RESUMO
The Iroquois genes code for homeodomain proteins that have been implicated in the neural development of Drosophila and vertebrates. We show here for the first time that Xiro-1, one of the Xenopus Iroquois genes, is expressed in the Spemann organizer from the start of gastrulation and that its overexpression induces a secondary axis as well as the ectopic expression of several organizer genes, such as chordin, goosecoid, and Xlim-1. Our results also indicate that Xiro-1 normally functions as a transcriptional repressor in the mesoderm. Overexpression of Xiro-1 or a chimeric form fused to the repressor domain of Engrailed cause similar phenotypes while overexpression of functional derivatives of Xiro-1 fused with transactivation domains (VP16 or E1A) produce the opposite effects. Finally, we show that Xiro-1 works as a repressor of bmp-4 transcription and that its effect on organizer development is dependent on BMP-4 activity. We propose that the previously observed down regulation of bmp-4 in the dorsal mesoderm during gastrulation can be explained by the repressor activity of Xiro-1 described here. Thus, Xiro-1 seems to have at least two different functions: control of neural plate and organizer development, both of which could be mediated by repression of bmp-4 transcription.
Assuntos
Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Mesoderma/fisiologia , Proteínas do Tecido Nervoso , Organizadores Embrionários/metabolismo , Fatores de Transcrição/fisiologia , Proteínas de Xenopus , Xenopus/embriologia , Animais , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/metabolismo , Embrião não Mamífero/fisiologia , Proteínas Repressoras/fisiologiaRESUMO
Calcium signals participate in the differentiation of electrically excitable and nonexcitable cells; one example of this differentiation is the acquisition of mature neuronal phenotypes. For example, transient elevations of the intracellular calcium concentration have been recorded in the ectoderm of early embryos, and this elevation has been proposed to participate in neural induction. Here, we present molecular evidence indicating that voltage-sensitive calcium channels (VSCC) are involved in early developmental processes leading to the establishment of the dorsoventral (D-V) patterning of a vertebrate embryo. We report that alpha1S VSCC are expressed selectively in the dorsal marginal zone at the early gastrula stage. The expression of the VSCC correlates with elevated intracellular calcium levels, as evaluated by the fluorescence of the intracellular calcium indicator Fluo-3. Misexpression of VSCC leads to a strong dorsalization of the ventral marginal zone and induction of the secondary axis but no direct neuralization of the ectoderm. Moreover, specific inhibition of VSCC by the use of calcicludine results in ventralization of the dorsal mesoderm. Together, these results indicate that calcium channels regulate mesodermal patterning by specificating the D-V identity of the mesodermal cells. The D-V patterning of the mesoderm has been shown to depend on a gradient of BMPs activity. We discuss the possibility that VSCC affect or act downstream of BMPs activity.
Assuntos
Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Gástrula/fisiologia , Mesoderma/fisiologia , Xenopus laevis/embriologia , Animais , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/metabolismo , Canais de Cálcio/efeitos dos fármacos , Venenos Elapídicos/farmacologia , Mesoderma/efeitos dos fármacos , Subunidades Proteicas , Transdução de Sinais/fisiologia , Proteínas de XenopusRESUMO
The Snail family of genes comprise a group of transcription factors with characteristic zinc finger motifs. One of the members of this family is the Slug gene. Slug has been implicated in the development of neural crest in chick and Xenopus by antisense loss of function experiments. Here, we have generated functional derivatives of Xslug by constructing cDNAs that encode the Xslug protein fused with the transactivation domain of the virus-derived VP16 activator or with the repressor domain of the Drosophila Engrailed protein. Our results suggest that Xslug normally functions as a transcriptional repressor and that Xslug-VP16 behaves as a dominant negative of Xslug. In the present work, we confirm and extend previous results that suggest that Xslug has an important function in neural crest development, by controlling its own transcription. In addition we have uncovered a new function for Xslug. We show that Xslug is expressed in the dorsal mesendoderm at the beginning of gastrulation, where is it able to upregulate the expression of dorsal genes. On the other hand when Xslug is expressed outside of the organizer it represses the expression of ventral genes. Our results indicate that this effect on mesodermal patterning depends on BMP activity, showing that Xslug can directly control the transcription of BMP-4.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Proteínas de Drosophila , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas , Peptídeos e Proteínas de Sinalização Intercelular , Mesoderma/fisiologia , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Proteínas de Xenopus , Proteínas de Peixe-Zebra , Animais , Biomarcadores , Proteína Morfogenética Óssea 4 , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ectoderma/metabolismo , Ectoderma/fisiologia , Proteínas de Homeodomínio/genética , Mesoderma/metabolismo , Crista Neural/metabolismo , Organizadores Embrionários/metabolismo , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Wnt , Xenopus laevis/embriologia , Dedos de ZincoRESUMO
The markers Xslug, Xsnail, and Xtwist all are expressed in the presumptive neural folds and are thought to delineate the presumptive neural crest. However, their interrelationship and relative spatiotemporal distributions are not well understood. Here, we present a detailed in situ hybridization analysis of the relative patterns of expression of these transcription factors from gastrulation through neurulation and post-neural crest migration. The three genes mark the prospective neural crest and roof plate, coming on sequentially, with Xsnail preceding Xslug preceding Xtwist. By combining gene expression analysis with a fate map of the same region using DiI labeling, we determined the correspondence between early and late domains of gene expression. At the beginning of gastrulation, Xsnail is present in a unique domain of expression in a lateral region of the embryo in both superficial and deep layers of the ectoderm, as are Xslug and Xtwist. During gastrulation and neurulation, the superficial layer moves faster toward the dorsal midline than the deep layer, producing a relative shift in these cell populations. By early neurula stage, the Xsnail domain is split into a medial domain in the superficial ectoderm (fated to become the roof plate) and a lateral domain in the deep layer of the ectoderm (fated to become neural crest). Xsnail is down-regulated in the most anterior neural plate and up-regulated in the posterior neural plate. Our results show that changes in the expression of Xsnail, Xslug, and Xtwist are a consequence of active cell movement in some regions coupled with dynamic changes in gene expression in other regions.
