Table of contents

Hedgehog, neural induction, migration and proliferation

Sonic hedgehog (Shh) is crucial for motoneuron development in chick and mouse. However, zebrafish embryos homozygous for a deletion of the shh locus have normal numbers of motoneurons, raising the possibility that zebrafish motoneurons may be specified differently. Unlike other vertebrates, zebrafish express three hh genes in the embryonic midline: shh, echidna hedgehog (ehh) and tiggywinkle hedgehog (twhh). Therefore, it is possible that Twhh and Ehh are sufficient for motoneuron formation in the absence of Shh. To test this hypothesis, all three Hh signals were eliminated, or severely reduced using mutations that directly or indirectly reduce Hh signaling and antisense morpholinos. This analysis shows that Hh signals are required for zebrafish motoneuron induction. However, each of the three zebrafish Hhs is individually dispensable for motoneuron development because the other two can compensate for its loss. These results also suggest that Twhh and Shh are more important for motoneuron development than Ehh (K. E. Lewis, 2001).

Even though Hh signaling is clearly required for the formation of at least the vast majority of motoneurons, it is still unclear exactly how Hh acts. Although it is difficult to explain how a few motoneurons might be independent of Hh signals if Hh directly and solely induces motoneurons, it is less paradoxical if Hh signaling induces motoneurons indirectly by, for example, inhibiting the repression of ventral fates by dorsal signals, or if Hh acts in concert with other signals that have very limited activity on their own to induce motoneurons. There is evidence for both of these scenarios. Motoneurons form in Shh;Gli3 double mutant mice, demonstrating that for a substantial number of mouse motoneurons Shh is only required to inhibit Gli3. However these results also demonstrate that differences exist between mouse motoneurons because half of the motoneurons in the lumbar region and most of the motoneurons in the brachial region still require Shh activity, even in the absence of Gli3. This could reflect redundancy between Gli2 and Gli3, or the presence of a second motoneuron-inducing factor in mice that is distributed differentially along the rostrocaudal axis. Retinoic acid (RA) is a good candidate for a second motoneuron-inducing factor because it has been shown, in vitro, to induce other ventral neuronal fates, specifically V0 and V1 interneurons, in a Shh-independent manner and it can induce motoneurons in chick neural explants and embryonic stem cells, although this may be an indirect effect as Shh is also induced in these experiments. However, Shh is also sufficient for induction of V0 and V1 interneurons, and is required for the development of some, but not all, of these neurons, suggesting that RA and Shh may act together to specify the full complement of these neurons. These interactions are still not properly understood, but as they are further elucidated it will be interesting to see whether any parallels can be drawn with motoneuron development (K. E. Lewis, 2001).

Hh signaling is required for initial islet1 expression in motoneurons. Thus, Hh signaling appears to be involved in motoneuron induction. This is consistent with the observation that both the islet1- and islet2-expressing subsets of primary motoneurons require Hh signals, suggesting that Hh signaling is required before these motoneurons assume their different identities. Although the possibilities that other signals might induce motoneuron fates and Hh might have a very early maintenance role cannot be ruled out, these experiments show that Hh signals are required from extremely early in motoneuron development. Shh signals appear to be required only during gastrulation for anterior primary motoneuron formation (K. E. Lewis, 2001).

Sonic hedgehog (Shh) is strongly implicated in the development of ventral structures in the nervous system. Addition of Sonic hedgehog protein to chick spinal cord explants induces floor plate and motoneuron development. Whether Shh acts directly to induce these cell types or whether their induction is mediated by additional factors is unknown. To further investigate the role of Shh in spinal neuron development, low-density cultures of murine spinal cord precursor cells were used. Shh stimulates neuronal differentiation; however, it does not increase the proportion of neurons expressing the first postmitotic motoneuron marker Islet-1. Moreover, Shh induces Islet-1 expression in neural tube explants, suggesting that it acts in combination with neural tube factors to induce motoneurons. Another factor implicated in motoneuron development is neurotrophin 3 (NT3): when assayed in isolated precursor cultures, it has no effect on Islet-1 expression. However, the combination of N-terminal Shh and NT3 induces Islet-1 expression in the majority of neurons in low-density cultures of caudal intermediate neural plate. Furthermore, in explant cultures, Shh-mediated Islet-1 expression is blocked by an anti-NT3 antibody. Previous studies have shown expression of NT3 in the region of motoneuron differentiation and that spinal fusimotor neurons are lost in NT3 knock-out animals. Taken together, these findings suggest that Shh can act directly on spinal cord precursors to promote neuronal differentiation, but induction of Islet-1 expression is regulated by factors additional to Shh, including NT3 (Dutton, 1999).

