Identification and Functional Characterization of Novel Genes Involved in Primary Neurogenesis in Xenopus laevis

Abstract

The South African claw-toed frog, Xenopus laevis, is a good model to study neurogenesis in vertebrates. Early on, at the open neural plate stage, the territory of primary neurons is nicely defined by a set of neural genes expressed in a typical pattern in the form of stripes which define three bilateral groups of cells giving rise to motor neurons, interneurons, and sensory neurons, respectively. One subset comprises genes whose expression pattern mimics that of N-tubulin, a neuronal differentiation marker. This subset has been refered to as N-tubulin synexpression group, thanks to the observation that members of this group are involved in the differentiation of primary neurons. Another subset, designated Delta synexpression group, includes genes whose expression pattern is similar to that of X-Delta-1. Members of this group regulate the local cell-cell interactions in the context of primary neurogenesis known as lateral inhibition. Overall, these patterns of expression are a valuable criterion to search for novel genes involved in primary neurogenesis. We have analysed 500 clones from a Xenopus tailbud stage head cDNA library by use of a systematic expression pattern screen. From the panel of neurally expressed genes, five were expressed in form of stripes. Three of these genes have not been described in Xenopus. These genes are XPak3, a serine/threonine protein kinase, and X-Mxi1, a basic helix-loop-helix leucine zipper protein, both belonging to the N-tubulin synexpression group, and XSeb4, a RRM-type RNA binding protein, belonging to the X-Delta-1 synexpression group. Based on the fact that XPak3 and XSeb4 define regulatory molecules with unknown function in neurogenesis, their functional characterization was carried out. By embryo microinjection experiments, XPak3 and XSeb4 were found to be transcriptionally activated by the proneural factor X-Neurogenin related-1 (X-Ngnr-1) and repressed by lateral inhibition. Comparative expression pattern analysis showed that XPak3 but not XPak1 or XPak2 is a neuronally expressed XPak isoform. Interestingly, overexpression of a constitutively active form of XPak3, XPak3-myr, induced premature neuronal differentiation, a phenotype correlating with the induction of cell cycle arrest. Conversely, XPak3 loss-of-function, generated by using a morpholino antisense oligonucleotide, blocked the formation of primary neurons and induced increased cell proliferation. This inhibition of neurogenesis was rescued by coinjection of XPak3-myr. In conclusion, we propose that XPak3 is induced by neurogenin to inhibit the mitosis of neuronally programmed cells and thereby allowing for their differentiation. Ectopic expression of high concentrations (>150 pg) of XSeb4 inhibits the formation of primary neurons. Unexpectedly, the suppression of XSeb4 expression, using morpholino antisense oligonucleotide, also led to inhibition of neurogenesis. Microinjection of low concentrations of XSeb4 mRNA showed no effect on the expression of N-tubulin. These contradictory findings require further systematic investigations.