cubitus interruptus

EVOLUTIONARY HOMOLOGS (part 2/3)

Cubitus interuptus homologs: Neural tube midline

Floor plate cells at the midline of the neural tube are specified by high-level activity of Sonic hedgehog (Shh), which is secreted by the notochord; in contrast, motor neurons are thought to be specified by a lower level activity of Shh, which is secreted, in turn, by floor plate cells. In Drosophila, the Gli zinc finger protein Cubitus interruptus functions as a transcription factor activating Hedgehog-responsive genes. The expression of known Shh-responsive genes such as Ptc and Gli1 is downregulated in mutant mice lacking Gli2 function. Gli2 mutants fail to develop a floor plate yet still develop motor neurons, which occupy the ventral midline of the neural tube. These results imply that Gli2 is required to mediate high level but not low level Shh activity and show that the development of motor neurons can occur in the absence of floor plate induction (Ding, 1998).

Induction of the floor plate at the ventral midline of the neural tube is one of the earliest events in the establishment of dorsoventral (d/v) polarity in the vertebrate central nervous system (CNS). The secreted molecule, Sonic hedgehog, has been shown to be both necessary and sufficient for this induction. In vertebrates, several downstream components of this signaling pathway have been identified, including members of the Gli transcription factor family. In this study, the d/v patterning of the CNS has been examined in Gli2 mouse mutants. The floor plate throughout the midbrain, hindbrain and spinal cord does not form in Gli2 homozygotes. Despite this, motoneurons and ventral interneurons form in their normal d/v positions at 9.5 to 12.5 days postcoitum (dpc). However, cells that are generated in the region flanking the floor plate, including dopaminergic and serotonergic neurons, are greatly reduced in number or absent in Gli2 homozygous embryos. These results suggest that early signals derived from the notochord can be sufficient for establishing the basic d/v domains of cell differentiation in the ventral spinal cord and hindbrain. Interestingly, the notochord in Gli2 mutants does not regress ventrally after 10.5 dpc, as in normal embryos. The spinal cord of Gli1/Gli2 zinc-finger-deletion double homozygous mutants appear similar to Gli2 homozygotes, indicating that neither gene is required downstream of Shh for the early development of ventral cell fates outside the ventral midline (Matise, 1998).

Within the neural tube of vertebrate embryos, the floor plate plays important roles in ventral pattern formation and axonal guidance. A critical event for floor plate development is the induction of a winged helix transcription factor, Hepatocyte Nuclear Factor-3ß (HNF-3ß, related to Drosophila Forkhead). The enhancer for floor plate expression of HNF-3ß is located 3' to the transcription unit and consists of multiple elements. HNF-3ß induction depends on the notochord-derived signal, Sonic hedgehog (Shh). A Gli-binding site is required for the activity of the minimal floor plate enhancer of HNF-3ß in vivo. Three Gli genes (Gli, Gli2 and Gli3) are differentially expressed in the developing neural tube. Gli expression is restricted to the ventral part, while Gli2 and Gli3 are expressed (respectively) throughout the neural tube and dorsally. Strong expression of Gli and Gli2, and weak expression of Gli3 transiently overlap with HNF-3ß at the time of its induction. Consistent with ventrally localized expression, Gli expression can be up-regulated by Shh. Finally, the Gli-binding site acts as a Shh responsive element, and human GLI (but not GLI3) can activate this binding site in tissue culture. Taken together, these findings suggest that Gli, and probably also Gli2, are good candidates for transcriptional activators of the HNF-3ß floor plate enhancer, and the binding site for Gli proteins is a key element for response to Shh signaling. These results also support the idea that Gli/Ci are evolutionarily conserved transcription factors in the Hedgehog signaling pathway (Sasaki, 1997).

