Cerebellar proteoglycans regulate sonic hedgehog responses during development

Sonic hedgehog promotes proliferation of developing cerebellar granule cells. As sonic hedgehog is expressed in the cerebellum throughout life it is not clear why proliferation occurs only in the early postnatal period and only in the external granule cell layer. We asked whether heparan sulfate proteoglycans might regulate sonic hedgehog-induced proliferation and thereby contribute to the specialized proliferative environment of the external granule cell layer. We identified a conserved sequence within sonic hedgehog that is essential for binding to heparan sulfate proteoglycans, but not for binding to the receptor patched. Sonic hedgehog interactions with heparan sulfate proteoglycans promote maximal proliferation of postnatal day 6 granule cells. By contrast, proliferation of less mature granule cells is not affected by sonic hedgehog-proteoglycan interactions. The importance of proteoglycans for proliferation increases during development in parallel with increasing expression of the glycosyltransferase genes, exostosin 1 and exostosin 2. These data suggest that heparan sulfate proteoglycans, synthesized by exostosins, may be critical determinants of granule cell proliferation.     

Sonic hedgehog regulates the growth and patterning of the cerebellum.

The molecular bases of brain development and CNS malignancies remain poorly understood. Here we show that Sonic hedgehog (Shh) signaling controls the development of the cerebellum at multiple levels. SHH is produced by Purkinje neurons, it is required for the proliferation of granule neuron precursors and it induces the differentiation of Bergmann glia. Blocking SHH function in vivo results in deficient granule neuron and Bergmann glia differentiation as well as in abnormal Purkinje neuron development. Thus, our findings provide a molecular model for the growth and patterning of the cerebellum by SHH through the coordination of the development of cortical cerebellar cell types. In addition, they provide a cellular context for medulloblastomas, childhood cancers of the cerebellum.     

Deficits in motor coordination with aberrant cerebellar development in mice lacking testicular orphan nuclear receptor 4

Since testicular orphan nuclear receptor 4 (TR4) was cloned, its physiological function has remained largely unknown. Throughout postnatal development, TR4-knockout (TR4-/-) mice exhibited behavioral deficits in motor coordination, suggesting impaired cerebellar function. Histological examination of the postnatal TR4-/- cerebellum revealed gross abnormalities in foliation; specifically, lobule VII in the anterior vermis was missing. Further analyses demonstrated that the laminations of the TR4-/- cerebellar cortex were changed, including reductions in the thickness of the molecular layer and the internal granule layer, as well as delayed disappearance of the external granule cell layer (EGL). These lamination irregularities may result from interference with granule cell proliferation within the EGL, delayed inward migration of postmitotic granule cells, and a higher incidence of apoptotis. In addition, abnormal development of Purkinje cells was observed in the postnatal TR4-/- cerebellum, as evidenced by aberrant dendritic arborization and reduced calbindin staining intensity. Expression of Pax-6, Sonic Hedgehog (Shh), astrotactin (Astn), reelin, and Cdk-5, genes correlated with the morphological development of the cerebellum, is reduced in the developing TR4-/- cerebellum. Together, our findings suggest that TR4 is required for normal cerebellar development.     

Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum

Purkinje cells (PCs) are the projection neurons of the cerebellar cortex. They receive two major types of synaptic input - that from the inferior olive via climbing fibres and that from the granule neurons via parallel fibres. The precursors of granule neurons proliferate at the surface of the developing cerebellumin the external granule layer (EGL), which persists until postnatal day 14 in the mouse [1]. PCs are thought to provide trophic support for granule neurons [2][3] and to stimulate the proliferation of cells in the EGL [4], but the signalling molecules that mediate these cell-cell interactions have not been identified. I show here that PCs in the developing mouse cerebellum express the gene encoding the morphogen Sonic hedgehog (Shh) and that dividing cells in the EGL express Patched (Ptc) and Gli1, two target genes of which expression is upregulated in response to Hedgehog signalling (see [5] and references therein). Treatment of developing mice with hybridoma cells that secrete neutralizing anti-Shh antibodies [6] disrupted cerebellar development and reduced bromodeoxyuridine (BrdU) incorporation in the EGL of neonatal mice, whereas treatment of dissociated granule neuron cultures with recombinant Shh stimulated BrdU incorporation. These results suggest that PC-derived Shh normally promotes the proliferation of granule neuron precursors in the EGL.     

