On molecular mechanisms of neural differentiation and forebrain development

Sammanfattning: Telencephalic development is an extraordinary complex process where neural structures of highly diverse cell compositions emerge as the result of multiple developmental strategies imposing tight regulation, balance and timing of gene expression. Embryonic telencephalic neural stem cells (NSCs) can form the major cell types found in the telencephalon, and are thus an excellent model for studying transcriptional control of lineage choice mechanisms in CNS development. In paper I, we show that a member of the BTB/POZ family, Zbtb20, is required for proper repression of genes involved in interneuron and oligodendrocyte differentiation as well as proliferation and cell cycle exit in NSCs. Zbtb20 was expressed in the subpallium in a Dlx and Mash1 overlapping fashion and interacted with the co-regulator SMRT. siRNA-mediated knockdown of Zbtb20 in NSCs modulated expression of key regulatory genes in control of differentiation and proliferation, including Dlx1/2/5 and p57kip2, thus affecting the outcome of cell fate acquisition in favor of oligodendrocytic differentiation. Zbtb20 protein was accumulated in regulatory regions of the Dlx1/2 bi-gene cluster along with a T3-dependent accumulation to the MBP M1 promoter. Furthermore, the zebrafish homolog to Zbtb20 appeared to be necessary for proper gastrulation and formation of neuroectoderm in vivo possibly due to precautious expression of p57kip2, analogous to the NSCs phenotype. In paper II, we suggest a novel role for the cyclin-dependent kinase inhibitor p57Kip2 as a context-dependent repressor of neurogenic transcription factors and telencephalic neuronal differentiation. p57Kip2 interacted with pro-neuronal basic helix-loop-helix factors such as Mash1. Increased levels of p57Kip2 inhibited Mash1 transcriptional activity in a cyclin-dependent kinase (CDK) independent manner and acted as a direct repressor in transcriptional assays. Proliferating telencephalic neural progenitors co-expressed basal levels of Mash1 and p57Kip2, and endogenous p57Kip2 accumulated transiently in the nuclei of NSCs during early stages of astrocyte differentiation, independent of cell-cycle exit and at times when Mash1 expression was still prominent. p57Kip2 repressed neuronal differentiation, but exerted little or no effect astroglial differentiation of NSCs. In paper III we propose that Zbtb45 is a novel regulator of glial differentiation. Zbtb45 mRNA was ubiquitously expressed in the developing CNS in mouse embryos but Zbtb45 mRNA knockdown in NSCs resulted in a rapid decrease in the expression of oligodendrocyte characteristic genes and significantly increased numbers of astrocytes. Zbtb45 mRNA knockdown in oligodendrocyte precursors completely inhibited oligodendrocyte differentiation upon mitogen withdrawal as assessed by a down-regulation of the expression of markers for oligodendrocytic differentiation such as CNPase, MBP and Sox10, whereas CD44 expression was found to be increased in all experiments. In paper IV we propose that the previously uncharacterized protein CXXC5 is acting as a BMP4 induced inhibitor of Wnt signaling in neural stem cells. CXXC5 expression overlapped with BMP4 adjacent to Wnt3a expression in the dorsal regions of the telencephalon. CXXC5 showed partial homology with Idax, shown to interact with the Wnt-signaling intermediate Dishevelled (Dvl). Indeed CXXC5 and Dvl colocalized in NSCs and interacted in vitro. CXXC5 over-expression attenuated Wnt signaling. Interestingly, BMP4-induced decrease in Axin2 levels was abolished by siRNA-mediated knockdown of CXXC5.