cubitus interruptus

EVOLUTIONARY HOMOLOGS (part 3/3)

Cubitus interruptus homologs: Limb development

During initiation of limb-bud outgrowth in vertebrate embryos, the polarizing region (limb-bud organizer) is established upon activation of the Sonic hedgehog (Shh) signaling molecule at the posterior limb-bud margin. Another hallmark of establishing anteroposterior limb-bud identities is the colinear activation of HoxD genes located at the 5' end of the cluster (5'HoxD genes, Hoxd11 to Hoxd13). The unique and shared functions of Gli3 and formin in these determinative events were genetically analyzed using single and double homozygous Extra-toes (Xt; disrupting Gli3) and limb deformity (ld; disrupting formin) mouse embryos. The formins, proteins involved in murine limb and kidney development, contain a proline-rich region that matches consensus sequences for ligands of Src homology 3 (SH3) domains (Zuniga, 1999 and references).

The positive Shh/Fgf-4 feedback loop controlling morphogeneisis of distal limb structures is disrupted in ld homozygous limb buds, which results in reduced polarizing activity. This disruption is due to a primary mesenchymal (mesodermal) defect preventing complete differentiation of the anterior ectodermal ridge (AER) and the induction of Fgf-4 in the posterior AER. Mesenchymal formin functions primarily in establishment of the signalling interactions between the polarizing region (Shh) and the AER (Fgf-4). Most likely as a consequence of disrupting these mesenchymal AER interactions, the transition from early to late 5'HOXD domains is delayed in ld/ld limb buds. Consistent with a genetic function in establishing the SHH/FGF4 feedback loop, formin is expressed in posterior and distal limb bud mesenchymal cells. High levels of formin transcripts are also expressed by the AER during limb-bud outgrowth, but transgene mediated formin re-expression in the ld mutant AER fails to rescue the Shh/Fgf-4 feedback loop and limb bud patterning. Furthermore, targeted disruption of the predominant formin isoform in the AER does not disrupt limb morphogenesis (Zuniga, 1999 and references).

Analysis of the limb skeletal phenotypes reveals genetic interaction of Gli2 and formin. In addition to loss of digit identity and varying degrees of polydactyly, proximal skeletal elements are severely shortened in Xt;ld double homozygous limbs. The underlying molecular defects affect both establishment of the polarizing region and posterior limb-bud identity. In particular, the synergism between Gli3- and formin-mediated mesenchyme-AER interactions positions the Shh signaling center at the posterior limb-bud margin. The present study shows that establishment and positioning of the polarizing region is regulated both by restriction of Shh through Gli3 and its positive feedback regulation through formin. Concurrently, Gli3 functions independent of formin during initial posterior nesting of 5' HoxD domains, whereas their subsequent distal restriction and anterior expansion depends on genetic interaction of Gli3 and formin, which act together to reinforce the posterior nesting of the 5'HoxD domains (Zuniga, 1999).

Two aspects of Gli3 and formin interaction during limb morphogenesis are noted: (1) both proximal (humerus and scapula) and distal (digits) limb skeletal elements are more severely affected in Xt;ld double than single homozygous limb buds, and (2) molecular analysis shows that both positioning of the Shh-expressing polarizing region and spatial regulation of 5'HoxD domains are disrupted in Xt;ld double mutant limbs. As Gli3 and formin are co-expressed in the posterior limb-bud mesenchyme, these two proteins with postulated nuclear functions might directly interact, but biochemical studies fail to detect direct molecular interactions. Therefore alterations in double mutant limb buds are most likely caused by disruption of two distinct, but genetically interacting cascades (Zuniga, 1999).

Sonic hedgehog expression in the developing limb is associated with the zone of polarizing activity (ZPA), and both are restricted to the posterior part of the limb bud. The expression patterns of Shh and Gli3, a member of the Gli-family believed to function in transcriptional control, appear to be mutually exclusive in limb buds of mouse embryos. In the polydactyly mouse mutant extra toes (Xt), possessing a null mutation of Gli3, Shh is additionally expressed in the anterior region of the limb bud. The transcript of Ptc, the putative receptor for Shh protein, can be detected anteriorly as well. Other genes known to be involved in limb outgrowth and patterning, like Fibroblast growth factor, Bone morphogenetic protein, and Hoxd are misexpressed in relation to the ectopic Shh expression domain in Xt limb buds. These data suggest that Gli3 is a negative regulator of Shh expression in mouse limb development. In the Xt mutant limbs both Ptc and Bmps are expressed despite the absence of Gli3. This differs from the regulatory hierarchy found in Drosophila, in which ptc and dpp function downstream of cubitus interruptus in wing patterning (Buscher, 1997)

