Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 73
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Dev Biol ; 459(2): 138-148, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31846624

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 laevis
2.
Development ; 145(12)2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29769220

RESUMO

Retinoic acid (RA) is required for pancreas specification in Xenopus and other vertebrates. However, the gene network that is directly induced by RA signalling in this context remains to be defined. By RNA sequencing of in vitro-generated pancreatic explants, we identified the genes encoding the transcription factor Hnf1ß and the Wnt-receptor Fzd4/Fzd4s as direct RA target genes. Functional analyses of Hnf1b and Fzd4/Fzd4s in programmed pancreatic explants and whole embryos revealed their requirement for pancreatic progenitor formation and differentiation. Thus, Hnf1ß and Fzd4/Fzd4s appear to be involved in pre-patterning events of the embryonic endoderm that allow pancreas formation in Xenopus.


Assuntos
Receptores Frizzled/biossíntese , Fator 1-beta Nuclear de Hepatócito/biossíntese , Organogênese/genética , Pâncreas/embriologia , Tretinoína/metabolismo , Proteínas de Xenopus/biossíntese , Xenopus laevis/embriologia , Animais , Diferenciação Celular/genética , Receptores Frizzled/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Silenciamento de Genes , Fator 1-beta Nuclear de Hepatócito/genética , Morfolinos/genética , Via de Sinalização Wnt/genética , Proteínas de Xenopus/genética
3.
Cereb Cortex ; 30(7): 3921-3937, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32147726

RESUMO

The balance of excitation and inhibition is essential for cortical information processing, relying on the tight orchestration of the underlying subcellular processes. Dynamic transcriptional control by DNA methylation, catalyzed by DNA methyltransferases (DNMTs), and DNA demethylation, achieved by ten-eleven translocation (TET)-dependent mechanisms, is proposed to regulate synaptic function in the adult brain with implications for learning and memory. However, focus so far is laid on excitatory neurons. Given the crucial role of inhibitory cortical interneurons in cortical information processing and in disease, deciphering the cellular and molecular mechanisms of GABAergic transmission is fundamental. The emerging relevance of DNMT and TET-mediated functions for synaptic regulation irrevocably raises the question for the targeted subcellular processes and mechanisms. In this study, we analyzed the role dynamic DNA methylation has in regulating cortical interneuron function. We found that DNMT1 and TET1/TET3 contrarily modulate clathrin-mediated endocytosis. Moreover, we provide evidence that DNMT1 influences synaptic vesicle replenishment and GABAergic transmission, presumably through the DNA methylation-dependent transcriptional control over endocytosis-related genes. The relevance of our findings is supported by human brain sample analysis, pointing to a potential implication of DNA methylation-dependent endocytosis regulation in the pathophysiology of temporal lobe epilepsy, a disease characterized by disturbed synaptic transmission.


Assuntos
Metilação de DNA/genética , Endocitose/genética , Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Inibição Neural/genética , Sinapses/metabolismo , Animais , Clatrina , Proteínas do Citoesqueleto/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Epigenoma , Epilepsia do Lobo Temporal/genética , Humanos , Potenciais Pós-Sinápticos Inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Técnicas de Patch-Clamp , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vesículas Sinápticas/metabolismo , Transcriptoma
4.
J Neurosci ; 38(42): 9105-9121, 2018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30143575

RESUMO

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/genética
5.
Development ; 142(19): 3416-28, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26443638

RESUMO

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/metabolismo
6.
Cereb Cortex ; 27(12): 5696-5714, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29117290

RESUMO

The proliferative niches in the subpallium generate a rich cellular variety fated for diverse telencephalic regions. The embryonic preoptic area (POA) represents one of these domains giving rise to the pool of cortical GABAergic interneurons and glial cells, in addition to striatal and residual POA cells. The migration from sites of origin within the subpallium to the distant targets like the cerebral cortex, accomplished by the adoption and maintenance of a particular migratory morphology, is a critical step during interneuron development. To identify factors orchestrating this process, we performed single-cell transcriptome analysis and detected Dnmt1 expression in murine migratory GABAergic POA-derived cells. Deletion of Dnmt1 in postmitotic immature cells of the POA caused defective migration and severely diminished adult cortical interneuron numbers. We found that DNA methyltransferase 1 (DNMT1) preserves the migratory shape in part through negative regulation of Pak6, which stimulates neuritogenesis at postmigratory stages. Our data underline the importance of DNMT1 for the migration of POA-derived cells including cortical interneurons.


