paired dorsal habenulae exhibit striking differences in size, neuroanatomical organization, and molecular properties. They also display differences in their efferent connections with the interpeduncular nucleus (IPN) and in their afferent input, with a subset of mitral cells distributed on both sides of the olfactory bulb innervating only the right habenula. Previous studies have implicated the dorsal habenulae in modulating fear/anxiety responses in juvenile and adult zebrafish. It has been suggested that the asymmetric olfactory-habenula pathway (OB-Ha), revealed by selective labeling from an lhx2a:YFP transgene, mediates fear behaviors elicited by alarm pheromone. Here we show that expression of the fam84b gene demarcates a unique region of the right habenula that is the site of innervation by lhx2a:YFP-labeled olfactory axons. Upon ablation of the parapineal, which normally promotes left habenular identity; the fam84b domain is present in both dorsal habenulae and lhx2a:YFP-labeled olfactory bulb neurons form synapses on the left and the right side. To explore the relevance of the asymmetric olfactory projection and how it might influence habenular function, we tested activation of this pathway using odorants known to evoke behaviors. We find that alarm substance or other aversive odors, and attractive cues, activate fos expression in subsets of cells in the olfactory bulb but not in the lhx2a:YFP expressing population. Moreover, neither alarm pheromone nor chondroitin sulfate elicited fos activation in the dorsal habenulae. The results indicate that L-R asymmetry of the epithalamus sets the directionality of olfactory innervation, however, the lhx2a:YFP OB-Ha pathway does not appear to mediate fear responses to aversive odorants.
Assuntos
Aprendizagem da Esquiva/fisiologia , Sinais (Psicologia) , Habenula/metabolismo , Bulbo Olfatório/metabolismo , Condutos Olfatórios/metabolismo , Proteínas Proto-Oncogênicas c-fos/biossíntese , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica , Habenula/citologia , Odorantes , Bulbo Olfatório/citologia , Condutos Olfatórios/citologia , Peixe-ZebraRESUMO
Preferential loading of the complementary bioluminescent (f-aequorin) and fluorescent (Calcium Green-1 dextran) Ca(2+) reporters into the yolk syncytial layer (YSL) of zebrafish embryos, revealed the generation of stochastic patterns of fast, short-range, and slow, long-range Ca(2+) waves that propagate exclusively through the external YSL (E-YSL). Starting abruptly just after doming (~4.5h post-fertilization: hpf), and ending at the shield stage (~6.0hpf) these distinct classes of waves propagated at mean velocities of ~50 and ~4µm/s, respectively. Although the number and pattern of these waves varied between embryos, their initiation site and arcs of propagation displayed a distinct dorsal bias, suggesting an association with the formation and maintenance of the nascent dorsal-ventral axis. Wave initiation coincided with a characteristic clustering of YSL nuclei (YSN), and their associated perinuclear ER, in the E-YSL. Furthermore, the inter-YSN distance (IND) appeared to be critical such that Ca(2+) wave propagation occurred only when this was <~8µm; an IND >~8µm was coincidental with wave termination at shield stage. Treatment with the IP3R antagonist, 2-APB, the Ca(2+) buffer, 5,5'-dibromo BAPTA, and the SERCA-pump inhibitor, thapsigargin, resulted in a significant disruption of the E-YSL Ca(2+) waves, whereas exposure to the RyR antagonists, ryanodine and dantrolene, had no significant effect. These findings led us to propose that the E-YSL Ca(2+) waves are generated mainly via Ca(2+) release from IP3Rs located in the perinuclear ER, and that the clustering of the YSN is an essential step in providing a CICR pathway required for wave propagation. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.
Assuntos
Blástula/metabolismo , Sinalização do Cálcio/fisiologia , Gema de Ovo/metabolismo , Embrião não Mamífero/metabolismo , Gástrula/metabolismo , Peixe-Zebra/embriologia , Equorina/metabolismo , Animais , Canais de Cálcio/metabolismo , Núcleo Celular/metabolismo , Gema de Ovo/citologia , Embrião não Mamífero/citologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Microscopia Confocal , Microscopia de Fluorescência por Excitação Multifotônica , Transdução de Sinais , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
In vertebrates, the animal-vegetal axis is determined during oogenesis and at ovulation, the egg is radially symmetric. In anamniotes, following fertilization, a microtubule-dependent movement leads to the displacement of maternal dorsal determinants from the vegetal pole to the future dorsal side of the embryo, providing the initial breaking of radial symmetry [Weaver C, Kimelman D (2004) Development 131:3491-3499]. These dorsal determinants induce ß-catenin nuclear translocation in dorsal cells of the blastula. Previous work in amphibians has shown that secreted Wnt11/5a complexes, regulated by the Wnt antagonist Dkk-1, are required for the initiation of embryonic axis formation [Cha et al. (2009) Curr Biol 29:1573-1580]. In the current study, we determined that the vegetal maternal dorsal determinant in fish is not the Wnt11/5a complex but the canonical Wnt, Wnt8a. Translation of this mRNA and secretion of the Wnt8a protein result in a dorsal-to-ventral gradient of Wnt stimulation, extending across the entire embryo. This gradient is counterbalanced by two Wnt inhibitors, Sfrp1a and Frzb. These proteins are essential to restrict the activation of the canonical Wnt pathway to the dorsal marginal blastomeres by defining the domain where the Wnt8a activity gradient is above the threshold value necessary for triggering the canonical ß-catenin pathway. In summary, this study establishes that the zebrafish maternal dorsal determinant, Wnt8a, is required to localize the primary dorsal center, and that the extent of this domain is defined by the activity of two maternally provided Wnt antagonists, Sfrp1a and Frzb.
