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Astrocytes are an essential component of the CNS, and recent evidence points to an increasing diversity of their functions. Identifying molecular pathways that mediate distinct astrocyte function is key to understanding how the nervous system operates in the intact and pathological states. In this study, we demonstrate that the Hedgehog (Hh) pathway, well known for its roles in the developing CNS, is active in astrocytes of the mature mouse forebrain in vivo. Using multiple genetic approaches, we show that regionally distinct subsets of astrocytes receive Hh signaling, indicating a molecular diversity between specific astrocyte populations. Furthermore, we identified neurons as a source of Sonic hedgehog (Shh) in the adult forebrain, suggesting that Shh signaling is involved in neuron-astrocyte communication. Attenuation of Shh signaling in postnatal astrocytes by targeted removal of Smoothened, an obligate Shh co-receptor, resulted in upregulation of GFAP and cellular hypertrophy specifically in astrocyte populations regulated by Shh signaling. Collectively, our findings demonstrate a role for neuron-derived Shh in regulating specific populations of differentiated astrocytes.
Emerging evidence indicates that astrocytes play active and diverse roles in the central nervous system (CNS). Astrocytes actively regulate cerebral blood flow (Takano et al., 2006), and respond to sensory stimuli in both the visual and somatosensory cortices (Wang et al., 2006; Schummers et al., 2008). During development, astrocytes play key roles in regulating synapse formation and function (Ullian et al., 2001), and promote maturation of dendritic spines (Nishida and Okabe, 2007). In addition, bidirectional communication between astrocytes and neurons is an important element of synaptic transmission (Zhang et al., 2003; Araque, 2008). Although it is becoming increasingly clear that astrocytes actively contribute to normal CNS function, the underlying molecular mechanisms that mediate the functional properties of astrocytes remain poorly understood.
Sonic hedgehog (Shh), a member of the Hh family of signaling proteins, is known to play critical roles in multiple aspects of CNS development. Patterning of the dorsal/ventral axis, specification of neuronal and oligodendroglial cell types, and proliferation of granule cell precursors in the cerebellum all rely on Shh signaling (Fuccillo et al., 2006). Components of this classical developmental signaling pathway are widely expressed throughout the parenchyma of the adult forebrain (Traiffort et al., 1998; 1999), but its functional significance in the adult is less well characterized. Proliferation and differentiation of adult neural stem cells are regulated by Shh (Balordi and Fishell, 2007a, b), however little is known about the role of Shh signaling in cells outside the neurogenic regions.
Several lines of evidence suggest that Shh signaling could play a role in astrocyte development and/or function. In the developing optic nerve, Shh mediates proliferation of astrocyte precursors (Wallace and Raff, 1999), and application of Shh agonists to early postnatal cortical astrocyte cultures upregulates Shh target genes (Atkinson et al., 2009). In addition, the transcription factors Gli1, 2, and 3, essential components of the Shh signaling pathway, are enriched in postnatal and adult astrocyte cultures (Cahoy et al., 2008). Moreover, a recent study suggests that reactive astrocytes produce and respond to Shh following cortical freeze injury (Amankulor et al., 2009). However, it remains unknown whether astrocytes in the mature CNS participate in Shh signaling in vivo, under normal physiological conditions.
Transduction of Hh signaling is mediated by the Gli family of transcription factors, Gli1, 2, and 3. Binding of Shh to the transmembrane receptor Patched (Ptc) relieves constitutive inhibition of a second transmembrane receptor, Smoothened (Smo), thereby initiating a cascade of events leading to induction of Gli1 transcription (Fuccillo et al., 2006). Gli1 transcription in the embryo is dependent on Hh signaling through Gli2 and Gli3 (Bai et al., 2004). Thus, the presence of Gli1 can be used as an indicator of cells actively responding to high levels of Hh signaling (Bai et al., 2002).
In this study, we show that regionally distinct populations of mature, differentiated astrocytes are the primary cell type responding to Shh in the adult forebrain. We further identified neurons as a source of Shh, suggesting a novel role for Shh signaling in neuron-astrocyte communication. Finally, we demonstrate that conditional deletion of Smo in postnatal astrocytes results in a mild astrogliosis in the cortex, suggesting that Shh is an important regulator of specific astrocyte populations.
The following mouse lines were used, Gli1nlacZ/+ (Bai et al., 2002), Gli1eGFP/+ (allele to be published, elsewhere), Gli2lacZ/+ (Bai et al., 2002), Gli3lacZ/+ (Bowers and Joyner, unpublished), PtclacZ/+ (Goodrich et al., 1997), Gli1CreER/+ (Ahn and Joyner, 2005), ShhCreER/+ (Harfe et al., 2004), Rosa26loxP-STOP-lacZ (Soriano, 1999), mGFAP-Cre (Garcia et al., 2004), Smofl/fl (Long et al., 2001). Animals were maintained on a 12-hour light/dark cycle, and allowed free access to food and water. Experiments were conducted according to protocols approved by Memorial Sloan-Kettering Cancer Center Institute for Animal Care and Use Committee and Research Animal Resource Center. Adult mice between 2 and 6 months of age were used for all experiments.
Tamoxifen (Sigma, T5648) was dissolved in corn oil to a final concentration of 20mg/ml. Adult Gli1CreER;R26lacZ and ShhCreER;R26R animals received 0.5ml tamoxifen by oral gavage for 3 consecutive days.