Sonic hedgehog (Shh) is a putative morphogen secreted by the floor plate and notochord; Shh specifies the fate of multiple cell types in the ventral aspect of the vertebrate nervous system. Since in Drosophila the actions of Hh have been shown to be transduced by Cubitus interruptus (Ci), a zinc finger transcription factor, an examination was carried out to determine whether a vertebrate homolog of this protein can mediate the functions of Shh in the vertebrate nervous system. Expression of Gli-1, one of three vertebrate homologs of Ci, can be induced by Shh in the neural tube. Further, ectopic expression of Gli-1 in the dorsal midbrain and hindbrain of transgenic mice mimics the effects of ectopically expressed Shh-N, leading to the activation of ventral neural tube markers such as Ptc, HNF-3beta, and Shh; to the suppression of dorsal markers such as Pax-3 and AL-1; and to the formation of ectopic dorsal clusters of dopaminergic and serotonergic neurons. These findings demonstrate that GLI-1 can reproduce the cell patterning actions of Shh in the developing nervous system and provide support for the hypothesis that it is a mediator of the Shh signal in vertebrates (Hynes, 1997).

Three cell types differentiate in the early frog neural plate: neural crest at the lateral edges; floorplate at the midline, and primary neurons in three bilateral stripes. Floorplate cells and ventral neurons are induced by Sonic hedgehog (Shh) and neural crest and dorsal neurons are induced by epidermal factors, such as bone morphogenetic proteins (BMPs). Neurogenesis in a subset of cells within the stripes involves lateral inhibition. However, the process by which pools of precursors are defined in stereotypic domains in response to inductive signals is unknown. Frog Zic2 encodes a zinc-finger transcription factor of the Gli superfamily that is expressed in stripes that alternate with those in which primary neurons differentiate and overlap the domains of floorplate and neural crest progenitors. Zic2 inhibits neurogenesis and induces neural crest differentiation. Conversely, Gli proteins are widely expressed, induce neurogenesis and inhibit neural crest differentiation. Zic2 is therefore a vertebrate pre-pattern gene, encoding anti-neurogenic and crest-inducing functions that counteract the neurogenic but not the floorplate-inducing activity of Gli proteins. It is proposed that the combined function of Gli/Zic genes responds to inductive signals and induces patterned neural cell differentiation (Brewster, 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).

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).

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).

Specialized cells at the midline of the central nervous system have been implicated in controlling axon projections in both invertebrates and vertebrates. Analysis has been made of the requirement for ventral midline cells in the provision of cues to commissural axons using Gli2 mouse mutants, which specifically lack the floor plate and immediately adjacent interneurons. A specific enhancer drives tau-lacZ expression in a subpopulation of commissural axons (C-axons) and it has been found that C-axons project to the ventral midline in Gli2 minus embryos. Netrin1 mRNA expression is detected in Gli2 minus embryos and, although much weaker than in wild-type embryos, is found in a dorsally decreasing gradient. Netrin1 mRNA expression in the VZ in Gli2 -/- mutant embryos is likely to be sufficient to attract C-axons to the midline in these embryos. While the floor plate can serve as a source of long-range cues for C-axons in vitro, it is not required in vivo for the guidance of commissural axons to the ventral midline in the mouse spinal cord. After reaching the ventral midline, most commissural axons remain clustered in Gli2 minus embryos, although some are able to extend longitudinally. Interestingly, some of the longitudinally projecting axons in Gli2 minus embryos extend caudally and others rostrally at the ventral midline, in contrast to normal embryos in which virtually all commissural axons turn rostrally after crossing the midline. This finding indicates a critical role for ventral midline cells in regulating the rostral polarity choice made by commissural axons after they cross the midline. In rodents, the turning of C-axons into the longitudinal axis is correlated with a switch in adhesion molecule localization from TAG-1 to L1 on these axons. Evidence is provided that interactions between commissural axons and floor plate cells are required to modulate the localization of Nr-CAM and TAG-1 proteins on axons at the midline. Finally, it has been shown that the floor plate is not required for the early trajectory of motoneurons or axons of the posterior commissure, whose projections are directed away from the ventral midline in both WT and Gli2 minus embryos, although they are less well organized in Gli2 minus mutants (Matise, 1999).

Cubitus interuptus homologs: Spinal cord ventral-to-dorsal patterning

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).

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).

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).