Gli genes are members of a small family, encoding zinc-finger proteins of the Kruppel-type. The family consists of Gli(1), Gli2, and Gli3, all of which are expressed in the developing mouse limb bud. To assess the role of the Gli family and Sonic hedgehog (Shh) in mouse limb development, the expression domains of all three Gli genes and of Shh were compared. Although each Gli gene has its own distinct expression pattern in limb buds, at 10.5-11.5 dpc, none of the three genes were found to be expressed in the posterior region, the presumptive Shh expression domain. This transient mutually exclusive expression suggests a potential interaction between Gli genes and Shh. To address this matter, the expression of Gli genes and Shh were examined in two polydactyly mouse mutants, Extra toes (Xt) and Hemimelic-extra toes (Hx), both of which express Shh ectopically in the anterior region of the limb field. Since Xt mice lack Gli3 expression, the ectopic Shh expression is genetically linked to the absence of Gli3. In Hx mice a down-regulation of Gli3 in the anterior region of the limb bud was found. In both mutants Gli2 expression pattern is not altered, whereas Gli1 expression is anteriorly up-regulated adjacent to the ectopic Shh domain. These results strongly suggest a positive regulation of Gli1 by Shh and a negative interaction between Shh and Gli3 (Buscher, 1998).

The bHLH transcription factor dHAND is required for establishment of SHH signaling by the limb bud organizer in posterior mesenchyme, a step crucial to development of vertebrate paired appendages. The transcriptional repressor GLI3 restricts dHAND expression to posterior mesenchyme prior to activation of SHH signaling in mouse limb buds. dHAND, in turn, excludes anterior genes such as Gli3 and Alx4 from posterior mesenchyme. Furthermore, genetic interaction of GLI3 and dHAND directs establishment of the SHH/FGF signaling feedback loop by restricting the BMP antagonist GREMLIN posteriorly. These interactions polarize the nascent limb bud mesenchyme prior to SHH signaling (te Welscher, 2002).

The patterning and growth of the embryonic vertebrate limb is dependent on Sonic hedgehog (Shh), a morphogen that regulates the activity of Gli transcription factors. However, Shh expression is not observed during the first 12 hr of limb development. During this phase, the limb bud is prepatterned into anterior and posterior regions through the antagonistic actions of transcription factors Gli3 and Hand2. This study demonstrates that precocious activation of Shh signaling during this early phase interferes with the Gli3-dependent specification of anterior progenitors, disturbing establishment of signaling centers and normal outgrowth of the limb. These findings illustrate that limb development requires a sweet spot in the level and timing of pathway activation that allows for the Shh-dependent expansion of posterior progenitors without interfering with early prepatterning functions of Gli3/Gli3R or specification of anterior progenitors (Zhulyn, 2014).

Cubitus interruptus homologs: Digit formation

Most mouse embryos homozygous for the Bmp4(tm1blh) (See Drosophila Decapentaplegic) null allele die around the time of gastrulation, with little or no mesoderm. Two independently derived Bmp4(tm1) mutations were backcrossed onto the C57BL/6 genetic background. Several independently expressed, incompletely penetrant abnormalities are observed in heterozygotes, including cystic kidney, craniofacial malformations, microphthalmia, and preaxial polydactyly of the right hindlimb. In addition, heterozygotes are consistently underrepresented at weaning. These results indicate that Bmp4 gene dosage is essential for the normal development of a variety of organs and for neonatal viability. Two mutations that enhance the penetrance and expressivity of the polydactylous phenotype were identified: Gli3(XtJ), a deletion mutation involving a gene encoding a zinc-finger protein related to Drosophila cubitus interruptus, and Alx4(tm1rwm), a targeted null mutation in a gene encoding a paired class homeoprotein related to Drosophila aristaless. All double Bmp4(tm1)/Gli3(XtJ) heterozygotes have extensive anterior digit abnormalities of both fore- and hindlimbs, while all double Bmp4(tm1)/Alx4(tm1) heterozygotes display ectopic anterior digits only on the hindlimbs. These genetic interactions suggest a model for the multigenic control of anterior digit patterning during vertebrate limb development. It is likely that both Alx4 and Gli3 are repressors of anterior Shh expression; loss-of-function mutations in either gene would lead to ectopic anterior Shh expression and to overproliferation of anterior mesenchyme in homozygous limbs. The addition of a weak Shh mitogenic signal to the reduced cell death resulting from decreased BMP4 levels would promote the formation of anterior supernumerary digits (Dunn, 1997).

talpid3 is an embryonic-lethal chicken mutation in a molecularly un-characterised autosomal gene. The recessive, pleiotropic phenotype includes polydactylous limbs with morphologically similar digits. Previous analysis established that hox-D and bmp genes [normally expressed posteriorly in the limb bud in response to a localized, posterior source of Sonic Hedgehog (Shh)] are expressed symmetrically across the entire anteroposterior axis in talpid3 limb buds. In contrast, Shh expression itself is unaffected. Expression of patched (ptc), which encodes a component of the Shh receptor and is probably itself a direct target of Shh signaling, was examined to establish whether talpid3 acts in the Shh pathway. ptc expression has been found to be significantly reduced in talpid3 embryos. talpid3 function is not required for Shh signal production but is required for normal response to Shh signals, implicating talpid3 in transduction of Shh signals in responding cells. Analysis of expression of putative components of the Shh pathway (gli1, gli3 and coupTFII) shows that genes regulated by Shh are either ectopically expressed or no longer responsive to Shh signals in talpid3 limbs, suggesting possible bifurcation in the Shh pathway. Genetic mapping of gli1, ptc, shh and smoothened in chickens is described. Co-segregation analysis confirms that none of these genes correspond to talpid3 (Lewis, 1999a).