Assuntos
Movimento Celular/fisiologia , Córtex Cerebral/embriologia , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Interneurônios/enzimologia , Células-Tronco Neurais/enzimologia , Área Pré-Óptica/embriologia , Animais , Animais Recém-Nascidos , Contagem de Células , Sobrevivência Celular/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/enzimologia , Metilação de DNA , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/enzimologia , Interneurônios/citologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neurais/citologia , Crescimento Neuronal/fisiologia , Área Pré-Óptica/citologia , Área Pré-Óptica/enzimologia , Técnicas de Cultura de Tecidos , Transcriptoma , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo
7.
Dev Biol ; 417(1): 77-90, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27341758

RESUMO

Canonical Wnt signaling plays a dominant role in the development of the neural crest (NC), a highly migratory cell population that generates a vast array of cell types. Canonical Wnt signaling is required for NC induction as well as differentiation, however its role in NC migration remains largely unknown. Analyzing nuclear localization of ß-catenin as readout for canonical Wnt activity, we detect nuclear ß-catenin in premigratory but not migratory Xenopus NC cells suggesting that canonical Wnt activity has to decrease to basal levels to enable NC migration. To define a possible function of canonical Wnt signaling in Xenopus NC migration, canonical Wnt signaling was modulated at different time points after NC induction. This was accomplished using either chemical modulators affecting ß-catenin stability or inducible glucocorticoid fusion constructs of Lef/Tcf transcription factors. In vivo analysis of NC migration by whole mount in situ hybridization demonstrates that ectopic activation of canonical Wnt signaling inhibits cranial NC migration. Further, NC transplantation experiments confirm that this effect is tissue-autonomous. In addition, live-cell imaging in combination with biophysical data analysis of explanted NC cells confirms the in vivo findings and demonstrates that modulation of canonical Wnt signaling affects the ability of NC cells to perform single cell migration. Thus, our data support the hypothesis that canonical Wnt signaling needs to be tightly controlled to enable migration of NC cells.


Assuntos
Movimento Celular/fisiologia , Crista Neural/citologia , Fatores de Transcrição TCF/metabolismo , Fator 3 de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Hibridização In Situ , Indóis/farmacologia , Organogênese/fisiologia , Oximas/farmacologia , Crânio/embriologia , beta Catenina/metabolismo
8.
Dev Biol ; 405(2): 214-24, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26164657

RESUMO

The localization of certain mRNAs to the vegetal cortex of Xenopus oocytes is of crucial importance for germ cell development and early embryonic patterning. Vegetal RNA localization is mediated by cis-acting RNA localization elements (LE). Several proteins assemble on the RNA LE and direct transport to the vegetal cortex. Although a number of localization RNP components have been identified, their full composition is unknown. In an RNA affinity purification approach, using the dead end 1 (dnd1) RNA LE, we identified Xenopus Celf1 as a novel component of vegetal localization RNP complexes. Celf1 is part of an RNP complex together with known vegetal localization factors and shows specific interactions with LEs from several but not all vegetally localizing RNAs. Immunostaining experiments reveal co-localization of Celf1 with vegetally localizing RNA and with known localization factors. Inhibition of Celf1 protein binding by localization element mutagenesis as well as Celf1 overexpression interfere with vegetal RNA localization. These results argue for a role of Celf1 in vegetal RNA localization during Xenopus oogenesis.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Oogênese/fisiologia , Proteínas de Ligação a RNA/fisiologia , RNA/metabolismo , Proteínas de Xenopus/fisiologia , Xenopus laevis/fisiologia , Animais , Deleção de Genes , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Hibridização In Situ , Oócitos/citologia , Fases de Leitura Aberta , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem , Xenopus laevis/embriologia
9.
Dev Biol ; 386(2): 340-57, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24370451