Assuntos
Biomarcadores , Movimento Celular , Regulação da Expressão Gênica no Desenvolvimento , Crista Neural/citologia , Animais , Hibridização In Situ , Crista Neural/metabolismo , Xenopus/embriologiaRESUMO
The forkhead type Brain Factor 2 from mouse and chicken help pattern the forebrain, optic vesicle and kidney. We have isolated a Xenopus homolog (Xbf2) and found that during gastrulation it is expressed in the dorsolateral mesoderm, where it helps specify this territory by downregulating BMP-4 and its downstream genes. Indeed, Xbf2 overexpression caused partial axis duplication. Interference with BMP-4 signaling also occurs in isolated animal caps, since Xbf2 induces neural tissue. Within the neurula forebrain, Xbf2 and the related Xbf1 gene are expressed in the contiguous diencephalic and telencephalic territories, respectively, and each gene represses the other. Finally, Xbf2 seems to participate in the control of neural crest migration. Our data suggest that XBF2 interferes with BMP-4 signaling, both in mesoderm and ectoderm.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Mesoderma/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Crista Neural/fisiologia , Prosencéfalo/embriologia , Proteínas de Xenopus , Xenopus/embriologia , Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Indução Embrionária , Fatores de Transcrição Forkhead , Galactosídeos/metabolismo , Expressão Gênica , Hibridização In Situ , Indóis/metabolismo , Dados de Sequência Molecular , Fatores de Tempo , Proteína 1 Relacionada a TwistRESUMO
The neural crest is a unique cell population among embryonic cell types, displaying properties of both ectodermal and mesodermal lineages. Most of the recent studies examining the neural crest have been performed in avian embryos. Only in the first half of this century were amphibians extensively used. We first summarize this important older source of information, reviewing studies made since the turn of the century. Due to the increasingly detailed in cellular and molecular knowledge of the early development of Xenopus laevis, the remainder of the review focuses on this species. We describe the route of migration and fate of the neural crest and propose a new model of neural crest induction in which prospective cells are induced independently of the neural plate by a double gradient of a morphogen that patterns the entire ectoderm. This model is also discussed in a more general context in connection with the dorsoventral patterning of the neural tube. Finally, we discuss some ideas concerning neural crest evolution and propose a novel hypothesis about its phylogenetic origin.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Crista Neural/embriologia , Xenopus laevis/embriologia , Xenopus laevis/genética , Anfíbios , Animais , Evolução Biológica , Padronização Corporal , Diferenciação Celular , Movimento Celular , Ectoderma , Modelos BiológicosRESUMO
We have analyzed the role of mesoderm in the induction of the neural crest in Xenopus using expression of neural plate (Xsox-2) and neural crest (Xslug and ADAM). Conjugation experiments using different kinds of mesoderm together with embryonic dissection experiments suggest that the dorsolateral mesoderm is capable of specifically inducing neural crest cells. Neural crest markers can be induced in competent ectoderm at varying distances from the inducing mesoderm, with dorsal tissue inducing neural crest at a distance while dorsolateral tissue only induces neural crest directly in adjacent ectoderm. The results suggest that dorsal mesoderm has a high level of inducer and dorsolateral mesoderm has a lower level, consistent with a inductive gradient. We explored the possible role of BMP and noggin in the generation of such a hypothetical gradient and found that: (1) progressively higher levels of BMP activity are sufficient for the specification of neural plate, neural crest, and nonneural cells, respectively; (2) progressively higher levels of noggin are able to induce neural crest at greater distances from the source of inducer; and (3) modification of the levels of BMP activity causes induction of the neural crest in absence of neural plate, suggesting independent induction of these two tissues. We propose a model in which a gradient of BMP activity is established in the ectoderm by interaction between BMP in the ectoderm and BMP inhibitors in the mesoderm. Neural crest is induced when a threshold level of BMP is attained in the ectoderm. The dorsolateral mesoderm produces either BMP inhibitors or a specific neural crest inducer, with low BMP activity inducing neural plate while high BMP activity induces epidermis.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Mesoderma/fisiologia , Crista Neural/fisiologia , Animais , Indução Embrionária , Feminino , Crista Neural/citologia , Xenopus/embriologiaRESUMO
