Regulation der Neurogenese durch bHLH-O-Proteine in Xenopus laevis

Abstract

Transcriptional activators and repressors of the bHLH protein family are involved in multiple developmental processes, including the formation of the nervous system in vertebrates and invertebrates. bHLH-O proteins play a particularly important role in the determination of neural cell fates and the regulation of neurogenesis. The Drosophila Hairy and E(spl)-related bHLH repressors can inhibit neural and neuronal differentiation through the repression of proneural bHLH protein activity. To gain further insight into the regulative mechanisms and the function of numerous bHLH-O proteins during neurogenesis, new members of the bHLH-O protein family in Xenopus were identified in silico and several of these proteins were further characterized.In this work, XHes2, a novel Hairy and E(spl)-related bHLH protein was identified that is closely related to the mammalian Hes2. The expression of XHes2 in the developing sensory organs and central nervous system implicates this gene as a potential regulator of neurogenesis. Studies analyzing the regulation of XHes2 in embryos revealed that XHes2 cannot be induced within the neural plate by the proneural bHLH protein X-Ngnr-1 or by the activation of the Notch signalling pathway. XHes2 expression in neural plate stages is restricted to the otic placode and some few cells within the neural plate. Taken together, these results indicate that XHes2, in contrast to Hes5 related genes such as ESR1 und ESR7, is not involved in the Notch-dependent lateral inhibition during primary neurogenesis. In misexpression studies using hormone inducible XHes2 variants in Xenopus embryos and animal caps, XHes2 inhibits neurogenesis similar to other bHLH-O proteins. This was obtained not only by the negative transcriptional regulation of X-Ngnr-1 und NeuroD, but also by the inhibition of NeuroD activity. The ectopic expression of NeuroD in late neurula stages upon injection of a XHes2 antisense morpholino oligonucleotide supports the hypothesis that XHes2 in developing sensory organs and forming CNS inhibits neuronal differentiation and thereby influences cell fate.While most of the mammalian bHLH-O proteins have only a single homologous protein in Xenopus, eight different mouse Hes5 related proteins could be identified in Xenopus. Out of these, the highly related proteins ESR8, ESR9 und ESR10 were analysed in more detail. During embryonic development, the expression domains of these Notch inducible genes are very similar. All three genes are expressed in the proneural domains, in the developing somites and in the pancreas. The expression pattern in neural plate stages implicates a regulatory function of these proteins during primary neurogenesis, probably by Notch dependent lateral inhibition. The misexpression of hormone inducible variants of ESR8, ESR9 and ESR10 in embryos from the midgastrula stage onwards support this hypothesis as the transcription of X-Ngnr-1 und N-tubulin is negatively regulated by inducible wildtype proteins and positively regulated by corresponding DNA binding mutants. Furthermore, ESR8 inhibits not only the transcription of X-Ngnr-1, but also the activity of X-Ngnr-1. Alternatively, the reduction of primary neurons upon injection of ESR8/ESR9/ESR10 specific antisense morpholino oligonucleotides in embryos suggests another function of these proteins, which takes place before lateral inhibition and demonstrates their necessity for the correct temporal and stereotypical differentiation of primary neurons.