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Hedgehog activation is required upstream of Wnt signalling to control neural progenitor proliferation

The canonical Wnt and sonic hedgehog pathways have been independently linked to cell proliferation in a variety of tissues and systems. However, interaction of these signals in the control of cell cycle progression has not been studied. This study demonstrates that in the developing vertebrate nervous system these pathways genetically interact to control progression of the G1 phase of the cell cycle. By in vivo loss-of-function experiments, the absolute requirement was demonstrated of an upstream Shh activity for the regulation of Tcf3/4 expression. In the absence of Tcf3/4, the canonical Wnt pathway cannot activate target gene expression, including that of cyclin D1, and the cell cycle is necessarily arrested at G1. In addition to the control of G1 progression, Shh activity controls the G2 phase through the regulation of cyclin E, cyclin A and cyclin B expression, and this is achieved independently of Wnt. Thus, in neural progenitors, cell cycle progression is co-ordinately regulated by Wnt and Shh activities (Alvarez-Medina, 2009).

Hedgehog and neural patterning: Neural tube ventral-to-dorsal patterning

In frog embryos, Gli1 is expressed transiently in the prospective floor plate during gastrulation and in cells lateral to the midline during late gastrula and neurula stages. In contrast, Gli2 and Gli3 are absent from the neural plate midline with Gli2 expressed widely and Gli3 in a graded fashion, with highest levels in lateral regions. In mouse embryos, the three Gli genes show a similar pattern of expression in the neural tube but are coexpressed throughout the early neural plate. Gli1 is the only Gli gene expressed in the prospective floor plate cells of frog embryos: it therefore seemed likely that this gene would be involved in ventral neural tube development. Sonic hedgehog (Shh) signaling activates Gli1 transcription and widespread expression of endogenous frog Gli1, but not Gli3, in developing frog embryos. This results in the ectopic differentiation of floor plate cells and ventral neurons within the neural tube. Floor-plate-inducing ability is retained when cytoplasmic Gli1 proteins are either forced into the nucleus or are fused to the VP16 transactivating domain. Gli1 induces HNF-3ß, a ventral marker, in the neural tube as well as in the epidermal ectoderm. In addition, ectopic expression of Gli1 induces the ectopic expression of Sonic hedgehog and the floor-plate-specific marker F-spondin. Embryos injected with Gli1 display ectopic cells in the midbrain resembling putative neurons. These ectopic cells can be identified as 5HT (serotonin) producing neurons These results identify Gli1 as a midline target of Shh and suggest that it mediates the induction of floor plate cells and ventral neurons when Shh acts as a transcriptional regulator (Lee, 1997).

Hedgehog (Hh) signaling plays a significant role in defining the polarity of a variety of tissue types along the anterior/posterior and dorsal/ventral axes in both vertebrate and invertebrate organisms. The pathway through which Hh transduces its signal is still obscure, however, recent data have implicated the cyclic AMP-dependent protein kinase A as a negative regulator of the Hh signal transduction pathway. One of the vertebrate Hh family members, Sonic hedgehog (Shh), can induce ventral neural cell types both in vivo and in vitro; high concentrations induce floor plate and lower concentrations motor neurons. To investigate whether PKA plays an active role in the suppression of ventral neural differentiation, transgenic embryos were generated expressing a dominant negative form of PKA (dnPKA) in primarily dorsal aspects of the mouse CNS. Induction of floor plate and motor neuron markers were observed in embryos expressing the dominant negative PKA transgene and the loss of dorsal gene expression at rostral levels. Thus suppression of PKA activity is sufficient to activate targets of the Shh signaling pathway in the vertebrate CNS suggesting that induction of ventral cell types occurs via the antagonistic action of Shh on PKA activity. Two mammalian target genes that are strongly expressed in ectopic dorsal locations in response to dnPKA are Ptc and Gli. As both of these are targets of Drosophila Hh signaling, these data point to an evolutionary conservation in both the mechanisms of signaling and the effectors of the signaling pathway (Epstein, 1996).

Experments with Drosophila indicate that hedgehog-related genes require the downstream activities of dpp-related signalling molecules for patterning functions related to hedgehog activity. But to date, there has been no example of an epistatic relation between HH-related members and dpp-related members during ventral neural tube formation in vertebrates. Dynamo, a new zebrafish DVR (dpp-vg-related) protein detected in the posterior neural plate from late gastrula on, becomes restricted to the ventral region of the trunk neural tube, exclusive of the most ventral cells (the floor plate and adjacent cells). Analysis of dynamo expression in zebrafish axial mutants such as cyclops, and floating head indicates that dynamo expression in the ventral region of the central nervous system (CNS) is induced by axial mesoderm and maintained by notochord, but is independent of a differentiated floor plate. Ectopic Sonic hedgehog expression can up-regulate dynamo expression in the posterior neural tube providing evidence that cells of the posterior neural tube are competent to respond to shh signaling and that the close relationship between DVR members and hedgehog-related genes might also apply to vertebrate CNS development (Bruneau, 1997).

The vertebrate spinal cord consists of a large number of different cell types in close proximity to one another. The identities of these cells appear to be specified largely by information acquired from their local environments. Local cell-cell interactions, mediated by zebrafish homologs of the Drosophila neurogenic gene Delta, regulate specification of diverse neuronal types in the ventral spinal cord. A novel zebrafish Delta gene, deltaA, has been identified that is expressed specifically in the nervous system. In zebrafish, cells that give rise to primary neurons of the trunk begin to exit the mitotic cycle once gastrulation is completed. The first of these cells known to differentiate are primary sensory Rohon Beard neurons (RBs) and primary neurons, which arise from lateral and medial regions of the neural plate, respectively. deltaA expression is initiated in the epiblast prior to completion of gastrulation. At the 2- to 3- somite stage (10.5 hours) low levels of deltaA RNA are distributed throughout the trunk CNS, with cells expressing higher levels found in the medial and lateral regions of the neural plate. These regions correspond to the positions at which primary motoneurons and RBs originate. Cells expressing high levels of deltaA RNA do not form contiguous domains. Rather, single cells or small clusters of several cells showing high expression are interspersed with cells having lower expression. deltaA is expressed in cells specified for neuronal fates such as presumptive primary motoneurons and presumptive RB neurons. By expressing a dominant negative form of Delta protein in embryos, it has been shown that Delta proteins mediate lateral inhibition in the zebrafish spinal cord. Delta function is important for specification of a variety of spinal cord neurons, suggesting that lateral inhibition serves to diversify neuronal fate during development of the vertebrate spinal cord (Appel, 1998).

Primary and secondary motoneurons are born beginning about 9-10 and 14-15 hours, respectively. Zebrafish shh expression is initiated in presumptive dorsal mesoderm at about 7 hours. In cyclops; floating head double mutant embryos, which lack differentiated notochord and floorplate, shh expression is initiated normally but not maintained after gastrulation; primary (but not secondary) motorneurons develop. Thus, early shh signaling may induce development of primary, but not secondary motorneurons. In chick, motoneuronal induction requires exposure to Shh for 1-2 cell cycles. The zebrafish cell cycle length during early neurogenesis is about 4 hours. Thus, secondary motoneurons are born 1-2 cell cycles after initiation of shh expression. Together, these observations suggest that secondary motoneurons are equivalent to chick motoneurons in their requirement for Shh signaling, but that primary motoneurons require only brief exposure. In the absence of lateral inhibition, too many primary motoneurons develop concomitant with the loss of secondary motoneurons. Thus it is proposed that Delta-Notch signaling specifies primary and secondary motoneuronal fates by regulating how neural precursor cells respond to Shh signaling. In this model, some cells of the neural plate respond to Shh by immediately developing as primary motoneurons. DeltaA is expressed at high levels in these cells and inhibits neighboring cells from responding to Shh in the same way. Later, as DeltaA is downregulated in primary motoneurons, neighboring cells are released from lateral inhibition and respond to Shh by adapting secondary neuronal fate. Alternatively, secondary motoneurons may be specified by a late-arising signal that acts with, or subsequent to, Shh (Appel, 1998 and references).