Cerebellar granule cells are the most abundant type of neuron in the brain, but the molecular mechanisms that control their generation are incompletely understood. The process of granule cell differentiation has been studied in detail. In contrast to most other neurons, which are born in the ventricular zone, granule cells are generated on the outside of the cerebellum, in a region known as the external germinal layer (EGL). For the first 2 to 3 weeks after birth, cells in the EGL undergo extensive proliferation and generate a large pool of granule cell precursors (GCPs). Developing GCPs then exit the cell cycle, extend axons (which will eventually synapse with Purkinje cell dendrites in the molecular layer), and migrate inward past the Purkinje cell bodies to their final destination, the internal granule layer (IGL). Sonic hedgehog (Shh), which is produced by Purkinje cells, regulates the division of granule cell precursors (GCPs). Treatment of GCPs with Shh prevents differentiation and induces a potent, long-lasting proliferative response. This response can be inhibited by basic fibroblast growth factor or by activation of protein kinase A. Blocking Shh function in vivo dramatically reduces GCP proliferation. These findings provide insight into the mechanisms of normal growth and tumorigenesis in the cerebellum (Wechsler-Reya, 1999).

The continued proliferation of cells in response to Shh raises the question of what normally stops GCP proliferation and allows cells to differentiate into mature granule cells. In light of an observation that forskolin inhibits the proliferative response of cerebellar cells to Shh, an alternative explanation for the termination of Shh-induced proliferation might be activation of PKA. One candidate for a physiologic activator of PKA in the cerebellum is pituitary adenylate cyclase-activating polypeptide (PACAP). PACAP is expressed at high levels in the developing cerebellum, and its receptors are expressed on GCPs in the external granule layer. In culture, PACAP promotes granule cell survival and neurite outgrowth in a PKA-dependent manner. Thus, Shh-induced proliferation of GCPs may be modulated by PACAP. Another important regulator of the response to Shh may be bFGF. In the developing cerebellum, bFGF is made by astrocytes and Purkinje cells and has been reported to stimulate proliferation as well as differentiation and neurite outgrowth in granule cells. The observation that bFGF is a weak mitogen on its own, but a potent inhibitor of the response to Shh, suggests that the primary role of bFGF may be in GCP differentiation. Further studies on the precise timing and spatial localization of Shh, PACAP, bFGF, and other growth factors may shed light on the mechanisms by which these factors cooperate to orchestrate granule cell generation in vivo (Wechsler-Reya, 1999 and references).

Purkinje cells (PCs) are the projection neurons of the cerebellar cortex. They receive two major types of synaptic input: from the inferior olive via climbing fibers and from the granule neurons via parallel fibers. The precursors of granule neurons proliferate at the surface of the developing cerebellum in the external granule layer (EGL), which persists until postnatal day 14 in the mouse. PCs are thought to provide trophic support for granule neurons and to stimulate the proliferation of cells in the EGL, but the signaling molecules that mediate these cell-cell interactions have not been identified. PCs in the developing mouse cerebellum express the gene encoding the morphogen Sonic hedgehog (Shh) and dividing cells in the EGL express Patched (Ptc) and Gli1, two target genes whose expression is upregulated in response to Hedgehog signaling. Treatment of developing mice with hybridoma cells that secrete neutralizing anti-Shh antibodies disrupt cerebellar development and reduced bromodeoxyuridine (BrdU) incorporation in the EGL of neonatal mice, whereas treatment of dissociated granule neuron cultures with recombinant Shh stimulates BrdU incorporation. These results suggest that PC-derived Shh normally promotes the proliferation of granule neuron precursors in the EGL (Wallace, 1999a).

Cerebellar tumors, or medulloblastomas (MBs), and basal cell carcinomas of the skin (BCCs) are two types of cancers overrepresented in patients with the Basal Cell Nevus or Gorlin’s Syndrome. These tumors can arise from activation of the Sonic hedgehog (Shh) signaling pathway through mutations in the membrane receptor components Patched or Smoothened. In addition, virtually all sporadic BCCs, representing the most common type of cancer, display activation of the Shh signaling pathway leading to expression of the transcription factor Gli1, a Shh-target and mediator that can itself induce epidermal tumors. Since BCCs are thought to recapitulate steps in hair follicle differentiation normally regulated by SHH, MBs could recapitulate SHH-regulated ontogenic steps of cerebellar development. This idea would be consistent with the expression of Ptc and Gli genes in the cerebellum and the function of Shh as a mitogen, or a differentiation or survival factor for different cell populations in the CNS. In addition, the majority of human MBs show expression of ZIC1, a zinc finger protein of the GLI superfamily, and other genes normally expressed in granule neurons or their precursors in the external germinal layer (EGL). However, Shh has not been reported to be expressed in the developing cerebellum and thus, a rational cellular context for MBs has been lacking (Dahmane, 1999 and references).

The cerebellum contains a large cortical region within which distinct cell types are positioned in a layered fashion. The outermost layer, the EGL, contains dividing granule neuron progenitors. Postmitotic granule cells leave the EGL and migrate inward to form the internal granular layer (IGL) where granule neurons terminally differentiate. Migrating neurons are guided by Bergmann radial glial fibers through the molecular layer and, before reaching the forming IGL, they pass through the Purkinje neuron layer (PL) containing the cell bodies of Purkinje neurons and Bergmann glia. Analyses of cerebellar granule cell development have shown (1) that EGL cells acquire a granule neuron fate from the onset of their migration in the rhombic lip; (2) that EGL proliferation requires cell contact and (3) is dependent on Purkinje neurons and (4) that this can be potentiated by Insulin-like and Epidermal growth factors. Moreover, mutual interactions between Purkinje neurons and their future presynaptic regulatory partners, the granule neurons, are required for the normal development of both cell types. Thus, whereas the cellular interactions involved in cerebellar development have been largely described, their molecular bases remain unknown (Dahmane, 1999 and references).

