Table of contents

Hedgehog and neural patterning: telencephalon, diencephalon and mesencephalon

The cortex and basal ganglia are the major structures of the adult brain derived from the embryonic telencephalon. Two morphologically distinct regions of the basal ganglia are evident within the mature ventral telencephalon: the globus pallidus (medially) and the striatum, which is positioned between the globus pallidus and the cortex. The globus pallidus develops from the medial ganglionic eminence (MGE), while the striatum develops from the lateral ganglionic eminence (LGE). These structures arise sequentially during development, with the MGE appearing immediately after anterior neuropore closure, followed later by the appearance of the LGE. Deletion of the Sonic Hedgehog gene in mice indicates that this secreted signaling molecule is vital for the generation of both these ventral telencephalic regions. Sonic hedgehog induces differentiation of ventral neurons characteristic of the medial ganglionic eminence, the embryonic structure which gives rise to the globus pallidus. While both Shh and Nkx2.1 are expressed strongly within the MGE, neither is present within the LGE. Sonic hedgehog induces ventral neurons with patterns of gene expression characteristic of the lateral ganglionic eminence. Although Ikaros is an LGE-specific marker, its expresssion only becomes detectable near birth, by in situ hybridization. Therefore, the absence or presence of Shh or Nkx2.1 in combination with the more widespred ventral telencephalic markers (Dlx, Evf-1, Islet-1/2 and GAD-16 ES) were used to distinguish LGE- and MGE-derived ventral neurons. An in vitro assay was used to evaluate the role of Shh in LGE induction. During a narrow window of competence, between E10.5-E11.5 of rat development (10 to 23 somites), either ventral telecephalic midline tissue or Shh protein can induce telencephalic tissue to express genes characteristic of the LGE. Even at the highest Shh concentration, the MGE/pallidal marker Nkx2.1 is not induced at this stage of development. Furthermore, if Shh is blocked between the time the MGE and LGE are induced, Dlx expression is greatly reduced. E11.5 explants in isolation are found to undergo a progressive loss of competence to express Dlx in response to Shh after one or two days in culture. These results suggest that temporally regulated changes in Sonic Hedgehog responsiveness are integral in the sequential induction of basal telencephalic structures (Kohtz, 1998).

The telencephalon is organized into distinct longitudinal domains: the cerebral cortex and the basal ganglia. The basal ganglia primarily consists of a dorsal region (striatum) and a ventral region (pallidum). Within the telencephalon, the anlage of the pallidum expresses the Nkx2.1 homeobox gene. Nkx2.1 expression is first detectable in the basal telencephalon of the mouse at approximately the 11 somite stage. As the basal telencephalon develops, Nkx2.1 is maintained in regions that form morphologically distinct structures such as the medial ganglionic eminence (MGE), as well as parts of the septum, anterior entopeduncular area and preoptic area (POA). Sonic hedgehog (SHH) secretion from the axial mesendoderm is required for patterning of the anteromedial neural plate, including the hypothalamus and basal telencephalon. In addition, SHH can induce markers of the basal telencephalon, such as Nkx2.1. Shh begins to be expressed in the VZ of the ventral-most regions of the basal telencephalon (preoptic and anterior entopeduncular areas; POA, AEP) between the 10-12 somite stage, at approximately the same time as Nkx2.1 begins to be expressed in the basal telencephalon. Subsequently, Shh expression spreads into the SVZ and mantle of the MGE. Because Shh is expressed early in MGE development and has a potential role in telencephalic patterning, its expression was assessed in the developing MGE of the Nkx2.1 mutant. Surprisingly, at E10.5 and E11.5, Shh expression is undetectable in the mutant basal telencephalon and hypothalamus, aside from trace levels of Shh in the rostral midline at E10.5. Shh expression in the midbrain and more posterior regions of the central nervous system appeared normal at all ages examined. To determine whether Nkx2.1 expression is required for induction or maintenance of Shh expression in the forebrain, E8.75-E9.5 Nkx2.1 mutant embryos were examined by whole-mount in situ hybridization. Even at these stages, which ordinarily show the expression of Shh in the forebrain, Shh transcripts in the hypothalamus and basal telencephalon are not detectable, while Shh expression in the anterior mesendoderm is normal (Sussel, 1999).

Sonic hedgehog (Shh) has a crucial role in the generation of ventral cell types along the entire rostrocaudal axis of the neural tube. At caudal levels of the neuraxis, Shh is secreted by the notochord and floor plate during the period that ventral cell fates are specified. However, at anterior prosencephalic levels that give rise to the telencephalon, neither the prechordal mesoderm nor the ventral neural tube expresses Shh at the time that the overt ventral character of the telencephalon becomes evident. Thus, the precise role and timing of Shh signaling relevant to the specification of ventral telencephalic identity remains unclear. By analyzing neural cell differentiation in chick neural plate explants, evidence is provided that neural cells acquire molecular properties characteristic of the ventral telencephalon in response to Shh signals derived from the anterior primitive streak/Hensen’s node region at gastrula stages. Exposure of prospective anterior prosencephalic cells to Shh at this early stage is sufficient to initiate a temporal program of differentiation that parallels that of neurons generated normally in the medial ganglionic eminence subdivision of the ventral telencephalon (Gunhaga, 2000).

An early phase of Shh signaling appears to initiate a program of differentiation that proceeds in the absence of further Shh exposure, but only at a later stage do cells progress to a state of ventral commitment. How do telencephalic cells acquire their early independence from Shh signaling? In vivo, the profile of expression of Nkx2.1, Dlx, and Isl1 defines temporally distinct phases in the differentiation of Isl1+ neurons. Nkx2.1 expression appears first and is confined to ventral progenitor cells; Dlx+ cells and Isl1+ cells are first detected approx. 15 hours later. At this stage, Dlx proteins are expressed both by proliferative progenitor cells and by post-mitotic neurons, whereas Isl1 is restricted to post-mitotic neurons, some of which also express Dlx proteins. The temporal patterns of generation of Nkx2.1+ , Dlx+ and Isl1 + cells in HH stage 6 T explants mimic closely those observed in vivo. The late onset of Dlx protein expression raises the possibility that Dlx expression defines a late step in the differentiation of ventral telencephalic progenitor cells, perhaps analogous to the onset of expression of MNR2 and Lim3 during the final division cycle of motor neuron progenitors at more caudal levels of the neuraxis. The sufficiency of a brief period of Shh exposure for the generation of ventral telencephalic character is in apparent contrast with results obtained at more caudal levels of the neuraxis, where a prolonged period of Shh exposure appears to be required for the specification of motor neuron differentiation. This difference may relate in part to the identity of the ventral cell type under study. Even at caudal levels of the neural tube, the inhibition of Shh signaling at late stages does not block the differentiation of V0 and V1 interneurons; neurons generated in the dorsalmost region of the ventral neural tube. Thus, at caudal levels of the neuraxis prolonged Shh exposure may be necessary for the differentiation of only certain ventral neuronal subtypes (Gunhaga, 2000).