Hedgehog signals regulate multiple aspects of gastrointestinal development

The gastrointestinal tract develops from the embryonic gut, which is composed of an endodermally derived epithelium surrounded by cells of mesodermal origin. Cell signaling between these two tissue layers appears to play a critical role in coordinating patterning and organogenesis of the gut and its derivatives. We have assessed the function of Sonic hedgehog and Indian hedgehog genes, which encode members of the Hedgehog family of cell signals. Both are expressed in gut endoderm, whereas target genes are expressed in discrete layers in the mesenchyme. It was unclear whether functional redundancy between the two genes would preclude a genetic analysis of the roles of Hedgehog signaling in the mouse gut. We show here that the mouse gut has both common and separate requirements for Sonic hedgehog and Indian hedgehog. Both Sonic hedgehog and Indian hedgehog mutant mice show reduced smooth muscle, gut malrotation and annular pancreas. Sonic hedgehog mutants display intestinal transformation of the stomach, duodenal stenosis (obstruction), abnormal innervation of the gut and imperforate anus. Indian hedgehog mutants show reduced epithelial stem cell proliferation and differentiation, together with features typical of Hirschsprung's disease (aganglionic colon). These results show that Hedgehog signals are essential for organogenesis of the mammalian gastrointestinal tract and suggest that mutations in members of this signaling pathway may be involved in human gastrointestinal malformations.     

Vitronectin regulates Sonic hedgehog activity during cerebellum development through CREB phosphorylation

During development of the cerebellum, Sonic hedgehog (SHH) is expressed in migrating and settled Purkinje neurons and is directly responsible for proliferation of granule cell precursors in the external germinal layer. We have previously demonstrated that SHH interacts with vitronectin in the differentiation of spinal motor neurons. Here, we analysed whether similar interactions between SHH and extracellular matrix glycoproteins regulate subsequent steps of granule cell development. Laminins and their integrin receptor subunit alpha6 accumulate in the outer most external germinal layer where proliferation of granule cell precursors is maximal. Consistent with this expression pattern, laminin significantly increases SHH-induced proliferation in primary cultures of cerebellar granule cells. Vitronectin and its integrin receptor subunits alpha(v) are expressed in the inner part of the external germinal layer where granule cell precursors exit the cell cycle and commence differentiation. In cultures, vitronectin is able to overcome SHH-induced proliferation, thus allowing granule cell differentiation. Our studies indicate that the pathway in granule cell precursors responsible for the conversion of a proliferative SHH-mediated response to a differentiation signal depends on CREB. Vitronectin stimulates phosphorylation of cyclic-AMP responsive element-binding protein (CREB), and over-expression of CREB is sufficient to induce granule cell differentiation in the presence of SHH. Taken together, these data suggest that granule neuron differentiation is regulated by the vitronectin-induced phosphorylation of CREB, a critical event that terminates SHH-mediated proliferation and permits the differentiation program to proceed in these cells.     

Sonic Hedgehog-Induced Histone Deacetylase Activation Is Required for Cerebellar Granule Precursor Hyperplasia in Medulloblastoma

Medulloblastoma, the most common pediatric brain tumor, is thought to arise from deregulated proliferation of cerebellar granule precursor (CGP) cells. Sonic hedgehog (Shh) is the primary mitogen that regulates proliferation of CGP cells during the early stages of postnatal cerebellum development. Aberrant activation of Shh signaling during this time has been associated with hyperplasia of CGP cells and eventually may lead to the development of medulloblastoma. The molecular targets of Shh signaling involved in medulloblastoma formation are still not well-understood. Here, we show that Shh regulates sustained activation of histone deacetylases (HDACs) and that this activity is required for continued proliferation of CGP cells. Suppression of HDAC activity not only blocked the Shh-induced CGP proliferation in primary cell cultures, but also ameliorated aberrant CGP proliferation at the external germinal layer (EGL) in a medulloblastoma mouse model. Increased levels of mRNA and protein of several HDAC family members were found in medulloblastoma compared to wild type cerebellum suggesting that HDAC activity is required for the survival/progression of tumor cells. The identification of a role of HDACs in the early steps of medulloblastoma formation suggests there may be a therapeutic potential for HDAC inhibitors in this disease.     