A possible endogenous role of SHH in cerebellar development has been investigaed. SHH is produced by chick and mouse Purkinje neurons and also transiently by early mouse EGL cells. The results of treating chick cerebellar explants or purified mouse cells with SHH or a blocking anti-SHH antibody show the requirement of SHH in the proliferation of granule neuron precursors. In addition, glial differentiation is induced by SHH. Blocking SHH signaling in vivo leads to the development of hypoplastic cerebella with abnormal foliation, in which Purkinje neurons are abnormally positioned and Bergmann glia and differentiated granule neurons are either reduced in number or absent. Together, these in vivo and in vitro results demonstrate previously unknown functions of SHH in the elaboration of pattern in the maturing central nervous system and provide a molecular model for the coordinate regulation of cortical development in the cerebellum. Moreover, they provide a basis for the development of brain tumors as deregulated or maintained Shh signaling in vivo, by any mutations activating this pathway. This is predicted to lead to the maintained and aberrant proliferation of granule neuron precursors (Dahmane, 1999).

Sonic hedgehog (Shh), an axis-determining secreted protein, is expressed during early vertebrate embryogenesis in the notochord and ventral neural tube. In this site it plays a role in the phenotypic specification of ventral neurons along the length of the CNS. For example, Shh induces the differentiation of motor neurons in the spinal cord and dopaminergic neurons in the midbrain. Shh expression, however, persists beyond this induction period, and this study asked whether the protein shows novel activities beyond phenotype specification. Using cultures derived from embryonic day 14.5 (E14. 5) rat ventral mesencephalon, it has been shown that Shh is also trophic for dopaminergic neurons. Interestingly, Shh not only promotes dopaminergic neuron survival, but also promotes the survival of midbrain GABA-immunoreactive (GABA-ir) neurons. In cultures derived from the E15-16 striatum, Shh promotes the survival of GABA-ir interneurons to the exclusion of any other cell type. Cultures derived from E15-16 ventral spinal cord reveal that Shh is again trophic for interneurons, many of which are GABA-ir and some of which express the Lim-1/2 nuclear marker, but it does not appear to support motorneuron survival. Shh does not support the survival of sympathetic or dorsal root ganglion neurons. Finally, using the midbrain cultures, it has been shown that in the presence of MPP+, a highly specific neurotoxin, Shh prevents dopaminergic neuron death that normally would have occurred. Thus Shh may have therapeutic value as a protective agent in neurodegenerative disease (Miao, 1997).

Loss of substantia nigra dopaminergic neurons, which develop from the ventral region of the midbrain, is associated with Parkinson's disease. During embryogenesis, induction of these and other ventral neurons is influenced by interactions with the induction of mesoderm of the notochord and the floor plate, which lies at the ventral midline of the developing CNS. Sonic hedgehog encodes a secreted peptide, which is expressed in notochord and floor plate cells and can induce appropriate ventral cell types in the basal forebrain and spinal cord. Sonic hedgehog is sufficient to induce dopaminergic and other neuronal phenotypes in chick mesencephalic explants in vitro. Sonic hedgehog is a general ventralizing signal in the CNS, the specific response being determined by the receiving cells. These results suggest that Sonic hedgehog may have utility in the induction of clinically important cell types (Wang, 1995).

During development of the cerebellum, Sonic hedgehog is expressed in migrating and settled Purkinje neurons and is directly responsible for proliferation of granule cell precursors in the external germinal layer. SHH interacts with vitronectin in the differentiation of spinal motor neurons. Whether similar interactions between SHH and extracellular matrix glycoproteins regulate subsequent steps of granule cell development has been examined. 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 alphav 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. 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. Although at the present time the different components mediating VN stimulation of CREB phosphorylation are not known, it has been reported that integrins stimulate Ca2+ influx through Calreticulin family members. Ca2+ is a pleiotrophic second messenger that activates a wide variety of kinases. 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 (Pons, 2001).

The influence of presenilins on the genetic cascades that control neuronal differentiation have been examined in Xenopus embryos. Resembling sonic hedgehog (shh) overexpression, presenilin mRNA injection reduces the number of N-tubulin plus primary neurons and modulates Gli3 and Zic2 according to their roles in activating and repressing primary neurogenesis, respectively. Presenilin increases shh expression within its normal domain, mainly in the floor plate, whereas an antisense X-presenilin-alpha morpholino oligonucleotide reduces shh expression. Both shh and presenilin promote cell proliferation and apoptosis, but the effects of shh are widely distributed, while those resulting from presenilin injection coincide with the range of shh signaling. It is suggested that presenilin may modulate primary neurogenesis, proliferation, and apoptosis in the neural plate, through the enhancement of shh signaling (Paganelli, 2001).