Most current models propose Sonic hedgehog (Shh) as the primary determinant of anteroposterior development of amniote limbs. Shh protein is said to be required to direct the formation of skeletal elements and to specify digit identity through dose-dependent activation of target gene expression. However, the identity of genes targeted by Shh, and the regulatory mechanisms controlling their expression, remain poorly understood. Gli3 (the gene implicated in human Greig cephalopolysyndactyly syndrome) is proposed to negatively regulate Shh by restricting its expression and influence to the posterior mesoderm. Genetic analyses in mice shows that Shh and Gli3 are dispensable for formation of limb skeletal elements: Shh^-/- Gli3^-/- limbs are distally complete and polydactylous, but completely lack wild-type digit identities. The effects of Shh signalling on skeletal patterning and ridge maintenance are necessarily mediated through Gli3. It is proposed that the function of Shh and Gli3 in limb skeletal patterning is limited to refining autopodial morphology, imposing pentadactyl constraint on the limb's polydactyl potential, and organizing digit identity specification, by regulating the relative balance of Gli3 transcriptional activator and repressor activities (Litingtung, 2002).

Sonic hedgehog (Shh) signaling regulates both digit number and identity, but how different distinct digit types (identities) are specified remains unclear. Shh regulates digit formation largely by preventing cleavage of the Gli3 transcription factor to a repressor form that shuts off expression of Shh target genes. The functionally redundant 5'Hoxd genes regulate digit pattern downstream of Shh and Gli3, through as yet unknown targets. Enforced expression of any of several 5'Hoxd genes causes polydactyly of different distinct digit types with posterior transformations in a Gli3(+) background, whereas, in Gli3 null limbs, polydactylous digits are all similar, short and dysmorphic, even though endogenous 5'Hoxd genes are broadly misexpressed. Hoxd12 interacts genetically and physically with Gli3, and can convert the Gli3 repressor into an activator of Shh target genes. Several 5'Hoxd genes, expressed differentially across the limb bud, interact physically with Gli3. It is proposed that a varying [Gli3]:[total Hoxd] ratio across the limb bud leads to differential activation of Gli3 target genes and contributes to the regulation of digit pattern. The resulting altered balance between 'effective' Gli3 activating and repressing functions may also serve to extend the Shh activity gradient spatially or temporally (Chen, 2004).

The results show a genetic interaction between a 5'Hoxd member and Gli3 in regulating digit formation. Biochemical and transfection analyses further indicate that the 5'Hoxd class protein interacts physically with Gli3 via the homeodomain, and can convert the truncated Gli3 repressor form into an activator of its target promoters. This suggests a model in which Gli3-responsive target promoter activity would depend, at least in part, on the ratio of Gli3 to total Hoxd protein expression at a given site. This model is consistent with the known functional overlap and additive effects of 5'Hoxd genes, as cumulative recruitment of Hoxd proteins to bound Gli3 repressor protein would modify the overall effect on Gli3 target promoters. Rather than a combinatorial Hox code, a quantitative Hox-activity gradient, determined by the total Hox protein relative to Gli3 protein at a particular site, would modify 'net' Gli3 function to regulate expression levels of Gli3 target promoters differentially, and thereby potentially activate downstream Shh pathway targets indirectly. The genetic evidence presented in this study suggests that Gli3-Hoxd interaction pertains mainly to the regulation of digit morphogenesis. This is not unexpected for an interaction with Gli3 shared among several posterior Hox proteins, given that some of the 5'Hoxd members normally only regulate digits physiologically (e.g., Hoxd13). In fact, the long bone shortening observed may represent a distinct dominant-negative effect independent of Gli3. Gli3-Hox interactions may represent a recent evolutionary acquisition that, together with the distal recruitment of 5'Hox genes, enables the development of the distal autopod with its multiple digits. Since the distal autopod is probably a neomorphic structure of tetrapod vertebrates, it is not surprising that an interaction between the homologous Drosophila Ci and AbdB proteins has not been described (Chen, 2004).

An interdigit signalling centre instructs coordinate phalanx-joint formation governed by 5'Hoxd-Gli3 antagonism

The number of phalanges and joints are key features of digit 'identity' and are central to limb functionality and evolutionary adaptation. Prior chick work indicated that digit phalanges and their associated joints arise in a different manner than the more sparsely jointed long bones, and their identity is regulated by differential signalling from adjacent interdigits. Currently, there is no genetic evidence for this model, and the molecular mechanisms governing digit joint specification remain poorly understood. Using genetic approaches in mouse, this study shows that functional 5'Hoxd-Gli3 antagonism acts indirectly, through Bmp signalling from the interdigital mesenchyme, to regulate specification of joint progenitors, which arise in conjunction with phalangeal precursors at the digit tip. Phalanx number, although co-regulated, can be uncoupled from joint specification. It is proposed that 5'Hoxd genes and Gli3 are part of an interdigital signalling centre that sets net Bmp signalling levels from different interdigits to coordinately regulate phalanx and joint formation (Huang, 2016).