BrdU (Sigma, B9285) was dissolved in 0.007N NaOH and administered by i.p. injection. Gli1CreER;R26lacZ animals received single injections of 200mg/kg/day for 3 days, beginning 1 day after the first tamoxifen administration, and were perfused 8 days, 1, 3, or 6 months later. Gli1nlacZ/+ animals received single injections of 50mg/kg/day for 12 days, and sacrificed 3 days later.
Animals were given an i.p. injection of sodium pentobarbital and transcardially perfused with 20–30 mls of PBS followed by 60 mls of 4% paraformaldehyde. Brains were dissected and post fixed for 2–4 hours, followed by cryoprotection in 30% sucrose and stored at 4°C until ready for sectioning. Tissue was sectioned on a cryostat at 40μm and serial sections were collected in 96 well plates containing 0.05% sodium azide, then stored at 4°C. Immunohistochemistry was performed on free floating sections using the following primary antibodies, rabbit anti-βGal (ICN, 55976), mouse anti-βGal (Promega, Z3793), chicken anti-®Gal (Abcam Inc., ab9361), rabbit anti-GFAP (DAKO, Z0334), rabbit anti-S100β (DAKO, Z0311), mouse anti-S100β (Abcam Inc. ab66028), rabbit anti-glutamine synthetase (Sigma, G2781), sheep anti-BrdU (Biodesign International, M20107S), sheep anti-CAII (Serotec, AHP206), mouse anti-APC-CC1 (EMD Chemicals Inc. OP80), mouse anti-NeuN (Chemicon, MAB377), rabbit anti-GFP (Invitrogen, A11122), and mouse anti-NG2 (Chemicon, MAB5384). For BrdU staining, sections were pre-treated with 2N HCl for 30 minutes and neutralized with PBS before incubation in primary antibody. For brightfield staining, species-specific, biotinylated secondary antibodies (Vector, Burlingame, CA) were used at 1:400 followed by incubation in avidin-biotin complex (ABC, Vector). Visualization was achieved using diaminobenzadine (Vector) as the developing agent. Double or triple labeling fluorescence immunohistochemistry was performed using species specific, AlexaFluor tagged secondary antibodies Alexa 488, and Alexa 555 (Invitrogen), and Cy5-conjugated secondary antibody (Jackson Immuno, 711-175-152), followed by counterstaining with DAPI (Invitrogen, D3571).
For X-gal staining, 30 μm thick brain sections were rinsed with X-gal washing buffer (2mM MgCl2, 0.1% Igepal Ca-30, 0.05% deoxycholate in PBS), then incubated in X-gal reaction buffer (0.17mM potassium ferrocyanide, 0.17mM potassium ferricyanide, 1mg/ml X-gal substrate) for 5 hours at 37°C. Sections were counterstained with nuclear fast red.
Stained sections were examined and photographed using brightfield and fluorescence microscopy using an inverted microscope (Zeiss) and Axiovision software. Single cell analysis of co-expression was evaluated on double stained, immunofluorescent tissues by taking optical sections with a 1μm slice distance with an Apotome (Zeiss) using a 40× objective. For each region examined, multiple z-stacks were collected from 2–3 sections from each of at least 3 brains, until a minimum of 50 cells from each brain was reached. Cell counts reflect the total number of cells analyzed from all sections from all brains.
βGal or S100β labeled cells were counted in the cortex using a modified optical fractionator (Gundersen et al., 1988) and stereological image analysis software (StereoInvestigator, Microbrightfield, USA) operating a computer-driven stage attached to an upright microscope (Zeiss). Cortical analysis was limited to cortex overlying the corpus callosum, with the ventral boundaries defined as the ventromedial and ventrolateral points of the corpus callosum, and anterior and posterior boundaries defined from Figure 18 and Figure 39, respectively, from Paxinos and Franklin (Paxinos and Franklin, 2001). One hemisphere from a minimum of 3 sections from at least 3 brains was analyzed for each group. The cortical area to be analyzed was traced at low power and counting frames were selected at random by the image analysis software. Cells were counted using a 100× objective and DIC optics. A target cell count of 300 cells was used to define scan grid and counting frame sizes, and 2μm guard zones were used. Estimated cell body volumes were determined in GFAP and S100β labeled cells using a nucleator probe (Moller et al., 1990; Howard and Reed, 1998) with 4 isotropic uniform random (IUR) rays emanating from the nucleus (StereoInvestigator, Microbrightfield, USA). For analysis of GFAP-labeled cells, only cells with a clear and distinct labeled cell body were analyzed. Cells from 3 brains each for mutants and controls were analyzed using a 63× oil objective and DIC optics. Statistical evaluations were performed using Prism® (GraphPad, San Diego, CA).
