RESUMO
The hormone Erythroferrone (ERFE) is a member of the C1q/TNF-related protein family that regulates iron homeostasis through the suppression of hamp. In a gain of function screen in Xenopus embryos, we identified ERFE as a potent secondary axis-inducing agent. Experiments in Xenopus embryos and ectodermal explants revealed that ERFE functions as a selective inhibitor of the BMP pathway and the conserved C1q domain is not required for this activity. Inhibition occurs at the extracelluar level, through the interaction of ERFE with the BMP ligand. During early Xenopus embryogenesis, erfe is first expressed in the ventral blood islands where initial erythropoiesis occurs and later in circulating blood cells. ERFE knockdown does not alter the expression of etv.2, aplnr and flt1 in tailbud stage embryos indicating endothelial cell specification is independent of ERFE. However, in tadpole embryos, defects of the vascular network and primitive blood circulation are observed as well as edema formation. RNAseq analysis of ERFE morphant embryos also revealed the inhibition of gja4 indicating disruption of dorsal aorta formation.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Sistema Cardiovascular/embriologia , Colágeno/metabolismo , Citocinas/metabolismo , Proteínas Musculares/metabolismo , Hormônios Peptídicos/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Colágeno/genética , Citocinas/genética , Ectoderma/metabolismo , Desenvolvimento Embrionário/genética , Eritrócitos/metabolismo , Eritropoese/genética , Feminino , Técnicas de Silenciamento de Genes , Masculino , Proteínas Musculares/genética , Hormônios Peptídicos/genética , RNA-Seq , Transdução de Sinais/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
In humans, many cases of congenital insensitivity to pain (CIP) are caused by mutations of components of the NGF/TrkA signaling pathway, which is required for survival and specification of nociceptors and plays a major role in pain processing. Mutations in PRDM12 have been identified in CIP patients that indicate a putative role for this transcriptional regulator in pain sensing. Here, we show that Prdm12 expression is restricted to developing and adult nociceptors and that its genetic ablation compromises their viability and maturation. Mechanistically, we find that Prdm12 is required for the initiation and maintenance of the expression of TrkA by acting as a modulator of Neurogenin1/2 transcription factor activity, in frogs, mice, and humans. Altogether, our results identify Prdm12 as an evolutionarily conserved key regulator of nociceptor specification and as an actionable target for new pain therapeutics.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurogênese/fisiologia , Nociceptores/citologia , Animais , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Transporte/genética , Linhagem Celular , Evolução Molecular , Feminino , Gânglios Sensitivos/citologia , Técnicas de Inativação de Genes , Células-Tronco Embrionárias Humanas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Crista Neural/citologia , Nociceptores/metabolismo , Receptor trkA/metabolismo , Tretinoína/fisiologia , Xenopus laevisRESUMO
Specification of dorsoventral regional identity in progenitors of the developing telencephalon is a first pivotal step in the development of the cerebral cortex and basal ganglia. Previously, we demonstrated that the two zinc finger doublesex and mab-3 related (Dmrt) genes, Dmrt5 (Dmrta2) and Dmrt3, which are coexpressed in high caudomedial to low rostrolateral gradients in the cerebral cortical primordium, are separately needed for normal formation of the cortical hem, hippocampus, and caudomedial neocortex. We have now addressed the role of Dmrt3 and Dmrt5 in controlling dorsoventral division of the telencephalon in mice of either sex by comparing the phenotypes of single knock-out (KO) with double KO embryos and by misexpressing Dmrt5 in the ventral telencephalon. We find that DMRT3 and DMRT5 act as critical regulators of progenitor cell dorsoventral identity by repressing ventralizing regulators. Early ventral fate transcriptional regulators expressed in the dorsal lateral ganglionic eminence, such as Gsx2, are upregulated in the dorsal telencephalon of Dmrt3;Dmrt5 double KO embryos and downregulated when ventral telencephalic progenitors express ectopic Dmrt5 Conditional overexpression of Dmrt5 throughout the telencephalon produces gene expression and structural defects