Mouse Model Reveals the Role of RERE in Cerebellar Foliation and the Migration and Maturation of Purkinje Cells

Nuclear receptors and their coregulators play a critical role in brain development by regulating the spatiotemporal expression of their target genes. The arginine-glutamic acid dipeptide repeats gene (Rere) encodes a nuclear receptor coregulator previously known as Atrophin 2. In the developing cerebellum, RERE is expressed in the molecular layer, the Purkinje cell layer and the granule cell layer but not in granule cell precursors. To study RERE''s role in cerebellar development, we used RERE-deficient embryos bearing a null allele (om) and a hypomorphic allele (eyes3) of Rere (Rere ^om/eyes3). In contrast to wild-type embryos, formation of the principal fissures in these RERE-deficient embryos was delayed and the proliferative activity of granule cell precursors (GCPs) was reduced at E18.5. This reduction in proliferation was accompanied by a decrease in the expression of sonic hedgehog (SHH), which is secreted from Purkinje cells and is required for normal GCP proliferation. The maturation and migration of Purkinje cells in Rere ^om/eyes3 embryos was also delayed with decreased numbers of post-migratory Purkinje cells in the cerebellum. During the postnatal period, RERE depletion caused incomplete division of lobules I/II and III due to truncated development of the precentral fissure in the cerebellar vermis, abnormal development of lobule crus I and lobule crus II in the cerebellar hemispheres due to attenuation of the intercrural fissure, and decreased levels of Purkinje cell dendritic branching. We conclude that RERE-deficiency leads to delayed development of the principal fissures and delayed maturation and migration of Purkinje cells during prenatal cerebellar development and abnormal cerebellar foliation and Purkinje cell maturation during postnatal cerebellar development.     

Widespread contribution of Gdf7 lineage to cerebellar cell types and implications for hedgehog-driven medulloblastoma formation

The roof plate is a specialized embryonic midline tissue of the central nervous system that functions as a signaling center regulating dorsal neural patterning. In the developing hindbrain, roof plate cells express Gdf7 and previous genetic fate mapping studies showed that these cells contribute mostly to non-neural choroid plexus epithelium. We demonstrate here that constitutive activation of the Sonic hedgehog signaling pathway in the Gdf7 lineage invariably leads to medulloblastoma. Lineage tracing analysis reveals that Gdf7-lineage cells not only are a source of choroid plexus epithelial cells, but are also present in the cerebellar rhombic lip and contribute to a subset of cerebellar granule neuron precursors, the presumed cell-of-origin for Sonic hedgehog-driven medulloblastoma. We further show that Gdf7-lineage cells also contribute to multiple neuronal and glial cell types in the cerebellum, including glutamatergic granule neurons, unipolar brush cells, Purkinje neurons, GABAergic interneurons, Bergmann glial cells, and white matter astrocytes. These findings establish hindbrain roof plate as a novel source of diverse neural cell types in the cerebellum that is also susceptible to oncogenic transformation by deregulated Sonic hedgehog signaling.     

Nmyc upregulation by sonic hedgehog signaling promotes proliferation in developing cerebellar granule neuron precursors

Hedgehog pathway activation is required for expansion of specific neuronal precursor populations during development and is etiologic in the human cerebellar tumor, medulloblastoma. We report that sonic hedgehog (Shh) signaling upregulates expression of the proto-oncogene Nmyc in cultured cerebellar granule neuron precursors (CGNPs) in the absence of new protein synthesis. The temporal-spatial expression pattern of Nmyc, but not other Myc family members, precisely coincides with regions of hedgehog proliferative activity in the developing cerebellum and is observed in medulloblastomas of Patched (Ptch) heterozygous mice. Overexpression of Nmyc promotes cell-autonomous G(1) cyclin upregulation and CGNP proliferation independent of Shh signaling. Furthermore, Myc antagonism in vitro significantly decreases proliferative effects of Shh in cultured CGNPs. Together, these findings identify Nmyc as a direct target of the Shh pathway that functions to regulate cell cycle progression in cerebellar granule neuron precursors.