Interactive Fly, Drosophila

Hedgehog homologs and limb patterning

Mice homozygous for the recessive limb deformity (ld) mutation display both limb and renal defects. The limb defects, oligodactyly and syndactyly, have been traced to improper differentiation of the apical ectodermal ridge (AER) and shortening of the anteroposterior limb axis. The renal defects, usually aplasia, are thought to result from failure of ureteric bud outgrowth. The ld locus gives rise to multiple RNA isoforms encoding several different proteins (termed 'formins'). Embryonic expression patterns of the four major ld mRNA isoforms were examined. Isoforms I, II and III (all containing a basic amino terminus) are expressed in dorsal root ganglia, cranial ganglia and the developing kidney (including the ureteric bud). Isoform IV (containing an acidic amino terminus) is expressed in the notochord, the somites, the apical ectodermal ridge (AER) of the limb bud and the developing kidney, also including the ureteric bud. Using a lacZ reporter assay in transgenic mice, it has been shown that this differential expression of isoform IV results from distinct regulatory sequences upstream of its first exon. These expression patterns suggest that all four isoforms may be involved in ureteric bud outgrowth, while isoform IV may be involved in AER differentiation. To define further the developmental consequences of the ld limb defect, the expression of a number of genes thought to play a role in limb development was examined. Although the AERs of ld limb buds express several AER markers, they do not express detectable levels of fibroblast growth factor 4 (Fgf-4), which has been proposed to be the AER signal to the mesoderm. Thus it is concluded that one or more formins are necessary to initiate and/or maintain Fgf-4 production in the distal limb. Since ld limbs form distal structures such as digits, it has been concluded that while Fgf-4 is capable of supporting distal limb outgrowth in manipulated limbs, it is not essential for distal outgrowth in normal limb development. In addition, ld limbs show a severe decrease in the expression of several mesodermal markers, including Sonic hedgehog, a marker for the polarizing region, and Hoxd-12, a marker for posterior mesoderm. It is proposed that incomplete differentiation of the AER in ld limb buds leads to reduction of polarizing activity and defects along the anteroposterior axis (Chan, 1995).

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)

Several 5' members of the Hoxd cluster are expressed in nested posterior-distal domains of the limb bud suggesting a role in regulating the anteroposterior pattern of skeletal elements. While loss-of-function mutants have demonstrated a regulatory role for these genes in the developing limb, extensive functional overlaps between various different Hox genes have hampered elucidation of the roles played by individual members. In particular, the function of Hoxd-12 (Drosophila homolog: Abdominal-B) in the limb remains obscure. Using a gain-of-function approach, it has been found that Hoxd-12 misexpression in transgenic mice produces apparent transformations of anterior digits to posterior morphology and digit duplications, while associated tibial hemimelia (shortening) and other changes indicate that formation/growth of certain skeletal elements is selectively inhibited. If the digital arch represents an anterior bending of the main limb axis, then the results are all reconcilable with a model in which Hoxd-12 promotes formation of postaxial chondrogenic condensations branching from this main axis (including the anteriormost digit) and selectively antagonizes formation of 'true' preaxial condensations that branch from this main axis (such as the tibia). Hoxd-12 misexpression can also induce ectopic Sonic hedgehog (Shh) expression, resulting in mirror-image polydactyly in the limb. Misexpression of Hoxd-12 in other lateral plate derivatives (sternum, pelvis) likewise phenocopies several luxoid/luxate class mouse mutants that all share ectopic Shh signaling. This suggests that feedback activation of Shh expression may be a major function of Hoxd-12. Hoxd-12 can bind to and transactivate the Shh promoter in vitro. Expression of either exogenous Hoxd-11 or Hoxd-12 in cultured limb bud cells, together with FGF, induces expression of the endogenous Shh gene. Together these results suggest that certain 5' Hoxd genes directly amplify the posterior Shh polarizing signal in a reinforcing positive feedback loop during limb bud outgrowth (Knezevic, 1997).

