Studying signaling regulators of neurogenesis during mammalian central nervous system development

Abstract

During neurogenesis of the mammalian central nervous system (CNS), multiple regulators of different signaling pathways cooperate to temporally and spatially control neural progenitor maintenance, proliferation and differentiation. The Sonic Hedgehog (Shh) and Notch signaling are two critical pathways due to their individual and cooperative functions in neurogenesis. Suppressor of fused (Sufu) is a negative regulator in Shh signaling and the recombination signal binding protein for immunoglobulin kappa J region (Rbpj) is an indispensable transcriptional mediator in canonical Notch signaling. In order to investigate the functions of Sufu and Rbpj during neurogenesis, mouse genetic approach was adopted to conditionally delete these genes in the developing CNS, including the mid-hindbrain and hindbrain regions.

To determine the role of Sufu in neurogenesis, a Pax2-Cre mouse line was used to conditionally inactivate Sufu in the mid-hindbrain region at around embryonic day 8.0 (E8.0). Sufu mutant embryos showed severe enlargement of the mid-hindbrain tissue at E11.5. By neurosphere assay, my results demonstrated that Sufuknockout cells had a higher primary sphere-forming ability, but not from the second passage onwards, indicating that Sufu did not affect neural stem cell proliferation in the in vitro condition. As an attempt to understand the functions of Sufu, the expression of the neurogenesis regulator GSK3,,was analyzed. Interestingly, ablation of Sufu led to decreased activity of GSK3lllGSK3GGis known to regulate Notch signaling, suggesting the possibility that Sufu also crosstalk with Notch signaling in regulating neurogenesis. Indeed, expression of the Notch target, Hes1, was expanded in the Sufu-depleted hindbrain. Therefore, Sufu could function as an intracellular mediator for multiple signaling pathways during neurogenesis.

As a central mediator in canonical Notch signaling, the function of Rbpj during CNS neurogenesis was also studied. B2-r4-Cre was employed to inactivate Rbpj in rhombomere 4 (r4) at around E8.5. Depletion of Rbpj caused premature differentiation of neural progenitors as shown by the reduced number of Sox2-positive progenitors and concomitant increased Tuj1-positive neurons. Interestingly, progenitors in different dorsal-ventral domains displayed varied responses to Rbpj deletion with the most severe phenotype in the Nkx2.2 domain. The Nkx2.2-positive progenitors started to lose their apical intercellular integrity at E10.5 and the premature facial branchiomotor neurons from the domain protruded into the ventricle at E11.5. The progenitor proliferation rate analyzed by short pulse BrdU labeling assay, however, was not affected at E10.5 in Rbpj mutants. Together, these results suggested that Rbpj was differentially required in different progenitor domains to maintain their undifferentiated status.

This study highlighted the important functions of Sufu in regulating neural progenitor proliferation and the role of Rbpj in neural progenitor maintenance. The specific functions of Sufu and Rbpj in other parts of the developing CNS, their cooperation in mediating neurogenesis will require further investigations. It would also be interesting to study the spatial and temporal difference in the functions of these two signaling modulators in CNS neurogenesis.