that are highly consistent with reduced GSX2 activity. Further, Emx2;Dmrt5 double KO embryos show a phenotype similar to Dmrt3;Dmrt5 double KO embryos, and both DMRT3, DMRT5 and the homeobox transcription factor EMX2 bind to a ventral telencephalon-specific enhancer in the Gsx2 locus. Together, our findings uncover cooperative functions of DMRT3, DMRT5, and EMX2 in dividing dorsal from ventral in the telencephalon.SIGNIFICANCE STATEMENT We identified the DMRT3 and DMRT5 zinc finger transcription factors as novel regulators of dorsoventral patterning in the telencephalon. Our data indicate that they have overlapping functions and compensate for one another. The double, but not the single, knock-out produces a dorsal telencephalon that is ventralized, and olfactory bulb tissue takes over most remaining cortex. Conversely, overexpressing Dmrt5 throughout the telencephalon causes expanded expression of dorsal gene determinants and smaller olfactory bulbs. Furthermore, we show that the homeobox transcription factor EMX2 that is coexpressed with DMRT3 and DMRT5 in cortical progenitors cooperates with them to maintain dorsoventral patterning in the telencephalon. Our study suggests that DMRT3/5 function with EMX2 in positioning the pallial-subpallial boundary by antagonizing the ventral homeobox transcription factor GSX2.
Assuntos
Proteínas de Homeodomínio/fisiologia , Células-Tronco Neurais/fisiologia , Neurônios/fisiologia , Telencéfalo/embriologia , Fatores de Transcrição/fisiologia , Animais , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Telencéfalo/metabolismo , Fatores de Transcrição/genéticaRESUMO
V1 interneurons are inhibitory neurons that play an essential role in vertebrate locomotion. The molecular mechanisms underlying their genesis remain, however, largely undefined. Here, we show that the transcription factor Prdm12 is selectively expressed in p1 progenitors of the hindbrain and spinal cord in the frog embryo, and that a similar restricted expression profile is observed in the nerve cord of other vertebrates as well as of the cephalochordate amphioxus. Using frog, chick and mice, we analyzed the regulation of Prdm12 and found that its expression in the caudal neural tube is dependent on retinoic acid and Pax6, and that it is restricted to p1 progenitors, due to the repressive action of Dbx1 and Nkx6-1/2 expressed in the adjacent p0 and p2 domains. Functional studies in the frog, including genome-wide identification of its targets by RNA-seq and ChIP-Seq, reveal that vertebrate Prdm12 proteins act as a general determinant of V1 cell fate, at least in part, by directly repressing Dbx1 and Nkx6 genes. This probably occurs by recruiting the methyltransferase G9a, an activity that is not displayed by the amphioxus Prdm12 protein. Together, these findings indicate that Prdm12 promotes V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes, and suggest that this function might have only been acquired after the split of the vertebrate and cephalochordate lineages.
Assuntos
Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Morfogênese/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Células de Renshaw/fisiologia , Xenopus/embriologia , Animais , Sequência de Bases , Embrião de Galinha , Imunoprecipitação da Cromatina , Biologia Computacional , Primers do DNA/genética , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Rombencéfalo/metabolismo , Análise de Sequência de RNA , Especificidade da Espécie , Medula Espinal/metabolismoRESUMO
The adult frog retina retains a reservoir of active neural stem cells that contribute to continuous eye growth throughout life. We found that Yap, a downstream effector of the Hippo pathway, is specifically expressed in these stem cells. Yap knock-down leads to an accelerated S-phase and an abnormal progression of DNA replication, a phenotype likely mediated by upregulation of c-Myc. This is associated with an increased occurrence of DNA damage and eventually p53-p21 pathway-mediated cell death. Finally, we identified PKNOX1, a transcription factor involved in the maintenance of genomic stability, as a functional and physical interactant of YAP. Altogether, we propose that YAP is required in adult retinal stem cells to regulate the temporal firing of replication origins and quality control of replicated DNA. Our data reinforce the view that specific mechanisms dedicated to S-phase control are at work in stem cells to protect them from genomic instability.