In vertebrates the neural tube, like most of the embryonic organs, shows discreet areas of programmed cell death at several stages during development. In the chick embryo, cell death is dramatically increased in the developing nervous system and other tissues when the midline cells, notochord and floor plate, are prevented from forming by excision of the axial-paraxial hinge (APH), i.e. caudal Hensen’s node and rostral primitive streak, at the 6-somite stage. One day after APH excision, when dramatic apoptosis is already present in the neural tube, the latter can be rescued from death by grafting a notochord or a floor plate fragment in its vicinity. The neural tube can also be recovered by transplanting it into a stage-matched chick embryo having one of these structures. In addition, cells engineered to produce Sonic hedgehog protein (SHH) can mimic the effect of the notochord and floor plate cells in in situ grafts and transplantation experiments. SHH can thus counteract a built-in cell death program and thereby contribute to organ morphogenesis, in particular in the central nervous system (Charrier, 2001).

Hedgehog pathway activation is required for expansion of specific neuronal precursor populations during development and is etiologic in the human cerebellar tumor, medulloblastoma. 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 G1 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 (Kenney, 2004).

Neuronal precursor cells in the developing cerebellum require activity of the sonic hedgehog (Shh) and phosphoinositide-3-kinase (PI3K) pathways for growth and survival. Synergy between the Shh and PI3K signaling pathways are implicated in the cerebellar tumor medulloblastoma. A mechanism through which these disparate signaling pathways cooperate to promote proliferation of cerebellar granule neuron precursors is described. Shh signaling drives expression of mRNA encoding the Nmyc1 oncoprotein (previously N-myc), which is essential for expansion of cerebellar granule neuron precursors. The PI3K pathway stabilizes Nmyc1 protein via inhibition of GSK3-dependent Nmyc1 phosphorylation and degradation. The effects of PI3K activity on Nmyc1 stabilization are mimicked by insulin-like growth factor, a PI3K agonist with roles in central nervous system precursor growth and tumorigenesis. These findings indicate that Shh and PI3K signaling pathways converge on N-Myc to regulate neuronal precursor cell cycle progression. Furthermore, they provide a rationale for therapeutic targeting of PI3K signaling in medulloblastoma (Kenney, 2004).

The trigeminal ganglia differentiate in part from specialized ectodermal structures in the embryonic head termed the trigeminal placodes. However, the signals which govern the migration of trigeminal precursors and the final morphology of the ganglia are poorly defined. Notochord or floor plate tissue can induce the formation of ectopic sensory ganglia adjacent to the developing dorsal mesencephalon. Neurons within these ganglia coexpress the transcription factors Brn3a and Islet, which together characterize primary sensory neurons throughout the developing embryo. The ectopic ganglia originate from Pax3-expressing regions of the surface ectoderm that normally contribute to the ophthalmic trigeminal (op5), and can only be induced at developmental stages during which op5 precursors are present in the mesencephalic region. The migration of trigeminal precursors is also blocked by a local source of recombinant Shh, while in mouse embryos lacking Shh, these cells continue to migrate until they fuse into a single ganglion at the ventral midline. Together, these results suggest that Shh acts to arrest the migration of sensory precursors rather than to induce sensory neurons de novo. Consistent with this hypothesis, Shh induces the expression of the proteoglycan PG-M/versican in the cranial mesoderm, which has been implicated in the regulation of the movement of sensory neural precursors (Fedtsova, 2003).

In vertebrates, the nervous system arises from a flat sheet of epithelial cells, the neural plate, that gradually transforms into a hollow neural tube. This process, called neurulation, involves sequential changes in cellular interactions that are precisely coordinated both spatially and temporally by the combined actions of morphogens. To gain further insight into the molecular events regulating cell adhesion during neurulation, whether the adhesive and migratory capacities of neuroepithelial cells might be modulated by Sonic hedgehog (Shh), a signaling molecule involved in the control of cell differentiation in the ventral neural tube, was investigated. When deposited onto extracellular matrix components in vitro, neural plates explanted from avian embryos at early neurulation readily dispersed into monolayers of spread cells, thereby revealing their intrinsic ability to migrate. In the presence of Shh added in solution to the culture medium, the explants still exhibited the same propensity to disperse. In contrast, when Shh was immobilized to the substrate or produced by neuroepithelial cells themselves after transfection, neural plate explants failed to disperse and instead formed compact structures. Changes in the adhesive capacities of neuroepithelial cells caused by Shh could be accounted for by inactivation of surface ß-integrins combined with an increase in N-cadherin-mediated cell adhesion. Furthermore, immobilized Shh promotes differentiation of neuroepithelial cells into motor neurons and floor plate cells with the same potency as soluble Shh. However, the effect of Shh on the neuroepithelial cell adhesion was discernible and apparently independent from its differentiation effect and was not mediated by the signaling cascade elicited by the Patched-Smoothened receptor and involving the Gli transcription factors. Thus, these experiments indicate that Shh is able to control sequentially adhesion and differentiation of neuroepithelial cells through different mechanisms, leading to a coordinated regulation of the various cell interactions essential for neural tube morphogenesis (Jarov, 2003).

