Sonic hedgehog controls stem cell behavior in the postnatal and adult brain

Abstract

Sonic hedgehog (Shh) signaling controls many aspects of ontogeny, orchestrating congruent growth and patterning. During brain development, Shh regulates early ventral patterning while later on it is critical for the regulation of precursor proliferation in the dorsal brain, namely in the neocortex, tectum and cerebellum. We have recently shown that Shh also controls the behavior of cells with stem cell properties in the mouse embryonic neocortex, and additional studies have implicated it in the control of cell proliferation in the adult ventral forebrain and in the hippocampus. However, it remains unclear whether it regulates adult stem cell lineages in an equivalent manner. Similarly, it is not known which cells respond to Shh signaling in stem cell niches. Here we demonstrate that Shh is required for cell proliferation in the mouse forebrain's subventricular zone (SVZ) stem cell niche and for the production of new olfactory interneurons in vivo. We identify two populations of Gli1+ Shh signaling responding cells: GFAP+ SVZ stem cells and GFAP- precursors. Consistently, we show that Shh regulates the self-renewal of neurosphere-forming stem cells and that it modulates proliferation of SVZ lineages by acting as a mitogen in cooperation with epidermal growth factor (EGF). Together, our data demonstrate a critical and conserved role of Shh signaling in the regulation of stem cell lineages in the adult mammalian brain, highlight the subventricular stem cell astrocytes and their more abundant derived precursors as in vivo targets of Shh signaling, and demonstrate the requirement for Shh signaling in postnatal and adult neurogenesis.

Fig. 1

Gli1 and Shh gene expression in the SVZ. (A,C) Expression of Shh mRNA in the lateral wall of the lateral ventricles (LV) of adult mice. At high magnification, Shh expression is clearly detected in SVZ cells (C). (B,D,F-H) Expression of Gli1 mRNA in the lateral wall of the lateral ventricle of adult (B,D) and postnatal (P3; G,H) mice. (E) Control section showing lack of hybridization of Shh antisense RNA probes in the fourth ventricle (4V) of an adult mouse. Sense probe controls gave no signal. (A-H) in situ hybridizations on cross sections. Arrows point to sites of expression. Dorsal is to the top. Scale bar in F: 400 μm for A,F; 200 μm for C-E; G, 150 μm; H, 30 μm.

Fig. 2

Gene expression analyses in cell populations in the postnatal and adult SVZ. (A) RT-PCR analyses of postnatal (P5) and adult sorted cells. Postnatal whole SVZ is also shown as control. (B) RT-PCR analyses of Shh expression in the SVZ and adjacent striatum (ST) from the same animal. Note that Shh is expressed in the adult SVZ but it is not detected in either B or E sorted cells (see text). All samples were tested with (+) or without (−) reverse transcriptase to control for any possible signal resulting from contaminating genomic DNA. (C) RT-PCR analyses of gene expression in P7 SVZ neurospheres (SVZ-NS). As positive control (+), RNA from a P7 brain was used. As a negative control P7 SVZ RNA without reverse transcriptase was used (−). As control for RT-PCR, all genes were found to be expressed in dissected but non-cell-sorted SVZ pieces. As control for RNA recovery and amounts of cDNA, the levels of the housekeeping gene Hprt were measured.

Fig. 3

Gene expression in single SVZ cells. (A) Schematic representation of the experimental procedure. Individual SVZ cells were randomly harvested with a patch-clamp pipette from fresh living slices, and were used for single cell multiplex RT-PCR. (B) Examples of RT-PCR assays showing gene expression in single SVZ cells. (C) Summary graph showing the expression of GFAP, Gli1 or both in single cells, shown as percentage of the total number of collected cells (n=65).