Assuntos
Divisão Celular , Período de Replicação do DNA , Instabilidade Genômica , Retina/citologia , Células-Tronco/fisiologia , Transativadores/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Xenopus , Proteínas de Sinalização YAPRESUMO
The basic helix-loop-helix (bHLH) transcriptional activator Ptf1a determines inhibitory GABAergic over excitatory glutamatergic neuronal cell fate in progenitors of the vertebrate dorsal spinal cord, cerebellum and retina. In an in situ hybridization expression survey of PR domain containing genes encoding putative chromatin-remodeling zinc finger transcription factors in Xenopus embryos, we identified Prdm13 as a histone methyltransferase belonging to the Ptf1a synexpression group. Gain and loss of Ptf1a function analyses in both frog and mice indicates that Prdm13 is positively regulated by Ptf1a and likely constitutes a direct transcriptional target. We also showed that this regulation requires the formation of the Ptf1a-Rbp-j complex. Prdm13 knockdown in Xenopus embryos and in Ptf1a overexpressing ectodermal explants lead to an upregulation of Tlx3/Hox11L2, which specifies a glutamatergic lineage and a reduction of the GABAergic neuronal marker Pax2. It also leads to an upregulation of Prdm13 transcription, suggesting an autonegative regulation. Conversely, in animal caps, Prdm13 blocks the ability of the bHLH factor Neurog2 to activate Tlx3. Additional gain of function experiments in the chick neural tube confirm that Prdm13 suppresses Tlx3(+)/glutamatergic and induces Pax2(+)/GABAergic neuronal fate. Thus, Prdm13 is a novel crucial component of the Ptf1a regulatory pathway that, by modulating the transcriptional activity of bHLH factors such as Neurog2, controls the balance between GABAergic and glutamatergic neuronal fate in the dorsal and caudal part of the vertebrate neural tube.
Assuntos
Diferenciação Celular/fisiologia , Neurônios GABAérgicos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Histona-Lisina N-Metiltransferase/metabolismo , Tubo Neural/embriologia , Proteínas de Xenopus/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Embrião de Galinha , Primers do DNA/genética , Eletroporação , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Imuno-Histoquímica , Imunoprecipitação , Hibridização In Situ , Camundongos , Tubo Neural/citologia , Fator de Transcrição PAX2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
Scratch genes (Scrt) are neural-specific zinc-finger transcription factors (TFs) with an unknown function in the developing brain. Here, we show that, in addition to the reported expression of mammalian Scrt2 in postmitotic differentiating and mature neurons in the developing and early postnatal brain, Scrt2 is also localized in subsets of mitotic and neurogenic radial glial (RGP) and intermediate (IP) progenitors, as well as in their descendants-postmitotic IPs and differentiating neurons at the border subventricular/intermediate zone. Conditional activation of transgenic Scrt2 in cortical progenitors in mice promotes neuronal differentiation by favoring the direct mode of neurogenesis of RGPs at the onset of neurogenesis, at the expense of IP generation. Neuronal amplification via indirect IP neurogenesis is thereby extenuated, leading to a mild postnatal reduction of cortical thickness. Forced in vivo overexpression of Scrt2 suppressed the generation of IPs from RGPs and caused a delay in the radial migration of upper layer neurons toward the cortical plate. Mechanistically, our results indicate that Scrt2 negatively regulates the transcriptional activation of the basic helix loop helix TFs Ngn2/NeuroD1 on E-box containing common target genes, including Rnd2, a well-known major effector for migrational defects in developing cortex. Altogether, these findings reveal a modulatory role of Scrt2 protein in cortical neurogenesis and neuronal migration.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Movimento Celular/genética , Neocórtex/fisiologia , Neurogênese/genética , Neurônios/fisiologia , Fatores de Transcrição/genética , Animais , Animais Recém-Nascidos , Linhagem Celular Transformada , Células Cultivadas , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Neocórtex/citologia , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Xenopus , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
BACKGROUND: Members of the vertebrate Numb family of cell fate determinants serve multiple functions throughout early embryogenesis, including an essential role in the development of the nervous system. The Numb proteins interact with various partner proteins and correspondingly participate in multiple cellular activities, including inhibition of the Notch pathway. RESULTS: Here, we describe the expression characteristics of Numb and Numblike (NumbL) during Xenopus development and characterize the function of NumbL during primary neurogenesis. NumbL, in contrast to Numb, is expressed in the territories of primary neurogenesis and is positively regulated by the Neurogenin family of proneural transcription factors. Knockdown of NumbL afforded a complete loss of primary neurons and did not lead to an increase in Notch signaling in the open neural plate. Furthermore, we provide evidence that interaction of NumbL with the AP-2 complex is required for NumbL function during primary neurogenesis. CONCLUSION: We demonstrate an essential role of NumbL during Xenopus primary neurogenesis and provide evidence for a Notch-independent function of NumbL in this context.