During development of the cerebellum, sonic hedgehog (Shh) is directly responsible for the proliferation of granule cell precursors in the external germinal layer. Signals able to regulate a switch from the Shh-mediated proliferative response to one that directs differentiation of granule neurons have been sought. Bone morphogenetic proteins (BMPs) are expressed in distinct neuronal populations within the developing cerebellar cortex. Bmp2 and Bmp4 are expressed in the proliferating precursors and subsequently in differentiated granule neurones of the internal granular layer, whereas Bmp7 is expressed by Purkinje neurones. In primary cultures, Bmp2 and Bmp4, but not Bmp7, are able to prevent Shh-induced proliferation, thereby allowing granule neuron differentiation. Furthermore, Bmp2 treatment downregulates components of the Shh pathway in proliferating granule cell precursors. Smad proteins, the only known BMP receptor substrates capable of transducing the signal, are also differentially expressed in the developing cerebellum: Smad1 in the external germinal layer and Smad5 in newly differentiated granule neurons. Among them, only Smad5 is phosphorylated in vivo and in primary cultures treated with Bmp2, and overexpression of Smad5 is sufficient to induce granule cell differentiation in the presence of Shh. A model is proposed in which Bmp2-mediated Smad5 signalling suppresses the proliferative response to Shh by downregulation of the pathway, and allows granule cell precursor to enter their differentiation programme (Rios, 2004).

Fate determination in the mammalian forebrain, where mature phenotypes are often not achieved until postnatal stages of development, has been an elusive topic of study despite its relevance to neuropsychiatric disease. In the ventral telencephalon, major subgroups of cerebral cortical interneurons originate in the medial ganglionic eminence (MGE), where the signaling molecule sonic hedgehog (Shh) continues to be expressed during the period of neuronogenesis. To examine whether Shh regulates cortical interneuron specification, mice were studied harboring conditional mutations in Shh within the neural tube. At embryonic day 12.5, NestinCre:ShhFl/Fl mutants have a relatively normal index of S-phase cells in the MGE, but many of these cells do not co-express the interneuron fate-determining gene Nkx2.1. This effect is reproduced by inhibiting Shh signaling in slice cultures, and the effect can be rescued in NestinCre:ShhFl/Fl slices by the addition of exogenous Shh. By culturing MGE progenitors on a cortical feeder layer, cell fate analyses suggest that Shh signaling maintains Nkx2.1 expression and cortical interneuron fate determination by MGE progenitors. These results are corroborated by the examination of NestinCre:ShhFl/Fl cortex at postnatal day 12, in which there is a dramatic reduction in cell profiles that express somatostatin or parvalbumin. By contrast, analyses of Dlx5/6Cre:SmoothenedFl/Fl mutant mice suggest that cell-autonomous hedgehog signaling is not crucial to the migration or differentiation of most cortical interneurons. These results combine in vitro and ex vivo analyses to link embryonic abnormalities in Shh signaling to postnatal alterations in cortical interneuron composition (Xu, 2005).

Sonic hedgehog and glial development

Spinal cord oligodendrocyte precursors arise in the ventral ventricular zone as a result of local signals. Over an extended developmental period, ectopic oligodendrocyte precursors can be induced by sonic hedgehog (Shh) in explants of chick dorsal spinal cord. However, the role of Shh during normal oligodendrocyte development is unclear. Shh is localized to the ventral spinal cord immediately prior to, and during the appearance of oligodendrocyte precursors. Continued expression of Shh is required for the appearance of spinal cord oligodendrocyte precursors because neutralization of Shh signaling both in vivo and in vitro during a defined developmental period blocks their emergence. The inhibition of oligodendrocyte precursor emergence in the absence of Shh signaling is not the result of inhibiting precursor cell proliferation, and the neutralization of Shh signaling after the emergence of oligodendrocyte precursors has no effect on the appearance of additional cells or on their subsequent differentiation. Similar concentrations of Shh induce motor neurons and oligodendrocytes in dorsal spinal cord explants. However, in explants from early embryos the motor neuron lineage is preferentially expanded while in explants from older embryos the oligodendrocyte lineage is preferentially expanded (Orentas, 1999).

Several lines of evidence suggest that the developmental of motor neurons and oligodendrocytes are closely linked. For example, both cell types arise in similar regions of the neural tube and both cell types can be induced in dorsal explants by Shh at similar concentrations. Based on these observations it has been proposed that the role of Shh may be to induce a common motor neuron/oligodendrocyte precursor. Several additional lines of evidence support this hypothesis. Retroviral cell lineage studies have demonstrated clones containing both motor neurons and glia, consistent with a lineage association between motor neurons and oligodendrocytes in the chick spinal cord. Recent studies demonstrate changes in the location of oligodendrocyte precursors and in the timing of development of both motor neurons and oligodendrocytes in the small eye (Pax6-knockout) mutant, suggesting Pax6 dependent alteration in the location of the common precursor cell in the ventricular zone. In the current study, additional support is found for the concept that motor neurons and oligodendrocytes are derived from a common precursor because virtually all dorsal explants that develop oligodendrocytes in response to Shh also contain motor neurons (Orentas, 1999 and references).

