Differential gene induction by genetic and ligand-mediated activation of the Sonic hedgehog pathway in neural stem cells

Abstract

Sonic hedgehog (Shh), a secreted morphogen and mitogen, is essential for nervous system development and neural stem cell (NSC) self-renewal. As the intracellular signal transduction of Shh in NSCs is largely unknown, we sought to characterize pathway targets using ligand stimulation and genetic models of activation. NSCs haploinsufficient for Patched (Ptc), a receptor repressive to Shh signaling, showed enhanced proliferation of a magnitude similar to Shh-treated wild-type (Wt) NSCs. Analysis of the Gli zinc-finger transcription factors, primary mediators of Shh activity, demonstrated differential induction between models of pathway activation. Gli1 was significantly induced in Wt NSCs exposed to Shh, whereas Gli2 was elevated and Gli1 expression did not change in Ptc(+/-) NSCs. Other Shh targets (Nmyc, Id factors) were induced under both conditions of pathway activation. Interestingly, Shh-treated Ptc(+/-) NSCs induced expression of Gli1 but failed to increase proliferation, suggesting that the NSCs may have reached a physiologic plateau in proliferative capacity. Thus, our data demonstrate that Ptc(+/-) mice have an expanded progenitor cell niche in vivo and that NSCs maintain a cell-intrinsic increase in basal proliferation in vitro that is sustained by a Gli transduction signature distinct from that of exogenous Shh stimulation. Additionally, Ptc(+/-) NSCs maintain tight control over mitosis and do not further augment proliferation in the presence of mitogenic stimulation.

Proteoglycan interactions with Sonic Hedgehog specify mitogenic responses

“…Whereas there were no discernable differences between Gli1 and Gli3 protein levels of wild type and mutant cerebella, clear differences were seen in Gli2 proteins. Gli2 is the major transcription factor driving Shh-induced proliferation41,48 and proliferation is reduced in multiple locations pre- and post-natally in Shh Ala / Ala animals. Gli2 proteins can function as either transcriptional activators or repressors; unprocessed Gli2 functions as a weak transcriptional activator37,39, while Gli2 cleavage and removal of the C-terminal activator domain generates a transcriptional repressor39.…”

“…Gli2 expression and processing are both Shh-regulated39,48. The ability of proteoglycans to affect the levels and proportions of the Gli2 isoforms in vivo and in vitro would alter Shh-dependent transcriptional activation and repression, and so could account for increases and decreases in Shh-target gene expression.…”

Reciprocal actions of ATF5 and Shh in proliferation of cerebellar granule neuron progenitor cells

“…cDNA was transcribed from total RNA with First-Strand cDNA Synthesis for Quantitative RT-PCR Kit (Marlingen Biosciences). Primers for Gli1 and GAPDH and qRT-PCR parameters are described in Galvin et al (2007). Analyses of amplification curves of real-time fluorescence and of melting curves were performed as described previously (Troy et al, 2001).…”

Therapeutic doses of cranial irradiation induce hippocampus-dependent cognitive deficits in young mice

“…To quantify the effects of irradiation on neurogenesis in the subgranular zone an equal number of irradiated and nonirradiated control mice (n = 3 in each group) were sacrificed at 72 h and 1 month post-radiation and tissue was processed as previously described [27]. For immunohistochemical characterization and quantification of cells within the hippocampus, sections of 8 lm thickness were collected at 50 lm intervals from three regions of the hippocampus, corresponding to 1.46 mm behind bregma (rostral) and to 2.54 mm behind Bregma (caudal) (Mouse Brain Atlas in Stereotactic Co-ordinates, Plaxinos & Franklin, second edition) as previously described [15,28].…”