Assuntos
Proteínas do Tecido Nervoso/fisiologia , Neurogênese/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevis/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Embrião não Mamífero , Feminino , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Genes Essenciais , Camundongos , Família Multigênica , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Placa Neural/metabolismo , Neurogênese/genética , Neurônios/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
The Neurogenin (Ngn 1-3) family of proneural basic helix-loop-helix (bHLH) transcription factors are key regulators of vertebrate neurogenesis. In the developing vertebrate nervous system, the Ngns are essential for the commitment to a neuronal fate and participate in the specification of neuronal cell-type identity. Xenopus laevis is widely used as a model system to study the early events of vertebrate neurogenesis, however, only Ngnr-1, which is most closely related to the mammalian Ngn2, has been described and characterized. Presently, we describe a comparative expression analysis of the Ngn1-3 in X. tropicalis and X. laevis embryos. The Xenopus Ngns are present in overlapping, as well as unique regions of the nervous system starting at gastrula stages, suggesting distinct roles for this important family of transcriptional factors in the establishment of the amphibian nervous system.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Embrião não Mamífero/metabolismo , Dados de Sequência Molecular , Filogenia , Xenopus/embriologia , Xenopus laevis/embriologia , Xenopus laevis/metabolismoRESUMO
VegT represents a localized maternal determinant essentially required for endoderm formation in Xenopus. Here, we report on the identification of the RNA-binding protein XSeb4R as a positive regulator of VegT. XSeb4R interacts directly with the 3'-untranslated region of VegT mRNA, stabilizes it, and stimulates translation. Ablation of XSeb4R activity results in impairment of endoderm and mesoderm formation, while ectopic expression of XSeb4R in ectodermal cells induces endodermal and mesodermal gene expression. These observations unravel a novel mode of VegT regulation at the post-transcriptional level that is essential for germ layer formation in Xenopus.
Assuntos
Camadas Germinativas/embriologia , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas com Domínio T/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Endoderma/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/metabolismo , Mesoderma/embriologia , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Xenopus laevis/metabolismoRESUMO
BACKGROUND: In recent years, considerable knowledge has been gained on the molecular mechanisms underlying retinal cell fate specification. However, hitherto studies focused primarily on the six major retinal cell classes (five types of neurons of one type of glial cell), and paid little attention to the specification of different neuronal subtypes within the same cell class. In particular, the molecular machinery governing the specification of the two most abundant neurotransmitter phenotypes in the retina, GABAergic and glutamatergic, is largely unknown. In the spinal cord and cerebellum, the transcription factor Ptf1a is essential for GABAergic neuron production. In the mouse retina, Ptf1a has been shown to be involved in horizontal and most amacrine neurons differentiation. RESULTS: In this study, we examined the distribution of neurotransmitter subtypes following Ptf1a gain and loss of function in the Xenopus retina. We found cell-autonomous dramatic switches between GABAergic and glutamatergic neuron production, concomitant with profound defects in the genesis of amacrine and horizontal cells, which are mainly GABAergic. Therefore, we investigated whether Ptf1a promotes the fate of these two cell types or acts directly as a GABAergic subtype determination factor. In ectodermal explant assays, Ptf1a was found to be a potent inducer of the GABAergic subtype. Moreover, clonal analysis in the retina revealed that Ptf1a overexpression leads to an increased ratio of GABAergic subtypes among the whole amacrine and horizontal cell population, highlighting its instructive capacity to promote this specific subtype of inhibitory neurons. Finally, we also found that within bipolar cells, which are typically glutamatergic interneurons, Ptf1a is able to trigger a GABAergic fate. CONCLUSION: Altogether, our results reveal for the first time in the retina a major player in the GABAergic versus glutamatergic cell specification genetic pathway.