Embryonic patterning in vertebrates is dependent on the balance of inductive signals and their specific antagonists. Noggin, which encodes a bone morphogenetic protein (BMP) antagonist expressed in the node, notochord, and dorsal somite, is required for normal mouse development. Noggin binds several BMPs with very high affinities, with a marked preference for BMP2 and BMP4 over BMP7. Although Noggin has been implicated in neural induction, examination of null mutants in the mouse indicates that Noggin is not essential for this process. However, Noggin is required for subsequent growth and patterning of the neural tube. Early BMP-dependent dorsal cell fates, such as the roof plate and neural crest, form in the absence of Noggin. However, there is a progressive loss of early, Sonic hedgehog (Shh)-dependent ventral cell fates despite the normal expression of Shh in the notochord. Somite differentiation is deficient in both muscle and sclerotomal precursors. Addition of BMP2 or BMP4 to paraxial mesoderm explants blocks Shh-mediated induction of Pax-1, a sclerotomal marker, whereas addition of Noggin is sufficient to induce Pax-1. Noggin and Shh induce Pax-1 synergistically. Use of protein kinase A stimulators blocks Shh-mediated induction of Pax-1, but not induction by Noggin, suggesting that induction is mediated by different pathways. Together these data demonstrate that inhibition of BMP signaling by axially secreted Noggin is an important requirement for normal patterning of the vertebrate neural tube and somite (McMahon, 1998).

Within the developing vertebrate nervous system, it is not known how progenitor cells interpret the positional information provided by inducing signals or how the domains are defined in which distinct groups of neural cells differentiate. Gli proteins may be involved in these processes. In the frog neural plate, the zinc finger transcription factor Gli1 is expressed in midline cells and mediates the effects of Shh inducing floor plate differentiation. In contrast, Gli2 and Gli3 are expressed throughout the neural plate except for the midline. Gli3 and Shh are shown to repress one another, whereas Gli2, like Gli1, is a target of Shh signaling. However, only Gli1 can induce the differentiation of floor plate cells. In addition, Gli2 and Gli3 repress the ectopic induction of floor plate cells by Gli1 in co-injection assays and inhibit endogenous floor plate differentiation. The definition of the floor plate domain, therefore, appears to be defined by the antagonizing activities of Gli2 and Gli3 on Gli1 function. Because both Gli1 and Gli2 are induced by Shh, these results establish a regulatory feedback loop triggered by Shh that restricts floor plate cells to the midline. The Gli genes have been shown to induce neuronal differentiation and this paper shows that the Gli proteins induce specific types of neurons. Only Gli1 induces Nkx2.1/TTF-1+ ventral forebrain neurons. Moreover, Gli2 and Gli3 inhibit neuron differentiation. In contrast, the differentiation of spinal motor neurons can be induced by the two ventrally expressed Gli genes, Gli1 and Gli2, suggesting that Gli2 directly mediates induction of motor neurons by Shh. In addition, Gli3 inhibits motor neuron differentiation by Gli2. Thus, combinatorial Gli function may pattern the neural tube, integrating positional information and cell type differentiation (Ruiz i Altaba, 1998).

Gli proteins may balance each other's functions in partially overlapping domains to create pattern. In this case, the Gli readout of a cell is predicted to be critical for determining its fate. Shh/Gli1 repress Gli3. At early gastrula stages prior to the onset of Shh expression in notochord precursors, low levels of Gli1 and Gli3 are detected in the dorsal animal cap (the prospective neural plate) before midline differentiation. As the notochord begins to express Shh and the midline begins to express floor plate markers, Gli3 expression is absent from the midline, consistent with its repression by Shh from the forming notochord. A first step in ventralizing the medial neural plate by Shh may therefore be the repression of Gli3. Studies on limb development further suggest that Shh/Gli1 and Gli3 have mutually repressive relationships. Ectopic Shh expression in the anterior chick limb bud induces Gli1 and represses Gli3, whereas in Gli3 mutant mouse embryos, this region displays ectopic Shh expression. Similarly, Gli3 represses Shh/Gli1 in the dorsal neural tube. This implicates Gli3 and Shh/Gli1 in a mutually repressive interaction that appears to be critical for pattern formation in different tissues. Gli3 must be absent for ventral cell type induction and patterning and Gli3 is involved in repressing Shh in dorsal regions, thus allowing dorsal development. Gli2 and Gli3 have redundant functions in repressing floor plate induction by Gli1. However, Gli2 and Gli3 are differently regulated and have different functions in neuronal patterning, because only Gli2 can induce spinal motor neurons. A partial redundancy between Gli2 and Gli3 has also been found in mouse skeletal patterning. Because the mutation introduced in the mouse Gli2 gene leaves intact the N-terminal region and the first two zinc fingers, it remains possible that such a mutation is not a null but rather a hypomorph if the N-terminal part of Gli proteins were to encode a repressive function like their Drosophila counterpart (Ruiz i Altaba, 1998 and references therein).

Gli2 represses the floor-plate-inducing function of Gli1 and endogenous floor plate differentiation. Gli2 could therefore normally restrict the ability of Gli1 to induce floor plate development in cells immediately adjacent to the midline. This interaction could provide a molecular basis for the spatial restrictions to the propagation of floor plate induction previously observed that occurs before neural cells lose their competence to respond to floor-plate-inducing signals. Shh signaling from the notochord may thus initiate a regulatory cascade by inducing Gli1 and Gli2 expression in different yet overlapping domains. The identity of midline cells as floor plate may be determined by Gli1 in the absence of Gli2. In contrast, the mediolateral (dorsoventral) extent of the floor plate may be determined by Gli2 acting to antagonize the floor-plate-inducing function of Gli1. This interaction would occur in Gli1+/Gli2+ cells adjacent to the Gli1+/Gli2- floor plate (Ruiz i Altaba, 1998 and references therein).

Different Gli proteins induce different types of neurons. Whereas Gli1 can induce a variety of ventral neuronal types including ventral forebrain neurons, hindbrain serotonergic neurons and spinal motor neurons, Gli2 can only induce a subset of these ventral neuronal classes. However, in the posterior CNS, Gli1 may induce many ventral neuronal types through the intermediate induction of floor plate cells, which themselves will express Shh, thus inducing Gli2 in adjacent cells. Because Gli1 is restricted to midline and adjacent ventral cells, Gli2 is predicted to be normally involved in inducing secondary motor neuron differentiation in response to Shh. How then can Gli2 induce motor neurons only in the ventral region? Two strategies appear to play a role in restricting the motor-neuron-inducing function of Gli2 to the ventral neural tube. Ventrally, even though Gli2 is activated by Shh, midline factors repress its expression in prospective floor plate cells. Dorsally, Gli2 and Gli3 expression overlaps and, at least in equivalent amounts, Gli3 represses the motor-neuron-inducing function of Gli2. Thus, Gli2 is transcriptionally repressed in midline cells and functionally repressed in dorsal cells, leaving only ventral non-midline cells free of Gli2 inhibitors. Gli3 cannot induce ventral neuronal types and is predicted to induce intermediate and/or dorsal neurons. In the forebrain, Gli2 and Gli3 also inhibit Gli1 function and Gli2 may normally induce the differentiation of a subset of ventral neurons. Gli2, however, is unable to induce ectopic differentiation of Nkx2.1/TTF-1+ neurons, suggesting that, in this case, Gli1 acts directly. Independent of which classes of forebrain neurons Gli2 may induce, its repressive action on Nkx2.1/TTF-1+ cells raises the possibility that in the forebrain, as in more posterior CNS regions, a regulatory feedback loop triggered by Shh patterns the ventral region. More dorsally, the same kinds of interactions between Gli2 and Gli3 proposed for the hindbrain and spinal cord may also be involved in neuronal patterning (Ruiz i Altaba, 1998).