In order to examine the distribution and identity of Hh-responding cells in the adult forebrain, we used Gli1nlacZ/+ mice in which nuclear lacZ is expressed from the Gli1 locus (Bai et al., 2002). In the developing neural tube, Gli1 is expressed predominantly in ventral populations of proliferating neuronal and oligodendrocyte precursors (Platt et al., 1997; Jessell, 2000). In contrast, we found that Gli1-expressing cells were distributed in the adult forebrain as far dorsally as the cortex, where they were localized primarily to layers 3, 4, and 5 (Fig. 1A). Dense populations of Gli1-expressing cells were also found in multiple basal forebrain nuclei, including the septum (data not shown) and globus pallidus, as well as in the thalamus, and hypothalamus (Fig. 1B–D). These results are consistent with a previous report that Ptc and Smo mRNA can be detected in the globus pallidus, thalamus and hypothalamus of the adult rat (Traiffort et al., 1999). However, whereas low levels of Ptc transcript were detected only in the piriform cortex (Traiffort et al., 1999), our findings indicate that cells in the entorhinal, motor and somatosensory cortex express Gli1, suggesting a more widespread cortical distribution of Hh-responding cells.
Shh signaling plays a critical role in regulating adult neural stem and progenitor cells in the adult forebrain (Han et al., 2008). Although constitutive neurogenesis does not occur outside the subependymal zone (SEZ) and subgranular zone (SGZ), glial progenitors proliferate throughout the parenchyma of the adult CNS (Nishiyama et al., 2002; Dawson et al., 2003). To examine whether Gli1-expressing cells outside the neurogenic regions correspond to glial progenitors, we used a second line of mice in which an inducible Cre recombinase (CreERT2) is expressed from the Gli1 locus (Gli1CreER/+; Ahn and Joyner, 2005). When combined with the Rosa26loxP-STOP-loxP-lacZ reporter allele (R26lacZ; Soriano, 1999), cells expressing CreERT2 from the Gli1 locus express lacZ following tamoxifen administration. LacZ expression is permanent and heritable, and because CreER is active for only ~36 hours (Nakamura et al., 2006), expression of the βGal reporter protein becomes a permanent marker of cells that were expressing Gli1 at the time of tamoxifen administration. Moreover, because βGal expression is cytoplasmic, it is possible to examine the morphology of marked cells.
We marked Gli1-expressing cells in adult Gli1CreER/+ animals that were homozygous for the reporter allele (Gli1CreER;R26lacZ) by administering tamoxifen for 3 consecutive days. We then examined βGal expression in the cortex 8 days, and 1, 3, and 6 months post-tamoxifen. In addition, the dividing cell marker, BrdU was administered for 3 days beginning 1 day after the first tamoxifen treatment (Fig. 2B). At day 8 post-tamoxifen, we detected fewer βGal-expressing cells in the cortex compared with the number of cells observed at later time points (Fig. 2C–F). In contrast, there was no apparent increase in the number of marked cells between 1, 3, and 6 months post-tamoxifen (Fig. 2D–F), suggesting that Gli1-expresing cells outside the neurogenic regions are not proliferating. Interestingly, the level of βGal in cells increased dramatically between 8 days and 3 months post-tamoxifen, while staining intensity remained constant between 3 and 6 months post-tamoxifen (Fig. 2E–F). These results suggest that an accumulation of βGal protein in Gli1CreER;R26lacZ mice is necessary before all cells marked by tamoxifen induction can be detected, and that this accumulation stabilizes after 3 months. The regional distribution of marked cells was the same at all time points (data not shown). Notably, marked cells in Gli1CreER;R26lacZ animals were found in the same regions as observed in Gli1nlacZ/+ animals (Fig. 1A–H). Specifically, multiple cortical regions including the prefrontal (Fig. 1E and 1I), motor, somatosensory, and cingulate cortex (Fig. 1J–N), contained many βGal positive cells, as did the globus pallidus, thalamus, and hypothalamus (Fig. 1F–H). Interestingly, white matter tracts including the corpus callosum, fimbria, and anterior commissure were devoid of any βGal-expressing cells in both Gli1nlacZ/+ (data not shown) and Gli1CreER;R26lacZ (Fig. 1J–N) mice, and the caudate putamen and hippocampus exhibited only a few scattered cells (Fig. 1J–N).
The absence of an apparent expansion of marked cells between 1 and 6 months post-tamoxifen suggested that Gli1-expressing cells are not proliferating. Consistent with this, analysis of double staining for BrdU and βGal in the cortex showed that βGal-expressing cells were not double labeled with BrdU at all time points examined (Fig. 2G). As expected, double labeled cells were readily observed in the SEZ (Fig. 2H), corresponding to adult neural stem and progenitor cells (Ahn and Joyner, 2005). However, since BrdU labeled cells were only rarely observed in the cortex, we could not rule out the possibility that some Gli1-expressing cells divided, but escaped detection due to insufficient BrdU. We therefore used a more extensive BrdU labeling protocol that has been shown to label glial progenitor cells throughout the adult forebrain and spinal cord (Horner et al., 2000). Adult Gli1nlacZ/+ animals were given 50mg/kg BrdU for 12 days, and examined 3 days after the last BrdU injection. Despite a greater number of BrdU-labeled cells than in the previous experiments, none of the cortical BrdU-positive cells co-expressed βGal (n=148 cells, Supp. Fig. 1). Glial progenitors in the adult forebrain and spinal cord express the proteoglycan, NG2 (Horner et al., 2000). Analysis of βGal and NG2 double labeling showed that none of the βGal-expressing cells corresponded to NG2-positive glial progenitor cells (Supp. Fig. 1). The apparent increase in βGal-expressing cells between day 8 and 1 month post-tamoxifen in Gli1CreER;R26lacZ forebrains therefore cannot be due to proliferation of glial progenitors, but instead must be due to accumulation of βGal protein within cells. Taken together, these data suggest that, with the exception of adult neural stem and progenitor cells, the vast majority of Gli1-expressing cells in the adult forebrain are terminally differentiated. Moreover, the regional distribution of cells expressing Gli1 indicates that Hh signaling occurs in discrete cell populations throughout the dorsal/ventral and anterior/posterior axes.