Assuntos
Linhagem da Célula , Neurônios/citologia , Retina/citologia , Fatores de Transcrição/fisiologia , Ácido gama-Aminobutírico/fisiologia , Animais , Sequência de Bases , Primers do DNA , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Xenopus laevisRESUMO
The collaspin response mediator proteins (CRMPs) are a family of cytosolic phosphoproteins which play a critical role in the establishment of neuronal polarity and growth cone guidance. Here, we describe the temporal and spatial expression of CRMP-4 during early Xenopus embryogenesis. CRMP-4 transcripts were first detected by whole mount in situ hybridization at the end of gastrulation in the prospective neuroectoderm. During open neural plate stages, CRMP-4 was expressed broadly throughout the anterior neural plate and in the three bilateral stripes of the posterior neural plate where primary neurons arise. The expression in the territories of primary neurogenesis prefigures that of the post-mitotic neuronal marker N-tubulin. At tadpole stages, expression was maintained throughout the central nervous system and in the retina of the eye. Consistent with the observed expression, CRMP-4 transcripts are positively regulated by X-ngnr-1 and negatively by Notch signaling. The observed expression and regulation of CRMP-4 differ from that of the CRMP-2, which is induced by the events of neural induction.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/embriologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Sequência de Aminoácidos , Animais , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Embrião não Mamífero , Gástrula , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Sistema Nervoso/citologia , Sistema Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fases de Leitura Aberta , Retina/embriologia , Retina/metabolismo , Homologia de Sequência de AminoácidosRESUMO
We have isolated and characterized Xenopus Mxi1, a member of the Myc/Max/Mad family of bHLHZip transcription factors. Xmxi1 transcripts are present during gastrulation and early neurula stages, earlier and in broader domains as compared to the neuronal determination factor neurogenin (X-ngnr-1). Consistent with an early role in neurogenesis, Xmxi1 is positively regulated by Sox3, SoxD, and proneural genes, as well as negatively by the Notch pathway. Loss-of-function experiments demonstrate an essential role for Xmxi1 in the establishment of a mature neural state that can be activated by factors that induce neuronal differentiation, such as SoxD and X-ngnr-1. Overexpression of Xmxi1 in Xenopus embryos results in ectopic activation of Sox3, an early pan-neural marker of proliferating neural precursor cells. Within the neural plate, the neuronal differentiation marker N-tubulin and cell cycle control genes such as XPak3 and p27(Xic1) are inhibited, but the expression of early determination and differentiation markers, including X-ngnr-1 and X-MyT1, is not affected. Inhibition of neuronal differentiation by Xmxi1 is only transient, and, at early tailbud stages, both endogenous and ectopic neurogenesis are observed. While Xmxi1 enhances cell proliferation and apoptosis in the early Xenopus embryo, both activities appear not to be required for the function of Xmxi1 in primary neurogenesis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Sistema Nervoso Central/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Organogênese , Proteínas Supressoras de Tumor/fisiologia , Xenopus/embriologia , Xenopus/genética , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , DNA Complementar , Embrião não Mamífero , Biblioteca Gênica , Hibridização In Situ , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