Although Sonic Hedgehog (Shh) plays a critical role in brain development, its actions on neural progenitor cell proliferation and differentiation have not been clearly defined. Transcripts for the putative Shh-receptor genes patched (Ptc) and smoothened (Smo) are expressed by embryonic, postnatal, and adult progenitor cells, suggesting that Shh can act directly on these cells. The recombinant human amino-terminal fragment of Shh protein (Shh-N) alone does not support the survival of cultured progenitor cells, but treatment with Shh-N in the presence of bFGF increases progenitor cell proliferation. Furthermore, treatment of embryonic rat progenitor cells propagated either in primary culture or after mitogen expansion significantly increases the proportions of both beta-tubulin- (neuronal marker) and O4- (oligodendroglial marker) immunoreactive cells and reduces the proportion of nestin- (uncommitted neural progenitor cell marker) immunoreactive cells. By contrast Shh-N has no effect on the elaboration of GFAP- (astroglial marker) immunoreactive cells. Cotreatment with Shh-N and bone morphogenetic protein-2 (BMP2) inhibits the anti-proliferative, astroglial-inductive, and oligodendroglial-suppressive effects of BMP2. These observations suggest that Shh-N selectively promotes the elaboration of both neuronal and oligodendroglial lineage species and inhibits the effects of BMP2 on progenitor cell proliferation and astroglial differentiation (Zhu, 1999).

In the chick metencephalon, oligodendrocyte precursors arise in distinct domains of the ventricular zone. During development, the earliest oligodendrocyte precursors appear in the metencephalic ventral ventricular zone adjacent to the midline, consistent with their location in the spinal cord. In contrast to spinal cord, however, distinct domains in the lateral and dorsal metencephalic ventricular zone subsequently generate oligodendrocyte precursors. All oligodendrogenic domains of the metencephalon appear in close apposition to regions that transiently express Sonic hedgehog. Inhibition studies demonstrate a functional requirement for Shh signaling in the early appearance of metencephalic oligodendrocyte precursors, while in vitro studies suggest a dose-dependent increase in the number of oligodendrocyte precursors in response to Shh. In purified cultures of oligodendrocyte precursors, Shh promotes cell survival and proliferation, suggesting that Shh can act directly on these cells. These data suggest that Shh may be responsible for the localized appearance of oligodendrocyte precursors throughout the CNS, irrespective of the dorso-ventral neural axis (Davis, 2001).

During development, basic helix-loop-helix (bHLH) proteins regulate formation of neurons from multipotent progenitor cells. However, bHLH factors linked to gliogenesis have not been described. A pair of oligodendrocyte lineage genes (Olg-1 and Olg-2: the Drosophila homolog is an uncharacterized gene designated CG5545) have been identified that encode bHLH proteins and are tightly associated with the development of oligodendrocytes in the vertebrate central nervous system (CNS). Ectopic expression of Olg-1 in rat cortical progenitor cell cultures promotes formation of oligodendrocyte precursors. In developing mouse embryos, Olg gene expression overlaps but precedes the earliest known markers of the oligodendrocyte lineage. Olg genes are expressed at the telencephalon-diencephalon border and adjacent to the floor plate, a source of the secreted signaling molecule Sonic hedgehog (Shh). Gain- and loss-of-function analyses in transgenic mice demonstrate that Shh is both necessary and sufficient for Olg gene expression in vivo (Lu, 2000).

Near the floor plate of the embryonic neural tube there is a group of neuroepithelial precursor cells that are specialized for production of the oligodendrocyte lineage. Experiments were performed to test whether specification of these neuroepithelial oligodendrocyte precursors, like other ventral neural cell types, depend on signals from the notochord and/or floor plate. Heterozygous Danforth's short tail (Sd/+) mutant mice, which lack a notochord and floor plate in caudal regions of the neural tube, were analyzed and oligodendrocyte precursors do not appear at the ventricular surface where there is no floor plate. Moreover, oligodendrocytes do not develop in explant cultures of Sd/+ spinal cord in the absence of a floor plate. When a second notochord is grafted into an ectopic position dorsolateral to the endogenous notochord of a chicken embryo, an additional floor plate is induced along with an ectopic focus of oligodendrocyte precursors at the ventricular surface. Oligodendrocytes develop in explants of intermediate neural tube only when they were cocultured with fragments of notochord or in the presence of purified Sonic hedgehog (Shh) protein. Thus, signals from the notochord/floor plate, possibly involving Shh, are necessary and sufficient to induce the development of ventrally derived oligodendroglia. These signals appear to act by specifying the future fate(s) of neuroepithelial cells at the ventricular surface rather than by influencing the proliferation or differentiation of prespecified progenitor cells in the parenchyma of the cord (Pringle, 1996).