Sonic hedgehog (Shh) plays a critical role in organizing cell pattern in the developing spinal cord. Gli proteins are thought to mediate Shh signaling, but their role in directing neural tube patterning remains unclear. A role for Gli3 transcriptional repressor activity in patterning the intermediate region of the spinal cord has been identified that complements the requirement for Gli2 in ventral regions. Moreover, blocking all Gli responses results in a complete dorsalization of ventral spinal cord, indicating that in addition to the specific roles of Gli2 and Gli3 in the neural tube, there is functional redundancy between Gli proteins. Finally, analysis of Shh/Gli3 compound mutant mice substantiates the idea that ventral patterning may involve a mechanism independent, or parallel, to graded Shh signaling. However, even in the absence of graded Shh signaling, Gli3 is required for the dorsal-ventral patterning of the intermediate neural tube. Together these data raise the possibility that Gli proteins act as common mediators integrating Shh signals, and other sources of positional information, to control patterning throughout the ventral neural tube (Persson, 2002).

The nature of the signal(s) conferring positional information independent of Shh remains unclear. It is possible that other Hh genes, such as Ihh, which is expressed in gastrulating embryos in regions of the embryo close to the forming neural plate, are able to partially substitute for Shh. Alternatively, it is possible that Wnt signals or BMP signals emanating from the dorsal neural tube and BMP antagonists expressed ventrally are sufficient to provide positional information. BMPs and Shh have opponent activity in the specification of ventral neuronal identity, and a reduction in BMP signaling leads to the expansion of ventral neural fates. Thus BMP signaling may be sufficient to provide positional information throughout the neural tube in embryos lacking Gli3 and Shh (Persson, 2002).

Together the data indicate that the signaling mechanisms that direct dorsal and ventral neural tube patterning are linked and that the limits of influence of ventral and dorsal signals are not clearly defined. Thus it seems likely that individual progenitor cells determine their gene expression profile by integrating the various dorsal and ventral extracellular signals that influence progenitor cell patterning. The data suggest that Gli proteins are part of this activity and may act as common mediators to integrate extracellular patterning signals. Consistent with this idea, as well as responding to Shh signaling, Gli proteins have been proposed to mediate Wnt signals; in addition, GSK3, a component of the Wnt signaling pathway, is implicated in influencing Ci activity in Drosophila. Moreover, BMPs are proposed to inhibit Shh signaling at a proximal point on the Shh signaling pathway, and there is evidence that Smads -- the transcriptional effectors of BMP signaling -- physically associate with Gli proteins. The level of Gli activity may therefore function as an intracellular correlate of positional information provided by extracellular patterning signals. In this model, Gli proteins act as pivotal intermediaries, interpreting patterning signals by directing the expression of class I and class II proteins that control neuronal subtype identity (Persson, 2002).

Sonic hedgehog (Shh) directs the development of ventral cell fates, including floor plate and V3 interneurons, in the mouse neural tube. The transcription factors Gli2 and Gli3, mediators of Shh signaling, are required for the development of the ventral cell fates but make distinct contributions to controlling cell fates at different locations along the rostral-caudal axis. Mutants lacking Patched1 (Ptc1), the putative receptor of Shh, were used to analyze Gli functions. Ptc1^-/- mutants develop floor plate, motor neuron, and V3 interneuron progenitors in lateral and dorsal regions, suggesting that the normal role of Ptc1 is to suppress ventral cell development in dorsal neural tube. The Ptc1^-/- phenotype is rescued, with restoration of dorsal cell types, by the lack of Gli2, but only in the caudal neural tube. In triple mutants of Gli2, Gli3, and Ptc1, dorsal and lateral cell fates are restored in the entire neural tube. These observations suggest that Gli2 is essential for ventral specification in the caudal neural tube, and that in more rostral regions, only Gli3 can promote development of ventral cells if Gli2 is absent. Thus, Shh signaling is mediated by overlapping but distinct functions of Gli2 and Gli3, and their relative contributions vary along the rostral-caudal axis (Moyoyama, 2003).

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 Gli2^3ki/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 Gli2^3ki/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 Gli2^3ki/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).

The three vertebrate Gli proteins play a central role in mediating Hedgehog (Hh)-dependent cell fate specification in the developing spinal cord; however, their individual contributions to this process have not been fully characterized. This issue has been addressed by examining patterning in the spinal cord of Gli2;Gli3 double mutant embryos, and in chick embryos transfected with dominant activator forms of Gli2 and Gli3. In double homozygotes, Gli1 is also not expressed; thus, all Gli protein activities are absent in these mice. Gli3 contributes activator functions to ventral neuronal patterning, and plays a redundant role with Gli2 in the generation of V3 interneurons. Motoneurons and three classes of ventral neurons are generated in the ventral spinal cord in double mutants, but develop as intermingled rather than discrete populations. Finally, evidence is proved that Gli2 and Gli3 activators control ventral neuronal patterning by regulating progenitor segregation. Thus, multiple ventral neuronal types can develop in the absence of Gli function, but require balanced Gli protein activities for their correct patterning and differentiation (Lei, 2004).

