RESUMO
An evolutionarily conserved subcircuit (kernel) dedicated to a specific developmental function is found at the top of the gene regulatory networks (GRNs) hierarchy. Here we comprehensively demonstrate that a pan-deuterostome endoderm specification kernel exists in zebrafish. We analyzed interactions among gata5, gata6, otx2 and prdm1a using specific morpholino knockdowns and measured the gene expression profiles by quantitative real-time RT-PCR and in situ hybridization. The mRNA rescue experiment validated the specificity of the morpholino knockdown. We found that the interactions among gata5, gata6, otx2 and prdm1a determine the initial specification of the zebrafish endoderm. Although otx2 can activate both gata5 and gata6, and the prdm1a/krox homologue also activates some endoderm transcription factors, a feedback loop from Gata to otx2 and prdm1a is missing. Furthermore, we found the positive regulation between gata5 and gata6 to further lock-on the mesendoderm specification by the Gata family. Chromatin immunoprecipitation was used to further validate the recruitment of Otx2 to the gata5 and gata6 loci. Functional assays revealed that module B of gata6 and the basal promoter of gata5 drive the gene at the mesendoderm, and mutational analysis demonstrated that Otx2 and Gata5/6 contribute to reporter gene activation. This is the first direct evidence for evolutionarily conserved endoderm specification across echinoderms and vertebrates.
Assuntos
Sequência Conservada/genética , Endoderma/embriologia , Evolução Molecular , Redes Reguladoras de Genes/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Sequência de Bases , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endoderma/efeitos dos fármacos , Endoderma/metabolismo , Epistasia Genética/efeitos dos fármacos , Fatores de Transcrição GATA/genética , Fatores de Transcrição GATA/metabolismo , Fator de Transcrição GATA5/genética , Fator de Transcrição GATA5/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Loci Gênicos/genética , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Fatores de Tempo , Proteínas de Peixe-Zebra/genéticaRESUMO
The genomic developmental program operates mainly through the regulated expression of genes encoding transcription factors and signaling pathways. Complex networks of regulatory genetic interactions control developmental cell specification and fates. Development in the zebrafish, Danio rerio, has been studied extensively and large amounts of experimental data, including information on spatial and temporal gene expression patterns, are available. A wide variety of maternal and zygotic regulatory factors and signaling pathways have been discovered in zebrafish, and these provide a useful starting point for reconstructing the gene regulatory networks (GRNs) underlying development. In this review, we describe in detail the genetic regulatory subcircuits responsible for dorsoanterior-ventroposterior patterning and endoderm formation. We describe a number of regulatory motifs, which appear to act as the functional building blocks of the GRNs. Different positive feedback loops drive the ventral and dorsal specification processes. Mutual exclusivity in dorsal-ventral polarity in zebrafish is governed by intra-cellular cross-inhibiting GRN motifs, including vent/dharma and tll1/chordin. The dorsal-ventral axis seems to be determined by competition between two maternally driven positive-feedback loops (one operating on Dharma, the other on Bmp). This is the first systematic approach aimed at developing an integrated model of the GRNs underlying zebrafish development. Comparison of GRNs' organizational motifs between different species will provide insights into developmental specification and its evolution. The online version of the zebrafish GRNs can be found at http://www.zebrafishGRNs.org.
Assuntos
Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Embrião não Mamífero/citologiaRESUMO
The type V transforming growth factor-beta (TGF-beta) receptor (TbetaR-V) is hypothesized to be involved in cellular growth inhibition by TGF-beta(1). Recently, TbetaR-V was found to be identical to low density lipoprotein receptor-related protein-1 (LRP-1). Here we demonstrate that TGF-beta(1) inhibits growth of wild-type CHO cells but not LRP-1-deficient mutant cells (CHO-LRP-1(-) cells). Stable transfection of CHO-LRP-1(-) cells with LRP-1 cDNA restores the wild-type morphology and the sensitivity to growth inhibition by TGF-beta(1). In addition, overexpression of LRP-1 minireceptors exerts a dominant negative effect and attenuates the growth inhibitory response to TGF-beta(1) in wild-type CHO cells. These results suggest that LRP-1/TbetaR-V is critical for TGF-beta(1)-mediated growth inhibition in CHO cells.
Assuntos
Células CHO/fisiologia , Divisão Celular/fisiologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Tamanho Celular , Cricetinae , Endocitose/fisiologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Fator de Crescimento Transformador beta1RESUMO
The type V TGF-beta receptor (TbetaR-V)/IGFBP-3 receptor mediates the IGF-independent growth inhibition induced by IGFBP-3. It also mediates the growth inhibitory response to TGF-beta1 in concert with other TGF-beta receptor types, and its loss may contribute to the malignant phenotype of human carcinoma cells. Here we demonstrate that TbetaR-V is identical to LRP-1/alpha2M receptor as shown by MALDI-TOF analysis of tryptic peptides of TbetaR-V purified from bovine liver. In addition, 125I-IGFBP-3 affinity-labeled TbetaR-V in Mv1Lu cells is immunoprecipitated by antibodies to LRP-1 and TbetaR-V. RAP, an LRP-1 antagonist, inhibits binding of 125I-TGF-beta1 and 125I-IGFBP-3 to TbetaR-V and diminishes IGFBP-3-induced growth inhibition in Mv1Lu cells. Absent or low levels of LRP-1, as with TbetaR-V, have been linked to the malignant phenotype of carcinoma cells. Mutagenized Mv1Lu cells selected for reduced expression of LRP-1 have an attenuated growth inhibitory response to TGF-beta1 and IGFBP-3. LRP-1-deficient mouse embryonic fibroblasts lack a growth inhibitory response to TGF-beta1 and IGFBP-3. On the other hand, stable transfection of H1299 human lung carcinoma cells with LRP-1 cDNA restores the growth inhibitory response. These results suggest that the LRP-1/TbetaR-V/IGFBP-3 receptor is required for the growth inhibitory response to IGFBP-3 and TGF-beta1.