During vertebrate development, the specification of distinct cell types is thought to be controlled by inductive signals acting at different concentration thresholds. The degree of receptor activation in response to these signals is a known determinant of cell fate, but the later steps at which graded signals are converted into all-or-none distinctions in cell identity remain poorly resolved. In the ventral neural tube, motor neuron and interneuron generation depends on the graded activity of the signaling protein Sonic hedgehog (Shh). These neuronal subtypes derive from distinct progenitor cell populations that express the homeodomain proteins Nkx2.2 or Pax6 in response to graded Shh signaling. In mice lacking Pax6, progenitor cells generate neurons characteristic of exposure to greater Shh activity. However, Nkx2.2 expression expands dosally in Pax6 mutants, raising the possibility that Pax6 controls neuronal pattern indirectly. Evidence that Nkx2.2 has a primary role in ventral neuronal patterning. In Nkx2.2 mutants, Pax6 expression is unchanged but cells undergo a ventral-to-dorsal transformation in fate and generate motor neurons rather than interneurons. Thus, Nkx2.2 has an essential role in interpreting graded Shh signals and selecting neuronal identity (Briscoe, 1999).

The axial midline mesoderm and the ventral midline of the neural tube, the floor plate, share the property of being a source of the secreted protein, Sonic hedgehog (Shh), which has the capacity to induce a variety of ventral cell types along the length of the mouse CNS. To gain insight into the mechanisms by which Shh transcription is initiated in these tissues, cis-acting sequences regulating Shh gene expression have been identified. As an approach, genomic clones encompassing 35 kb of the Shh locus were tested for their ability to direct a lacZ reporter gene to the temporally and spatially restricted confines of the Shh expression domains in transgenic mice. Three enhancers were identified that direct lacZ expression to distinct regions along the anteroposterior axis including the ventral midline of the spinal cord, hindbrain, rostral midbrain and caudal diencephalon, suggesting that multiple transcriptional regulators are required to initiate Shh gene expression within the CNS. In addition, regulatory sequences have also been identified that directed reporter expression to the notochord, albeit, under limited circumstances. Sequence analysis of the genomic clones responsible for enhancer activity from a variety of organisms, including mouse, chicken and human, have identified highly conserved binding sites for the hepatocyte nuclear factor 3 (Hnf3) family of transcriptional regulators in some, but not all, of the enhancers. A Shh floor plate enhancer (SFPE1) has been identified within a 1.1 kb genome fragment located approximately 8 kb upstream of the Shh transcriptional start site. Intronic enhancers direct reporter gene expression to cranial and spinal cord regions. The location of an Shh brain enhancer (SBE1) was delineated to within a 2.2 kb segment within the second intron. SFPE1 activity is regulated independently of Hnf3 function, while SBE1 and SFPE2, a second intronic floor plate enhancer directing expression to hindbrain and spinal cord, both rely on Hnf3 function for midline but not lateral Shh expression. Moreover, the generation of mutations in the Hnf3-binding sites shows their requirement in certain, but not all, aspects of Shh reporter expression. Taken together, these results support the existence of Hnf3-dependent and -independent mechanisms in the direct activation of Shh transcription within the CNS and axial mesoderm (Epstein, 1999).

Sonic hedgehog signaling controls the differentiation of motor neurons in the ventral neural tube, but the intervening steps are poorly understood. A differential screen of a cDNA library derived from a single Shh-induced motor neuron has identified a novel homeobox gene, MNR2, expressed by motor neuron progenitors and transiently by postmitotic motor neurons. The ectopic expression of MNR2 in neural cells initiates a program of somatic motor neuron differentiation characterized by the expression of homeodomain proteins, by neurotransmitter phenotype, and by axonal trajectory. These results suggest that the Shh-mediated induction of a single transcription factor, MNR2, is sufficient to direct somatic motor neuron differentiation. At what step in the pathway of somatic motor neuron differentiation does MNR2 act? MNR2 is expressed by ventral progenitor cells 4-5 hr prior to the generation of the first postmitotic motor neurons, and the cell cycle time of ventral progenitor cells is 8 hr. Thus, it appears that MNR2 expression is initiated during the final division cycle of motor neuron progenitors. The onset of MNR2 expression by motor neuron progenitors coincides with the time that they attain independence of Shh signaling. This observation and the ability of MNR2 to activate its own expression provide a potential molecular basis for the transition of Shh-dependent ventral progenitor cells into Shh-independent, committed somatic motor neuron progenitors. MNR2 is induced rapidly by Shh, prior to the expression of other somatic motor neuron transcription factors. It is unclear, however, whether MNR2 is a direct target for the conserved Hedgehog (Hh) signal transduction pathway mediated by the Ci/Gli class of transcription factors. Gli proteins have been implicated in floor plate differentiation, but it is uncertain whether they are also involved directly in the generation of motor neurons (Tanabe, 1998).

Netrins, a family of growth cone guidance molecules (see Drosophila Netrins), are expressed both in the ventral neural tube and in subsets of mesodermal cells. In an effort to better understand the regulation of netrins, the expression of netrin-1a was examined in mutant cyclops, no tail, and floating head zebrafish embryos; such mutants show perterbances in their axial midline structures. Netrin-1a expression requires signals present in notochord and floor plate cells. In the myotome, but not the neural tube, netrin-1a expression requires sonic hedgehog. In embryos lacking sonic hedgehog (the sonic-you locus) netrin-1a expression is reduced or absent in the myotomes but present in the neural tube. Embryos lacking sonic hedgehog express tiggy-winkle hedgehog in the floor plate, suggesting that, in the neural tube, tiggy-winkle hedgehog can compensate for the lack of sonic hedgehog by inducing netrin-1a expression. Ectopic expression of sonic hedgehog, tiggy-winkle hedgehog, or echidna hedgehog induces ectopic netrin-1a expression in the neural tube, and ectopic expression of either sonic hedgehog or tiggy-winkle hedgehog (but not echidna hedgehog) induces ectopic netrin-1a expression in somites. These data demonstrate that in vertebrates netrin expression is regulated by Hedgehog signaling (Lauderdale, 1998).

Nkx2.1 is related to the Drosophila gene ventral nervous system defective (vnd). vnd encodes the NK2 homeodomain protein that is expressed in the ventral part of central nervous system. The CNS in fly embryos lacking vnd has a ventral-to-dorsal transformation, analogous to the phenotype in the Nkx2.1 mutants. There are several Nkx genes expressed in the ventral CNS of vertebrates, including Nkx2.2 and Nkx6.1. Mutations of Nkx2.2 and Nkx6.1 also have ventral-to-dorsal transformations. In both of these cases, Shh expression is unaffected, suggesting that Nkx2.2 and Nkx6.1, like vnd, have primary roles in ventral specification. It is uncertain that Nkx2.1 alone is necessary for fate specification of the MGE, because Shh expression, which is essential for ventral specification, is also reduced in the Nkx2.1 knockout mice. However, the described functions of vnd, Nkx2.2 and Nkx6.1 support the hypothesis that Nkx2.1 has a primary role in regional specification, like its homologs. Furthermore, in all regions of the CNS, the Nkx genes are expressed before Shh, supporting the model that Nkx2.1 is upstream of Shh in the basal telencephalon. Finally, in Gli2 mutant mice, Shh is not expressed in the floor plate, yet Nkx2.2 is expressed and motor neurons form. Thus, while Shh expression in the axial mesendoderm is essential for ventral specification of the CNS, Shh expression in neural tissue may not have a major role in regionalization (Sussel, 1999 and references).

The Nodal and Hedgehog signaling pathways influence dorsoventral patterning at all axial levels of the CNS, but it remains largely unclear how these pathways interact to mediate patterning. In zebrafish, Nodal signaling is required for induction of the homeobox genes nk2.1a in the ventral diencephalon and nk2.1b in the ventral telencephalon. Hedgehog signaling is also required for telencephalic expression but may not be essential to establish diencephalic nk2.1a expression. Furthermore, Shh does not restore ventral diencephalic development in embryos lacking Nodal activity. In contrast, Shh does restore telencephalic nk2.1b expression in the absence of Nodal activity; this suggests that Shh acts downstream of Nodal activity to pattern the ventral telencephalon. Thus, the Nodal pathway regulates ventral forebrain patterning through both Hedgehog signaling-dependent and -independent mechanisms (Rohr, 2001).