Recent evidence indicates that oligodendrocytes originate initially from the ventral neural tube. The effect of early ventralization of the dorsal neural tube on oligodendrocyte differentiation has been analyzed in chick embryos. Notochord or floor plate grafted at stage 10 in a dorsal position induces the development of oligodendrocyte precursors in the dorsal spinal cord. In vitro, oligodendrocytes differentiate from medial but not intermediate neural plate explants, suggesting that the ventral restriction of oligodendrogenesis is established early. Furthermore, quail fibroblasts overexpressing the ventralizing signal Sonic Hedgehog induce oligodendrocyte differentiation in both the intermediate neural plate and the E4 dorsal spinal cord. These results strongly suggest that the emergence of the oligodendrocyte lineage are related to the establishment of the dorso-ventral polarity of the neural tube (Poncet, 1996).

There is increasing evidence that neurons and glial cells influence each other’s development. The optic nerve is an attractive place to study such neuron-glia interactions, since it is one of the simplest parts of the central nervous system (CNS), being composed mainly of retinal ganglion cell (RGC) axons and two types of glial cells: astrocytes and oligodendrocytes. The astrocytes develop from neuroepithelial cells that form the optic stalk, whereas the oligodendrocytes develop from precursor cells that migrate into the developing nerve from the brain. Axons in the optic nerve are known to signal to the glial cells in a number of ways: they promote the survival of oligodendrocytes, the proliferation and/or survival of oligodendrocyte precursors, and the proliferation of astrocytes. Sonic hedgehog Shh is expressed in RGCs and the Hedgehog receptor gene Patched (Ptc) is expressed in the adjacent neuroblast layer in the embryonic and postnatal mouse retina. From E12 onward, Ptc, but not Shh, is expressed in the optic nerve. Since oligodendrocyte precursors do not enter the optic nerve until after E14, it seems likely that the Ptc + cells in the nerve at E14 are cells of the astrocyte lineage, either astrocyte precursor cells and/or differentiated astrocytes. The cells in the nerve at this age, however, do not contain mRNA encoding the astrocyte marker glial fibrillary acidic protein (GFAP), suggesting that the Ptc+ cells are astrocyte precursors rather than differentiated astrocytes. At postnatal day 0, Ptc and GFAP are expressed all along the length of the optic nerve, whereas oligodendrocyte precursors expressing the platelet-derived growth factor alpha receptor (PDGFalphaR) were confined to the chiasm end of the optic nerve (Wallace, 1999b).

The following findings suggest that Shh is one of the signals that promote the survival of oligodendrocytes. (1) RGCs express both Shh mRNA and protein, whereas the optic nerve contains the protein but not the mRNA. (2) Astrocytes and their precursors in the developing optic nerve express the Hedgehog (Hh) receptor gene Patched (Ptc), suggesting that they are being signalled by an Hh protein. (3) Ptc expression in the nerve is greatly decreased by either nerve transection or by treatment with neutralizing anti-Shh antibodies, suggesting that it depends on axon-derived Shh. (4) Astrocyte proliferation in the developing nerve is reduced by treatment with anti-Shh antibodies, suggesting that Shh normally helps stimulate this proliferation (Wallace, 1999b).

The results suggest that Shh protein is transported to the RGC axon from the soma. If the protein simply diffuses in the extracellular fluid from the neural retina into the nerve, one might expect that it would induce a gradient of Ptc expression along the nerve, with the highest levels of expression closest to the eye. It is found, however, that the level of Ptc expression is uniform along the developing nerve. It is also unlikely that the Shh in the nerve comes via retrograde transport from the target cells that the RGCs innervate: although Shh is expressed in adult RGCs, Shh expression in the superior colliculus of adult mice cannot be detected. How then does Shh get from the RGC soma to the axon in a form that can signal to astrocyte lineage cells in the nerve? The simplest possibility is that it is inserted into the plasma membrane of the soma and diffuses along the surface of the axon to the nerve. Shh cannot be immunolocalized on the surface of RGC axons, however, perhaps because the protein is present at amounts too low to detect in this way. Another possibility is that Shh is transported within vesicles in the axon and is released by exocytosis, either at the nerve terminals, followed by retrograde transport along the axonal surface back to the nerve, or in the nerve itself. The results do not distinguish between these possibilities, but the previous finding that an intraocular injection of colchicine inhibits mitogenic signaling between axons and astrocytes in the optic nerve raises the possibility that microtubule-dependent processes may be involved in the transport of Shh to the nerve (Wallace, 1999b).