The ventral half of the prospective telencephalon contains two major regional subdivisions at early stages in its differentiation: these are the medial ganglionic eminence (MGE) and the lateral ganglionic eminence (LGE), regions that later give rise to the globus pallidum and the corpus striatum, respectively (Smart and Sturrock, 1979). At a molecular level, cells that contribute to the MGE can be distinguished from LGE cells by their expression of the Nkx class homeobox gene Nkx2.1. Moreover, Nkx2.1 function is required for the specification of cells of MGE character. In Nkx2.1 mutant mouse embryos the MGE fails to differentiate and cells of LGE character are found at a more ventral position of the neural tube. This early distinction between the MGE and LGE appears to depend, at least in part, on a limitation in the range of Shh signaling. In support of this idea, Nkx2.1 is expressed along most of the dorsoventral axis of the telencephalon in mice lacking Ptc function, a situation in which neural cells receive high level activation from the Shh signal transduction pathway. This finding implies that cells arrayed along the entire dorsoventral axis of the telencephalon are initially competent to generate an MGE-like character if exposed at an early stage to Shh signaling (Gunhaga, 2000).

The spatial limitation in generation of cells of MGE character could reflect exposure of more dorsal telencephalic cells to a Shh concentration insufficient to induce a MGE fate. Alternatively, dorsal cells may be exposed to signals that antagonize Shh signaling. BMPs have been shown to antagonize Shh signaling at caudal levels of the neural tube and are normally required to induce dorsal interneurons. BMP4 can block the expression of Nkx2.1 by cells in explants of stage 6 and stage 9 ventral telencephalon, and telencephalic progenitor cells become resistant to this action of BMP4 during an approx. 5 hours period, between stages 12 and 15. BMPs are expressed in the dorsal region of the forebrain and in the surrounding epidermal ectoderm at these stages. BMPs have also been shown to promote the expression of Msx1/Msx2 and thus may act to specify dorsal cell fates in the telencephalon as in the spinal cord and hindbrain. During development of the telencephalon, the ability of BMP signaling to antagonize Shh signaling could therefore contribute to the generation of cell types of more dorsal character (Gunhaga, 2000).

In addition, early exposure of prospective ventral telencephalic cells to FGF prevents the generation of Isl1+ neurons. FGF signaling enhances the incorporation of BrdU and prevents the generation of Isl1+ neurons, but does not affect the generation of Nkx2.1+ progenitor cells in explants of the ventral telencephalon. The FGF family members, Fgf8, Fgf15, Fgf17 and Fgf8, are expressed in the anterior neural ridge and later in the ventral telencephalon. Reduced levels of expression of Fgf8 in mouse lead to an impairment in the growth of the telencephalon. Moreover, in chick, Fgf8 starts to be expressed by cells in the anterior neural ridge by stage 9, approx. 30 hours before Isl1+ neurons start to accumulate in the ventral telencephalon. One interpretation of these observations is that FGFs normally have a role in promoting the proliferation and in delaying the differentiation of forebrain progenitor cells. In this respect the actions of FGFs in the ventral telencephalon parallel the role of FGF signaling in the developing pituitary gland. Here Fgf 8 is expressed in the infundibular region of the ventral diencephalon, which later develops into the posterior pituitary. Isl1 is expressed by cells in the adjacent anterior pituitary and FGF signaling can block the expression of Isl1 in cells in the dorsal region of the anterior pituitary. Thus FGFs may act to inhibit the expression of Isl1 in both neuroepithelial and neuroendocrine cells (Gunhaga, 2000).

Midbrain consists of two major components along the dorsoventral (DV) axis: the tectum and the tegmentum. It has been shown in chick-quail chimera that the optic tectum derives exclusively from the midbrain alar plate. After day 3 of development (E3), the alar plate undergoes rapid cell proliferation to form an enlarged tectum. In contrast, the basal plate gives rise to the midbrain tegmentum. Instead of massive cell proliferation, neural differentiation takes place in this ventral area to produce many nuclei related to motor function. Although these two structures undergo quite different developmental programs, it is largely unknown how the differences are molecularly controlled along the DV axis. It is largely unknown how the differences between these two structures are molecularly controlled during the midbrain development. The secreted protein Sonic hedgehog (Shh) produced in the notochord and floor plate induces differentiation of ventral cell types of the central nervous system. To evaluate the role of Shh in the establishment of dorsoventral polarity in the developing midbrain, Shh was ectopically expressed unilaterally in the brain vesicles, including whole midbrain of E1.5 chick embryos in ovo. Ectopic Shh represses normal growth of the tectum, producing a dorsally enlarged tegmentum region. In addition, the expression of several genes crucial for tectum formation is strongly suppressed in the midbrain and isthmus. Markers for midbrain roof plate are inhibited, indicating that the roof plate was not fully generated (Watanabe. 2000).