Comparative analysis of the mouse and fish nk2.1 genes indicates that the expression domains of the two zebrafish nk2.1 genes cumulatively equate to those of the single mouse gene. Mouse nkx2.1 is expressed in the medial ganglionic eminence of the ventral telencephalon, the hypothalamus, lung, and thyroid gland. Zebrafish nk2.1a is expressed in the hypothalamus and the developing thyroid gland but not the ventral telencephalon. In contrast, nk2.1b is expressed strongly in the telencephalon, weakly in the hypothalamus, and not at all in the thyroid gland. Thus, both nk2.1 genes in fish have retained some sites of ancestral gene expression and lost others. The duplication-degeneration-complementation model of gene evolution suggests that both copies of duplicated genes may be retained if each gene accumulates mutations in regulatory regions that disrupt specific temporal or spatial patterns of expression. In this way, the functions of the ancestral gene are partitioned between the two paralogs, and both are retained within the genome. The two nk2.1 genes described in this study provide one of the most striking known examples of the segregation of expression domains between duplicated and retained orthologs of a single mammalian gene. The discrete expression domains of each gene provide an explanation as to why both copies have been retained over the several hundred million years since the genome duplication event is assumed to have occurred and provide compelling evidence in support of the duplication-degeneration-complementation model (Rohr, 2001).

Distinct classes of neurons are generated at defined positions in the ventral neural tube in response to a gradient of Sonic Hedgehog (Shh) activity. A set of homeodomain transcription factors expressed by neural progenitors act as intermediaries in Shh-dependent neural patterning. These homeodomain factors fall into two classes: class I proteins are repressed by Shh and class II proteins require Shh signaling for their expression. The profile of class I and class II protein expression defines five progenitor domains, each of which generates a distinct class of postmitotic neurons. Cross-repressive interactions between class I and class II proteins appear to refine and maintain these progenitor domains. The combinatorial expression of three of these proteins -- Nkx6.1, Nkx2.2, and Irx3 -- specifies the identity of three classes of neurons generated in the ventral third of the neural tube (Briscoe, 2000).

The expression of certain class I (Pax7, Dbx1, Dbx2, and Pax6) and class II (Nkx2.2) proteins is controlled by Shh signaling in vitro. The expression of class I proteins is repressed by Shh signaling, and the more ventral the boundary of class I protein expression in vivo, the higher is the concentration of Shh required for repression of protein expression in vitro. Conversely, Shh signaling is required to induce expression of the class II protein Nkx2.2 in vitro. Repression of Irx3 requires ~3 nM Shh-N, a concentration greater than that required for repression of Pax7, Dbx1, and Dbx2 expression, but less than that required for complete repression of Pax6. Conversely, induction of Nkx6.1 requires ~0.25 nM Shh-N -- a concentration lower than that required for induction of Nkx2.2 (3-4 nM). Thus, the link between the domains of expression of class I and class II proteins in vivo and the Shh concentration that regulates their expression in vitro extends to Irx3 and Nkx6.1. These findings support the idea that the differential patterns of expression of all class I and class II proteins depend initially on graded Shh signaling (Briscoe, 2000).

The boundaries of progenitor domains are sharply delineated in vivo, raising questions about the steps that operate downstream of Shh signaling to establish the nongraded domains of expression of class I and class II proteins. It was asked whether the domain of expression of class I proteins might be constrained by the action of the class II protein that abuts the same domain boundary, or vice versa. To test this, individual homeodomain proteins were misexpressed in the chick neural tube in mosaic fashion, and the resulting pattern of class I and class II protein expression was examined. Examined was the interaction between the class I protein Pax6 and the class II protein Nkx2.2 -- proteins that exhibit complementary domains of expression at the pMN/p3 boundary. To assess the influence of Pax6 on Nkx2.2, Pax6 was misexpressed ventral to its normal limit and the resulting pattern of Nkx2.2 expression was examined. After electroporation of Pax6, small clusters of ectopic Pax6+ cells were detected within the p3 domain. These cells lack Nkx2.2 expression, whereas expression of Nkx2.2 is maintained by neighboring p3 domain cells that lack ectopic Pax6 expression, arguing for a cell-autonomous action of Pax6. The expression of other class I and class II proteins is not affected by the deregulated expression of Pax6. Thus, Pax6 acts selectively to repress Nkx2.2 expression in p3 domain cells. These results complement studies showing a requirement for Pax6 activity in defining the dorsal limit of the p3 domain in vivo. The loss of Nkx6.1 function results in a ventral expansion in the extent of the p1 domain, without any change in Shh signaling. It is noteworthy that the boundaries of each of the five progenitor domains are sharply defined, yet class II proteins have been identified only at the pMN/p3 and p1/p2 boundaries. Thus, additional class II proteins may exist, with patterns of expression that complement the three orphan class I proteins (Briscoe, 2000).

This study has relied on ectopic expression methods to address the roles of Nkx6.1, Nkx2.2, and Irx3 in specifying the fate of V2 neurons, MNs, and V3 neurons. The results show that Nkx2.2 activity is sufficient to induce V3 neurons, that Nkx6.1 activity in the absence of Irx3 induces MNs, whereas Nkx6.1 activity in the presence of Irx3 induces V2 neurons. The inferences derived from these gain-of-function studies are supported by the switches in neuronal fate that occur in mice in which individual class I and class II proteins have been inactivated by gene targeting. In mice lacking Pax6 activity, the dorsal expansion in the domain of Nkx2.2 expression is accompanied by an expansion in the domain of V3 neuron generation, and by the loss of MNs. Conversely, the loss of Nkx2.2 results in the loss of V3 neurons and in the ectopic generation of MNs within the p3 domain. In addition, the loss of Nkx6.1 activity depletes the ventral neural tube of many MNs and V2 neurons (Briscoe, 2000 and references therein).

How do class I and class II proteins control neuronal subtype identity? The final cell division of certain ventral progenitors is accompanied by the onset of expression of a distinct set of homeodomain proteins, notably MNR2 and Lim3. Ectopic expression of MNR2 is able to induce MN differentiation independent of dorsoventral position, and ectopic expression of Lim3 induces V2 neurons. The studies indicate that class I and class II proteins function upstream of MNR2 and Lim3. Thus, within the pMN and p2 domains, the actions of progenitor homeodomain proteins in specifying neuronal subtype identity are likely to be mediated through MNR2 and Lim3. Subtype determinant factors with equivalent functions may therefore be expressed by cells in the other ventral progenitor domains (Briscoe, 2000).

A set of seven homeodomain proteins defines five neural progenitor domains with a fundamental role in the organization of ventral neural pattern. The analysis of these homeodomain proteins suggests that ventral patterning proceeds in three stages: (1) the regulation of class I and class II proteins by graded Shh signals; (2) the refinement and maintenance of progenitor domain identity by cross-repressive interactions between homeodomain proteins, and (3) the translation of a homeodomain protein code into neuronal subtype identity. The central features of this model may apply to other vertebrate tissues in which cell pattern is regulated by local sources of extrinsic signals. Consistent with this idea, cross-regulatory interactions between transcription factors have been suggested to refine cell pattern in the embryonic mesoderm and in the pituitary gland. The principles of the model of ventral patterning outlined here resemble those involved in subdividing the Drosophila embryo. Graded Shh signaling subdivides the ventral neural tube into five domains, just as graded levels of the dorsal protein establish five distinct regions of the early Drosophila embryo, suggesting an upper limit to the number of distinct cell fates that can be generated in response to a single gradient signaling system. In addition, the graded anterioposterior distribution of maternally supplied factors in the Drosophila embryo is known to initiate the expression of a set of proteins encoded by the gap genes. Subsequent cross-regulatory interactions establish and maintain sharp boundaries in the expression of gap proteins, and their activities within individual domains control later aspects of cell pattern. Thus, in the neural tube and the Drosophila embryo, the cross-repression of genes whose initial expression is controlled by graded upstream signals provides an effective mechanism for establishing and maintaining progenitor domains and for imposing cell type identity (Briscoe, 2000 and references therein).