The role of Sonic hedgehog (Shh) in promoting the generation of oligodendrocytes in the mouse telencephalon is addressed in this study. In the forebrain, expression of the early oligodendrocyte markers Olig2, plp/dm20 and PDGFRalpha corresponds to regions of Shh expression. To directly test if Shh can induce the development of oligodendrocytes within the telencephalon, retroviral vectors were used to ectopically express Shh within the mouse embryonic telencephalon. Infections with Shh-expressing retrovirus at embryonic day 9.5 result in ectopic Olig2 and PDGFRalpha expression by mid-embryogenesis. By postnatal day 21, cells expressing ectopic Shh overwhelmingly adopt an oligodendrocyte identity. To determine if the loss of telencephalic Shh correspondingly results in the loss of oligodendrocyte production, Nkx2.1 mutant mice were studied, in which telencephalic expression of Shh is selectively lost. In accordance with Shh playing a role in oligodendrogenesis, within the medial ganglionic eminence of Nkx2.1 mutants, the early expression of PDGFRalpha is absent and the level of Olig2 expression is diminished in this region. In addition, in these same mutants, expression of both Shh and plp/dm20 is lost in the hypothalamus. Notably, in the prospective amygdala region where Shh expression persists in the Nkx2.1 mutant, the presence of plp/dm20 is unperturbed. Further supporting the idea that Shh is required for the in vivo establishment of early oligodendrocyte populations, expression of PDGFRalpha can be partially rescued by virally mediated expression of Shh in the Nkx2.1 mutant telencephalon. Interestingly, despite the apparent requirement for Shh for oligodendrocyte specification in vivo, all regions of either wild-type or Nkx2.1 mutant telencephalon are competent to produce oligodendrocytes in vitro. Furthermore, analysis of CNS tissue from Shh null animals definitively shows that, in vitro, Shh is not required for the generation of oligodendrocytes. It is proposed that oligodendrocyte specification is negatively regulated in vivo and that Shh generates oligodendrocytes by overcoming this inhibition. Furthermore, it appears that a Shh-independent pathway for generating oligodendrocytes exists (Nery, 2001).

Most studies on the origin of oligodendrocyte lineage, the myelin-forming cells in the central nervous system, have been performed in the spinal cord. By contrast, molecular mechanisms that regulate the appearance of the oligodendroglial lineage in the brain have not yet attracted much attention. Evidence exists for three distinct sources of oligodendrocytes in the mouse telencephalon. In addition to two subpallial ventricular foci (the anterior entopeduncular area and the medial ganglionic eminence), the rostral telencephalon also gives rise to oligodendrocytes. Oligodendrocytes in the olfactory bulb are generated within the rostral pallium from ventricular progenitors characterized by the expression of proteolipid protein (Plp). Evidence is provided that these Plp oligodendrocyte progenitors do not depend on signal transduction mediated by platelet-derived growth factor receptors (PDGFRs), and therefore it is proposed that they belong to a different lineage from that of the PDGFRalpha-expressing progenitors. Moreover, induction of oligodendrocytes in the telencephalon is dependent on sonic hedgehog signaling, as in the spinal cord. In all these telencephalic ventricular territories, oligodendrocyte progenitors are detected at about the same developmental stage as in the spinal cord. However, both in vivo and in vitro, the differentiation into O4-positive pre-oligodendrocytes is postponed by 4-5 days in the telencephalon in comparison with the spinal cord. This delay between determination and differentiation appears to be intrinsic to telencephalic oligodendrocytes, since it is not shortened by diffusible or cell-cell contact factors present in the spinal cord (Spassky, 2001).

Sonic hedgehog (SHH) and fibroblast growth factor 2 (FGF2) can both induce neocortical precursors to express the transcription factor OLIG2 and generate oligodendrocyte progenitors (OLPs) in culture. The activity of FGF2 is unaffected by cyclopamine, which blocks Hedgehog signalling, demonstrating that the FGF pathway to OLP production is Hedgehog independent. Unexpectedly, SHH-mediated OLP induction is blocked by PD173074, a selective inhibitor of FGF receptor (FGFR) tyrosine kinase. SHH activity also depends on mitogen-activated protein kinase (MAPK) but SHH does not itself activate MAPK. Instead, constitutive activity of FGFR maintains a basal level of phosphorylated MAPK that is absolutely required for the OLIG2- and OLP-inducing activities of SHH. Stimulating the MAPK pathway with a retrovirus encoding constitutively active RAS shows that the requirement for MAPK is cell-autonomous, i.e. MAPK is needed together with SHH signalling in the cells that become OLPs. It seems likely that SHH needs a basal level of active MAPK in order to function, and that constitutive FGFR activity in cortical cultures provides the necessary stimulus (Kessaris, 2004).

Sonic hedgehog is a negative regulator of growth cone movement

Retinal ganglion cell (RGC) axons grow towards the diencephalic ventral midline during embryogenesis guided by cues whose nature is largely unknown. In vitro and in vivo evidence is provided for a novel role of Sonic hedgehog (SHH) as a negative regulator of growth cone movement. SHH suppresses both the number and the length of neurites emerging from the chick retina but not from neural tube or dorsal root ganglia explants, without interfering with their rate of proliferation and differentiation. Similarly, retroviral-mediated ectopic expression of Shh along the chick visual pathway greatly interferes with the growth of RGC axons. Upon SHH addition to grown neurites, the intracellular level of cAMP decreases, suggesting that the dampening of growth cone extension mediated by SHH may involve interaction with its receptor Patched, which is expressed by RGC. Based on these findings, it is proposed that Shh expression at the chiasm border defines a constrained pathway within the ventral midline, which serves to guide the progression of RGC axons (Troussel, 2001).

Table of contents

hedgehog continued: Biological Overview | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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