The Sonic Hedgehog SHH pathway

The Sonic Hedgehog (SHH) pathway is active in the adult SEZ, where it has been proposed to regulate cell proliferation (Ruat et al., 2012; Ahn and Joyner, 2005; Machold et al., 2003) and to modulate the migration of neuroblasts exiting the niche (Angot et al., 2008). The mosaic inactivation of the Smoothened (SMO) receptor in cell types expressing the neuroepithelial marker NESTIN suggested the requirement of this transducer of SHH signal for maintenance of the NSC population (Balordi and Fishell, 2007). Patched (PTC) is the main SHH receptor and is considered an antagonist of the pathway (Briscoe and Thérond, 2013). Embryonic deletion of Ptc in multipotent stem pelitinib of human glial fibrillary acidic protein (hGFAP)-Cre;Ptc mice results in medulloblastoma. The tumors do not manifest until the cells have committed to the neuronal lineage (Yang et al., 2008). However, the effects of Ptc inactivation in adult NSCs of the SEZ remain yet unknown. Here, we used a tamoxifen-inducible Cre transgene under the control of the astrocyte-specific glutamate transporter (GLAST) expressed in astrocyte-like NSCs (Mori et al., 2006) and took advantage of a conditional Ptc knockout (Ptcfl/fl) mouse line that has loxP recombinase recognition sites within the Ptc gene (Uhmann et al., 2007). We show that Ptc inactivation in the adult NSCs leads to a dramatic decrease of the neurogenic process and to a marked expansion of NSCs in the SEZ. Neurogenesis blockade was related to a shift in NSC division mode from asymmetric to symmetric, leading to a decrease in the differentiation process and involving NOTCH signaling. Thus, we report a role for PTC in the regulation of adult NSC self-renewal mechanisms.
Results
Discussion Conditional Ptc inactivation in the adult NSCs allowed the study of HH signaling activation in a ligand-independent, cell-intrinsic fashion in the SEZ in vivo. This activation is likely due to the suspension of the inhibitory effect of PTC on SMO and may promote the generation of symmetric GLAST+/EGFR− pairs as observed in vitro. Moreover, our data suggest a likely alteration of the transition from aNSCs to TAPs, which might account for the high decrease of TAPs and subsequently of neuroblasts. The expression of NOTCH signaling and high-mobility group box transcription factors SOX2 and SOX9, all known to prevent neuronal differentiation (Scott et al., 2010; Ables et al., 2011), was also increased. Consistent with the alteration of the transition between aNSCs and TAPs, we also observed that the Ptc-deficient NSCs do not regenerate the SEZ properly after an antimitotic treatment. Nevertheless, our data do not allow excluding the potential existence of additional mechanisms such as, for instance, the alteration of TAP sensitivity to EGF, which would be consistent with the previously reported EGF-mediated induction of TAP proliferation (Doetsch et al., 2002). Sustained HH signaling activation in the SEZ of deficient mice maintains multipotency but influences the fate of SEZ-derived precursors toward either the oligodendroglial phenotype in the cc or the glutamatergic and dopaminergic phenotypes, two populations of OB periglomerular interneurons, the specification of which may be of high interest in the context of brain repair (Brill et al., 2009; Lledo et al., 2006). The long-lasting high activation of HH signaling is still detected 1 year after the recombination. However, the absence of major alteration of OB morphology we observed in the present work is consistent with previous reports showing that adult neurogenesis in the SEZ allows only a partial turnover of newborn neurons in the OB (Imayoshi et al., 2008; Ninkovic et al., 2007). Our results point out a likely interaction between HH and NOTCH signaling pathways in adult neurogenesis, suggesting that both should contribute to properly maintain adult NSCs, preventing them to become TAPs (Aguirre et al., 2010; Imayoshi et al., 2013; Kawaguchi et al., 2013). Such a crosstalk was proposed to be required for the regulation of neurogenic divisions and thus for proper corticogenesis in development (Dave et al., 2011). The constitutive activation of HH signaling through Ptc deletion in enteric neural crest cells also robustly activates NOTCH pathway via Hes1 upregulation and promotes premature gliogenesis, impairing these cells to form ganglia in the hindgut during enteric nervous system development (Ngan et al., 2011). In agreement with HH and NOTCH crosstalk in the adult SEZ, NSCs are both HH (Ahn and Joyner, 2005)- and NOTCH (Aguirre et al., 2010)-responding cells. If HES transcription factors are generally regarded as canonical NOTCH pathway effectors (Imayoshi et al., 2013), a NOTCH-independent HH/HES1 activity was reported in the progenitor cell proliferation of the retina (Wall et al., 2009). We do not know whether Hes1 is a direct target of HH signaling in the adult NSCs, but our results indicate that the effect of active HH signaling on NSCs is NOTCH dependent. TAPs were reported to regulate NSC self-renewal in vivo, and an interaction between EGFR and NOTCH pathways was proposed to have selective roles in this process with EGFR being an upstream regulator of NOTCH through a nonautonomous mechanism (Aguirre et al., 2010). Because the conditional Ptc deletion leads to a huge decrease in EGFR-expressing TAPs in the SEZ, the subsequent EGFR decrease might amplify NOTCH signaling induction and thus also participate in the observed effects. Likewise, SOX9 increase in the conditional Ptc mutant is consistent with data reporting that active HH signaling induces Sox9 transcription and results in the precocious ability of neuroepithelial cells to form neurospheres (Scott et al., 2010).