RESUMO

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 laevis
10.
Cereb Cortex ; 24(3): 754-72, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23180754

RESUMO

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/metabolismo
11.
Proc Natl Acad Sci U S A ; 109(22): 8594-9, 2012 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-22592794

RESUMO

Liver and ventral pancreas develop from neighboring territories within the endoderm of gastrulae. ventral pancreatic precursor 1 (vpp1) is a marker gene that is differentially expressed in a cell population within the dorsal endoderm in a pattern partially overlapping with that of hematopoietically expressed homeobox (hhex) during gastrulation. In tail bud embryos, vpp1 expression specifically demarcates two ventral pancreatic buds, whereas hhex expression is mainly restricted to the liver diverticulum. Ectopic expression of a critical dose of hhex led to a greatly enlarged vpp1-positive domain and, subsequently, to the formation of giant ventral pancreata, putatively by conversion of intestinal to ventral pancreatic precursor cells. Conversely, antisense morpholino oligonucleotide-mediated knockdown of hhex resulted in a down-regulation of vpp1 expression and a specific loss of the ventral pancreas. Furthermore, titration of hhex with a dexamethasone-inducible hhex-VP16GR fusion construct suggested that endogenous hhex activity during gastrulation is essential for the formation of ventral pancreatic progenitor cells. These observations suggest that, beyond its role in liver development, hhex controls specification of a vpp1-positive endodermal cell population during gastrulation that is required for the formation of the ventral pancreas.


Assuntos
Embrião não Mamífero/metabolismo , Proteínas de Homeodomínio/genética , Mucosa Intestinal/metabolismo , Pâncreas/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Embrião não Mamífero/embriologia , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Gástrula/embriologia , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Hibridização In Situ , Intestinos/embriologia , Dados de Sequência Molecular , Pâncreas/embriologia , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Transfecção , Xenopus laevis/embriologia
12.
BMC Dev Biol ; 13: 36, 2013 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-24125469

RESUMO

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/genética
13.
Proc Natl Acad Sci U S A ; 107(37): 16148-53, 2010 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-20805475

RESUMO

Segregation of the future germ line defines a crucial cell fate decision during animal development. In Xenopus, germ cells are specified by inheritance of vegetally localized maternal determinants, including a group of specific mRNAs. Here, we show that the vegetal localization elements (LE) of Xenopus Dead end (XDE) and of several other germ-line-specific, vegetally localized transcripts mediate germ cell-specific stabilization and somatic clearance of microinjected reporter mRNA in Xenopus embryos. The part of XDE-LE critical for somatic RNA clearance exhibits homology to zebrafish nanos1 and appears to be targeted by Xenopus miR-18 for somatic mRNA clearance. Xenopus Elr-type proteins of the vegetal localization complex can alleviate somatic RNA clearance of microinjected XDE-LE and endogenous XDE mRNA. ElrB1 synergizes with Xenopus Dead end protein in the stabilization of XDE-LE mRNA. Taken together, our findings unveil a functional link of vegetal mRNA localization and the protection of germ-line mRNAs from somatic clearance.


Assuntos
MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Sequência de Bases , Proteína Semelhante a ELAV 2 , Embrião não Mamífero/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Células Germinativas/metabolismo , MicroRNAs/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Transcrição Gênica , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética
14.
Dev Biol ; 349(2): 169-78, 2011 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-21029731

RESUMO

Primordial germ cell (PGC) development in Xenopus embryos relies on localised maternal determinants. We report on the identification and functional characterisation of such one novel activity, a germ plasm associated mRNA encoding for the Xenopus version of a kinesin termed KIF13B. Modulations of xKIF13B function result in germ cell mismigration and in reduced numbers of such cells. PGCs explanted from Xenopus embryos form bleb-like protrusions enriched in PIP3. Knockdown of xKIF13B results in inhibition of blebbing and PIP3 accumulation. Interference with PIP3 synthesis leads to PGC mismigration in vivo and in vitro. We propose that xKIF13B function is linked to polarized accumulation of PIP3 and directional migration of the PGCs in Xenopus embryos.