The secretion of Sonic hedgehog (Shh) from the notochord and floor plate appears to generate a ventral-to-dorsal gradient of Shh activity that directs progenitor cell identity and neuronal fate in the ventral neural tube. In principle, the establishment of this Shh activity gradient could be achieved through the graded distribution of the Shh protein itself, or could depend on additional cell surface or secreted proteins that modify the response of neural cells to Shh. Cells of the neural plate differentiate from a region of the ectoderm that has recently expressed high levels of BMPs, raising the possibility that prospective ventral neural cells are exposed to residual levels of BMP activity. Whether modulation of the level of BMP signaling regulates neural cell responses to Shh, and thus might contribute to the patterning of cell types in the ventral neural tube, has been examined. Based on results from an in vitro assay of neural cell differentiation, BMP signaling has been shown to markedly alter neural cell responses to Shh signals, eliciting a ventral-to-dorsal switch in progenitor cell identity and neuronal fate (Reim, 2000).

BMP signaling is regulated by secreted inhibitory factors, including noggin and follistatin, both of which are expressed in or adjacent to the neural plate. Conversely, follistatin but not noggin produces a dorsal-to-ventral switch in progenitor cell identity and neuronal fate in response to Shh both in vitro and in vivo. Since patched is likely to be a direct target of Shh signaling, the ability of BMPs to block the Shh-induced elevation in ptc expression provides one line of evidence that the modulatory action of BMPs on neural cell responses to Shh is exerted at a proximal step in the Shh signal transduction pathway. Exposure of neural cells to BMPs also blocks the Shh-mediated induction of HNF3 beta, thought to be induced as a direct response to hedgehog signaling. These results suggest that the specification of ventral neural cell types depends on the integration of Shh and BMP signaling activities. The net level of BMP signaling within neural tissue may be regulated by follistatin and perhaps other BMP inhibitors secreted by mesodermal cell types that flank the ventral neural tube (Riem, 2000).

In the vertebrate spinal cord, oligodendrocytes (OL) arise from the ventral part of the neuroepithelium, a region also known to generate somatic motoneurons. The emergence of oligodendrocytes, like that of motoneurons, depends on an inductive signal mediated by Sonic hedgehog. The precise timing of oligodendrocyte progenitor specification in the cervico-brachial spinal cord of the chick embryo has been determined. Ventral neuroepithelial explants, isolated at various development stages, are unable to generate oligodendrocytes in culture until E5 but become able to do so in an autonomous way from E5.5. This indicates that the induction of oligodendrocyte precursors is a late event that occurs between E5 and E5.5, precisely at the time when the ventral neuroepithelium stops producing somatic motoneurons. Analysis of the spatial restriction of oligodendrocyte progenitors, evidenced by their expression of O4 or PDGFRalpha, indicate that they always lie within the most ventral Nkx2.2-expressing domain of the neuroepithelium, and not in the adjacent domain characterized by Pax6 expression from which somatic motoneurons emerge. Shh is necessary between E5 and E5.5 to specify oligodendrocyte precursors but is no longer required beyond this stage to maintain ongoing oligodendrocyte production. Furthermore, Shh is sufficient to induce oligodendrocyte formation from ventral neuroepithelial explants dissected at E5. Newly induced oligodendrocytes express Nkx2.2 but not Pax6, correlating with the in vivo observation. Altogether, these results show that, in the chick spinal cord, oligodendrocytes originate from Nkx2.2-expressing progenitors (Soula, 2001).

Bone morphogenetic protein-7 (BMP-7) is a member of the 60A branch of the BMP family, less closely related to Dpp than is BMP2 or BMP-7. The expression pattern of BMP-7 in the hindbrain region of the headfold and early somite stage of the developing mouse embryo suggests a role for BMP-7 in the patterning of this part of the cranial CNS. In chick embryos it is thought that BMP-7 is one of the secreted molecules that mediates the dorsalizing influence of surface ectoderm on the neural tube. Mouse surface ectoderm has been shown to have a similar dorsalizing effect. While BMP-7 is expressed in the surface ectoderm of mouse embryos at the appropriate time to dorsalize the neural tube, at early stages of hindbrain development BMP-7 transcripts are present in paraxial and ventral tissues, within and surrounding the hindbrain neurectoderm; only later does expression become restricted to a dorsal domain. To determine more directly the effect that BMP-7 may have on the developing hindbrain, COS cells expressing BMP-7 were grafted into the ventrolateral mesoderm abutting the neurectoderm in order to prolong BMP-7 expression in the vicinity of ventral hindbrain. Three distinct actvities of BMP-7 are apparent. (1) BMP-7 can promote dorsal characteristics in the neural tube. (2)BMP-7 can also attenuate the expression of sonic hedgehog (Shh) in the floorplate without affecting Shh expression in the notochord, and (3) ectopic BMP-7 appears to promote growth of the neurectoderm. It seems that BMP-7 is involved in timing mechanisms necessary for the coordination of hindbrain dorsoventral patterning (Arkell, 1997).

The floor plate plays crucial roles in the specification and differentiation of neurons along the dorsal-ventral (DV) axis of the neural tube. In the mesencephalon, the dorsal part gives rise to the tectum, which is the major visual center in lower vertebrates, and the ventral part to the tegmentum. The transplantation of the mesecephalic floor plate (mfp) into the dorsal mesencephalon in chick embryos alters the fate of the mesencephalon adjacent to the transplant from the tectum to the tegmentum. In this study, to test whether the mfp is involved in the specification of the DV polarity of the tectum and affects the projection patterns of retinal fibers to the tectum along the DV axis, quail mfp was transplanted into the dorsal mesencephalon of chick embryos, and projection patterns of dorsal and ventral retinal fibers to the tectum were analyzed. In the embryos with the mfp graft, dorsal retinal fibers grow into the dorsal part of the tectum, which is the original target for ventral but not dorsal retinal fibers, and form tight focuses there. In contrast, ventral retinal fibers do not terminate at any part of the tectum. Transplantation of Sonic hedgehog (Shh)-secreting quail fibroblasts into the dorsal mesencephalon also induces the ectopic tegmentum and alters the retinotectal projection along the DV axis, as does the mfp graft. These results suggest that some factors from the mesencephalic floor plate or the tegmentum, or Shh itself, play a crucial role in the establishment of the DV polarity of the tectum and the retinotectal projection map along the DV axis (Nomura, 2000).

The elaboration of distinct cell types during development is dependent on a small number of inductive molecules. Among these inducers is Sonic hedgehog (Shh), which, in combination with other factors, patterns the dorsoventral (DV) axis of the nervous system. The response of a cell is dependent in part on its complement of cyclic nucleotides. cAMP antagonizes Shh signaling, and the influence of cGMP on the Shh response was examined. Cells in chick neural plate explants respond to Shh by differentiating into ventral neural-cell types. Exposure of intermediate-zone explants to cGMP analogs enhances their response to Shh in a dose-dependent manner. The Shh response is also enhanced in dorsal-zone explants exposed to chick natriuretic peptide (chNP), which stimulates cGMP production by membrane-bound guanylate cyclase (mGC). Addition of chNP to intermediate-zone explants does not enhance the Shh response, consistent with a reported lack of mGC in this region of the neural tube. Finally, the presence of a nitric oxide (NO)-sensitive guanylate cyclase (GC) was established by demonstrating cGMP immunoreactivity in neural tissue following NO stimulation of whole chick embryos. Intracellular levels of cGMP and cAMP may thus provide a mechanism through which other factors modulate the Shh response during neural development (Robertson, 2001).

Members of the GATA transcription factor gene family have been implicated in a variety of developmental processes, including those involved in vertebrate central nervous system development. However, the role of GATA proteins in spinal cord development remains unresolved. The expression and function of two GATA proteins, GATA2 and GATA3, were examined in the developing chick spinal cord. Both proteins are expressed by a distinct subpopulation of ventral interneurons that share the same dorsoventral position as CHX10-positive V2 interneurons. However, no coexpression is observed between the two GATA proteins and CHX10. By in vivo notochord grafting and cyclopamine treatment, it has been demonstrated that the spatially restricted pattern of GATA3 expression is regulated, at least in part, by the signaling molecule Sonic hedgehog. In addition, Sonic hedgehog induces GATA3 expression in a dose-dependent manner. Using in ovo electroporations, it has been demonstrated that GATA2 is upstream of GATA3 in the same epigenetic cascade and that GATA3 is capable of inducing GATA2 expression in vivo. Furthermore, the ectopically expressed GATA proteins can repress differentiation of other ventral cell fates, but not the development of progenitor populations identified by PAX protein expression. Taken together, these findings strongly suggest an important role for GATA2 and GATA3 proteins in the establishment of a distinct ventral interneuron subpopulation in the developing chick spinal cord (Karunaratne, 2002).