After E5, the tectum territory of the Shh-transfected side has been significantly reduced and is fused with that of the untransfected side. Thus, Shh inhibits the tectum formation and its subdivision into optic lobes. From E3 onward, dorsal midbrain continues rapid expansion to form the optic tectum. In the E5.5 embryo, the tectum has just started to subdivide into right and left hemispheres at the dorsal midline. In Shh-transfected embryos, reduction of the tectum size is apparent. Subdivision of the tectum into two hemispheres is not observed at the dorsal midline. Most importantly, the transfected tectum is significantly reduced in size, while the ventral tegmentum region is dorsally enlarged. Thereafter the tectum becomes completely separated into the right and the left hemispheres to form optic lobes, which later rotate between E7 and E12 so that the rostrocaudal axis of the tectum accords with the ventrodorsal axis of the body. At E12.5, the rotation is almost complete and the two optic lobes are situated dorsolaterally to the tegmentum. In contrast, the tectum of Shh-transfected embryos is not separated, no matter on which side Shh is introduced. Rotation of the tectum is also incomplete; the fused tectum is located at the dorsal side of the tegmentum. Histologically, the fused tectum still has the laminar structure of the tectum. At E18.5, total brain size is reduced to some extent in transfected embryos and the fused tectum is in a dorsal position. Ectopic Shh induces a considerable number of SC1-positive motor neurons, overlapping markers such as HNF-3 beta (floor plate), Isl-1 (postmitotic motor neuron) and Lim1/2. Dopaminergic and serotonergic neurons are also generated in the dorsally extended region. These changes indicate that ectopic Shh changes the fate of the mesencephalic alar plate to that of the basal plate, suppressing the massive cell proliferation that normally occurs in the developing tectum. Taken together these results suggest that Shh signaling restricts the tectum territory by controlling the molecular cascade for tectum formation along the dorsoventral axis and by regulating neuronal cell diversity in the ventral midbrain (Watanabe, 2000).

The expressions of genes related to the tectum formation were examined in Shh-transfected embryos at E3.5. The transcription factor HNF-3 beta required for the differentiation of floor plate cells is expressed in the midline of the neural plate after stage 5. At E3.5 HNF-3 beta expression is observed in the zona limitans intrathalamica (ZLI) and the floor plate posterior to the diencephalon. On the transfected side, ectopic expression of HNF-3 beta is exclusively induced in the introduced Shh-positive region of diencephalon, midbrain and hindbrain. In diencephalon, Pax-6 is expressed at the dorsal side complementary to Shh expression at ventral side. Pax-6 expression totally disappears in the ectopic Shh-positive region, making a distinct boundary between Shh- and Pax-6-positive domains as in ZLI. Otx-2 transcripts are restricted anterior to the isthmus in an early phase of embryogenesis. Genetic alterations of Otx genes in mice suggests that they are required to establish the midbrain-hindbrain boundary along the AP axis. Misexpression of Shh in these experiment does not affect the Otx-2 expression domain and its posterior limit is conserved. En-2 is also involved in midbrain and hindbrain development together with En-1. The gradient of En-2 expression is a determinant of AP polarity in the midbrain. The AP gradient of En-2 expression in the midbrain and isthmus is diminished in Shh-transfected area. PAX-7 localization is detected in the pretectum, tectum and dorsal hindbrain. PAX-7 expression in the dorsal midbrain is suppressed by the heterotopic grafting of the midbrain floor plate, producing tegmentum-like structure instead of the tectum. Ectopic Shh extinguishes expression of PAX-7 in dorsal midbrain. Pax-2 and Pax-5 are involved in the organization of the tectum. Expression of both genes in the isthmus is inhibited by Shh transfection. Another gene of the isthmic organizer, Fgf8, is localized in the narrow band at the midbrain-hindbrain boundary. Interestingly, in the transfected embryo, the band of Fgf8 expression shifts to the rhombic lip, while it is almost extinguished in the isthmus. Wnt-1 and Msx-1 transcripts are normally localized on the dorsal midline from posterior diencephalon to midbrain. In the Shh-transfected embryos, caudal expression of both genes in the midbrain is totally absent, with diminished expression rostral to the midbrain. These changes caused by Shh transfection are already detected at E2.5, indicating that Shh can suppress the expression of genes involved in early tectal development. Moreover, the loss of dorsal midline markers suggests that the roof plate might not have been fully generated when the ectopic Shh was introduced in the lateral hemisphere. The expression of Shh-target genes Ptc and Gli-1 was examined. Both genes are expressed in the neural tube adjacent to the Shh-positive domain, which reflects the Shh signal reception. At the midbrain level, Ptc is expressed in a longitudinal line complementary to Shh expression. In Shh-transfected embryos, Ptc expression is induced in the domain where Shh has been introduced. Moreover, another line of ectopic Ptc expression appears at the dorsal side complementary to the introduced Shh expression. In contrast, the Gli-1 transcripts that are observed in the ventral half of the midbrain in the normal embryos are absent with ectopic Shh expression (Watanabe, 2000).

The mechanisms that regulate the growth of the brain remain unclear. Sonic hedgehog (Shh) is expressed in a layer-specific manner in the perinatal mouse neocortex and tectum, whereas the Gli genes, which are targets and mediators of SHH signaling, are expressed in proliferative zones. In vitro and in vivo assays show that SHH is a mitogen for neocortical and tectal precursors and that it modulates cell proliferation in the dorsal brain. Together with its role in the cerebellum, these findings indicate that SHH signaling unexpectedly controls the development of the three major dorsal brain structures. A variety of primary human brain tumors and tumor lines consistently express the GLI genes and cyclopamine, a SHH signaling inhibitor, inhibits the proliferation of tumor cells. Using the in vivo tadpole assay system, it has been further shown that misexpression of GLI1 induces CNS hyperproliferation that depends on the activation of endogenous Gli1 function. SHH-GLI signaling thus modulates normal dorsal brain growth by controlling precursor proliferation, an evolutionarily important and plastic process that is deregulated in brain tumors (Dahmane, 2001).

Hedgehog signaling is required for multiple aspects of brain development, including growth, the establishment of both dorsal and ventral midline patterning and the generation of specific cell types such as oligodendrocytes and interneurons. To identify more precisely when during development hedgehog signaling mediates these events, the removal of hedgehog signaling within the brain was directed by embryonic day 9 of development, using a FoxG1Cre driver line to mediate the removal of a conditional smoothened null allele. A loss of ventral telencephalic patterning was observed that appears to result from an initial lack of specification of these structures rather than by changes in proliferation or cell death. A further consequence of the removal of smoothened in these mice is the near absence of both oligodendrocytes and interneurons. Surprisingly, the dorsal midline appears to be patterned normally in these mutants. Together with previous analyses, the present results demonstrate that hedgehog signaling in the period between E9.0 and E12 is essential for the patterning of ventral regions and the generation of cell types that are thought to largely arise from them (Puccillo, 2004).

