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
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
), 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
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
) 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 and ). 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
, 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 CKO
s 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.