Assuntos
Proteínas do Citoesqueleto/genética , Glicoproteínas/genética , Proteínas Wnt/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Sequência de Bases , Padronização Corporal/genética , Proteínas do Citoesqueleto/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Microscopia Confocal , Dados de Sequência Molecular , Morfolinos/genética , Nocodazol/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Tempo , Moduladores de Tubulina/farmacologia , Proteínas Wnt/metabolismo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Zebrafish (Danio rerio) can serve as a model system to study heritable skin diseases. The skin is rapidly developed during the first 5-6 days of embryonic growth, accompanied by expression of skin-specific genes. Transmission electron microscopy (TEM) of wild-type zebrafish at day 5 reveals a two-cell-layer epidermis separated from the underlying collagenous stroma by a basement membrane with fully developed hemidesmosomes. Scanning electron microscopy (SEM) reveals an ordered surface contour of keratinocytes with discrete microridges. To gain insight into epidermal morphogenesis, we have employed morpholino-mediated knockdown of the abca12 and snap29 genes, which are crucial for secretion of lipids and intracellular trafficking of lamellar granules, respectively. Morpholinos, when placed on exon-intron junctions, were >90% effective in preventing the corresponding gene expression when injected into one- to four-cell-stage embryos. By day 3, TEM of abca12 morphants showed accumulation of lipid-containing electron-dense lamellar granules, whereas snap29 morphants showed the presence of apparently empty vesicles in the epidermis. Evaluation of epidermal morphogenesis by SEM revealed similar perturbations in both cases in the microridge architecture and the development of spicule-like protrusions on the surface of keratinocytes. These morphological findings are akin to epidermal changes in harlequin ichthyosis and CEDNIK syndrome, autosomal recessive keratinization disorders due to mutations in the ABCA12 and SNAP29 genes, respectively. The results indicate that interference of independent pathways involving lipid transport in the epidermis can result in phenotypically similar perturbations in epidermal morphogenesis, and that these fish mutants can serve as a model to study the pathomechanisms of these keratinization disorders.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Grânulos Citoplasmáticos/metabolismo , Epiderme/embriologia , Ictiose/patologia , Morfogênese , Proteínas SNARE/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Transporte Biológico/efeitos dos fármacos , Sequência Conservada/genética , Grânulos Citoplasmáticos/efeitos dos fármacos , Modelos Animais de Doenças , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Epiderme/efeitos dos fármacos , Epiderme/patologia , Epiderme/ultraestrutura , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Genoma/genética , Humanos , Ictiose/embriologia , Hibridização In Situ , Larva/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Morfogênese/efeitos dos fármacos , Morfolinos/farmacologia , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas SNARE/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
In vertebrates, embryonic structures present at the dorsal midline, prechordal plate, notochord, hypochord and floor plate share a common embryonic origin. In zebrafish, they derive from a pool of progenitors located within the embryonic shield at the onset of gastrulation. The molecular mechanisms responsible for the common development of these structures remain unknown. Based on their spatial and temporal expression, transcription factors of the Forkhead box A (FoxA) family appeared to be good candidates to play such a role. In agreement with this hypothesis, we found that simultaneous knockdown of FoxA2 and FoxA3 abolish the formation of all axial derivatives, while overexpression of these transcription factors strongly enlarges dorsal mesodermal territories. We establish that, in FoxA2-FoxA3 double morphants, precursors of axial tissues are correctly induced at early gastrula stage, but their dorsal midline identity is not maintained during development and we found that progenitors of these tissues are cell-autonomously re-specified to form muscle fibers as well as cells of the ventral neural tube. Our study provides the first example of a specific loss of all dorsal midline tissues and demonstrates that members of the FoxA family have redundant functions essential to maintain the axial identity of prechordal plate, notochord, floor plate and hypochord progenitors during gastrulation.
Assuntos
Fatores de Transcrição Forkhead/fisiologia , Fator 3-gama Nuclear de Hepatócito/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Gastrulação , Mesoderma/fisiologiaRESUMO
Heritable skin diseases represent a broad spectrum of clinical manifestations due to mutations in â¼500 different genes. A number of model systems have been developed to advance our understanding of the pathomechanisms of genodermatoses. Zebrafish (Danio rerio), a freshwater vertebrate, has a well-characterized genome, the expression of which can be easily manipulated. The larvae develop rapidly, with all major organs having developed by 5-6 days post-fertilization, including the skin, consisting of the epidermis comprising two cell layers and separated from the dermal collagenous matrix by a basement membrane. This perspective highlights the morphological and ultrastructural features of zebrafish skin, in the context of cutaneous gene expression. These observations suggest that zebrafish provide a useful model system to study the molecular aspects of skin development, as well as the pathogenesis and treatment of select heritable skin diseases.