The zona limitans intrathalamica (ZLI) is located at the border between the prospective ventral thalamus and dorsal thalamus, and functions as a diencephalic signaling center. Little is known about the mechanism controlling ZLI formation. Using a combination of fate-mapping studies and in vitro assays, it was shown that the differentiation of the ZLI from progenitor cells in the alar plate is initiated by a Shh-dependent signal from the basal plate. The subsequent dorsal progression of ZLI differentiation requires ongoing Shh signaling, and is constrained by inhibitory factors derived from the dorsal diencephalon. These studies demonstrate that self-organizing signals from the basal plate regulate the formation of a potential patterning center in the ZLI in an orthogonal orientation in the alar plate, and thus create the potential for coordinated thalamic patterning in two dimensions (Zeltser, 2005).

The specification of rostrocaudal neural identity appears to obey a fragmentary logic, with distinct organizing centers operating over different rostrocaudal domains of the neural tube. Three major signaling centers are known to control rostrocaudal neural pattern: the anterior neural ridge (ANR), the isthmic organizer (IsO) and the node. The ANR is positioned at the rostral extreme of the neural tube and directs rostrocaudal cell fates in the telencephalon, in part through the actions of Fgf8. The IsO is positioned at the junction between the midbrain and hindbrain, and regulates the specification of cell types in the midbrain and rostral hindbrain, through the secretion of Wnt1 and Fgf8. At more caudal levels of the neural tube that give rise to the spinal cord, rostrocaudal positional identity is influenced by node-derived signals, and Fgf8 is a major component of activity of the node. Thus, FGF signaling is a common feature of the activity of three distinct rostrocaudal organizing centers (Zeltser, 2005).

Within the diencephalon, however, the rostrocaudal patterning of cell types occurs independently of signals provided by the ANR and the IsO, and is likely to depend on signals provided by the ZLI, a prominent structure that protrudes from the basal plate at the boundary between the prospective ventral thalamus and the dorsal thalamus. Shh is expressed within the ZLI, and the acquisition of post-mitotic neural identities in adjacent thalamic tissues emerges in the wake of the ventral to dorsal progression of Shh expression within the ZLI. Recent studies have demonstrated that Shh signals are required for the specification of thalamic identitites, and that the likely source of these signals is the ZLI. Thus, the ZLI, as with the IsO and the ANR, is aligned perpendicular to the main axis of the neural tube, but as with a dorsoventral organizing center, the floor plate expresses Shh. In some respects then, the ZLI may be an organizing center with properties characteristic of both rostrocaudal and dorsoventral patterning centers (Zeltser, 2005).

Although the ZLI has been implicated in diencephalic patterning, it is as yet unclear how the ZLI is formed. Some evidence about the early patterning mechanisms that govern the initial position of ZLI formation has emerged. At neural plate stages, cross-repressive interactions between the transcription factors Six3 and Irx3 establish a boundary that anticipates the position of formation of the ZLI. After neural tube closure, Wnt8b expression marks the prospective ZLI and is flanked by domains of Lunatic fringe (Lfng) expression. Furthermore, ectopic Lfng expression represses ZLI formation, suggesting that the lateral limits of the ZLI are normally constrained by these adjacent domains of Lfng expression. Despite these insights, however, the factors responsible for inducing the ZLI, in the context of these positional constraints, remain unknown (Zeltser, 2005).

This study examines the molecular mechanisms that control ZLI formation in an avian forebrain explant system. Through fate-mapping experiments, it is shown that the ZLI differentiates in the alar plate of the diencephalon in response to inductive signals derived from the basal plate. Shh signaling from the basal plate is required to initiate Shh expression within the ZLI, and, subsequently, long-range Shh signals provided by the basal plate and ZLI are needed for its dorsal progression. Finally, the dorsal limit of progression of the ZLI appears to be constrained in part by a limit in the range of Shh action, and in part by an opponent signal emanating from the dorsal diencephalon (Zeltser, 2005).

In the developing ventral midbrain, the signaling molecule sonic hedgehog (SHH) is sufficient to specify a striped pattern of cell fates (midbrain arcs). This study asked whether and precisely how hedgehog (HH) signaling might be necessary for ventral midbrain patterning. By blocking HH signaling by in ovo misexpression of Ptc1Deltaloop2, it was shown that HH signaling is necessary and can act directly at a distance to specify midbrain cell fates. Ventral midbrain progenitors extinguish their dependence upon HH in a spatiotemporally complex manner, completing cell-fate specification at the periphery by stage 13. Thus, patterning at the lateral periphery of the ventral midbrain is accomplished early, when the midbrain is small and the HH signal needs to travel relatively short distances (approximately 30 cell diameters). Interestingly, single-cell injections demonstrate that patterning in the midbrain occurs within the context of cortex-like radial columns of cells that can share HH blockade and are cytoplasmically connected by gap junctions. HH blockade results in increased cell scatter, disrupting the spatial coherence of the midbrain arc pattern. Finally, HH signaling is required for the integrity and the signaling properties of the boundaries of the midbrain (e.g., the midbrain-hindbrain boundary, the dorsoventral boundary), its perturbations resulting in abnormal cell mixing across 'leaky' borders (Bayly, 2007).

The forebrain is patterned along the dorsoventral (DV) axis by Sonic Hedgehog (Shh). However, previous studies have suggested the presence of an Shh-independent mechanism. This study identifies Wnt/β-catenin (activated from the telencephalic roof) as an Shh-independent pathway that is essential for telencephalic pallial (dorsal) specification during neurulation. The transcription factor Foxg1 coordinates the activity of two signaling centers: Foxg1 is a key downstream effector of the Shh pathway during induction of subpallial (ventral) identity, and it inhibits Wnt/β-catenin signaling through direct transcriptional repression of Wnt ligands. This inhibition restricts the dorsal Wnt signaling center to the roof plate and consequently limits pallial identities. Concomitantly to these roles, Foxg1 controls the formation of the compartment boundary between telencephalon and basal diencephalon. Altogether, these findings identify a key direct target of Foxg1, and uncover a simple molecular mechanism by which Foxg1 integrates two opposing signaling centers (Danesin, 2009).

