Persistent sonic hedgehog signaling in adult brain determines neural stem cell positional identity - PubMed (original) (raw)

Persistent sonic hedgehog signaling in adult brain determines neural stem cell positional identity

Rebecca A Ihrie et al. Neuron. 2011.

Abstract

Neural stem cells (NSCs) persist in the subventricular zone (SVZ) of the adult brain. Location within this germinal region determines the type of neuronal progeny NSCs generate, but the mechanism of adult NSC positional specification remains unknown. We show that sonic hedgehog (Shh) signaling, resulting in high gli1 levels, occurs in the ventral SVZ and is associated with the genesis of specific neuronal progeny. Shh is selectively produced by a small group of ventral forebrain neurons. Ablation of Shh decreases production of ventrally derived neuron types, while ectopic activation of this pathway in dorsal NSCs respecifies their progeny to deep granule interneurons and calbindin-positive periglomerular cells. These results show that Shh is necessary and sufficient for the specification of adult ventral NSCs.

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Figures

Figure 1. Gli1 Expression is Primarily Ventral

(A–F) Dorsal and ventral areas shown in images A–F are indicated in the cartoon at left. In situ hybridization for the three Gli family members reveals that gli1 mRNA is present in the ventral SVZ (D), but not the dorsal SVZ (A). gli2 and gli3 mRNA are present at low levels in both the dorsal and ventral SVZ (B,C,E,F). Scale bars: 50 microns. (G–J) LacZ staining in either Ptc-lacZ (G, I) or gli1-nlacZ (H, J) reporter strains is strongest in the ventral SVZ (I, J) and is reduced or absent in the dorsal SVZ (G, H). Red dashed lines indicate the lateral ventricle. (K) qRT-PCR detection of Gli family members in microdissected ventral SVZ and septum recapitulates the expression observed in in situ hybridization. Values shown are relative transcript expression compared to microdissected dorsal SVZ. Nkx2.1 and Nkx6.2, which are known to be upregulated in ventral SVZ, are highly expressed in both ventral SVZ samples (blue bars) and RNA derived from the septum (red bars). gli1 is expressed more highly in the ventral SVZ and septum than in the dorsal SVZ. gli2 and gli3 transcript expression did not differ significantly between the dorsal and ventral SVZ, but both genes were expressed to higher levels in the septum. Asterisks = p < 0.05.

Figure 2. High Hedgehog Pathway Activity is Limited to the Ventral SVZ

(A–L) Lineage tracing of Gli1-expressing cells in the SVZ. Schematic at top indicates the experimental timeline. Immediately after 5 days of tamoxifen administration in Gli1CreERT2; R26YFP animals, a small number of YFP/GFAP double-positive cells are present in the ventral SVZ (arrowhead and inset, D). YFP-labeled cells are absent from the dorsal SVZ, RMS, and OB (A, G, J). At 5 days after tamoxifen administration, YFP-labeled cells are present in the ventral SVZ, and some YFP labeling coincides with GFAP labeling (arrowheads and inset, E). YFP-labeled cells are also present in the dorsal SVZ (B), medial septum (right side in E), RMS (H), and the core of the OB, where doublecortin (Dcx)-expressing migrating neuroblasts enter this structure (K). One month after tamoxifen administration, there is widespread YFP labeling in the SVZ (C, F), RMS (I), and OB (L). Scale bars: 50 microns. (M–O) Quantification of the localization of labeled granular layer neurons in Gli1CreERT2; R26YFP animals at one month after tamoxifen administration (red bars, M). The neuronal progeny of labeled cells tend to localize in the deep OB granular layer close to the core, a phenotype that is consistent with an origin in the ventral SVZ. The distributions of cells resulting from dorsal (dark blue) or ventral (light blue) viral injection are shown for comparison. Data shown are average +/− SEM (n in figure is number of animals). This localization can be observed consistently in multiple samples (N, O). The OB core (dashed line) is located at the bottom of both images.

