RESUMO
Smad-interacting protein-1 (SIP1), also known as deltaEF2, ZEB2 and zfhx1b, is essential for the formation of the neural tube and the somites. Overexpression of Xenopus SIP1 causes ectopic neural induction via inhibition of bone morphogenetic protein (BMP) signaling and inhibition of Xbra expression. Here, we report the functional analyses of 4 domain-deletion mutants of XSIP1. Deletion of the N-terminus zinc finger domain suppressed neural induction and BMP inhibition, but these were not affected by deletion of the other domains (the Smad binding domain, the DNA-binding homeodomain together with the CtBP binding site and the C-terminus zinc finger). Therefore SIP1 does not inhibit BMP signaling by binding to Smad proteins. In contrast, all of the deletion constructs inhibited Xbra expression. These results suggest that the N-terminus zinc finger domain of XSIP1 has an important role in neural induction and that Xbra suppression occurs via a mechanism separate from the neural inducing activity.
Assuntos
Indução Embrionária , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Sistema Nervoso/embriologia , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas com Domínio T/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Animais , Embrião não Mamífero , Proteínas de Homeodomínio/genética , Mutação , Sistema Nervoso/metabolismo , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Proteínas com Domínio T/genética , Xenopus/embriologia , Proteínas de Xenopus/genética , Dedos de ZincoRESUMO
Nodal and Nodal-related proteins activate the Activin-like signal pathway and play a key role in the formation of mesoderm and endoderm in vertebrate development. Recent studies have shown additional activities of Nodal-related proteins apart from the canonical Activin-like signal pathway. Here we report a novel function of Nodal-related proteins using cleavage mutants of Xenopus nodal-related genes (cmXnr2 and cmXnr5), which are known to be dominant-negative inhibitors of nodal family signaling. cmXnr2 and cmXnr5 inhibited both BMP signaling and Wnt signaling without activating the Activin-like signal in animal cap assays. Pro region construct of Xnr2 and Xnr5 did not inhibit Xwnt8, and pro/mature region chimera mutant cmActivin-Xnr2 and cmActivin-Xnr5 also did not inhibit Xwnt8 activity. These results indicate that the pro domains of Xnr2 and Xnr5 are necessary, but not sufficient, for Wnt inhibition, by Xnr family proteins. In addition, Western blot analysis and immunohistochemistry analysis revealed that the unprocessed Xnr5 protein is stably produced and secreted as effectively as mature Xnr5 protein, and that the unprocessed Xnr5 protein diffused in the extracellular space. These results suggest that unprocessed Xnr2 and Xnr5 proteins may be involved in inhibiting both BMP and Wnt signaling and are able to be secreted to act on somewhat distant target cells, if these are highly produced.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Animais , Western Blotting , Proteínas Desgrenhadas , Embrião não Mamífero/metabolismo , Imuno-Histoquímica , Ligantes da Sinalização Nodal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Xenopus/genética , Xenopus/metabolismoRESUMO
Neural differentiation is induced by inhibition of BMP signaling. Secreted inhibitors of BMP such as Chordin from the Spemann organizer contribute to the initial step of neural induction. Xenopus Smad-interacting protein-1 gene (XSIP1) is expressed in neuroectoderm from the early gastrula stage through to the neurula stage. XSIP1 is able to inhibit BMP signaling and overexpression of XSIP1 induces neural differentiation. To clarify the function of XSIP1 in neural differentiation, we performed a loss-of-function study of XSIP1. Knockdown of XSIP1 inhibited SoxD expression and neural differentiation. These results indicate that XSIP1 is essential for neural induction. Furthermore, loss-of-function experiments showed that SoxD is essential for XSIP1 transcription and for neural differentiation. However, inhibition of XSIP1 translation prevented neural differentiation induced by SoxD; thus, SoxD was not sufficient to mediate neural differentiation. Expression of XSIP1 was also required for inhibition of BMP signaling. Together, these results suggest that XSIP1 and SoxD interdependently function to maintain neural differentiation.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Indução Embrionária/fisiologia , Proteínas de Homeodomínio/metabolismo , Sistema Nervoso/embriologia , Neurônios/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Diferenciação Celular/fisiologia , Regulação para Baixo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mesoderma/metabolismo , Sistema Nervoso/metabolismo , Oligodesoxirribonucleotídeos Antissenso/metabolismo , Transcrição Gênica/fisiologia , XenopusRESUMO
Lefty, antivin and related genes act in a feedback inhibition mechanism for nodal signaling at a number of stages of vertebrate embryogenesis. To analyze the function of the feedback inhibitor of nodal signaling, Xantivin in Xenopus embryos, we designed a morpholino antisense oligonucleotide (XatvMO) for this gene. XatvMO caused the expansion of mesodermal tissue and head defects. XatvMO-injected gastrulae showed up-regulated expression of the mesodermal markers Xbra, Xwnt8, Xnot, and Chordin, suggesting expansion of the trunk-tail organizer. As expected, depletion of Xantivin also up-regulated nodal signaling as confirmed by the enhanced ectopic expression of Xantivin mRNA, a known target gene of nodal signaling. Furthermore, we investigated the relationship between Xantivin and the EGF-CFC gene FRL-1, which is a component of the nodal receptor. In animal cap assays, FRL-1 could not induce expression of nodal-responsive genes, but could up-regulate expression of these genes when FRL-1 was coinjected with a low dose of Xnr1; coinjection of Xantivin suppressed this up-regulation by FRL-1. We also found that Xantivin can rescue the caudalized phenotype induced by overexpression of FRL-1. Co-immunoprecipitation assays showed that Xantivin interacted with the EGF-CFC proteins, FRL-1 and cripto. Taken together, these results suggest that Xantivin opposes the activity of EGF-CFC genes and thereby antagonizes nodal signaling.