Sonic hedgehog (Shh) is a key signal in the specification of ventral cell identities along the length of the developing vertebrate neural tube. In the presumptive hindbrain and spinal cord, dorsal development is largely Shh independent. By contrast, Shh is required for cyclin D1 expression and the subsequent growth of both ventral and dorsal regions of the diencephalon and midbrain in early somite-stage mouse embryos. It is proposed that a Shh-dependent signaling relay regulates proliferation and survival of dorsal cell populations in the diencephalon and midbrain. Evidence is presented that Fgf15 shows Shh-dependent expression in the diencephalon and may participate in this interaction, at least in part, by regulating the ability of dorsal neural precursors to respond to dorsally secreted Wnt mitogens (Ishibashi, 2002).

The hedgehog signaling pathway organizes the developing ventral neural tube by establishing distinct neural progenitor fates along the dorsoventral axis. In the embryonic spinal unique and partially overlapping patterns of expression of several families of homeodomain-containing transcriptional regulators define five neural progenitor populations in the ventral half of the neural tube. From ventral to dorsal, these are the p3, pMN, p2, p1, and p0 progenitors. pMN progenitors later give rise to motorneurons, and p3, p2, p1, and p0 progenitors give rise to v3, v2, v1, and v0 interneurons, respectively. Smoothened (Smo) is essential for all Hedgehog (Hh) signaling, and genetic inactivation of Smo cells autonomously blocks the ability of cells to transduce the Hh signal. Using a chimeric approach, the behavior of Smo null mutant neural progenitor cells was examined in the developing vertebrate spinal cord; direct Hh signaling is essential for the specification of all ventral progenitor populations. Further, Hh signaling extends into the dorsal half of the spinal cord including the intermediate Dbx expression domain. Surprisingly, in the absence of Sonic hedgehog (Shh), the presence of a Smo-dependent Hh signaling activity operating in the ventral half of the spinal cord is observed that most likely reflects Indian hedgehog (Ihh) signaling originating from the underlying gut endoderm. Comparative studies of Shh, Smo, and Gli3 single and compound mutants reveal that Hh signaling acts in part to specify neural cell identity by counteracting the repressive action of Gli3 on p0, p1, p2, and pMN formation. However, whereas these cell identities are restored in Gli3/Smo compound mutants, correct stratification of the rescued ventral cell types is lost. Thus, Hh signaling is essential for organizing ventral cell pattern, possibly through the control of differential cell affinities (Wijgerde, 2002).

One question of considerable importance is the actual range of Hh action. Although all ventral progenitors (p0, p1, p2, pMN, and p3) can be induced by distinct concentrations of Shh in vitro, it is not clear that there is a direct requirement for Hh signaling in the specification of all ventral cell fates. By genetically preventing all Hh responsiveness in a subpopulation of cells within the putative Hh target field, it has been shown that specification of all ventral progenitors requires Hh signaling in the embryo. Indeed, this requirement actually extends into a domain of Pax7-, Dbx1/2-expressing cells directly above the postulated ventral p0 domain. Although these cells (dorsal p5: the most dorsally located ventral interneuron precursor) have not been thought to produce Evx1/2 neurons, there is no direct evidence that this is the case. Thus, all Dbx1+ cells, regardless of Pax7 expression, may generate Evx1/2 interneuron precursors, an issue that remains to be resolved. Thus although this study establishes a direct long-range requirement for Hh signaling in neural tube patterning this study does not establish the actual range of action of the signaling process, nor does it address the concentration dependence of Hh signaling (Wijgerde, 2002).

With respect to the issue of the range of the signaling process in ventral patterning, this depends on knowing when each of the specific progenitor populations is first established in response to a Hh input and when the maintenance of a given cell fate becomes Hh-independent. Already at the 15-somite stage, at neural tube levels that later give rise to rostral spinal cord regions, there is a well organized ventral pattern with specification of even the most ventral p3, Nkx2.2-producing, progenitor cells. At this axial level, floor plate induction has not occurred; hence the principle source of Shh is the notochord underlying the neural tube. Dbx1 induction is Hedgehog-dependent, and Dbx1 is induced in cells that extend 15-20 cell diameters from the ventral midline at the 15-somite stage. That Shh might act over this distance is certainly consistent with the actual distribution of Shh ligand, the transcriptional upregulation of primary targets such as Ptch1 and Gli1, and studies of Shh signaling in other systems such as the vertebrate limb (Wijgerde, 2002).

Further, although the data indicate that there is a direct Hh input for the establishment of all ventral cell identities, they do not address whether Hedgehog action is concentration-dependent. For example, Hedgehog signaling might define a domain of ventral competence, whereas other factors might play a more direct role in the induction of each individual cell fate. Repression of Pax7 in ventral cells is thought to be a critical first step in the induction of ventral cell identities, and the ventral limit of Pax7-expressing cells has long been seen as the limit for Shh signaling. Thus, repression of Pax7 might define a ventral competence domain. All Smo-/- cells that lie ventral to the normal ventral limit of Pax7 expression at 10.5 dpc maintain Pax7 expression, consistent with there being an absolute requirement for a Hh input to repress Pax7 (Wijgerde, 2002).

The results also demonstrate that the presence of v0 and v1 progenitors observed in Shh mutants reflects the presence of low-level Hh signaling as indicated by the absence of Pax7 expression and the upregulation of Ptch1 at the ventral midline of the Shh mutant neural tube, where these ventral interneuron precursors arise. Since Ihh is expressed in both the node and the gut endoderm that underlies the notochord, it is speculated that Ihh signaling is responsible for the limited ventralization in Shh mutants. Interestingly, Shh and Ihh play semiredundant roles in patterning somite domains that lie adjacent to the neural tube. Whether Ihh plays any normal role in patterning the ventral neural tube in the context of an active Shh signal is doubtful (Wijgerde, 2002).

Examining the subsequent fate of Pax7-producing cells provides an insight into the assignment of ventral cell fates in the neural tube. Based on the expression profile of ventral Smo-/- neural progenitor cells (Pax7on, Dbx1on, Dbx2on, Irx3on, and Pax6on where 'on refers to cells expressing the particular transcription factor) and the neuron types generated by Smo-/- progenitors (Lim1/2on, Pax2on), it appears that many ventral Smo-/- cells adopt a dorsal identity, possibly dorsal p5. However, a few cells also express Evx1/2, indicating that at least some Smo-/- progenitors give rise to v0 precursors. In contrast, as discussed above, Smo-/- cells are unable to generate Dbx-producing neural progenitor cells or v0 precursors where these cells normally arise, in the intermediate domains of the spinal cord. How can these paradoxical results be explained (Wijgerde, 2002)?

One possibility is that Hh signaling, though required for the specification of a v0 fate, does not directly specify that fate. For example, a Hh signaling input may counteract the inhibitory activity of another signaling pathway that is active at the dorsal-ventral (DV) interface, an inhibitory activity that does not normally extend into the ventral half of the neural tube. The most likely candidates for these presumably dorsal signals would be members of the TGF-ß superfamily that are responsible for the induction of dorsal neural cell identities. Indeed, the TGF-ß family members Bmp2, -4 and -7 are potent inhibitors of Dbx1/2 expression and can block the generation of Evx1/2 and En1 interneurons. Further, addition of low concentrations of Shh inhibits the expression of BMPs in neural explants in vitro. Recent ablation studies using the chick spinal cord indicate that dorsally derived BMP signaling extends into the ventral half of the neural tube at neural groove stages, and it is clear that Shh signaling extends into dorsal regions (as defined by Pax7 expression). Thus, it is likely that BMP and Hh signaling overlap in the intermediate region of the spinal cord and that Shh signaling either counteracts or collaborates with the activity of BMPs. Recent studies on the modification of the response of intermediate explants to Shh signaling by BMPs suggest that the former is more likely. Additional signaling by retinoids may then be required for the specification of v0 fates. If so, the production of a retinoid signal would not appear to depend upon the induction of other ventral progenitor populations (Wijgerde, 2002).