Figure 3. Sonic Hedgehog-Producing Cells Localize to the Ventral Forebrain and Can Be Labeled Via the SVZ or Lateral Ventricles

(A–D) Colorimetric staining for YFP expression in coronal sections of ShhCreER; R26YFP animals 4 days after tamoxifen treatment. Clusters of labeled cells are visible in the medial septal nucleus (red box in left section in A, enlarged in B), the preoptic nuclei (middle section in A), and the bed nuclei of the stria terminalis (red boxes in middle section of A, enlarged in D). A small number of labeled cells approached the ventral regions of the lateral ventricles (D). We did not observe YFP-labeled processes or cell bodies in the dorsal SVZ (red box in middle section of A, enlarged in C). Scale bars throughout figure: 50 microns. (E–G) Labeled cells in ShhCreER; R26YFP animals extend processes towards the ventral SVZ. Frontal sections of the ventral tip of the SVZ (delineated by red dashed lines) are shown in F and G with accompanying computerized tracing of processes shown in white. En-face visualization of a whole-mount specimen is shown with tracing of processes in G. (H–J) Labeled cells in ShhCreER; R26YFP animals express the neuronal marker NeuN (H). A subset of these neurons express the neurotransmitter GABA (I) or the cholinergic cell marker choline acetyltransferase (ChAT, shown in J). No colabeling of YFP and the neuronal subtype marker tyrosine hydroxylase (TH) was observed (not shown). (K–M) Colabeling of Shh-producing cells in the septum with Fluorogold retrograde tracer. At 4 days after tamoxifen treatment and 2 days after injection of Fluorogold into the lateral ventricle of ShhCreER; R26YFP mice, many Fluorogold-labeled cells (L) can be observed in the medial septum. YFP-positive cells are present in this region (K), and these cells are also labeled with Fluorogold (arrow, M). (N–R) Shh protein is present in the septum and SVZ. (N) Antibody against Shh protein robustly labels the Purkinje cells of P5 cerebellum, a known source of Shh ligand. In tamoxifen-treated ShhCreER; tdTomato mice, Shh protein is found around the ventral SVZ, and is found in the cell body of an RFP-labeled neuron (arrow, O). Within the septum, Shh is present in the neuropil and in cell bodies associated with NeuN-positive nuclei (arrows, P). In the dorsal SVZ, minimal staining is observed, although infrequent punctuate staining is present (Q). In contrast, higher levels of Shh protein can be observed in and around the ventral SVZ (red dashed line, R).

Figure 4. Loss of Shh Alters the Production of Olfactory Interneurons

(A–F) Treatment of ShhCreER/Shhfl animals with tamoxifen results in the loss of Shh protein and gli1 expression. Schematic at top shows the timeline of tamoxifen and BrdU administration. At six weeks after tamoxifen administration, Shh protein is present in the septum and ventral SVZ of control animals (A, C). The SVZ is outlined by a red dashed line. In situ hybridization for gli1 shows the expected pattern of ventral expression in Shhfl/+ animals (arrowhead, E). Shh protein is largely absent in ShhCreER/Shhfl animals (B, D). gli1 expression is also substantially reduced or absent in ShhCreER/Shhfl animals, indicating that pathway activity is compromised (arrowhead, F). White scale bars throughout figure: 50 microns. Black scale bars: 500 microns. (G) The distribution of newly born granule neurons in the OB is altered in the absence of Shh. At three weeks after a one-week pulse of BrdU in Shhfl/+ animals, labeled cells are distributed throughout the granular layers of the OB, with a greater percentage of the total present in the deep granule layers close to the core (blue bars, G). However, in ShhCreER/Shhfl animals, BrdU-labeled cells were less frequent in the deep granule layers and more prevalent in the superficial layers of the OB. Quantification of the distribution of BrdU-labeled cells by genotype shows a statistically significant decrease in the number of deep granule cells and concomitant increase in the number of superficial granule cells (* - p = 0.01, unpaired t test). Data shown are average +/− SEM. N indicates number of biological replicates counted, with at least 100 OB granular layer cells counted per animal. (H–J) The production of calbindin-positive periglomerular cells is decreased in the absence of Shh. At three weeks after a one-week pulse of BrdU, a subpopulation of BrdU-labeled cells in the periglomerular layer of Shhfl/+ OBs are calbindin-positive (colabeling shown in I). This population is substantially reduced in ShhCreER/Shhfl animals (p = 0.0033, unpaired t test).