Holoprosencephaly (HPE) is the most common congenital malformation of the forebrain in human. Several genes with essential roles during forebrain development have been identified because they cause HPE when mutated. Among these are genes that encode the secreted growth factor Sonic hedgehog (Shh) and the transcription factors Six3 and Zic2. In the mouse, Six3 and Shh activate each other's transcription, but a role for Zic2 in this interaction has not been tested. This study demonstrates that in zebrafish, as in mouse, Hh signaling activates transcription of six3b in the developing forebrain. zic2a is also activated by Hh signaling, and represses six3b non-cell-autonomously, i.e. outside of its own expression domain, probably through limiting Hh signaling. Zic2a repression of six3b is essential for the correct formation of the prethalamus. The diencephalon-derived optic stalk (OS) and neural retina are also patterned in response to Hh signaling. This study shows that zebrafish Zic2a limits transcription of the Hh targets pax2a and fgf8a in the OS and retina. The effects of Zic2a depletion in the forebrain and in the OS and retina are rescued by blocking Hh signaling or by increasing levels of the Hh antagonist Hhip, suggesting that in both tissues Zic2a acts to attenuate the effects of Hh signaling. These data uncover a novel, essential role for Zic2a as a modulator of Hh-activated gene expression in the developing forebrain and advance understanding of a key gene regulatory network that, when disrupted, causes HPE (Sanek, 2009).

Dorsoventral (DV) specification is a crucial step for the development of the vertebrate telencephalon. Clarifying the origin of this mechanism will lead to a better understanding of vertebrate central nervous system (CNS) evolution. Based on the lamprey, a sister group of the gnathostomes (jawed vertebrates), three lamprey Hedgehog (Hh) homologues were identified, that are thought to play central signalling roles in telencephalon patterning. However, unlike in gnathostomes, none of these genes, nor Lhx6/7/8, a marker for the migrating interneuron subtype, was expressed in the ventral telencephalon, consistent with the reported absence of the medial ganglionic eminence (MGE) in this animal. Homologues of Gsh2, Isl1/2 and Sp8, which are involved in the patterning of the lateral ganglionic eminence (LGE) of gnathostomes, were expressed in the lamprey subpallium, as in gnathostomes. Hh signalling is necessary for induction of the subpallium identity in the gnathostome telencephalon. When Hh signalling was inhibited, the ventral identity was disrupted in the lamprey, suggesting that prechordal mesoderm-derived Hh signalling might be involved in the DV patterning of the telencephalon. By blocking fibroblast growth factor (FGF) signalling, the ventral telencephalon was suppressed in the lamprey, as in gnathostomes. It is concluded that Hh- and FGF-dependent DV patterning, together with the resultant LGE identity, are likely to have been established in a common ancestor before the divergence of cyclostomes and gnathostomes. Later, gnathostomes would have acquired a novel Hh expression domain corresponding to the MGE, leading to the obtainment of cortical interneurons (Sugahara, 2011).

The prospective white matter (PWM) in the nascent cerebellum contains a transient germinal compartment that produces all postnatally born GABAergic inhibitory interneurons and astrocytes. However, little is known about the molecular identity and developmental potential of resident progenitors or key regulatory niche signals. This study shows that neural stem-cell-like primary progenitors (TncYFP-low CD133+) generate intermediate astrocyte (TncYFP-low CD15+) precursors and GABAergic transient amplifying (Ptf1a+) cells. Interestingly, these lineally related but functionally divergent progenitors commonly respond to Sonic hedgehog (Shh), and blockade of reception in TNCYFP-low cells attenuates proliferation in the PWM, reducing both intermediate progenitor classes. Furthermore, Shh produced from distant Purkinje neurons was shown to maintain the PWM niche independently of its classical role in regulating granule cell precursor proliferation. These results indicate that Purkinje neurons maintain a bidirectional signaling axis, driving the production of spatially and functionally opposed inhibitory and excitatory interneurons important for motor learning and cognition (Fleming, 2013).

During development, region-specific patterns of regulatory gene expression are controlled by signaling centers that release morphogens providing positional information to surrounding cells. Regulation of signaling centers themselves is therefore critical. The size and the influence of a Shh-producing forebrain organizer, the zona limitans intrathalamica (ZLI), are limited by Pax6 (see Drosophila Eyeless). By studying mouse chimeras, this study found that Pax6 acts cell autonomously to block Shh expression in cells around the ZLI. Immunoprecipitation and luciferase assays indicate that Pax6 can bind the Shh promoter and repress its function. An analysis of chimeras suggests that many of the regional gene expression pattern defects that occur in Pax6-/- diencephalic cells result from a non-cell-autonomous position-dependent defect of local intercellular signaling. Blocking Shh signaling in Pax6-/- mutants reverses major diencephalic patterning defects. It is concluded that Pax6's cell-autonomous repression of Shh expression around the ZLI is critical for many aspects of normal diencephalic patterning (Caballero, 2014: PubMed).

Anterior/posterior patterning

The posteriorizing agent retinoic acid can accelerate anterior neuronal differentiation in Xenopus laevis embryos. To elucidate the role of retinoic acid in the primary neurogenesis cascade, an investigation was carried out to see whether retinoic acid treatment of whole embryos can change the spatial expression of a set of genes known to be involved in neurogenesis. Retinoic acid expands the N-tubulin, X-ngnr-1, X-MyT1, X-Delta-1 and Gli3 domains and inhibits the expression of Zic2 and sonic hedgehog in the neural ectoderm, whereas a retinoid antagonist produces the opposite changes. In contrast, sonic and banded hedgehog overexpression reduce the N-tubulin stripes, enlarge the neural plate at the expense of the neural crest, downregulate Gli3 and upregulate Zic2. Thus, retinoic acid and hedgehog signaling have opposite effects on the prepattern genes Gli3 and Zic2 and on other genes acting downstream in the neurogenesis cascade. In addition, retinoic acid cannot rescue the inhibitory effect of NotchICD, Zic2 or sonic hedgehog on primary neurogenesis. These results suggest that retinoic acid acts very early, upstream of sonic hedgehog, and a model is proposed for regulation of differentiation and proliferation in the neural plate, showing that retinoic acid might be activating primary neurogenesis by repressing sonic hedgehog expression (Franco, 1999).