Fig. 4

Cyclopamine inhibits SVZ cell proliferation in vivo. (A,B,E) Cross section through the forebrain SVZ of an adult mouse showing normal BrdU incorporation (arrows in A,B) or the expression of GFAP and Nestin (E) following one week’s injection of HBC carrier (cyclodextrin) alone. Animals were perfused ~12–24 hours after the last injection. (C,D,F) Decrease of BrdU⁺ cells in adult mice treated with cyclopamine for one week (C,D) does not lead to the loss of Nestin⁺ or GFAP⁺ cells (F). (G) Quantification of the number of BrdU⁺ cells in the SVZ of control and cyclopamine-treated adult mice. Counts are averaged and shown per section. Error bars=s.e.m., n=13 for control HBC-injected mice and n=18 for cyclopamine-injected mice in four independent experiments pooled together. Out of 18 cyclopamine-injected mice, three animals did not respond, five animals decreased the number of BrdU⁺ cells by ~50%, and ten animals reduced incorporation by ~100%. No reduction was observed in the HBC-injected mice. (H) RT-PCR of fresh SVZ tissue from adult control or cyclopamine-treated mice, dissected 4 hours after the last injection. Hprt levels are used as loading controls.

Fig. 5

Reduced number of newborn interneurons in the adult olfactory bulb after cyclopamine treatment. (A) Experimental procedure. BrdU injections are done during cyclopamine or vehicle treatment. One month post-injection, the number of newborn neurons is quantified after BrdU staining. (B,C,D,E) Photographs of the BrdU staining in the olfactory bulb of vehicle- (B,C) or cyclopamine- (D,E) treated mice. (F) The number of BrdU⁺ cells in the olfactory bulb of cyclopamine-treated mice (open circle, n=5) is significantly reduced in comparison to vehicle-treated mice (filled circle, n=5) along the antero-posterior axis. Error bars=s.e.m.

Fig. 6

Shh signaling regulates SVZ proliferation and neurogenesis. (A) Quantification of the effects of Shh on the proliferation of dissociated P5 SVZ cells plated on a quiescent astrocytic monolayer. BrdU incorporation was quantified by immunofluorescence. Under these conditions, SVZ precursors proliferate and generate new neurons, as they normally do in vivo. (B) Quantification of the effects of blocking anti-Shh monoclonal antibody (5E1) on the proliferation of P5 SVZ cells after dissociation and reaggregation. Cell proliferation was measured by radioactive thymidine incorporation. (C) Quantification of the effect of Shh on neurogenesis in dissociated adult SVZ cells plated on an astrocytic monolayer. Generation of new neurons was measured by co-labeling with Tuj1, identifying neurons, and anti-BrdU antibodies, identifying cells that replicated after BrdU addition. (D) Quantification of the effects of Shh on isolated type A SVZ neuroblasts. Type A cells were purified from P5 mice and cultured with or without Shh. At 3 and 7 days, the number of Tuj1+ cells in Shh-treated cultures were compared to control cultures. Error bars=s.e.m. (E) Immunocytochemistry of a 7-day SVZ cell culture on an astrocytic monolayer showing the labeling of neurons with Tuj1 (red) and recently divided cells with anti-BrdU (green) antibodies. Note the large number of doubly labeled (yellow) cells representing newly born neurons. (F) Nomarski optics image of the same panel shown in E.

Fig. 7

Shh regulates proliferation and neurosphere formation in cooperation with EGF. (A) Image of neurospheres from adult SVZ cultures. (B) Quantification of the number of primary neurospheres formed from cultures of SVZ cells previously grown on astrocytic monolayers with or without exogenous Shh (5 nM). Shh was not added to the neurosphere cultures. (C) Synergism of Shh and EGF on neurosphere proliferation. The assay was done with a constant dose of EGF at 1 ng/ml, and varying doses of Shh at 5 or 0.5 nM (left) or with a constant does of Shh at 5 nM and varying doses of EGF at 5 and 0.5 ng/ml (right). Treatments were for 48 hours. (D,E) Quantification of proliferation as measured by the percentage of BrdU⁺ cells (D) and the number of clones obtained in cloning assays (E) in adult SVZ neurospheres treated with cyclopamine (5 μM) or treated with an equal dose of ethanol used as carrier for in vitro work. In all cases, error bars indicate s.e.m. of triplicate cultures.