RESUMEN
Developmental control of proliferation relies on tight regulation of protein expression. Although this has been well studied in early embryogenesis, how the cell cycle is regulated during organogenesis is not well understood. Bruno-Like RNA binding proteins bind to consensus sequences in the 3'UTR of specific mRNAs and repress protein translation, but much of this functional information is derived from studies on mainly two members, Drosophila Bruno and vertebrate BrunoL2 (CUGBP1). There are however, six vertebrate and three Drosophila Bruno family members, but less is known about these other family members, and none have been shown to function in the endoderm. We recently identified BrunoL1 as a dorsal pancreas enriched gene, and in this paper we define BrunoL1 function in Xenopus endoderm development. We find that, in contrast to other Bruno-Like proteins, BrunoL1 acts to enhance rather than repress translation. We demonstrate that BrunoL1 regulates proliferation of endoderm cells through translational control of cyclin A2 mRNA. Specifically BrunoL1 enhanced translation of cyclin A2 through binding consensus Bruno Response Elements (BREs) in its 3'UTR. We compared the ability of other Bruno-Like proteins, both vertebrate and invertebrate, to stimulate translation via the cyclin A2 3'UTR and found that only Drosophila Bru-3 had similar activity. In addition, we also found that both BrunoL1 and Bru-3 enhanced translation of mRNAs containing the 3'UTRs of Drosophila oskar or cyclin A, which have been well characterized to mediate repression. Lastly, we show that it is the Linker region of BrunoL1 that is both necessary and sufficient for this activity. These results are the first example of BRE-dependent translational enhancement and are the first demonstration in vertebrates of Bruno-Like proteins regulating translation through BREs.
Asunto(s)
Ciclina A2/genética , Proteínas ELAV/fisiología , Endodermo/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/fisiología , Proteínas de Xenopus/fisiología , Regiones no Traducidas 3' , Animales , Proliferación Celular , Ciclina A2/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Embrión no Mamífero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Xenopus , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMEN
In mammals, it has been well established that gastrointestinal and pancreatic endocrine cells are specified by a cascade of different transcription factors, but whether these same pathways (or linear relationships) operate in Xenopus is currently unknown. We recently identified the endocrine-specific zinc finger transcription factor insulinoma associated protein 1 (insm1) as a dorsal-enriched gene. We found that insm1 is expressed in the dorsal pancreas as early as NF28, making it one of the earliest markers to be localized to the dorsal pancreas. Through morpholino-mediated knockdown, we demonstrate that insm1 is essential for proper specification of both gastrointestinal and pancreatic endocrine cells. In addition, we place insm1 downstream of ngn3 and upstream of pax6 and neuroD in the endocrine cell transcription factor cascade. These are the first results showing that the endodermal endocrine cell development in Xenopus uses the same transcriptional cascade as in mammals.
Asunto(s)
Células Enteroendocrinas , Tracto Gastrointestinal , Páncreas , Factores de Transcripción/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis , Animales , Biomarcadores/metabolismo , Células Enteroendocrinas/citología , Células Enteroendocrinas/fisiología , Tracto Gastrointestinal/citología , Tracto Gastrointestinal/embriología , Tracto Gastrointestinal/metabolismo , Regulación del Desarrollo de la Expresión Génica , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Páncreas/citología , Páncreas/embriología , Páncreas/metabolismo , Factores de Transcripción/genética , Proteínas de Xenopus/genética , Xenopus laevis/anatomía & histología , Xenopus laevis/embriología , Xenopus laevis/metabolismoRESUMEN
During embryogenesis, the pancreas develops from separate dorsal and ventral buds, which fuse to form the mature pancreas. Little is known about the functional differences between these two buds or the relative contribution of cells derived from each region to the pancreas after fusion. To follow the fate of dorsal or ventral bud derived cells in the pancreas after fusion, we produced chimeric Elas-GFP transgenic/wild-type embryos in which either dorsal or ventral pancreatic bud cells expressed GFP. We found that ventral pancreatic cells migrate extensively into the dorsal pancreas after fusion, whereas the converse does not occur. Moreover, we found that annular pancreatic tissue is composed exclusively of ventral pancreas-derived cells. To identify ventral pancreas-specific genes that may play a role in pancreatic bud fusion, we isolated individual dorsal and ventral pancreatic buds, prior to fusion, from NF38/39 Xenopus laevis tadpoles and compared their gene expression profiles (NF refers to the specific stage of Xenopus development). As a result of this screen, we have identified several new ventral pancreas-specific genes, all of which are expressed in the same location within the ventral pancreas at the junction where the two ventral pancreatic buds fuse. Morpholino-mediated knockdown of one of these ventral-specific genes, transmembrane 4 superfamily member 3 (tm4sf3), inhibited dorsal-ventral pancreatic bud fusion, as well as acinar cell differentiation. Conversely, overexpression of tm4sf3 promoted development of annular pancreas. Our results are the first to define molecular and behavioral differences between the dorsal and ventral pancreas, and suggest an unexpected role for the ventral pancreas in pancreatic bud fusion.