RA treatment can accelerate neuronal differentiation in the anterior neural plate of whole embryos. Could RA also alter neuronal differentiation in the posterior neural plate where endogenous RA might mainly play its role and where primary neurogenesis occurs? It has been shown that RA exposure during gastrulation greatly expands the normal domains of N-tubulin expression at the neural plate stage. In contrast, retinoic acid antagonist Ro treatments decrease N-tubulin expression, in agreement with the loss of primary neurons produced by the microinjection of dominant negative forms of retinoic acid receptors. RA treatment increases the domains of genes previously shown to promote neuronal differentiation, such as X-ngnr-1, X-MyT1 and Gli3. The deletion of spacing between the stripes of X-ngnr-1 and X-MyT1 suggests that RA changes the activity of prepattern genes, thus directing the neural plate toward a uniform proneural territory. Indeed, RA produces a widespread Gli3 expansion in the posterior neural plate and a dramatic downregulation of Zic2, a gene proposed to inhibit neuronal differentiation. The involvement of endogenous retinoids in this regulatory hierarchy was confirmed by blocking RA signaling with Ro, which produced opposite changes in the expression patterns of these genes (Franco, 1999).

Because X-Delta-1 appears to be expressed in the future primary neurons themselves, they should be the source of the inhibitory signal that activates X-Notch-1 in the neighboring cells, thus preventing them from undergoing neuronal differentiation, inhibiting their own X-Delta-1 expression and decreasing their ability to inhibit the original signaling cell. This would generate a feedback loop that reinforces contrasts between adjacent cells. Here it is shown that RA treatment enhances the density of X-Delta-1-positive cells and it is presumed that, in this way, it impairs the developing distinction between adjacent cells, allowing more precursors to become neurons. Since X-ngnr-1 overexpression leads to X-Delta-1 overproduction, RA could be activating X-Delta-1 expression through X-ngnr-1 induction (Franco, 1999).

Evidence is presented that endogenous retinoids downregulate the expression of genes that inhibit neurogenesis, like Zic2 and X-shh. While RA treatment reduces their expression, after blocking RA signaling, X-shh expression is increased along the dorsal midline and Zic2 expression becomes dispersed over the mediolateral axis of the neural plate, accounting for the inhibition of primary neurogenesis by Ro. Because previous work in chicken limb and zebrafish fin buds has demonstrated an induction of Shh expression in response to RA, it was surprising that X-shh expression is downregulated by RA at neurula stage both in the notochord and floor plate. These results agree with the very early transient downregulation observed in developing and regenerating axolotl limbs. Furthermore, the upstream region of zebrafish shh contains a retinoic acid responsive element (RARE), implying a direct regulation of the shh gene by RA. Thus there is solid evidence for a link between X-shh and RA at the molecular level (Franco, 1999).

The suppression of primary neurogenesis produced by the overexpression of X-shh and X-bhh is not due to inhibition of neural development, because the neural plate is expanded on the injected side, as shown with the general neural marker nrp-1. When compared to RA treatments, X-shh and X-bhh overexpression produce opposite changes in the expression patterns of different members of the neurogenesis cascade that resemble Ro effects, suggesting that a counterbalance exists between retinoid and hedgehog signaling to restrict primary neurogenesis to the normal sites. Precursors of the primary and secondary neurons arise from different layers of the neural plate. The superficial layer contains predominantly secondary precursors, whereas the deep layer contains both types of precursors at a similar density. Although the fate of the cells inhibited to differentiate by X-shh and X-bhh was not followed, they probably participate in subsequent waves of neurogenesis, as suggested by the fact that both hedgehog members later expande the number of cells expressing Xsal-1, a marker of ventral motor and intermediate neurons in the neural tube of tadpoles (Franco, 1999).

The evident expansion of the neural ectoderm and the paraxial mesoderm together with the increase in cell number are consistent with X-shh and X-bhh playing a proliferative role in both germ layers, but an inhibition of cell death cannot be excluded. Indeed, Shh promotes proliferation in the sclerotome and prevents differentiation, and induces a proliferative response in cerebellar cells. Therefore, it is proposed that both hedgehog members produce a differential effect on primary and secondary neuronal precursors, perhaps withdrawing cells from premature differentiation, holding their proliferative state and precluding them from subsequent waves of neuron formation (Franco, 1999).

RA downregulates X-shh expression whereas Ro produces the opposite change. It is proposed that, in the normal embryo, X-shh expression in the dorsal midline should be controlled by positive and negative regulators. When negative regulation of X-shh is impaired by Ro, the equilibrium is displaced toward a gain-of-function of shh that correlates with decreased primary neuron differentiation (Franco, 1999).

Because RA treatments could not rescue the inhibitory effect of X-shh on neuronal differentiation, while X-shh overexpression produces a widespread expansion of Zic2 and suppresses Gli3, it is suggested that a cascade of interactions occurs, wherein endogenous retinoids act far upstream, promoting primary neurogenesis by inhibiting X-shh expression in the dorsal midline. This in turn changes the balance of prepattern genes (activation of Gli3 and reduction of Zic2), thus altering the expression of other intermediary genes, ultimately leading to N-tubulin activation. Because in the normal embryo X-shh is expressed along the dorsal midline, it is evident that endogenous retinoids do not completely block shh signaling. This fact suggests that a precise balance between retinoid and hedgehog signaling must be established, resulting in the normal primary neurogenesis pattern. While endogenous retinoids constitute an early signal that promotes primary neuron formation by inclining the entire neural plate towards a uniform proneural territory, shh signaling is necessarily required at the same time and at an accurate level, limited at least by endogenous retinoids, to save a pool of neuronal precursors from premature differentiation by retinoid signaling, keeping them in a mitotic, undifferentiated state for subsequent waves of neurogenesis (Franco, 1999).

Hedgehog and cerebellum development

The cerebellum consists of a highly organized set of folia that are largely generated postnatally during expansion of the granule cell precursor (GCP) pool. Since the secreted factor sonic hedgehog (Shh) is expressed in Purkinje cells and functions as a GCP mitogen in vitro, it is possible that Shh influences foliation during cerebellum development by regulating the position and/or size of lobes. How Shh and its transcriptional mediators, the Gli proteins, regulate GCP proliferation in vivo was studied, and whether they influence foliation was tested. Shh expression correlates spatially and temporally with foliation. Expression of the Shh target gene Gli1 is also highest in the anterior medial cerebellum, but is restricted to proliferating GCPs and Bergmann glia. By contrast, Gli2 is expressed uniformly in all cells in the developing cerebellum except Purkinje cells and Gli3 is broadly expressed along the anteroposterior axis. Whereas Gli mutants have a normal cerebellum, Gli2 mutants have greatly reduced foliation at birth and a decrease in GCPs. In a complementary study using transgenic mice, it was shown that overexpressing Shh in the normal domain does not grossly alter the basic foliation pattern, but does lead to prolonged proliferation of GCPs and an increase in the overall size of the cerebellum. Taken together, these studies demonstrate that positive Shh signaling through Gli2 is required to generate a sufficient number of GCPs for proper lobe growth (Corrales, 2004).