The formation of skeletal elements from proximal to distal (hip to toe) proceeds by progressive branching and segmentation of chondrogenic condensations to produce the more distal elements (e.g., the femur branches distally to produce tibia and fibula). Comparisons of the branching pattern in various tetrapods has genearated a model in which the autopod forms by an anterior bending of the main (metapterygial-like) limb axis: the distal row of carpal/tarsals and the digits all arise as successive postaxial branching events from the continuation of the main limb axis along a curving 'digital arch'. In this view, the digits (including digit 1) and the distal row of tarsals are in fact all 'postaxial' structures whereas the tibia or the radius are true preaxial branches. The effects of Hoxd-12 misexpression in the limb are compatible with a model in which Hoxd-12 promotes formation of postaxial condensations branching from the main limb axis, while selectively inhibiting formation or growth of preaxial condensations (Knezevic, 1997 and citations).

Sonic hedgehog (Shh) is expressed in the posterior vertebrate limb bud mesenchyme and directs anteroposterior patterning and growth during limb development. Reported here is an analysis of the pectoral fin phenotype of zebrafish sonic you mutants, which disrupt the shh gene. Shh is required for the establishment of some aspects of anteroposterior polarity, while other aspects of anteroposterior polarity are established independent of Shh, and only later come to depend on Shh for their maintenance. shh activity is required for the posterior activation of ptc, hoxd-13 and hoxa-13, and for the posterior repression of msx-c and hoxc-6 in the pectoral fin bud. The maintenance of posterior aspects of expression of hoxa-9 and hoxa-11 also depends on shh activity. In contrast, the posterior activation of hoxd-11, hoxd-12, hoxa-10 and bmp-2 does not depend on shh activity, although it is slightly delayed and weaker than in wild-type fin buds. After the Shh-independent activation of hoxd-11, hoxd-12, hoxa-10 and bmp-2, the maintenance of these genes becomes dependent on Shh. Shh is required for the activation of posterior HoxD genes by retinoic acid. Shh is required for normal development of the apical ectodermal fold, for growth of the fin bud, and for formation of the fin endoskeleton (Neumann, 1999).

This study aimed at characterizing (in newt limbs) the Sonic hedgehog(shh) gene that encodes a signaling molecule of the zone of polarizing activity (ZPA) responsible for determining the anterior-posterior axis in embryonic chicken and mouse limbs. The reverse transcription-PCR showed that adult newt regenerating limbs express shh genes. In situ hybridization experiments demonstrate that shh genes are expressed in mesenchymal cells of the posterior region of both embryonic buds and regenerating blastemas of newt limbs, strongly suggesting the presence of ZPA in these tissues. Experiments of the axial reversal graft of blastemas further support this suggestion. The grafted blastemas regenerate supernumerary limbs; this has been explained by three models: the polar coordinate model, the boundary model, and the polarizing zone model. In favor of the third model, the shh gene is expressed not only in the original region (new anterior region) of the graft, but also ectopically in the other region (new posterior region) of the same graft. This study implies that the regenerating limb blastema produces ZPA as the signaling center of the AP patterning as in the developing limb bud, supporting the notion that the limb regeneration recapitulates the limb development (Imokawa, 1997).

The distribution of Hoxb-8 (Drosophila homolog: Antennapedia) transcripts through the chick flank and early forelimb mirrors the distribution of polarizing activity in the flank at these early stages. Polarizing activity displayed by Hoxb-8-expressing tissue is only realized when placed adjacent to the apical ectodermal ridge (AER) and appears to be mediated through Shh induction, suggesting that Hoxb-8 may lie genetically upstream of Shh. Accordingly, Hoxb-8 expression is rapidly induced by retinoic acid (RA) treatment in the anterior of the forelimb in a spatial and temporal manner, consistent with the induction of Shh and formation of the ZPA. Inhibition of RA synthesis in the flank downregulates the expression of endogenous Hoxb-8 and results in the loss of Shh expression. However, once the ZPA has become established the posterior limb mesoderm displays resistance to the induction of Hoxb-8 expression. Grafting of ZPA cells to the anterior of a host limb renders the host anterior tissue resistant to RA-induced Hoxb-8 expression. These results indicate that Hoxb-8 expression may be regulated by the established ZPA through a negative feedback loop. The anterior AER also secretes an inhibitory factor, preventing RA-induced or already established Hoxb-8 expression in the cells immediately underneath the AER. Consistent with a role for Hoxb-8 in positioning of the forelimb ZPA, Hoxb-8 expression is not seen in RA-induced duplications at the anterior of the hindlimb. However, grafting of Hoxb-8-expressing tissue to the hindlimb can lead to Shh expression and similar duplications, suggesting that factors mediating ZPA formation are very similar in both wing and leg (Stratford, 1997).