Cubitus interruptus homologs: Muscle and mesodermal development

Sonic hedgehog (Shh) is a secreted signaling molecule for tissue patterning and stem cell specification in vertebrate embryos. Shh mediates both long-range and short-range signaling responses in embryonic tissues through the activation and repression of target genes by its Gli transcription factor effectors. Despite the well-established functions of Shh signaling in development and human disease, developmental target genes of Gli regulation are virtually unknown. The role of Shh signaling has been examined in the control of Myf5, a skeletal muscle regulatory gene for specification of muscle stem cells in vertebrate embryos. Shh is required for Myf5 expression in the specification of dorsal somite epaxial muscle progenitors. However, these studies did not distinguish whether Myf5 is a direct target of Gli regulation through long-range Shh signaling, or alternatively, whether Myf5 regulation is a secondary response to Shh signaling. To address this question, transgenic analysis with lacZ reporter genes was used to characterize an Myf5 transcription enhancer that controls the activation of Myf5 expression in the somite epaxial muscle progenitors in mouse embryos. This Myf5 epaxial somite (ES) enhancer is Shh-dependent, as shown by its complete inactivity in somites of homozygous Shh mutant embryos, and by its reduced activity in heterozygous Shh mutant embryos. Furthermore, Shh and downstream Shh signal transducers specifically induce ES enhancer/luciferase reporters in Shh-responsive 3T3 cells. A Gli-binding site located within the ES enhancer is required for enhancer activation by Shh signaling in transfected 3T3 cells and in epaxial somite progenitors in transgenic embryos. These findings establish that Myf5 is a direct target of long-range Shh signaling through positive regulation by Gli transcription factors, providing evidence that Shh signaling has a direct inductive function in cell lineage specification (Gustafsson, 2002).

Indian hedgehog (Ihh) controls multiple aspects of endochondral skeletal development, including proliferation and maturation of chondrocytes, osteoblast development and cartilage vascularization. Although it is known that Gli transcription factors are key effectors of hedgehog signaling, it has not been established which Gli protein mediates Ihh activity in skeletal development. This study shows that removal of Gli3 in Ihh-null mouse embryos restores normal proliferation and maturation of chondrocytes, but only partially rescues the defects in osteoblast development and cartilage vascularization. Remarkably, in both Ihh^-/- and Ihh^-/-; Gli3^-/- embryos, vascularization promotes osteoblast development in perichondrial progenitor cells. These results not only establish Gli3 as a critical effector for Ihh activity in the developing skeleton, but also identify an osteogenic role for a vasculature-derived signal, which integrates with Ihh and Wnt signals to determine the osteoblast versus chondrocyte fate in the mesenchymal progenitors (Hilton, 2005).

Hedgehog (Hh)-Patched1 (Ptch1) signaling plays essential roles in various developmental processes, but little is known about its role in postnatal homeostasis. This study demonstrate regulation of postnatal bone homeostasis by Hh-Ptch1 signaling. Ptch1-deficient (Ptch1^+/-) mice and patients with nevoid basal cell carcinoma syndrome show high bone mass in adults. In culture, Ptch1^+/- cells showed accelerated osteoblast differentiation, enhanced responsiveness to the runt-related transcription factor 2 (Runx2), and reduced generation of the repressor form of Gli3 (Gli3rep). Gli3rep inhibited DNA binding by Runx2 in vitro, suggesting a mechanism that could contribute to the bone phenotypes seen in the Ptch1 heterozygotes. Moreover, systemic administration of the Hh signaling inhibitor cyclopamine decreased bone mass in adult mice. These data provide evidence that Hh-Ptch1 signaling plays a crucial role in postnatal bone homeostasis and point to Hh-Ptch1 signaling as a potential molecular target for the treatment of osteoporosis (Ohba, 2008).

Cubitus interruptus homologs: Lung development

In murine embryonic lung, all three Gli genes are strongly expressed at the pseudoglandular stage, in distinct but overlapping domains of the mesoderm. Expression of Gli and Gli3, but not of Gli2, is subsequently downregulated at the canalicular stage, coincident with a decline in the expression of sonic hedgehog and the Hedgehog receptor gene, patched. Overexpression of Shh in the lung results in increased levels of Ptc mRNA. Gli, but not Gli2, is also upregulated, suggesting a differential involvement of the Gli genes in the regulation of Ptc by SHH during lung development. Gli3 is not upregulated by Shh overexpression. However, it is important for lung development: Gli3XtJ embryos, homozygous for a mutation involving a deletion of the Gli3 gene, have a stereotypic pattern of abnormalities in lung morphogenesis. The pulmonary defects in these embryos, consisting of localized shape changes and size reductions, correlate with normal Gli3 expression. Thus, the data indicate that one of the Gli genes, Gli3, is essential for normal lung development, and that another, Gli, can be placed downstream of Shh signaling in the lung (Grindley, 1997).

Cubitus interruptus homologs: Skeletal development

Gli2 mutant mice exhibit severe skeletal abnormalities including cleft palate, tooth defects, absence of vertebral body and intervertebral discs, and shortened limbs and sternum. Interestingly, Gli2 and Gli3 mutant mice exhibit different subsets of skeletal defects indicating that they implement specific functions in the development of the neural crest, somite and lateral plate mesoderm derivatives. Although Gli2 and Gli3 are not functionally equivalent, double mutant analysis indicates that, in addition to their specific roles, they also serve redundant functions during skeletal development (Mo, 1997).