Assuntos
Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Células Germinativas/fisiologia , Cinesinas/genética , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro/metabolismo , Xenopus laevis/embriologia , Animais , Movimento Celular/genética , Clonagem Molecular , Técnicas de Silenciamento de Genes , Hibridização In Situ , Cinesinas/metabolismo
15.
RNA Biol ; 8(5): 873-82, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21788733

RESUMO

Grip2.1 is a conserved PDZ-domain protein with a function in the context of primordial germ cell development and migration in Xenopus embryos. Its mRNA is maternally supplied and found to be associated with the germ plasm, located at the tip of the vegetal cortex in Xenopus oocytes. Here, we demonstrate that the 3'-UTR of XGrip2.1 contains a 211 nucleotide RNA signal sequence that promotes localization to the mitochondrial cloud via the early localization pathway upon injection into stage I oocytes. The same element is also capable of using the late transport pathway if injected into stage III/IV oocytes. In vitro protein interaction studies reveal binding to ElrA/B, Vg1RBP and VgRBP60, proteins that have previously been associated with the vegetal localization machinery. Mutational interference with Vg1RBP and VgRBP60 binding severely reduces early and late localization activity. Selective interference with Vg1RBP binding significantly reduces late localization while having only a mild effect on localization to the mitochondrial cloud, indicating that the signal sequences and protein machinery required for early and late pathway localization though overlapping are not identical.


Assuntos
Proteínas de Transporte/genética , Oócitos/metabolismo , Sinais Direcionadores de Proteínas/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Regiões 3' não Traduzidas , Animais , Proteínas de Transporte/metabolismo , Proteína Semelhante a ELAV 2 , Peptídeos e Proteínas de Sinalização Intracelular , Mitocôndrias/genética , Mutação , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Transdução de Sinais/genética , Proteínas de Xenopus/metabolismo
16.
Methods ; 51(1): 146-51, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20178845

RESUMO

Localized mRNAs have been identified in a large variety of cell types where they contribute to the establishment of cell asymmetries and can function as cell fate determinants. In Xenopus, RNAs that localize to the vegetal cortex during oogenesis function in early embryonic patterning as well as in the development of primordial germ cells. Based on their temporal and spatial localization patterns, vegetally localizing RNAs are referred to as either early-pathway RNAs which transiently localize in the mitochondrial cloud, or as late-pathway RNAs. Vegetal RNA-localization is driven by cis-acting signal sequences that, in most cases, were found to reside in the 3'-UTRs and which are recognized by trans-acting localization factors. Here we describe the methods of how vegetal RNA-localization elements can be identified by injection of fluorescently-labeled or tagged RNAs.


Assuntos
Técnicas Citológicas , Oócitos/citologia , Xenopus/metabolismo , Regiões 3' não Traduzidas , Animais , Endopeptidase K/metabolismo , Células Germinativas/citologia , Óperon Lac , Microinjeções , Microscopia de Fluorescência/métodos , Mitocôndrias/metabolismo , Hibridização de Ácido Nucleico , Oócitos/metabolismo , RNA/metabolismo , Ribonucleases/metabolismo
17.
Dev Biol ; 329(2): 327-37, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19285490

