RESUMO
In vertebrates, the periodic formation of somites from the presomitic mesoderm (PSM) is driven by the molecular oscillator known as the segmentation clock. The hairy-related gene, hes6/her13.2, functions as a hub by dimerizing with other oscillators of the segmentation clock in zebrafish embryos. Although hes6 exhibits a posterior-anterior expression gradient in the posterior PSM with a peak at the tailbud, the detailed mechanisms underlying this unique expression pattern have not yet been clarified. By establishing several transgenic lines, we found that the transcriptional regulatory region downstream of hes6 in combination with the hes6 3'UTR recapitulates the endogenous gradient of hes6 mRNA expression. This downstream region, which we termed the PT enhancer, possessed several putative binding sites for the T-box and Ets transcription factors that were required for the regulatory activity. Indeed, the T-box transcription factor (Tbx16) and Ets transcription factor (Pea3) bound specifically to the putative binding sites and regulated the enhancer activity in zebrafish embryos. In addition, the 3'UTR of hes6 is required for recapitulation of the endogenous mRNA expression. Furthermore, the PT enhancer with the 3'UTR of hes6 responded to the inhibition of retinoic acid synthesis and fibroblast growth factor signaling in a manner similar to endogenous hes6. The results showed that transcriptional regulation by the T-box and Ets transcription factors, combined with the mRNA stability given by the 3'UTR, is responsible for the unique expression gradient of hes6 mRNA in the posterior PSM of zebrafish embryos.
Assuntos
Regiões 3' não Traduzidas/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Elementos Facilitadores Genéticos/genética , Mesoderma/embriologia , Proteínas Repressoras/genética , Somitos/embriologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Padronização Corporal/genética , Embrião não Mamífero/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Dados de Sequência Molecular , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Somitos/efeitos dos fármacos , Somitos/metabolismo , Cauda/embriologia , Tretinoína/farmacologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
In the zebrafish segmentation clock, hairy/enhancer of split-related genes her1, her7, and hes6 encodes components of core oscillators. Since the expression of cyclic genes proceeds rapidly in the presomitic mesoderm (PSM), these hairy-related mRNAs are subject to strict post-transcriptional regulation. In this study, we demonstrate that inhibition of the CCR4-NOT deadenylase complex lengthens poly(A) tails of hairy-related mRNAs and increases the amount of these mRNAs, which is accompanied by defective somite segmentation. In transgenic embryos, we show that EGFP mRNAs with 3'UTRs of hairy-related genes exhibit turnover similar to endogenous mRNAs. Our results suggest that turnover rates of her1, her7, and hes6 mRNAs are differently regulated by the CCR4-NOT deadenylase complex possibly through their 3'UTRs in the zebrafish PSM.