Figure 5. Dorsal SVZ Cells Are Resistant to Pathway Activation

(A–H) Infusion of Hedgehog pathway agonists in the SVZ. Schematic at top illustrates pump cannula placement, allowing infusion of solution into the SVZ and CSF. Infusion of vehicle (sterile saline) does not alter the predominantly ventral distribution of cells labeled by the Gli1CreERT2; R26YFP reporter (A,E). Infusion of either the small-molecule antagonist cyclopamine (B,F) or an anti-Shh antibody (C,G) results in a reduction in YFP-positive cells. Administration of the small-molecule agonist SAG causes a robust increase in YFP labeling in the ventral SVZ (H), but does not cause a similar upregulation of YFP expression in the dorsal SVZ (D). Scale bars: 50 microns.

Figure 6. High Pathway Activity Causes Relocalization of Neuronal Progeny

(A–L) Ad:GFAPpCre was injected into the dorsal or ventral SVZ of R26R and SmoM2-YFP/R26R animals and brains were collected one month later to analyze labeled progeny. LacZ labeling within the SVZ (A, D, G, J) confirmed the site of virus injection. Dorsal injections in control animals primarily generated superficial labeled cells (B) that were distant from the core of the OB (indicated by a dashed line). Injections in the dorsal SVZ of SmoM2 animals gave rise to deep cells located close to the core of the OB (E), similar to those derived from ventral viral injections in either genotype (H,K). β-galactosidase-expressing progeny in both control and SmoM2 animals coexpressed the NeuN protein, a marker of mature neurons (arrows in C,F,I,L). Black scale bars: 50 microns. White scale bars: 14 microns. (M) Quantification of the spatial distribution of labeled progeny in R26R and SmoM2-YFP; R26R animals after dorsal or ventral Ad:GFAPpCre injection. Ventral virus injection in either genotype (pale red and pale blue bars) results in cells that are mostly located close to the core of the OB. Dorsal virus injection in R26R animals (bright blue bars) results in many cells that are distant from the core. However, dorsal virus injection in SmoM2; R26R animals results in cells that are located close to the core (bright red bars), with a distribution similar to that from ventral injections. Data shown are average +/− SEM, with total number of animals indicated on graph. Asterisks = p < 0.01.

Figure 7. SmoM2 Expression Results in Relocalization, Altered Dendritic Projections, and Ventral Marker Expression

(A–F) Dorsal injection of Ad:GFAPpCre in SmoM2-YFP; CAG animals (D–F) also resulted in relocalization of labeled progeny close to the core of the OB (dashed line) when compared to CAG controls (A–C). GFP-expressing progeny of both genotypes expressed NeuN (arrows, B and E) and the neurotransmitter GABA (arrows, C and F), consistent with their status as differentiated inhibitory interneurons. Scale bars: 50 microns. (G, H). Camera lucida tracings of individual GFP-labeled cells in CAG and SmoM2; CAG animals show the projections of these cells into the external plexiform layer of the OB (delineated by gray shading). Dashed lines indicate the mitral cell layer (lower line) and periglomerular layer (upper line) of the OB. While dorsal SVZ-derived cells in CAG animals typically project to the outer half of the EPL (G), dorsal SVZ-derived cells from SmoM2; CAG animals project to the inner half of the EPL, a feature associated with a deep granule interneuron identity (H). (I,J,L,M,N,P,R) At one month after dorsal injection of Ad:GFAPpCre in CAG animals, many GFP-labeled cells are present at the site of injection in the dorsal SVZ (I). However, the transcription factor Pbx3a is more prevalent in the ventral SVZ (J). After a similar injection in SmoM2-YFP; CAG animals, Pbx3a is present at similar frequency in the uninjected cells of the ventral SVZ (M), but is also enriched in dorsal GFP-positive cells (L). str = striatum, sep = septum. This enrichment is quantified in (R) – graph shows average +/− SEM for three independent injections per genotype (p = 0.0025, unpaired t test). In concert with the increased expression of the ventral marker Pbx3a, we also observed a loss of the dorsal marker Pax6 – while many dorsal GFP-positive cells normally express Pax6 (arrows, N), this expression is absent in SmoM2/CAG animals (P). (O,Q,S) Within the OBs of CAG animals at one month after dorsal Ad:GFAPpCre injection, few GFP-positive periglomerular cells (O,S) expressed the marker calbindin (shown in red). However, we did observe an increase in the number of CalB/GFP double-positive cells in periglomerular cells derived from injections in SmoM2-YFP; CAG animals (Q,S). Graph shows average +/− SEM for three independent injections per genotype.

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Persistent sonic hedgehog signaling in adult brain determines neural stem cell positional identity - PubMed (original) (raw)