The Myc-Max-Mad network of transcription factors plays an essential role in many cellular processes such as proliferation, differentiation, and apoptosis. The Mad proteins heterodimerize with Max, function as transcriptional repressors, and are capable of antagonizing the transforming activity of Myc. We report on the isolation of Xmad1, Xmad3, and Xmnt, novel Xenopus genes belonging to the Mad family. We also describe their temporal and spatial expression patterns during Xenopus embryogenesis. Xmad1 expression is found primarily in cells that have undergone terminal differentiation including the notochord, floor plate, and cement gland. Xmad3 transcripts are expressed broadly throughout the central nervous system and the eye, starting at neurula stages. In contrast, Xmnt expression in the CNS was localized anteriorly and, in addition, is present in the migrating neural crest cells. This study demonstrates the Mads are expressed in specific and mostly nonoverlapping patterns, suggesting distinct roles during embryogenesis.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Fúngicas/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteínas Repressoras/genética , Proteínas de Xenopus/genética , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/isolamento & purificação , Embrião não Mamífero/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/isolamento & purificação , Biblioteca Gênica , Dados de Sequência Molecular , Especificidade de Órgãos , Proteínas Repressoras/biossíntese , Proteínas Repressoras/isolamento & purificação , Alinhamento de Sequência , Proteínas de Xenopus/biossíntese , Proteínas de Xenopus/isolamento & purificação , Xenopus laevisRESUMO
The HIVEP gene family encodes for very large sequence-specific DNA binding proteins containing multiple zinc fingers. Three mammalian paralogous genes have been identified, HIVEP1, -2 and -3, as well as the closely related Drosophila gene, Schnurri. These genes have been found to directly participate in the transcriptional regulation of a variety of genes. Mammalian HIVEP members have been implicated in signaling by TNF-alpha and in the positive selection of thymocytes, while Schnurri has been shown to be an essential component of the TGF-beta signaling pathway. In this study, we describe the isolation of Xenopus HIVEP1, as well as partial cDNAs of HIVEP2 and -3. Analysis of the temporal and spatial expression of the XHIVEP transcripts during early embryogenesis revealed ubiquitous expression of the transcripts. Assays using Xenopus oocytes mapped XHIVEP1 domains that are responsible for nuclear export and import activity. The DNA binding specificity of XHIVEP was characterized using a PCR-mediated selection and gel mobility shift assays.
Assuntos
DNA Complementar/isolamento & purificação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição , Proteínas de Xenopus , Xenopus/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Núcleo Celular/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Expressão Gênica , Genes , Proteínas de Homeodomínio/genética , Dados de Sequência Molecular , Oócitos/metabolismo , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas , RNA Mensageiro/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Transcrição Gênica , Xenopus/metabolismo , Dedos de Zinco/genéticaRESUMO
Members of the Xvent-2 homeodomain transcription factor family are immediate response genes of BMP-4 signaling. The bone morphogenetic protein response element (BRE) of Xvent-2B was previously identified and characterized with respect to Smad1 and Smad4 binding sites. In this study, we further report on the transcriptional regulation of Xvent-2B. We provide evidence that Xvent-2B (Xvent-2) maintains its own expression through autoregulation. This activity was demonstrated for the endogenous gene by reverse transcriptase-PCR analysis and was found to be insensitive to cycloheximide. Localized by DNase I footprinting were several Xvent-2 binding sites within the proximal upstream region including the BRE. In the early Xenopus embryo, the BRE was shown to be sufficient to drive expression of a green fluorescent protein reporter in a similar pattern compared with the endogenous gene. Furthermore, Xvent-2B was able to activate the BRE in luciferase reporter assays, and in co-injection experiments Xvent-2B and Smad1 were found to synergistically activate the BRE. Moreover, glutathione S-transferase pull-down experiments demonstrated that Xvent-2B directly and specifically interacts with Smad1. This association was mediated by the MH1 domain of Smad1 and required the C-terminal domain of Xvent-2. The failure of an Xvent-2 mutant lacking the C terminus to stimulate the BRE underlines the significance of the C-terminal domain in the described autoregulatory loop.