Skeletal abnormalities are described that appeared in Zic1-deficient mice. These mice show multiple abnormalities in the axial skeleton. The deformities are severe in the dorsal parts of the vertebrae (the vertebral arches), but less so in the vertebral bodies (spina bifida occulta). The proximal ribs are deformed by ectopic processes. The abnormalities found in the vertebral arches can be traced back to disturbed segmental patterns of dorsal sclerotome. The Zic1/Gli3 double mutants show severe abnormalities of vertebral arches that are not found in single mutants. The abnormalities in the vertebral arches are less severe in Zic1/Pax1 (Pax1 is a homolog of Drosophila Pax-meso) mutants than in Zic1/Gli3 mutants, but significantly more pronounced than in Zic1 single mutants. The three genes may act synergistically in the development of the vertebral arches (Aruga, 1999).

The vertebral arch phenotypes in the Zic1/Gli3 and Zic1/Pax1 combined mutants indicate that these two sets of the genes cooperate in the AP patterning of the dorsal sclerotome. Based on these results, the role of the three genes in the development and compartmentalization of the sclerotome can be considered. In terms of the expression in the axial structures, Zic1/Gli3 co-expression is observed in the dorsolateral sclerotome, the dorsal dermomyotome and the dorsal neural tube whereas Zic1/Pax1 co-expression is detected in the intermediate part of the sclerotome. Zic1/Gli3 synergism may be related to the Zic1/Gli3 coexpression in the dorsolateral sclerotomes and/or in the dorsal spinal cord. Although it is not yet known whether the neural tube is involved in the segmental organization of the axial skeleton, it is possible that the loss of the most dorsal vertebral arches secondarily affects the segmental organization of the vertebral laminae. A detailed examination of the spinal cord of these mutants should clarify the roles of Zic and Gli genes in the interactions between spinal cord and sclerotome (Aruga, 1999).

From a molecular point of view, Zic1 and Gli3 proteins might be functionally redundant during vertebral arch development, since Gli3 expression is not affected in Zic1 mutants and Zic1 expression is not affected in Gli3 mutants. The two proteins may interact with the same target sequences in vivo and cooperate in the transcriptional regulation of the same target genes. It is also possible that the same target molecules other than DNA are recognized by the conserved zinc finger domain. Although this study suggests synergism between the Zic and the Gli proteins, Xenopus Zic2 and Gli genes counteract one another during neurogenesis. Different set of downstream genes or associating factors for Zic and Gli proteins might function in neurogenesis versus vertebral arch development (Aruga, 1999).

The sclerotome cells expressing both Zic1 and Pax1 may contribute to the formation of vertebral arches. In support of this possibility, the vertebral arches are known to be derived in part from lateral sclerotome. Interestingly, the mitosis of sclerotome cells is most active in the Zic1/Pax1 co-expressing region. In the Pax1-/- single mutant, the impaired proliferation in this sclerotome subregion leads to insufficient expansion of sclerotome cells. Therefore, Zic1 protein could be involved in the proliferation of the sclerotome cells in this region in collaboration with Pax1 protein. As an alternative explanation, Zic1 and Pax1 proteins might synergistically affect the responsiveness of sclerotome cells to vertebral arch-inductive signals, such as BMP4 and PDGF (Aruga, 1999).

Recent studies showed that Shh signal is essential for the development of the vertebral column by establishing the dorsoventral axis of the somite and subsequently that of the sclerotome. Gli genes are considered to act as a downstream factor of the Shh-mediated signaling cascade. Although the role of Gli3 has not been clarified, involvement in the Shh-mediated signaling cascade is possible, judging from the phenotypes of the Gli2/Gli3 mutant. Pax1 is also a mediator of Shh signals. Genetic interactions of Zic1 mutation with Gli3 and Pax1 suggest the involvement of Zic1 gene product in the Shh-mediated signaling cascade (Aruga, 1999).

Loss of Twist gene function arrests the growth of the limb bud shortly after its formation. In the Twist^-/- forelimb bud, Fgf10 expression is reduced, Fgf4 is not expressed, and the domain of Fgf8 and Fgfr2 expression is altered. This is accompanied by disruption of the expression of genes (Shh, Gli1, Gli2, Gli3, and Ptch) associated with SHH signaling in the limb bud mesenchyme, the down-regulation of Bmp4 in the apical ectoderm, the absence of Alx3, Alx4, Pax1, and Pax3 activity in the mesenchyme, and a reduced potency of the limb bud tissues to differentiate into osteogenic and myogenic tissues. Development of the hindlimb buds in Twist^-/- embryos is also retarded. The overall activity of genes involved in SHH signaling is reduced. Fgf4 and Fgf8 expression is lost or reduced in the apical ectoderm, but other genes (Fgf10, Fgfr2) involved with FGF signaling are expressed in normal patterns. Twist^+/-;Gli3^+/XtJ mice display more severe polydactyly than that seen in either Twist^+/- or Gli3^+/XtJ mice, suggesting that there is genetic interaction between Twist and Gli3 activity. Twist activity is therefore essential for the growth and differentiation of the limb bud tissues as well as regulation of tissue patterning via the modulation of SHH and FGF signal transduction. The finding of the down-regulation of the Gli genes in the Twist mutant limb mesenchyme is concordant with the observation that the expression of a Gli-related gene (lame duck) is also altered by the loss of Twist function in the Drosophila embryo (O'Rourke, 2002).