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).

Cubitus interuptus homologs: Primary neurogenesis

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 Notch^ICD, 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 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).

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

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

GLI and neural stem cell properties

Stem cells are crucial for normal development and homeostasis, and their misbehavior may be related to the origin of cancer. Progress in these areas has been difficult because the mechanisms regulating stem cell lineages are not well understood. The role of the SHH-GLI pathway in the developing mouse neocortex has been investigated. The results show that SHH signaling endogenously regulates the number of embryonic and postnatal mouse neocortical cells with stem cell properties, and controls precursor proliferation in a concentration-dependent manner in cooperation with EGF signaling. Shh^-/- mice die at birth showing overt signs of cyclopia and lacking all ventral CNS cell types. Their dorsal-only CNS comprises an Emx1⁺, Tbr1⁺ forebrain cortex. The Shh^-/- cortex produced neurosphere (nsp) cultures in full media, but these were fewer and smaller than those from wild-type cortices, and contained fewer BrdU⁺ cells. Analyses of gene expression confirmed the loss of Shh transcripts in the few Shh^-/- nsps that formed (representing a small pool of viable cells). A decrease in Ptch1 and Dhh expression was detected, whereas the expression of Ihh, Gli1 and Gli2 were unchanged, and the expression of Gli3 expression was slightly higher. These Shh^-/- nsps expressed nestin and were tripotential, as judged by the ability to differentiate as Tuj1⁺ neurons, GFAP⁺ astrocytes or O4⁺ oligodendrocytes. Cloning assays showed that Shh^-/- nsps contain approximately one quarter of the number of nsp-forming stem cells of wild-type nsps at E15.5. At E18.5, there were very few, if any, mutant nsps. Gli2^-/- mice also die at birth, displaying defects in multiple organs (Palma, 2004).

Novel dorsal brain phenotypes of Gli2^-/- mice were found at mid and late gestation stages in an outbred background. Gli2 null embryos present a variably penetrant severe phenotype, displaying excencephaly by E13.5 (also seen at E17-E18.5), and a consistent milder phenotype characterized by expanded but thinner telencephalic vesicles, most clearly seen posteriorly, and an overtly reduced tectum and cerebellum. Focus was placed on the non-exencephalic Gli2^-/- mice. Histological analyses showed that E18.5 Gli2^-/- telencephalic vesicles have a thinner proliferative zone (an ~30-50% reduction of the vz/svz). Gli2^-/- mice have fewer BrdU⁺ precursors in the cortex at mid and late gestation periods, suggesting defects in neuronal as well as glial cell populations. The decrease is most notable in the deeper proliferative area (the svz). Local variations without a clear pattern in the density of BrdU⁺ nuclei were also observed, indicating an additional degree of neocortical disorder in these mutant mice. TUNEL and activated caspase 3 analyses did not show an increase in apoptosis (Palma, 2004).

Gli2^-/- neocortices gave rise to nsps, containing Nestin⁺ cells that were tripotential. However, at late embryonic stages mutant nsps progressively became smaller, more delicate, and showed more blebbing than wild-type nsps. Gli2^-/- nsps decreased in numbers during culture and, after a few passes, they were rare, and all died soon after. The Gli2^-/- nsps surviving at passage ~2-4 lacked Gli1 expression and showed downregulation of Ihh and Dhh expression. Shh expression was unchanged, whereas Gli3 and Ptch1 expression was reduced. The expression of Egfr was also reduced. Cloning assays in the presence of EGF and FGF showed that there was an ~10-fold decrease in the number of Gli2^-/- cells able to form secondary nsps, as compared with wild-type cells. These findings identify a crucial mechanism for the regulation of the number of cells with stem cell properties that is unexpectedly conserved in different stem cell niches (Palma, 2004).

Cubitus interruptus homologs: Limb development

Continued: Cubitus interruptus Evolutionary homologs part 3/3 | back to part 1/3