Mesoderm induction requires interaction between cells of the animal and vegetal hemispheres of the embryo. Several molecules have been proposed as candidates for mesoderm-inducing signals, with activin a particularly strong candidate. However, it has not been possible to inhibit mesoderm formation in vivo by specifically blocking activin action. Follistatin is able to inhibit the action of activin but not that of the mature region of Vg1, a member of the transforming growth factor beta family. Follistatin therefore provides a useful tool for distinguishing between signalling by these two factors. We have overexpressed Xenopus follistatin mRNA and analysed the expression of several mesodermal markers. Our results show an inhibition of mesodermal formation by follistatin in a concentration-dependent manner, showing the requirement of activin for mesodermal induction.
Assuntos
Proteínas Fetais , Glicoproteínas/farmacologia , Inibidores do Crescimento/farmacologia , Proteínas de Homeodomínio , Mesoderma/efeitos dos fármacos , Proteínas Repressoras , Proteínas com Domínio T , Proteínas de Peixe-Zebra , Animais , Técnicas de Cultura , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Indução Embrionária/efeitos dos fármacos , Folistatina , Marcadores Genéticos , Glicoproteínas/genética , Proteína Goosecoid , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro , Proteínas Recombinantes/farmacologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Proteínas Wnt , XenopusRESUMO
The Drosophila homeoproteins Ara and Caup are members of a combination of factors (prepattern) that control the highly localized expression of the proneural genes achaete and scute. We have identified two Xenopus homologs of ara and caup, Xiro1 and Xiro2. Similarly to their Drosophila counterparts, they control the expression of proneural genes and, probably as a consequence, the size of the neural plate. Moreover, Xiro1 and Xiro2 are themselves controlled by noggin and retinoic acid and, similarly to ara and caup, they are overexpressed by expression in Xenopus embryos of the Drosophila cubitus interruptus gene. These and other findings suggest the conservation of at least part of the genetic cascade that regulates proneural genes, and the existence in vertebrates of a prepattern of factors important to control the differentiation of the neural plate.
Assuntos
Proteínas de Drosophila , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas do Tecido Nervoso , Sistema Nervoso/embriologia , Fatores de Transcrição/genética , Proteínas de Xenopus , Sequência de Aminoácidos , Animais , Clonagem Molecular , Ectoderma/metabolismo , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Xenopus/embriologia , Xenopus/genéticaRESUMO
A study of the molecules noggin and fibroblast growth factor (FGF) and its receptor in the induction of the prospective neural crest in Xenopus laevis embryos has been carried out, using the expression of the gene Xslu as a marker for the neural crest. We show that when a truncated FGF receptor (XFD) was expressed ectopically in order to block FGF signaling Xslu expression was inhibited. The effect of XFD on Xslu was specific and could be reversed by the coinjection of the wild-type FGF receptor (FGFR). Inhibition of Xslu expression by XFD is not a consequence of neural plate inhibition, as was shown by analyzing Xsox-2 expression. When ectoderm expressing XFD was transplanted into the prospective neural fold region of embryos Xslu induction was inhibited. The neural crest can also be induced by an interaction between neural plate and epidermis. As this induction is suppressed by the presence of XFD in the neural plate and not in the epidermis, it suggests that the neural crest is induced by FGF from the epidermis. However, treatment of neural plate with FGF was not able to induce Xslug expression, showing that in addition to FGF other non-FGF factors are also required. Previously we have suggested that the ectopic ventral expression of Xslu produced by overexpression of noggin mRNA resulted from an interaction of noggin with a ventral signal. Overexpression of XFD inhibits this effect, suggesting that FGF could be one component involved in this ventral signaling. Overexpression of FGFR produced a remarkable increase in the expression of Xslu in the posterior neural folds and around the blastopore. Injections in different blastomeres of the embryo suggest that the target cells of this effect are the ventral cells. Finally, we proposed a model in which the induction of the neural crests at the border of the neural plate requires functional FGF signaling, which possibly interacts with a neural inducer such as noggin.