During endochondral ossification, the secreted growth factor Indian hedgehog (Ihh) regulates several differentiation steps. It interacts with a second secreted factor, parathyroid hormone-related protein (PTHrP), to regulate the onset of hypertrophic differentiation, and it regulates chondrocyte proliferation and ossification of the perichondrium independently of PTHrP. To investigate how the Ihh signal is translated in the different target tissues, the role of the zinc-finger transcription factor Gli3, which acts downstream of hedgehog signals in other organs, was analyzed. Loss of Gli3 in Ihh mutants restores chondrocyte proliferation and delays the accelerated onset of hypertrophic differentiation observed in Ihh^–/– mutants. Furthermore the expression of the Ihh target genes patched (Ptch) and PTHrP is reactivated in Ihh^–/–;Gli3^–/– mutants. Gli3 seems thus to act as a strong repressor of Ihh signals in regulating chondrocyte differentiation. In addition, loss of Gli3 in mice that overexpress Ihh in chondrocytes accelerates the onset of hypertrophic differentiation by reducing the domain and possibly the level of PTHrP expression. Careful analysis of chondrocyte differentiation in Gli3^–/– mutants revealed that Gli3 negatively regulates the differentiation of distal, low proliferating chondrocytes into columnar, high proliferating cells. The results suggest a model in which the Ihh/Gli3 system regulates two distinct steps of chondrocyte differentiation: (1) the switch from distal into columnar chondrocytes is repressed by Gli3 in a PTHrP-independent mechanism; (2) the transition from proliferating into hypertrophic chondrocytes is regulated by Gli3-dependent expression of PTHrP. Furthermore, by regulating distal chondrocyte differentiation, Gli3 seems to position the domain of PTHrP expression (Koziel, 2005).

Cubitus interruptus homologs: Hair development

Proper patterning of self-renewing organs, like the hair follicle, requires exquisite regulation of growth signals. Sonic hedgehog (Shh) signaling in skin controls the growth and morphogenesis of hair follicle epithelium in part through regulating the Gli transcription factors. While ectopic induction of Shh target genes leads to hair follicle tumors, such as basal cell carcinomas, how Shh signaling normally functions during the cyclic process of hair development is unknown. During the hair cycle, Shh expression and the ability of skin cells to respond to Shh signaling is spatially and temporally regulated. Induction of Shh target genes normally occurs only in the anagen (the growth phase of follicular epithelium) hair follicle in response to expression of Shh. However, in patched1 heterozygous mice, putative tumor precursors form with concomitant induction of Shh target gene transcription only during anagen in follicular and interfollicular keratinocytes. Ectopic production of Gli1 accumulates Gli protein and induces Shh target genes and epithelial tumors at anagen but not other stages, pointing to a restricted competence occurring at the level of Gli protein accumulation. Delivery and reception of growth signals among multipotent cells are restricted in time and space to facilitate cyclic pattern formation (Oro, 2003).

Cubitus interruptus homologs: Prostate development

Ductal budding in the developing prostate is a testosterone-dependent event that involves signaling between the urogenital sinus epithelium (UGE) and urogenital sinus mesenchyme (UGM). Ductal bud formation is associated with focused expression of Sonic hedgehog (Shh) in the epithelium of nascent prostate buds and in the growing tips of elongating prostate ducts. This pattern of localized Shh expression occurs in response to testosterone stimulation. The gene for the Shh receptor, Ptc1, is expressed in the UGM, as are the members of the Gli gene family of transcriptional regulators (Gli1, Gli2, and Gli3). Expression of Ptc1, Gli1, and Gli2 is localized primarily to mesenchyme surrounding prostate buds, whereas Gli3 is expressed diffusely throughout the UGM. A strong dependence of Gli1 (and Ptc1) expression on Shh signaling is demonstrated by induction of expression in both the intact urogenital sinus and the isolated UGM by exogenous SHH peptide. A similar dependence of Gli2 and Gli3 expression on Shh is not observed. Nonetheless, the chemical inhibitor of Shh signaling, cyclopamine, produced a graded inhibition of Gli gene expression (Gli1->Gli2->Gli3) in urogenital sinus explants that was paralleled by a severe inhibition of ductal budding. It is concluded that Shh activates mesenchymal Gli1 expression during prostate ductal bud formation (Lamm, 2002).