Interestingly, whereas v0 precursors are appropriately positioned in the neural tube of Smo/Gli3 compound mutants, v1, v2, and MN precursors, which normally do not overlap, now extend over much of the ventral half of the neural tube. Thus, a direct Hh signaling input is required for the normal stratification of ventral progenitor populations within separate domains of the ventral neural tube. This indicates that a central role of the hedgehog-Gli3 signaling axis is to refine the size and position of ventral progenitor pools rather than specifying the individual identity of MN, v2, v1, and v0 precursors. During normal DV patterning, each precursor population forms a sharp boundary with its neighbor, suggesting that there may be some Hh-dependent mechanism that prevents their mixing. In Drosophila, Hh signaling maintains a sharp anterior-posterior (AP) compartment boundary, preventing the mixing of cells at the AP interface. Further, analysis of Smo-/- cells in the abdomen of the fly suggests that a gradient of Hedgehog signaling specifies graded levels of cell affinity. Thus, it is tempting to speculate that Hh signaling in the mammalian neural tube might regulate the precise separation of precursor domain boundaries along the DV axis through the control of cell affinities (Wijgerde, 2002).

If a DV gradient of affinities normally contributes to the segregation of progenitors, then dorsalized Smo-/- cells in ventral positions might be expected to cluster with each other, minimizing contacts with their ventral neighbors, similar to the behavior of clones of Smo-/- clones in the anterior compartment of the fly wing or abdomen. In this regard it is noted that whereas Smo+/- cells show a fine-grained mosaicism in the neural tube of chimeras, Smo-/- cells tend to cluster in patches, a finding observed in other Hh target fields. Functionally, modifying differences in cell affinities could prevent cells from mixing freely within a morphogenetic field as cell identities are being specified, a mechanism that may contribute to precision and stability in the induction of different progenitor domains. Presumably, once neurons are generated from progenitor cells their identity is fixed. At this time they may mix 'freely' to participate in the formation of appropriate neural circuits (Wijgerde, 2002).

An important question is how the gradient of Hedgehog is interpreted by cells at the level of the Gli transcription factors. The full range of Gli activity and its dependence on Hh have not been determined, although the Gli2 activator and Gli3 repressor have been implicated. Using the spinal cord as a model system, it has been demonstrated that Gli3 can transduce Hedgehog signaling as an activator. All expression of the Hh target gene Gli1 is dependent on both Gli2 and Gli3. Unlike Gli2, however, Gli3 requires endogenous Gli1 for induction of floor plate and V3 interneurons. Strikingly, embryos lacking all Gli function develop motor neurons and three ventral interneuron subtypes, similar to embryos lacking Hh signaling and Gli3. Therefore, in the spinal cord all Hh signaling is Gli dependent. Furthermore, a combination of Gli2 and Gli3 is required to regulate motor neuron development and spatial patterning of ventral spinal cord progenitors (Bai, 2004).

By using a sensitive marker for Shh positive function, it has been demonstrated that loss of Gli3 results in reduced Hh signaling throughout the CNS at E9.5 and E10.5, as well as in the limb buds at E10.5. An overlap in activator functions between Gli2 and Gli3 is demonstrated using two in vivo approaches. First, in Gli2;Gli3 double homozygous mutant embryos, all Gli1-lacZ expression is absent, whereas it is only reduced in either single mutant. Given that Gli1-lacZ is a readout of the positive action of Hh signaling, this demonstrates a loss of the activator functions of both Gli2 and Gli3 in these embryos. The activator function of Gli3 during normal Hh signaling was directly tested in the spinal cord by expressing human GLI3 in place of the endogenous Gli2 gene. This study demonstrates that Gli3 protein expressed from the endogenous Gli3 allele, along with GLI3 protein from the Gli23ki/3ki knockin allele, function as activators to induce the expression of Gli1 and development of some FP cells and many V3 interneurons (Bai, 2004).

Although an internal deletion of the N terminus turns both Gli2 and Gli3 into strong activators of the Hh target Hnf3β, these studies show that the inherent activator function of Gli3 is not as potent as Gli2. First, Gli3 cannot fully rescue FP and V3 interneuron induction in the spinal cord of Gli2 mutants. Second, unlike Gli2 (or Gli1), Gli3 cannot induce FP and V3 interneurons in the absence of the endogenous Gli1 gene when expressed like Gli2. Furthermore, Gli3 cannot completely rescue other Gli2 mutant phenotypes, such as the lung defects, and Gli23ki/3ki pups live for only 2 days after birth. Consistent with these results, Gli3 appears to only weakly contribute to positive Hh signaling in Ptc mutants in which the pathway is highly activated. Removal of Gli2, but not Gli3, rescues the expanded FP and V3 interneuron phenotype in Ptc mutants, whereas removal of both Gli2 and Gli3 leads to a suppression of MN development, at least at E9.5 (Bai, 2004).

The fact that Gli3 cannot fully rescue many Gli2 defects demonstrates that Gli2 and Gli3 proteins have distinct intrinsic biochemical properties. One possibility is that Gli2 and Gli3 recognize different subsets of target genes with different affinities. Indeed, Gli1 and Gli3 have different affinities for Gli DNA binding sites. Alternatively, or in addition, Gli3 but not Gli2 forms a potent repressor and thus there may be some Gli3 repressor made in the ventral spinal cord of Gli23ki/3ki embryos that competes with any activator produced. Consistent with this, Western blot analysis shows that a substantial amount of the Gli3 expressed from the Gli2 locus is in the repressor form. Thus, changing the expression domain of Gli3 to that of Gli2 is not sufficient to remove all Gli3 repressor (Bai, 2004).

A central argument for the morphogen model of Shh activity is the fact that particular neurons are induced at specific Shh concentrations in spinal cord explants. How the cells read this gradient at the molecular level has not been clear. A surprising finding was that when Gli3 is removed from Smo mutant embryos MNs and V0-2 interneurons form, albeit in abnormal positions. Thus an essential requirement for Hh signaling in these four ventral cell types must be to inhibit formation of the Gli3 repressor. Since Hh signaling is lost in these mutants, it is possible that formation of Gli2 or Gli3 activators is also required in these cells. In addition, because Gli2 is expressed in the double mutant background, it was not known whether Gli2 has an Hh-independent function in generating the remaining ventral cell types. Furthermore, it was not known whether Gli2 and Gli3 play redundant roles in MN and V0-V2 development, since although a recent study concluded that Gli2 and Gli3 are required for MN development, the analysis was only done at E9.5 and V0-V2 interneurons were not examined. These questions are resolved by demonstrating that Gli proteins are not required for generation of many MNs and V0-V2 interneurons at E10.5, but instead they are required to regulate normal MN differentiation, as well as for correct spatial patterning of ventral cell types (Bai, 2004).

Distinct classes of serotonergic (5-HT) neurons develop along the ventral midline of the vertebrate hindbrain. A Sonic hedgehog (Shh)-regulated cascade of transcription factors has been identified that acts to generate a specific subset of 5-HT neurons. This transcriptional cascade is sufficient for the induction of rostral 5-HT neurons within rhombomere 1 (r1) that project to the forebrain, but not for the induction of caudal 5-HT neurons, which largely terminate in the spinal cord. Within the rostral hindbrain, the Shh-activated homeodomain proteins Nkx2.2 and Nkx6.1 cooperate to induce the closely related zinc-finger transcription factors Gata2 and Gata3. Gata2 in turn is necessary and sufficient to activate the transcription factors Lmx1b and Pet1, and to induce 5-HT neurons within r1. In contrast to Gata2, Gata3 is not required for the specification of rostral 5-HT neurons and appears unable to substitute for the loss of Gata2. These findings reveal that the identity of closely related 5-HT subclasses occurs through distinct responses of adjacent rostrocaudal progenitor domains to broad ventral inducers (Craven, 2004).