We next examined the cell types that express Gli1 in the adult forebrain. We primarily analyzed Gli1CreER;R26lacZ animals because the cytoplasmic localization of βGal permitted morphological analysis. In addition, since our previous experiments indicated that reporter expression stabilizes by 3 months post-tamoxifen, we restricted our analysis to adult Gli1CreER;R26lacZ animals given tamoxifen 3 months earlier. Throughout the cortex, Gli1-expressing cells exhibited small cell bodies with an elaborate branching morphology (Fig. 1I and and3B).3B). The processes were highly ramified and very fine, creating a bushy appearance, consistent with the morphology of protoplasmic astrocytes. In addition, some labeled cells extended processes to nearby blood vessels (Fig. 3B), further suggesting that these cells correspond to astrocytes.
We performed double labeling immunohistochemistry for βGal and multiple cell type specific markers to determine the identity of Gli1-expressing cells in the cortex. Many βGal-labeled cells co-expressed the astrocyte specific marker Glial fibrillary acidic protein (GFAP, Fig. 3C). However, since relatively few astrocytes express GFAP under normal conditions, (Stichel et al., 1991; Walz, 2000), we also examined colocalization of βGal and the pan-astrocytic marker S100β (Cahoy et al., 2008; Ludwin et al., 1976) which is localized to the cell body making assessment of double labeling with βGal more reliable. Single cell analysis of βGal-positive cells indicated that 98.1% (n=468 cells) of cortical Gli1-expressing cells co-labeled with S100β (Fig. 3D). Because some studies have reported expression of S100β in cells of the oligodendrocyte lineage, we also examined colocalization between βGal and a third astrocytic marker, glutamine synthetase (Herrmann et al., 2008). Consistent with our previous analyses, most βGal-labeled cells co-expressed glutamine synthetase (Supp. Fig. 2C). Analysis of S100β and the oligodendrocyte marker CAII (Ghandour et al., 1980) showed that very few S100β cells co-labeled with CAII (Supp. Fig. 2), ruling out high levels of non-specific expression of S100β in olidgodendrocytes, and further supporting a predominantly astrocytic identity of Gli1-expressing cells. Double labeling and analysis of βGal and CAII (Fig. 3F) or APC-CC1 (Supp. Fig. 2) indicated that few cells co-expressed either of these oligodendrocyte markers (1.3% of βGal-positive cells co-labeled with CAII, n=316 cells) providing additional evidence that astrocytes, and not oligodendrocytes, are the predominant cells expressing Gli1 in the adult forebrain. Strikingly, no βGal-expressing cells in the cortex co-labeled with the neuronal marker NeuN (n=246 cells, Fig. 3E). Examination of other forebrain regions containing Gli1-expressing cells, such as the globus pallidus and hypothalamus also showed a high degree of colocalization between βGal and GFAP (Fig. 3G–H) or S100β (data not shown). These data indicate that the predominant cells expressing Gli1 in the adult forebrain correspond to astrocytes.
Interestingly, although the majority of Gli1-positive cells are astrocytes, not all astrocytes express Gli1. In addition, the proportion of astrocytes that express Gli1 differs between specific forebrain regions. In the cortex of tamoxifen-treated Gli1CreER;R26lacZ mice, only 24% of the S100β-positive cells were marked with βGal (n=1932 cells), whereas 56% (n=1233 cells) and 80% (n=1489 cells) of S100β-expressing cells in the globus pallidus and hypothalamus respectively, were βGal-positive (Fig. 4). In contrast, the caudate putamen and area CA1 of the hippocampus exhibit few βGal-positive cells (Fig. 1), indicating that the vast majority of astrocytes in these regions do not express Gli1. Similarly, white matter astrocytes do not express Gli1, as indicated by the lack of βGal staining in the corpus callosum, anterior commissure, and fimbria (Fig. 1F–J). Inducible Cre lines, such as the one used in this study, provide the advantage of temporal control over recombination. However, it is possible that incomplete recombination might account for some of the astrocytes that appear to be Gli1-negative. Although we cannot rule out this possibility within regions containing a high proportion of Gli1-positive cells such as the cortex, it is unlikely to be the case in regions containing few or no Gli1-positive cells. The regional distribution of βGal-positive cells was similar in both Gli1CreER;R26lacZ and Gli1nlacZ/+ lines, and between all mice examined. This suggests that specific subsets of astrocytes in different regions of the forebrain are regulated by high level Hh signaling.