While prostate gland development is dependent on androgens, other hormones including retinoids and estrogens can influence this process. Brief exposure to high-dose estrogen during the neonatal period in rats leads to permanent, lobe-specific aberrations in the prostate gland, a phenomenon referred to as developmental estrogenization. This response is mediated through alterations in steroid receptor expression; however, further downstream mechanisms remain unclear. Sonic hedgehog (Shh)-patched (ptc)-gli was investigated in the developing rat prostate gland, its role in branching morphogenesis, and the effects of neonatal estrogens on its expression and localization to determine whether a disturbance in this signaling pathway is involved in mediating the estrogenized phenotype. Shh is expressed in epithelial cells at the distal tips of elongating ducts in discreet, heterogeneous foci, while ptc and gli1–3 are expressed in the adjacent mesenchymal cells in the developing gland. The addition of Shh protein to cultured neonatal prostates reduces ductal growth and branching, decreases Fgf10 transcript, and increases Bmp4 expression in the adjacent mesenchyme. Shh-induced growth suppression is reversed by exogenous Fgf10, but not noggin, indicating that Fgf10 suppression is the proximate cause of the growth inhibition. A model is proposed to show how highly localized Shh expression along with regulation of downstream morphogens participates in dichotomous branching during prostate morphogenesis. Neonatal exposure to high-dose estradiol suppresses Shh, ptc, gli1, and gli3 expressions and concomitantly blocks ductal branching in the dorsal and lateral prostate lobes specifically. In contrast, ventral lobe branching and Shh-ptc-gli expression are minimally affected by estrogen exposure. Organ culture studies with lateral prostates confirms that estradiol suppresses Shh-ptc-gli expression directly at the prostatic level. Taken together, the present findings indicate that lobe-specific decreases in Shh-ptc-gli expression are involved in mediating estradiol-induced suppression of dorsal and lateral lobe ductal growth and branching during prostate morphogenesis (Pu, 2004).

Cubitus interruptus homologs: Tooth development

The expression of genes involved in the Sonic Hedgehog signalling pathway, including Shh, Ptc, Smo, Gli1, Gli2 and Gli3, were found to be expressed in temporal and spatial patterns during early murine tooth development, suggestive of a role in early tooth germ initiation and subsequent epithelial-mesenchymal interactions. Of these, all but Shh (Ptc, Smo, Gli1, Gli2 and Gli3) are expressed in epithelium and mesenchyme whereas Shh is only detected in epithelium. This suggests that Shh is involved in both lateral (epithelial-mesenchymal) and planar (epithelial-epithelial) signaling in early tooth development. Ectopic application of Shh protein to mandibular mesenchyme induces the expression of Ptc and Gli1. Addition of exogenous Shh protein directly into early tooth germs and adjacent to tooth germs, results in abnormal epithelial invagination, indicative of a role for Shh in epithelial cell proliferation. In order to assess the possible role of this pathway, tooth development in Gli2 and Gli3 mutant embryos was investigated. Gli2 mutants have abnormal development of maxillary incisors, probably resulting from a mild holoprosencephaly, whereas Gli3 mutants have no major tooth abnormalities. Gli2/Gli3 double homozygous mutants do not develop any normal teeth and do not survive beyond embryonic day 14.5; however, Gli2(-/-); Gli3(+/-) mice survive until birth and have small molars and mandibular incisors, whereas maxillary incisor development is arrested as a rudimentary epithelial thickening. These results show an essential role for Shh signaling in tooth development that involves functional redundancy of downstream Gli genes (Hardcastle, 1998).

Opposing gradients of Gli repressor and activators mediate Shh signaling along the dorsoventral axis of the inner ear

Organization of the vertebrate inner ear is mainly dependent on localized signals from surrounding tissues. Previous studies demonstrated that sonic hedgehog (Shh) secreted from the floor plate and notochord is required for specification of ventral (auditory) and dorsal (vestibular) inner ear structures, yet it was not clear how this signaling activity is propagated. To elucidate the molecular mechanisms by which Shh regulates inner ear development, embryos were examined with various combinations of mutant alleles for Shh, Gli2 and Gli3. This study shows that Gli3 repressor (R) is required for patterning dorsal inner ear structures, whereas Gli activator (A) proteins are essential for ventral inner ear structures. A proper balance of Gli3R and Gli2/3A is required along the length of the dorsoventral axis of the inner ear to mediate graded levels of Shh signaling, emanating from ventral midline tissues. Formation of the ventral-most otic region, the distal cochlear duct, requires robust Gli2/3A function. By contrast, the formation of the proximal cochlear duct and saccule, which requires less Shh signaling, is achieved by antagonizing Gli3R. The dorsal vestibular region requires the least amount of Shh signaling in order to generate the correct dose of Gli3R required for the development of this otic region. Taken together, these data suggest that reciprocal gradients of GliA and GliR mediate the responses to Shh signaling along the dorsoventral axis of the inner ear (Bok, 2007).