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/genética , Fator de Crescimento Transformador beta/fisiologia , Proteínas de Xenopus/genética , Proteínas de Xenopus/fisiologia , Animais , Sequência de Bases , Feminino , Técnicas In Vitro , Fatores de Determinação Direita-Esquerda , Mesoderma/efeitos dos fármacos , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Fenótipo , Transdução de Sinais , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/genética , Xenopus/embriologia , Xenopus/genética , Xenopus/fisiologia , Proteínas de Xenopus/antagonistas & inibidoresRESUMO
A gamete is derived initially from a presumptive primordial germ cell (pPGC) and transmits genetic potential to the next generation. Xenopus tropicalis, which is a close relative of Xenopus laevis, has a diploid genome and advantages for genetic and genomic research; however, little is known about the developmental mechanism of its germinal lineage. Here, we identified the Xenopus tropicalis DAZ-like gene (Xtdazl), which encodes RNA-binding proteins homologous to Xdazl in Xenopus laevis and examined the expression patterns of Xtdazl transcripts during embryogenesis. In this work, we showed that Xtdazl mRNA was localized in the germ plasm and was expressed from the previtellogenic oocyte to early tadpole, in testis and ovary. The same localization patterns have been reported in Xenopus laevis germ plasm and pPGCs. These results indicate that Xtdazl mRNA is the first specific marker of germ plasm and pPGCs in Xenopus tropicalis and is very useful to trace Xenopus tropicalis pPGCs, including germ plasm until the early tadpole stage.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Oogênese/genética , Proteínas de Ligação a RNA/genética , Espermatogênese/genética , Proteínas de Xenopus/genética , Xenopus/embriologia , Sequência de Aminoácidos , Animais , Diferenciação Celular/genética , Citoplasma/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Hibridização In Situ , Dados de Sequência Molecular , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Xenopus/genética , Proteínas de Xenopus/biossínteseRESUMO
In vertebrates, nodal-related genes are crucial for specifying mesendodermal cell fates. Six nodal-related genes have been identified in Xenopus, but only one, nodal, has been identified in the mouse. The Xenopus nodal-related gene 3 (Xnr3), however, lacks the mesoderm-inducing activity of the other five nodal-related genes in Xenopus, and can directly induce neural tissue in animal caps by antagonizing BMP signals. In this study, we isolated three clones of the Xenopus (Silurana) tropicalis nodal-related gene 3 (Xtnr3) and analyzed their function. The Xtnr3 genes show high homology to Xnr3 and have the same activity. Southern blot and genomic PCR analyses indicate that the X. tropicalis genome has duplications in the Xtnr3 gene sequences and our three clones represent separate gene loci. We also found a partial clone of Xtnr3 that coded for the N-terminal part of its pro-region. Surprisingly, this sequence also induced neural tissue by antagonizing BMP signals, and its coded protein physically associated with BMP4 mature protein. Furthermore, we showed that the pro-region of Xnr5 has the same activity. Together, these findings indicate that the pro-region of nodal-related genes acts antagonistically towards BMP signals, which identifies a novel mechanism for the inhibition of BMP signaling.
Assuntos
Indução Embrionária/fisiologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/genética , Proteínas de Xenopus , Xenopus/embriologia , Animais , Sequência de Bases , Southern Blotting , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/fisiologia , Primers do DNA , Hibridização In Situ , Microinjeções , Dados de Sequência Molecular , Testes de Precipitina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNA , Xenopus/genéticaRESUMO
Recent studies indicate an essential role for the EGF-CFC family in vertebrate development, particularly in the regulation of nodal signaling. Biochemical evidence suggests that EGF-CFC genes can also activate certain cellular responses independently of nodal signaling. Here, we show that FRL-1, a Xenopus EGF-CFC gene, suppresses BMP signaling to regulate an early step in neural induction. Overexpression of FRL-1 in animal caps induced the early neural markers zic3, soxD and Xngnr-1, but not the pan-mesodermal marker Xbra or the dorsal mesodermal marker chordin. Furthermore, overexpression of FRL-1 suppressed the expression of the BMP-responsive genes, Xvent-1 and Xmsx-1, which are expressed in animal caps and induced by overexpressed BMP-4. Conversely, loss of function analysis using morpholino-antisense oligonucleotides against FRL-1 (FRL-1MO) showed that FRL-1 is required for neural development. FRL-1MO-injected embryos lacked neural structures but contained mesodermal tissue. It was suggested previously that expression of early neural genes that mark the start of neuralization is activated in the presumptive neuroectoderm of gastrulae. FRL-1MO also inhibited the expression of these genes in dorsal ectoderm, but did not affect the expression of chordin, which acts as a neural inducer from dorsal mesoderm. FRL-1MO also inhibited the expression of neural markers that were induced by chordin in animal caps, suggesting that FRL-1 enables the response to neural inducing signals in ectoderm. Furthermore, we showed that the activation of mitogen-activated protein kinase by FRL-1 is required for neural induction and BMP inhibition. Together, these results suggest that FRL-1 is essential in the establishment of the neural induction response.