Although transcriptional activation of Gli1 has been shown to be a reliable readout of Hh signaling in the developing CNS (Lee et al., 1997), it is possible that signaling pathways other than Hh might activate Gli1 transcription in the adult. In order to address whether Hh signaling is responsible for Gli1 expression in adult forebrain astrocytes, we took several approaches. First, we examined whether astrocytes express additional components of the Hh signaling pathway by examining βGal expression in adult PtclacZ/+ knock-in reporter mice (Goodrich et al., 1997). Since transcription of Ptc is positively regulated by Hh (Goodrich and Scott, 1998), Ptc expression can also be used as a read out of Hh activity. In adult PtclacZ/+ mice, many βGal-positive cells also expressed GFAP or S100β in all forebrain regions examined, including the cortex, globus pallidus, and hypothalamus (Fig. 5A–C). In addition, PtclacZ/+;Gli1GFP/+ mice showed that all Gli1-expressing cells were Ptc-positive, and many Ptc-positive cells also expressed Gli1 (Supp. Fig. 3). Ptc-positive cells that were Gli1-negative (Supp. Fig. 3), exhibited a lower intensity of βGal staining than Ptc-positive/Gli1-positive double-labeled cells (Supp. Fig. 3). This result is due to a lower threshold of induction for Ptc trasncription by Hh signaling than Gli1. Since Gli1 transcription requires both Gli2 and Gli3, we further examined βGal expression in Gli2lacZ/+ (Bai et al., 2002) and Gli3lacZ/+ (Bowers and Joyner, unpublished) knock-in reporter mice. In both lines, βGal-labeled cells throughout the forebrain co-expressed GFAP or S100β (Fig. 5D–I). Thus, astrocytes possess the key machinery to respond to Shh signaling.
We next disrupted Hh signaling by conditional deletion of Smo using a loxP flanked allele (SmoloxP; (Long et al., 2001) and a transgene in which Cre expression is driven by the full length mouse GFAP promoter (mGFAP-Cre, line 73.12, (Garcia et al., 2004). In the absence of functional Smo, Hh signaling cannot be effectively transduced within the cell, and Hh target genes, including Gli1, fail to be expressed (Long et al., 2001). In mGFAP-Cre;R26lacZ mice, X-gal staining shows that Cre activity is low in the forebrain at P0 and P3 (Supp. Fig. 4), consistent with the low levels of endogenous GFAP expression at these ages (Lewis and Cowan, 1985). At P7, there was a substantial increase in X-gal staining, and by P14 the expression pattern was similar to that seen in adult tissues (Supp. Fig. 4), suggesting that gene deletion in SmoloxP/loxP;mGFAP-Cre mice (referred to as mGFAP-Smo CKO mice) begins between P0 and P7 and is largely complete by P14. GFAP expression is limited to subsets of astrocytes in the adult forebrain. However the number and distribution of labeled cells in mGFAP-Cre;R26lacZ animals was greater than that of GFAP-expressing cells. Double labeling of βGal and S100β or GFAP showed that most βGal positive cells are astrocytes (Supp. Fig. 4). This suggests that in these mice, Cre is likely expressed in a population of early postnatal neural precursors that generate most astrocytes, thus enabling effective disruption of Hh signaling in both GFAP-positive and negative astrocytes throughout the forebrain.
In order to monitor Gli1 expression in mGFAP-Smo CKO mice, Gli1nlacZ/+ was bred onto the mutant background. mGFAP-Smo CKO mice exhibited no gross behavioral defects, and the general morphology and cytoacrhitecture of the forebrain appeared normal (Supp. Fig. 5). Throughout the forebrains of adult mGFAP-Smo CKO;Gli1nlacZ/+ mice, the number of Gli1-expressing cells was dramatically lower than in littermate controls (Fig. 6). Most of the remaining Gli1-positive cells expressed both S100β and CAII, likely corresponding to the minor population of S100β-expressing oligodendrocytes (Supp. Fig. 2) that express Gli1 in Gli1CreER;R26lacZ and Gli1nlacZ/+ tissue. The remaining Gli1-positive cells expressed S100β, but not CAII, suggesting that some astrocytes never express mGFAP-Cre and therefore do not undergo Cre-mediated recombination. Indeed, in mGFAP-Cre;R26lacZ mice, a minor population of S100β-positive cells are βGal-negative (Supp. Fig. 4E). Thus, our data demonstrate that targeted deletion of Smo in mGFAP-Cre expressing cells results in a dramatic reduction in Gli1 expression in astrocytes, suggesting that Gli1 transcription is dependent on Smo, and is a sensitive readout of Shh signaling.
In order to address whether Shh is a critical ligand for Gli1 transcription in astrocytes, we analyzed mice heterozygous for a null mutation in Shh (ShhCreER; (Harfe et al., 2004), and thus have reduced levels of Shh. There were significantly fewer βGal positive cells in ShhCreER/+;R26YFP/+;Gli1nlacZ/+ mutant mice compared with littermate controls (Shh+/+;R26YFP/+;Gli1nlacZ/+). Stereological quantification of the number of βGal positive cells in the cortex indicated an 80% reduction in the number of Gli1-expressing cells in mutant (36,641 cells, n=5) versus control (165,088 cells, n=4) mice (Fig. 6G, p<0.01). While the other vertebrate homologues of Hh, Indian (Ihh) and Desert hedgehog (Dhh), can activate Gli1 transcription, only Shh has been shown to be highly expressed in the adult CNS (Traiffort et al., 1998). Thus, the reduction in the number of Gli1-expressing cells in mice with reduced Shh suggests that only a small number of astrocytes can respond to a reduction in Shh in the forebrain. Taken together, these data demonstrate that Gli1 expression in adult forebrain astrocytes is activated specifically by Shh.
In order to identify the source of Shh ligand in the adult forebrain, we marked Shh-expressing cells in the brains of adult ShhCreER;R26lacZ or ShhCreER;R26YFP mice, and examined βGal or GFP expression 1 month after tamoxifen. In contrast to Gli1CreER;R26lacZ animals in which the staining intensity of βGal positive cells was relatively weak at 1 month post-tamoxifen, ShhCreER;R26lacZ mice exhibited strong immunoreactivity for βGal even at 1 month after tamoxifen (Fig. 7). This suggests that following Cre-mediated recombination, expression of lacZ from the R26 promoter is differentially regulated in various cell types. Indeed, it has been reported that lacZ expression from the R26 allele is poor in astrocytes, whereas fate mapped neurons exhibit strong immunoreactivity for βGal (Zhuo et al., 2001; Malatesta et al., 2003).
Analysis of βGal or GFP staining showed that Shh-expressing cells in the cortex were localized primarily to layer 4/5 (Fig. 7A), although a small number of cells were found in layer 2 (data not shown). In addition, basal forebrain nuclei such as the globus pallidus and medial septum, as well as the hypothalamus and amygdala, exhibited a large number of Shh-expressing cells (Supp. Fig. 6), consistent with the high density of Gli1-expressing cells in these regions (see Fig. 1). Interestingly, marked cells exhibited a neuronal morphology, and double staining with NeuN confirmed their neuronal phenotype (Fig. 7B–D). 81% of the Shh-expressing cells throughout the forebrain double labeled with NeuN (n=377 cells from 5 animals). Although some marked cells did not express NeuN, those cells nevertheless exhibited a clear neuronal morphology (Supp. Fig. 6). Thus, our data indicate that neurons are the source of Shh in the mature forebrain.
During development, Shh plays a critical role in specification of neuronal and oligodendrocyte precursors (Jessell, 2000). Shh has also been implicated in regulating proliferation of astrocytes in the developing optic nerve (Wallace and Raff, 1999; Dakubo et al., 2008). In order to investigate whether Shh plays a role in generating forebrain astrocytes, we examined S100β staining in mGFAP-Smo CKO mice. Since many astrocytes in the intact CNS do not express GFAP, and because GFAP, when expressed in normal astrocytes, is localized primarily to processes and rarely labels cell bodies, quantification based on GFAP expression is not a reliable measure of the total number of astrocytes. Thus, the number of astrocytes in mGFAP-Smo CKO mutants and controls was evaluated based on qualitative and quantitative analysis of S100β-labeled cells. Stereological quantification of S100β-expressing cells in the cortex of mGFAP-Smo CKOs indicated no difference in the number of astrocytes between mutants (430,076 cells, n=3) and littermate controls (392,652 cells, n=4, Fig. 8). This suggests that in contrast to astrocytes in the developing optic nerve, the production of forebrain astrocytes is not dependent on Shh signaling.
Although disruption of Shh signaling did not interfere with the number of astrocytes generated in mGFAP-Smo CKO mice, immunostaining for GFAP in mGFAP-Smo CKO animals revealed a striking elevation in GFAP expression. mGFAP-Smo CKO animals exhibited a greater intensity of GFAP immunostaining in individual cells compared to littermate controls (Fig. 9A–B), as well as an increase in the number of cells expressing GFAP (Fig. 9A–B). Examination of individual cells at high magnification in the cortex revealed that many cells exhibited enlarged cell bodies and thicker GFAP-stained processes (insets in Fig. 9B), suggesting that astrocytes become reactive when Shh signaling is disrupted. Importantly, the upregulation of GFAP was specific to forebrain regions that normally contain many Gli1-positive cells, such as the cortex and the globus pallidus (Fig. 9A–B and data not shown). In contrast, the caudate putamen, which contains few Gli1-expressing cells, showed no obvious change in GFAP expression (Fig. 9C–D).
To confirm the cellular hypertrophy observed in mGFAP-Smo CKO mutants, we measured the volume of the cell bodies of GFAP- or S100β-stained cells in the cortex and caudate putamen using the nucleator method (Fig. 9E–F), (Gundersen, 1988). In the cortices of mGFAP-Smo CKO mutants, there was a significant increase in volume compared to controls of both GFAP (411.7μm3, n=157 cells and 129.4μm3, n=259 cells for mutants and controls, respectively) and S100β-stained cells (299.6μm3, n=354 cells and 159.5μm3, n=412 cells for mutants and controls, respectively). In contrast, there was no difference in the volume of cells in the caudate putamen between mutants and controls for both markers (GFAP, 181.4 μm3, n=278 cells and 172.6μm3, n=195 cells for mutants and controls, respectively and S100β, 131.2μm3, n=329 cells and 143.9μm3, n=371 cells for mutants and controls, respectively). In addition, triple labeling of the few remaining Gli1-expressing cells in the cortex of mGFAP-Smo CKO;Gli1nlacZ/+ animals with βGal, S100β, and GFAP showed that none of the Gli1-expressing cells expressed GFAP (Fig. 6). Thus, any remaining Gli1-expressing astrocytes in mGFAP-Smo CKO mutants maintain Shh signaling and do not show signs of astrogliosis. These data demonstrate that perturbing Shh signaling in astrocytes results in specific physiological consequences indicating an important role for Shh signaling in regulating cellular function. Moreover, the absence of cellular hypertrophy or of changes in GFAP expression in caudate putamen astrocytes indicates a highly specific response of distinct astrocyte populations to loss of Smo.
Upregulation of GFAP and hypertrophy are hallmarks of reactive astrogliosis, which occurs in response to injury or disease (Eng and Ghirnikar, 1994). Reactive astrocytes have also been shown to undergo increased proliferation and synthesis of nestin and vimentin (Sofroniew, 2009). However BrdU labeling showed no change in proliferation between mutants and controls (data not shown), and staining for nestin and vimentin showed no change in expression of these intermediate filaments (data not shown). Taken together, these data show that the cellular response to interrupting Shh signaling includes key characteristics of reactive astrogliosis and indicates that Shh signaling plays an important role in mediating intracellular properties of specific astrocyte populations.
In this study, we show that high level Shh signaling in the adult CNS occurs in regionally distinct populations of mature, differentiated astrocytes. Our data demonstrate that neurons are a source of Shh, suggesting a novel signaling pathway involved in direct neuron-astrocyte communication. Furthermore, we provide evidence that Shh signaling is required to maintain normal cellular functions in specific astrocyte populations. Taken together, our data are the first to demonstrate a critical role for Shh signaling in neuron-astrocyte communication in vivo, in the adult CNS.
The roles of Shh in regulating proliferation and differentiation of neural precursors in the developing and adult CNS are well characterized (Jessell, 2000; Fuccillo et al., 2006). However our results show that the predominant cells receiving Shh in the adult CNS are not proliferating. Using 2 different BrdU labeling protocols and two transgenic mouse lines, we found no evidence for proliferation of Gli1-expressing cells localized outside the neurogenic regions of the adult forebrain. Our finding that the number of marked cells in Gli1CreER;R26lacZ mice remains constant between 1 and 6 months post-tamoxifen further supports the idea that Shh signals primarily to differentiated cells in the adult CNS.
Although GFAP-expressing adult neural stem cells have been shown to respond to Shh (Ahn and Joyner, 2005; Balordi and Fishell, 2007a, b), the vast majority of Gli1-expressing cells in the adult forebrain are post-mitotic, protoplasmic astrocytes. The identification of astrocytes as the predominant cell population expressing Gli1 was based on expression of well-known astrocyte-specific markers, GFAP, S100β, and glutamine synthetase. S100β expression has been reported in oligodendrocyte precursors or in myelinating oligodendrocytes of the white matter (Hachem et al., 2005). However mature oligodendrocytes in cortical gray matter downregulate S100β (Dyck et al., 1993), and our own analysis of S100β and CAII in the cortex indicated a low incidence of colocalization between these two markers. The astrocytic identity of Gli1-expresing cells is further supported by the morphology of marked cells in Gli1CreER;R26lacZ mice. Although we cannot rule out non-canonical mechanisms of Shh signaling, our data demonstrate that in the adult forebrain, astrocytes are the predominant cells utilizing Gli1-mediated Shh signaling.
Astrocytes are comprised of cells with diverse characteristics. They exhibit varying morphologies (Emsley and Macklis, 2006), and differ in their expression of intermediate filaments such as GFAP and vimentin (Eng et al., 2000; Kimelberg, 2004). In this study, we demonstrate that regionally distinct populations of astrocytes express Gli1, showing an additional layer of astrocyte diversity based on underlying signaling mechanisms. The molecular and morphological diversity that characterizes different neuronal populations has long been the basis of important functional differences between distinct neuronal classes. The diversity of astrocytes, and the functional consequences of such diversity however, are poorly understood. Nevertheless, a growing body of evidence supports the idea that subpopulations of astrocytes exhibit specific functional properties. In the hippocampus, subpopulations of astrocytes have been identified with distinct electrophysiological properties (Steinhauser et al., 1992; 1994) and gap junctional coupling (Wallraff et al., 2004). In addition, astrocytes differ in their expression of gluatmate receptors and transporters (Zhou and Kimelberg, 2001; Matthias et al., 2003). It is likely that intracellular signaling pathways regulate regional and/or functional identity. Our results point to Shh signaling as one mechanism by which different astrocyte populations might gain specific functional properties.
Using an antibody to Shh, a recent report indicated that reactive astrocytes express Shh following a cortical freeze injury model (Amankulor et al., 2009). In contrast, our data show that in the uninjured CNS, neurons are the primary source of Shh signaling to astrocytes, and we found no evidence for astrocyte expression of Shh under normal, physiological conditions. All marked cells in ShhCreER;R26R animals expressed the neuronal marker NeuN and/or exhibited a neuronal morphology. Moreover, although the ROSA promoter is weaker in astrocytes than in neurons, recombined cells with a clear astrocytic morphology are readily observed in Gli1CreER;R26lacZ animals at 1 month post tamoxifen, but not in ShhCreER;R26R animals at the same time point (compare Fig. 6 and Fig. 2). Although we cannot exclude the possibility that astrocytes express very low levels of Shh, we find no evidence for cell autonomous, high level Shh signaling in the uninjured forebrain.
In the developing CNS, cells expressing the Shh target gene Gli1 are localized adjacent to the source of Shh (Platt et al., 1997). Similarly, we found a major population of Gli1-expressing astrocytes in conjunction with Shh-expressing neurons in the hypothalamus. However, our data suggest that in the mature forebrain, Gli1 transcription is not necessarily dependent on local sources of Shh. In the cortex, for example, we found few Shh-expressing neurons, despite the large population of Gli1-expressing cells. It is possible that projections from subcortical Shh-expressing neurons induce cortical Gli1. In support of this hypothesis, Shh is transported axonally in the developing and adult rodent optic nerve (Wallace and Raff, 1999; Traiffort et al., 2001; Dakubo et al., 2008). Notably, Shh-expressing cells were found in the caudate (Supp. Fig. 5) despite a relatively low level of Gli1 expression, further suggesting that Shh does not necessarily induce local Gli1 expression. Alternatively, signaling pathways other than Shh may be responsible for Gli1 transcription in some cells. However several lines of evidence suggest against this explanation. First, astrocytes express all the critical components of the Shh signaling pathway, including Ptc, Gli2, and Gli3, suggesting that astrocytes possess the machinery to respond to Shh. Moreover, both reduced levels of Shh in Shh+/− mice, as well as targeted deletion of Smo in mGFAP-Smo CKO mutants leads to concomitant reductions in the number of Gli1-expressing cells throughout the forebrain. Taken together, these data provide strong evidence that Shh is the critical signal regulating Gli1 transcription. In addition, our data raise the possibility that both local and long-distance Shh signaling might occur in the adult forebrain.
Astrocytes are known to be sensitive to disturbances in CNS homeostasis, and become reactive in response to various insults to the CNS (Sofroniew, 2009). The severity of the reactive astrogliosis response is dependent on the nature and severity of the initial insult. Dramatic upregulation of GFAP and cellular hypertrophy are key characteristics of reactive astrocytes. The observation that cortical astrocytes in mGFAP-Smo CKO mutants exhibit increased GFAP expression and cell volume, without concomitant changes in cell proliferation or expression of nestin or vimentin indicates a mild gliosis in response to disruptions in Shh signaling, and suggests that Shh plays an important role in maintaining normal CNS function. Moreover, our observation that cortical, but not striatal, astrocytes exhibit reactive gliosis indicates a specific response of cortical, Gli1-expressing astrocytes to disruptions in Shh signaling. This result argues against a global defect in homeostasis in mGFAP-Smo CKO mutants, and instead supports the hypothesis that neuronal-derived Shh regulates specific subsets of astrocytes in the adult forebrain.
In addition to injury or disease, it has been shown that astrocytes become reactive in response to neuronal hyperactivity (Steward et al., 1991; Torre et al., 1993). A tempting speculation therefore, is that the reactive astrocyte phenotype observed in mGFAP-Smo CKOs reflects abnormal synaptic activity resulting from aberrant gliotransmission from mutant astrocytes to neighboring wild type neurons. Activation of G protein-coupled receptors (GPCRs) on astrocytes elicits Ca2+-dependent release of various gliotransmitters, including glutamate, ATP, and D-Serine (Fiacco and McCarthy, 2004; Perea and Araque, 2007), which can in turn modulate neuronal activity (Pascual et al., 2005b; Fellin, 2009). Interestingly, Smo is a 7-pass transmembrane receptor, and has been shown to stimulate multiple G proteins (Kasai et al., 2004; Masdeu et al., 2006). In addition, Shh can increase intracellular Ca2+ in mouse embryonic stem cells, and rat gastric mucosal cells (Osawa et al., 2006; Heo et al., 2007). Thus insufficient Ca2+ signaling as a result of impaired Smo function in astrocytes might lead to aberrant intercellular communication events between astrocytes and neurons. Subsequently, inappropriate synaptic activity due to impaired gliotransmission would feed back to neighboring astrocytes, resulting in a reactive response. In support of this hypothesis are studies demonstrating that application of Shh to brain slices reduces neuronal activity (Bezard et al., 2003; Pascual et al., 2005a). Notably, Bezard et al., (2003) only observed Shh-mediated neuronal responses after a 3 minute delay, a time scale that is consistent with secondary signaling mechanisms, rather than a direct effect of Shh on neuronal ion channels. In this scenario, the molecular events that regulate astrocyte intercellular communication likely would be coincident with, but independent of, Gli1 transcription. Alternatively, Gli-dependent transcription might be mediating intracellular mechanisms that lead to impaired glial function and subsequently, reactive astrocytosis. Future experiments are needed to examine whether Gli-dependent or independent mechanisms govern the intracellular events leading to the reactive astrocytosis observed in mGFAP-Smo CKO mutants. Our data nevertheless indicate that Shh plays an important role in intercellular communication between specific neuronal and astrocyte populations of the adult forebrain, demonstrating a novel role for Shh signaling. Moreover, our data support an emerging paradigm in which astrocytes, like neurons, are molecularly and functionally diverse.
We are grateful to M. Sofroniew for mGFAPCre mice and for critical reading of the manuscript; T. Kelly, S. Wilson and E. Legue for insightful discussion and critical reading of the manuscript. This work was supported by grants to ALJ from the New York State spinal cord injury research program and the NCI (R01CA128158).