Cubitus interruptus homologs and cancer

The oncogene GLI is amplified and expressed in some cases of human malignant glioma and undifferentiated childhood sarcoma and is the prototype for a gene family characterized by a highly conserved set of five tandem zinc fingers and a consensus cysteine-histidine link. This zinc finger motif has been shown to bind DNA with sequence specificity and may mediate transcriptional regulation. GLI is expressed in embryonal carcinoma cell lines but not in most normal adult tissues. Transcripts of the mouse homologue of human GLI were demonstrated on days 10 through 18 of mouse embryonic development as well as in normal adult uterus, brain, testis, and limb. Tissue expression of gli during gestation was demonstrated in Meckel's precartilage mesenchyme, the basis occipitus, rib mesenchymal condensations, primordial vertebral bodies, digital mesenchymal condensations in forefoot and hindfoot plates, the ependymal layer of the spinal cord, and the mesoderm of the gastrointestinal tract. Expression persisted throughout gestation in developing bone and cartilage of the extremities, the ribs, and the vertebral bodies, as well as the gastrointestinal tract mesoderm. These findings support a role for gli family genes in normal craniofacial and digital development in mammals first suggested by the demonstration of translocation breakpoints within the GLI3 gene in families with the Greig cephalopolysyndactylyl syndrome and subsequently by reduced gli3 expression in the mouse mutant extra toes. It is surprising that a single gene would be expressed in such a wide range of mesenchymal structures (Walterhouse, 1993).

Sporadic basal cell carcinoma (BCC) is the most common type of malignant cancer in fair-skinned adults. Familial BCCs and a fraction of sporadic BCCs have lost the function of Patched (Ptc), a Sonic hedgehog (Shh) receptor that acts negatively on this signaling pathway. Overexpression of Shh can induce BCCs in mice. Ectopic expression of the zinc-finger transcription factor Gli1 in the embryonic frog epidermis results in the development of tumours that express endogenous Gli1. Shh and the Gli genes are normally expressed in hair follicles, and human sporadic BCCs consistently express Gli1 but not Shh or Gli3. Because Gli1, but not Gli3, acts as a target and mediator of Shh signalling, these results suggest that expression of Gli1 in basal cells induces BCC formation. Loss of Ptc or overexpression of Shh cannot be the sole causes of Gli1 induction and sporadic BCC formation, since they do not occur consistently. Thus any mutations leading to the expression of Gli1 in basal cells are predicted to induce BCC formation (Dahmane, 1997).

Temporally and spatially constrained Hedgehog (Hh) signaling regulates cyclic growth of hair follicle epithelium while constitutive Hh signaling drives the development of basal cell carcinomas (BCCs), the most common cancers in humans. Using mice engineered to conditionally express the Hh effector Gli2, it was shown that continued Hh signaling is required for growth of established BCCs. Transgene inactivation leads to BCC regression accompanied by reduced tumor cell proliferation and increased apoptosis, leaving behind a small subset of nonproliferative cells that could form tumors upon transgene reactivation. Nearly all BCCs arise from hair follicles, which harbor cutaneous epithelial stem cells, and reconstitution of regressing tumor cells with an inductive mesenchyme leads to multilineage differentiation and hair follicle formation. These data reveal that continued Hh signaling is required for proliferation and survival of established BCCs, provide compelling support for the concept that these tumors represent an aberrant form of follicle organogenesis, and uncover potential limitations to treating BCCs using Hh pathway inhibitors (Hutchin, 2005).

Cancer stem cells are rare tumor cells characterized by their ability to self-renew and to induce tumorigenesis. They are present in gliomas and may be responsible for the lethality of these incurable brain tumors. In the most aggressive and invasive type, glioblastoma multiforme (GBM), an average of about one year spans the period between detection and death. The resistence of gliomas to current therapies may be related to the existence of cancer stem cells. Human gliomas display a stemness signature and demonstrate that Hedgehog (Hh)-Gli signaling regulates the expression of stemness genes in and the self-renewal of CD133+ glioma cancer stem cells. Hh-Gli signaling is also required for sustained glioma growth and survival. It displays additive and synergistic effects with temozolomide (TMZ), the current chemotherapeutic agent of choice. TMZ, however, does not block glioma stem cell self-renewal. Finally, interference of Hh-Gli signaling with cyclopamine or through lentiviral-mediated silencing demonstrates that the tumorigenicity of human gliomas in mice requires an active pathway. These results reveal the essential role of Hh-Gli signaling in controlling the behavior of human glioma cancer stem cells and offer new therapeutic possibilities (Clement, 2007).

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the deregulation of the hedgehog signaling pathway. The Sonic Hedgehog ligand (Shh), absent in the normal pancreas, is highly expressed in pancreatic tumors and is sufficient to induce neoplastic precursor lesions in mouse models. This study investigated the mechanism of Shh signaling in PDAC carcinogenesis by genetically ablating the canonical bottleneck of hedgehog signaling, the transmembrane protein Smoothened (Smo), in the pancreatic epithelium of PDAC-susceptible mice. Multistage development of PDAC tumors is not affected by the deletion of Smo in the pancreas, demonstrating that autocrine Shh-Ptch-Smo signaling is not required in pancreatic ductal cells for PDAC progression. However, the expression of Gli target genes is maintained in Smo-negative ducts, implicating alternative means of regulating Gli transcription in the neoplastic ductal epithelium. In PDAC tumor cells, Gli transcription is decoupled from upstream Shh-Ptch-Smo signaling and is regulated by TGF-beta and the proto-oncogene KRAS, and Gli1 is shown to be required both for survival and for the KRAS-mediated transformed phenotype of cultured PDAC cancer cells (Nolan-Stevaux, 2009).

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