RESUMO

Canonical Wnt signalling is known to be involved in the regulation of differentiation and proliferation in the context of endodermal organogenesis. Wnt mediated beta-catenin activation is understood to be modulated by secreted Frizzled-related proteins, such as XsFRP5, which is dynamically expressed in the prospective liver/ventral pancreatic precursor cells during late neurula stages, becoming liver specific at tailbud stages and shifting to the posterior stomach/anterior duodenum territory during tadpole stages of Xenopus embryogenesis. These expression characteristics prompted us to analyse the function of XsFRP5 in the context of endodermal organogenesis. We demonstrate that XsFRP5 can form a complex with and inhibit a multitude of different Wnt ligands, including both canonical and non-canonical ones. Knockdown of XsFRP5 results in transient pancreatic hypoplasia as well as in an enlargement of the stomach. In VegT-injected animal cap explants, XsFRP5 can induce expression of exocrine but not endocrine pancreatic marker genes. Both, its expression characteristics as well as its interactions with XsFRP5, define Wnt2b as a putative target for XsFRP5 in vivo. Knockdown of Wnt2b results in a hypoplastic stomach as well as in hypoplasia of the pancreas. On the basis of these findings we propose that XsFRP5 exerts an early regulatory function in the specification of the ventral pancreas, as well as a late function in controlling stomach size via inhibition of Wnt signalling.


Assuntos
Glicoproteínas/fisiologia , Organogênese/fisiologia , Xenopus laevis/embriologia , Animais , Sequência de Bases , Padronização Corporal , Diferenciação Celular , Primers do DNA , Técnicas de Silenciamento de Genes , Glicoproteínas/genética , Imunoprecipitação , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteínas Wnt/metabolismo
18.
Front Cell Dev Biol ; 8: 639, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32793592

RESUMO

Increased life expectancy in modern society comes at the cost of age-associated disabilities and diseases. Aged brains not only show reduced excitability and plasticity, but also a decline in inhibition. Age-associated defects in inhibitory circuits likely contribute to cognitive decline and age-related disorders. Molecular mechanisms that exert epigenetic control of gene expression contribute to age-associated neuronal impairments. Both DNA methylation, mediated by DNA methyltransferases (DNMTs), and histone modifications maintain neuronal function throughout lifespan. Here we provide evidence that DNMT1 function is implicated in the age-related loss of cortical inhibitory interneurons. Dnmt1 deletion in parvalbumin-positive interneurons attenuates their age-related decline in the cerebral cortex. Moreover, conditional Dnmt1-deficient mice show improved somatomotor performance and reduced aging-associated transcriptional changes. A decline in the proteostasis network, responsible for the proper degradation and removal of defective proteins, is implicated in age- and disease-related neurodegeneration. Our data suggest that DNMT1 acts indirectly on interneuron survival in aged mice by modulating the proteostasis network during life-time.

19.
Methods Mol Biol ; 561: 65-72, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19504064

RESUMO

The possibility of generating transgenic animals is of obvious advantage for the analysis of gene function in development and disease. One of the established vertebrate model systems in developmental biology is the amphibian Xenopus laevis. Different techniques have been successfully applied to create Xenopus transgenics; in this chapter, the so-called meganuclease method is described. This technique is not only technically simple, but also comparably efficient and applicable to both Xenopus laevis and Xenopus tropicalis. The commercially available endonuclease I-SceI (meganuclease) mediates the integration of foreign DNA into the frog genome after coinjection into fertilized eggs. Tissue-specific gene expression, as well as germline transmission, has been observed.


Assuntos
Animais Geneticamente Modificados , Desoxirribonucleases de Sítio Específico do Tipo II/administração & dosagem , Fertilização in vitro/métodos , Técnicas de Transferência de Genes , Proteínas de Saccharomyces cerevisiae/administração & dosagem , Transgenes/fisiologia , Xenopus laevis/genética , Animais , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Embrião não Mamífero/metabolismo , Feminino , Mutação em Linhagem Germinativa , Microinjeções/métodos , Oócitos/citologia , Oócitos/fisiologia , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Distribuição Tecidual , Xenopus laevis/embriologia
20.
Cell Rep ; 26(13): 3522-3536.e5, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30917309

RESUMO

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 laevis
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA