Genetic regulation of mouse hindbrain neural progenitor development by suppressor of fused (Sufu) and hedgehog signaling

Abstract

Hedgehog (Hh)-Gli signaling regulates multiple aspects of nervous system development such as progenitor specification and proliferation. Suppressor of fused (Sufu) negatively modulates mammalian Hh signaling by restraining Gli activity. Spop is an adaptor protein required for Gli2 and Gli3 degradation, which is antagonized by Sufu. Besides Gli, Sufu prevents β-catenin nuclear localization. Sufu mutations caused many defects in murine neural development, all relating to mis-specification, altered proliferation or differentiation of neural progenitors. Correct neural tube development is based on precise establishment and maintenance of neural progenitor pools that demand spatial-temporally regulated cell proliferation and differentiation, of which the genetic regulatory mechanisms are unclear. This study aims to investigate the functions of Sufu and its downstream genetic network, especially regarding Spop, Gli and β-catenin, in organizing neural progenitor specification, proliferation and differentiation during mouse hindbrain neurogenesis.

Sufu was deleted using Hoxb2-r4-Cre in developing mouse hindbrain rhombomere 4 (r4Cre;Sufu f/f) and via CRISPR-Cas9 technology in P19 embryonic teratocarcinoma stem cells. Proliferation analysis and computational modeling was performed across multiple stages to determine proliferation periods and cell cycle parameters of progenitors along D-V axis of r4. Pro-neural genes were examined by in situ hybridizations. Sufu mutant dorsal Pax6+ progenitors exhibited better progenitor maintenance than control Pax6+ cells, manifested by lengthened proliferation period, longer S phase duration and disrupted Mash1 expression. Upon retinoic acid treatment, Sufu mutant P19 cells showed compromised neuronal differentiation, consistent with the notion that genetic removal of Sufu inhibited neurogenesis.

In r4Cre;Sufu f/f embryos, more ventral-fated Nkx2.2+ progenitors were identified than control, yet the proliferation changes occurred later and milder in mutant Nkx2.2+ than Pax6+ cells, which was associated with the enriched expressions of Gli2/3 in dorsal r4. R4Cre;Sufu f/f; Gli2 f/- brains, where major Gli activator was removed besides Sufu, showed restored dorsal-ventral patterning, Pax6+ cell proliferation and Mash1 expression. In r4Cre;Sufu f/f;Spop f/f brains, cell cycle defects were exacerbated in Pax6+ domain but not in Nkx2.2+ domain lacking Gli expression. These findings argued that Sufu regulated Gli during r4 neurogenesis, and the Gli availability at different D-V positions was critical to coordinate progenitor patterning and maintenance in r4Cre;Sufu f/f brains. To tackle the cross-talk between Sufu and β-catenin in r4 neurogenesis, a gain-of-function model of β-catenin was utilized. The third exon encoding β-catenin phosphodegron was removed upon Hoxb2-r4-Cre expression (r4Cre;Ex3 f/+). The resulting mutant, stabilized β-catenin increased proliferation, decreased cell cycle exit and inhibited differentiation, as was observed in r4Cre;Sufu f/f embryos. In r4Cre;Ex3 f/+ brains, Sufu could not affect neural progenitor proliferation, suggesting an underlying genetic regulatory hierarchy between Sufu and β-catenin. However, lack of Pax6 and Neurogenin1/2 were observed in r4Cre;Ex3 f/+ brains only, arguing that β-catenin was not the main mediator of Sufu in r4. Taken together, this study examined the functions of Sufu-Gli signaling in the spatial-temporal coordination of neural progenitor specification, proliferation and differentiation in hindbrain. Stabilization of β-catenin caused a lack of patterning and pro-neural genes expression in hindbrain, implying a specific mechanism through which β-catenin suppressed differentiation. This study furthers the understanding towards the regulation of neural patterning and progenitor maintenance by molecular signaling network.