Cerebellar development is a carefully orchestrated process that produces an exquisitely foliated structure with a simple layered cytoarchitecture. In mammals, the cerebellum is divided into three regions with distinct anteroposterior (AP) foliation patterns: a central vermis and two bilaterally symmetric hemispheres. The most abundant neurons in the cerebellum, as well as the entire brain, are the granule cells. Whereas Purkinje cells and cerebellar interneurons originate in the ventricular neuroepithelium, cerebellar granular cell precursors (GCPs) arise from a germinal zone in the rhombic lip situated in dorsal posterior rhombomere 1. The GCPs begin to leave the rhombic lip at approximately embryonic day (E) 13 and migrate over the cerebellar anlage to form the external granule layer (EGL). Although the EGL is formed by E15, GCPs in the EGL remain mitotically active until 2 weeks postnatal. Granule cells start to exit the cell cycle after birth and as part of their differentiation program migrate internally past the Purkinje cells to form the inner granule layer (IGL). Over the course of the first two postnatal weeks, cerebellar folia form, suggesting the increase in granule cells is largely responsible for foliation. The process of foliation begins with the formation of four principal fissures, which divide the cerebellum into five cardinal lobes. As GCP proliferation continues, these lobes expand and are further subdivided to give rise to the species-specific foliation pattern observed in the mature cerebellum. The fissures that divide the central cardinal lobe into lobes VI-VIII are among the last to form in the vermis (Corrales, 2004).

It has been shown that an interaction between Purkinje cells and GCPs is important for granule cell proliferation and foliation. For example, when Purkinje cells are ablated or as in mouse mutants that lack Purkinje cells, such as Lurcher and Staggerer, the GCP population is diminished and foliation is arrested. One key GCP mitogen expressed in Purkinje cells is sonic hedgehog (Shh), since it can induce proliferation of GCPs in culture, and injection of Shh antibodies into the cerebellum reduces granule cell proliferation. Shh signaling is mediated by the Gli family of transcription factors. In the spinal cord Gli2 is the primary activator of Shh signaling, whereas Gli3 functions mainly as a repressor but is also a weak activator. By contrast, in the limb only Gli3 is required for digit patterning and to regulate a normal level of proliferation. An important question, therefore, is whether Shh functions in the cerebellum primarily by inhibiting the Gli3 repressor as in the limb, and/or by inducing the activator Gli2. Due to the embryonic lethality of Gli2 and Gli3 mutants, the in vivo requirements for these two genes during postnatal cerebellum development have not been addressed. Gli1 (Gli: Mouse Genome Informatics) however, is not required for mouse development, although it plays a redundant activator function with Gli2, which is revealed only in Gli2 heterozygotes. Furthermore, unlike that of Gli2 and Gli3, Gli1 transcription is regulated by Shh signaling. In particular, all transcription of Gli1 is absolutely dependent on induction of Gli2 and Gli3 activators by Hh signaling. Since Gli1 is a transcriptional target of Shh signaling, lacZ expression in Gli-lacZ knock-in mice (Gli1lz/+) is a readout of positive Shh signaling (Corrales, 2004).

Gli1-lacZ mice were utilized to characterize the precise spatial and temporal pattern of positive Shh signaling in the developing cerebellum. Strikingly, Shh expression and signaling (Gli-lacZ expression) in the developing vermis is spatially patterned from E18 to P10 with highest levels in anterior lobes (III-VIa) and the most posterior lobe (X). Both Gli1 and Gli2 are primarily excluded from Purkinje cells, and Gli expression is strongest in Bergmann glia and in the GCPs in the outer layer of the EGL. Gli3 is expressed in most cell types along the AP axis. In the absence of Gli2, normal expansion of GCPs in the EGL is impaired, and foliation is reduced at birth. Gli1-lacZ expression is undetectable in Gli2 mutants, demonstrating that Gli2 is the major activator required to transduce Shh-positive signaling in the developing cerebellum. In support of this, the thickness of the EGL appears normal in Gli3 mutants. In transgenic mice overexpressing Shh in a normal pattern in the cerebellum, the basic pattern of cerebellum foliation is maintained, although the entire cerebellum is enlarged and the lobes that normally express higher levels of Shh have an irregular IGL. In addition, the EGL persists longer than normal in transgenics. This study utilizes in vivo experiments to establish a role for positive Shh signaling in regulating expansion of the cerebellar lobes by regulating GCP proliferation, and demonstrates that Gli2 is a required mediator for this signaling (Corrales, 2004).

Development of the postnatal cerebellum relies on the tight regulation of cell number by morphogens that control the balance between cell proliferation, survival and differentiation. This study has analyzed the role of the serine-protease inhibitor protease nexin 1 (PN-1; SERPINE2) in the proliferation and differentiation of cerebellar granular neuron precursors (CGNPs) via the modulation of their main mitogenic factor, sonic hedgehog (SHH). These studies show that PN-1 interacts with low-density lipoprotein receptor-related proteins (LRPs) to antagonize SHH-induced CGNP proliferation and that it inhibits the activity of the SHH transcriptional target GLI1. The binding of PN-1 to LRPs interferes with SHH-induced cyclin D1 expression. CGNPs isolated from Pn-1-deficient mice exhibit enhanced basal proliferation rates due to overactivation of the SHH pathway and show higher sensitivity to exogenous SHH. In vivo, the Pn-1 deficiency alters the expression of SHH target genes. In addition, the onset of CGNP differentiation is delayed, which results in an enlarged outer external granular layer. Furthermore, the Pn-1 deficiency leads to an overproduction of CGNPs and to enlargement of the internal granular layer in a subset of cerebellar lobes during late development and adulthood. It is proposed that PN-1 contributes to shaping the cerebellum by promoting cell cycle exit (Vaillant, 2007).