Graded Hedgehog (Hh) signaling patterns the spinal cord dorsoventral axis by inducing and positioning distinct precursor domains, each of which gives rise to a different type of neuron. These domains also generate glial cells, but the full range of cell types that any one precursor population produces and the mechanisms that diversify cell fate are unknown. By fate mapping and clonal analysis in zebrafish, it has been shown that individual ventral precursor cells that express olig2 can form motoneurons, interneurons and oligodendrocytes. However, olig2+ precursors are not developmentally equivalent, but instead produce subsets of progeny cells in a spatially and temporally biased manner. Using genetic and pharmacological manipulations, evidence is provided that these biases emerge from Hh acting over time to set, maintain, subdivide and enlarge the olig2+ precursor domain and subsequently specify oligodendrocyte development. These studies show that spatial and temporal differences in Hh signaling within a common population of neural precursors can contribute to cell fate diversification (Park, 2004).

In the vertebrate embryo, spinal cord elongation requires FGF signaling that promotes the continuous development of the posterior nervous system by maintaining a stem zone of proliferating neural progenitors. Those escaping the caudal neural stem zone initiate ventral patterning in the neural groove before starting neuronal differentiation in the neural tube. The integration of D-type cyclins, known to govern cell cycle progression under the control of extracellular signals, in the program of spinal cord maturation was investigated. In chicken embryo, it was found that cyclin D2 is preferentially expressed in the posterior neural plate, whereas cyclin D1 appears in the neural groove. Loss- and gain-of-function experiments demonstrate that FGF signaling maintains cyclin D2 in the immature caudal neural epithelium, while Shh activates cyclin D1 in the neural groove. Moreover, forced maintenance of cyclin D1 or D2 in the neural tube favors proliferation at the expense of neuronal differentiation. These results contribute to the understanding of how the cell cycle control can be linked to the patterning programs to influence the balance between proliferation and neuronal differentiation in discrete progenitors domains (Labjois, 2004)

The ventral neural tube of vertebrates consists of distinct neural progenitor domains positioned along the dorsoventral (DV) axis that develop different types of motorneurons and interneurons. Several signalling molecules, most notably Sonic Hedgehog (Shh), retinoic acid (RA) and fibroblast growth factor (FGF) have been implicated in the generation of these domains. Shh is secreted from the floor plate, the ventral most neural tube structure that consists of the medial (MFP) and the lateral floor plate (LFP). While the MFP is well characterized, organization and function of the LFP remains unclear. The homeobox gene nkx2.2b is strongly expressed in the trunk LFP of zebrafish and thus represents a unique marker for the characterization of LFP formation and the identification of LFP deficient mutants. nkx2.2b and its paralog nkx2.2a (formerly known as nk2.2 and nkx2.2) arose by gene duplication in zebrafish. Both duplicates show significant differences in their expression patterns. For example, while prominent nkx2.2a expression has been described in the ventral brain, hardly any expression can be found in the trunk LFP, which is in contrast to nkx2.2b. Overexpression, mutant and inhibitor analyses show that nkx2.2b expression in the LFP is up-regulated by Shh, but repressed by retinoids and ectopic islet-1 (isl1) expression. In contrast to previously described zebrafish trunk LFP markers, like e.g. tal2 or foxa2, nkx2.2b is exclusively expressed in the LFP. Thus, nkx2.2b represents a unique tool to analyse the mechanisms of ventral neural tube patterning in zebrafish (Schafer, 2005).

During development, many signaling factors behave as morphogens, long-range signals eliciting different cellular responses according to their concentration. In ventral regions of the spinal cord, Sonic Hedgehog is such a signal and controls the emergence, in precise spatial order, of distinct neuronal subtypes. The Gli family of transcription factors plays a central role in this process. A gradient of Gli activity has been show to be sufficient to mediate, cell-autonomously, the full range of Shh responses in the neural tube. The incremental two- to three-fold changes in Shh concentration, that determine alternative neuronal subtypes, are mimicked by similar small changes in the level of Gli activity, indicating that a gradient of Gli activity represents the intracellular correlate of graded Shh signaling. Moreover, this analysis suggests that cells integrate the level of signaling over time, consistent with the idea that signal duration, in addition to signal strength, is an important parameter controlling dorsal-ventral patterning. Together, these data indicate that Shh signaling is transduced, without amplification, into a gradient of Gli activity that orchestrates patterning of the ventral neural tube (Stamatiki, 2005).

Dynamics of Sonic hedgehog signaling in the ventral spinal cord are controlled by intrinsic changes in source cells requiring Sulfatase 1

In the ventral spinal cord, generation of neuronal and glial cell subtypes is controlled by Sonic hedgehog (Shh). This morphogen contributes to cell diversity by regulating spatial and temporal sequences of gene expression during development. This study reports that establishing Shh source cells is not sufficient to induce the high-threshold response required to specify sequential generation of ventral interneurons and oligodendroglial cells at the right time and place in zebrafish. Instead, it was shown that Shh-producing cells must repeatedly upregulate the secreted enzyme Sulfatase1 (Sulf1) at two critical time points of development to reach their full inductive capacity. Evidence is provided that Sulf1 triggers Shh signaling activity to establish and, later on, modify the spatial arrangement of gene expression in ventral neural progenitors. Arguments are further presented in favor of Sulf1 controlling Shh temporal activity by stimulating production of active forms of Shh from its source. This work, by pointing out the key role of Sulf1 in regulating Shh-dependent neural cell diversity, highlights a novel level of regulation, which involves temporal evolution of Shh source properties (Oustah, 2014).

Sonic hedgehog is an axonal chemoattractant in midline axon guidance

Developing axons are guided to their targets by attractive and repulsive guidance cues. In the embryonic spinal cord, the floor plate chemoattractant Netrin-1 is required to guide commissural neuron axons to the midline. However, genetic evidence suggests that other chemoattractant(s) are also involved. The morphogen Sonic hedgehog (Shh) can mimic the additional chemoattractant activity of the floor plate in vitro and can act directly as a chemoattractant on isolated axons. Cyclopamine-mediated inhibition of the Shh signaling mediator Smoothened (Smo) or conditional inactivation of Smo in commissural neurons indicates that Smo activity is important for the additional chemoattractant activity of the floor plate in vitro and for the normal projection of commissural axons to the floor plate in vivo. These results provide evidence that Shh, acting via Smo, is a midline-derived chemoattractant for commissural axons and shows that a morphogen can also act as an axonal chemoattractant (Charron, 2003).

In non-mammalian vertebrates, the relatively homogeneous population of retinal ganglion cells (RGCs) differentiates and projects entirely to the contralateral side of the brain under the influence of Sonic hedgehog (Shh). In mammals, by contrast, there are two different RGC types: the Zic2-positive ipsilateral projecting and the Isl2-positive contralateral projecting. It was asked whether the axons of these two populations respond to Shh and if their response differs. Also, whether midline- and RGC-derived Shh contributes to the growth of the axons in the proximal visual pathway was analysed. These two RGC types are characterised by a differential expression of Shh signalling components, and they respond differently to Shh when challenged in vitro. In vivo blockade of Shh activity, however, alters the path and distribution mostly of the contralateral projecting RGC axons at the chiasm, indicating that midline-derived Shh participates in funnelling contralateral visual fibres in this region. Furthermore, interference with Shh signalling in the RGCs themselves causes abnormal growth and navigation of contralateral projecting axons in the proximal portion of the pathway, highlighting a novel cell-autonomous mechanism by which Shh can influence growth cone behaviour (Sánchez-Camacho, 2008).

Sonic hedgehog plays essential roles in developmental events such as cell fate specification and axon guidance. Shh induces cell fate specification through canonical Shh signaling, mediated by transcription. However, the mechanism by which Shh guides axons is unknown. To study this, an in vitro assay was developed for axon guidance, in which neurons can be imaged while responding to a defined gradient of a chemical cue. Axons of dissociated commissural neurons placed in a Shh gradient turned rapidly toward increasing concentrations of Shh. Consistent with this rapid response, attraction by Shh was shown not to require transcription. Instead, Shh stimulates the activity of Src family kinase (SFK) members in a Smoothened-dependent manner. Moreover, SFK activity is required for Shh-mediated guidance of commissural axons, but not for induction of Gli transcriptional reporter activity. Together, these results indicate that Shh acts via a rapidly acting, noncanonical signaling pathway to guide axons (Yam, 2009).

Table of contents

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

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