Heterozygous deletions encompassing the ZIC1;ZIC4 locus have been identified in a subset of individuals with the common cerebellar birth defect Dandy-Walker malformation (DWM). Deletion of Zic1 and Zic4 in mice produces both cerebellar size and foliation defects similar to human DWM, confirming a requirement for these genes in cerebellar development and providing a model to delineate the developmental basis of this clinically important congenital malformation. This study shows that reduced cerebellar size in Zic1 and Zic4 mutants results from decreased postnatal granule cell progenitor proliferation. Through genetic and molecular analyses, it was shown that Zic1 and Zic4 have Shh-dependent function promoting proliferation of granule cell progenitors. Expression of the Shh-downstream genes Ptch1, Gli1 and Mycn was downregulated in Zic1/4 mutants, although Shh production and Purkinje cell gene expression were normal. Reduction of Shh dose on the Zic1+/-;Zic4+/- background also resulted in cerebellar size reductions and gene expression changes comparable with those observed in Zic1-/-;Zic4-/- mice. Zic1 and Zic4 are additionally required to pattern anterior vermis foliation. Zic mutant folial patterning abnormalities correlate with disrupted cerebellar anlage gene expression and Purkinje cell topography during late embryonic stages; however, this phenotype is Shh independent. In Zic1+/-;Zic4+/-;Shh+/-, normal cerebellar anlage patterning and foliation was observed. Furthermore, cerebellar patterning was normal in both Gli2-cko and Smo-cko mutant mice, where all Shh function was removed from the developing cerebellum. Thus, these data demonstrate that Zic1 and Zic4 have both Shh-dependent and -independent roles during cerebellar development and that multiple developmental disruptions underlie Zic1/4-related DWM (Blank, 2011).

Shh signaling protects Atoh1 from degradation mediated by the E3 ubiquitin ligase Huwe1 in neural precursors

Signaling networks controlled by Sonic hedgehog (SHH) and the transcription factor Atoh1 regulate the proliferation and differentiation of cerebellar granule neuron progenitors (GNPs). Deregulations in those developmental processes lead to medulloblastoma formation, the most common malignant brain tumor in childhood. Although the protein Atoh1 is a key factor during both cerebellar development and medulloblastoma formation, up-to-date detailed mechanisms underlying its function and regulation have remained poorly understood. This study reports that SHH regulates Atoh1 stability by preventing its phosphodependent degradation by the E3 ubiquitin ligase Huwe1. These results reveal that SHH and Atoh1 contribute to a positive autoregulatory loop promoting neuronal precursor expansion. Consequently, Huwe1 loss in mouse SHH medulloblastoma illustrates the disruption of this developmental mechanism in cancer. Hence, the crosstalk between SHH signaling and Atoh1 during cerebellar development highlights a collaborative network that could be further targeted in medulloblastoma (Forget, 2014).

Sonic hedgehog and the hypothalamus

Despite its evolutionary conservation and functional importance, little is known of the signaling pathways that underlie development of the hypothalamus. In zebrafish, fate mapping experiments have shown that hypothalamic precursors originate closer to the organizer than other forebrain derivatives and move rostrally within the neural plate to their final location in the anterior CNS. Thus, early steps of hypothalamic development involve regulation both of the induction of hypothalamic identity and the migration of hypothalamic precursors. Although embryological manipulations have revealed the importance of mesodermal and/or endodermal tissues that underlie the brain in these processes, little is known of the molecular pathways involved in the specification and patterning of the hypothalamus. Tissue ablation experiments have shown that organizer-derived mesendodermal tissues are a source of inducing signals required for hypothalamic development. The secreted protein Sonic Hedgehog (Shh) is proposed to be one such signal, because hypothalamic tissue is absent in mice lacking Shh function and increased Shh activity leads to ectopic expression of hypothalamic markers in fish. However, induction of the hypothalamus still occurs in fish that lack the function of Smoothened, an essential transmembrane modulator of Hh activity. This raises the possibility that at least in fish, Hh signaling may play a more important role in the maintenance and/or later development of hypothalamic tissue than in its induction. Thus, although mutations affecting Nodal and Hedgehog signaling disrupt hypothalamic development in zebrafish, the time and site of action and the exact roles of these pathways remain very poorly understood. Unexpectedly, cell-autonomous reception of Nodal signals is neither required for the migration of hypothalamic precursors within the neural plate, nor for further development of the anterior-dorsal hypothalamus. Nodal signaling is, however, cell-autonomously required for establishment of the posterior-ventral hypothalamus. Conversely, Hedgehog signaling antagonizes the development of posterior-ventral hypothalamus, while promoting anterior-dorsal hypothalamic fates. In addition, their distinct roles in the regionalization of the diencephalon, this study reveals cooperation between Nodal and Hedgehog pathways in the maintenance of the anterior-dorsal hypothalamus. It is the prechordal plate and not the head endoderm that provides the early signals essential for establishment of the hypothalamus (Mathieu, 2002).

In the developing chick hypothalamus, Shh and BMPs are expressed in a spatially overlapping, but temporally consecutive, manner. This study demonstrates how the temporal integration of Shh and BMP signalling leads to the late acquisition of Pax7 expression in hypothalamic progenitor cells. These studies reveal a requirement for a dual action of BMPs: first, the inhibition of GliA function through Gli3 upregulation; and second, activation of a Smad5-dependent BMP pathway. Previous studies have shown a requirement for spatial antagonism of Shh and BMPs in early CNS patterning; this study proposes that neural pattern elaboration can be achieved through a versatile temporal antagonism between Shh and BMPs (Ohyama, 2008).

This analysis makes a number of key points. First, it provides a novel insight into how cellular diversity can be achieved within the embryo in response to a limited repertoire of signalling molecules. In addition to the well-accepted view that BMP signal opposes Shh activity in a spatial manner, ventrally derived Bmp7 signalling can oppose Shh signalling in a temporal manner to specify ventral progenitors within the hypothalamus. The deployment of the two signals in this versatile temporal manner in turn leads to novel modules of transcription factor expression, in order to achieve elaborate cellular diversity. This work adds to the growing body of data suggesting that cell fate in the neural tube is governed through the temporal integration of, and adaptation to, signalling ligands (Ohyama, 2008)

Table of contents

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

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.