A Japanese chick wingless mutant (Jwg) has been analyzed to elucidate the molecular mechanism underlying wing development. The expression patterns of eleven marker genes were studied to characterize the mutant. In Jwg mutants, expression of Fgf8, a marker gene for the apical ectodermal ridge (AER), is delayed and shortly disappears in the wing as the AER regresses. Likewise, Shh, which is expressed in the posterior mesoderm of the normal chick limb by late stage 18, is considerably weaker in stage 19/20 mutant wing buds; Shh is expressed normally expressed in the posterior mesenchyme of the leg bud of the same mutant embryo. Fgf4 expression, which is normally induced in the posterior domain of the AER by Shh is not detected in the Jwg mutant wing bud at stage 19 and thereafter. Expressions of limb dorsoventral (DV) patterning genes such as Wnt7a and Lmx1 and mesenchymal marker genes such as Msx2 and Lh2 (a LIM homeodomain protein) are intact in nascent Jwg limb buds. Later in development, ventral expression of dorsal marker genes Wnt7a and Lmx1 indicate that the wing bud without the AER becomes bi-dorsal. The posterior mesoderm becomes defective, as deduced from the impaired expression patterns of Sonic hedgehog, Msx1, and Prx1. Rescue of the wing was attempted by implanting Fgf8-expressing cells into the Jwg wing bud. FGF8 can rescue outgrowth of the wing bud by maintaining Shh expression. Thus, the Jwg gene seems to be involved in maintenance of the Fgf8 expression in the wing bud. Further, it is suggested that the AER is required for maintenance of the DV boundary and the polarizing activity of the established wing bud (Ohuchi, 1997).

The signaling molecule encoded by Sonic hedgehog participates in the patterning of several embryonic structures including limbs. During early fin development in zebrafish, a subset of cells in the posterior margin of pectoral fin buds express shh. Regulation of shh in pectoral fin buds is consistent with a role in mediating the activity of a structure analogous to the zone of polarizing activity (ZPA). During growth of the bony rays of both paired and unpaired fins, and during fin regeneration, there does not seem to be a region equivalent to the ZPA and one would predict that shh would play a different role, if any, during these processes specific to fish fins. The expression of shh was examined in the developing fins of 4-week old larvae and in regenerating fins of adults. A subset of cells in the basal layer of the epidermis in close proximity to the newly formed dermal bone structures of the fin rays, the lepidotrichia, express both shh and ptc1 (which is thought to encode the receptor of the SHH signal). The expression domain of ptc1 is broader than that of shh; adjacent blastemal cells releasing the dermal bone matrix also express ptc1. Further observations indicate that the bmp2 gene, in addition to being expressed in the same cells of the basal layer of the epidermis as shh, is also expressed in a subset of the ptc1-expressing cells of the blastema. Amputations of caudal fins immediately after the first branching point of the lepidotrichia, and global administration of all-trans-retinoic acid, two procedures known to cause fusion of adjacent rays, result in a transient decrease in the expression of shh, ptc1 and bmp2. The effects of retinoic acid on shh expression occur within minutes after the onset of treatment, suggesting direct regulation of shh by retinoic acid. These observations suggest a role for shh, ptc1 and bmp2 in the patterning of the dermoskeleton of developing and regenerating teleost fins (Laforest, 1998).

Mice deficient for FgfR2-IIIb were generated by placing translational stop codons and an IRES-LacZ cassette into exon IIIb of FgfR2. Expression of the alternatively spliced receptor isoform, FgfR2-IIIc, is not affected in mice deficient for the IIIb isoform. FgfR2-IIIb ^-/-lacZ mice survive to term but show dysgenesis of the kidneys, salivary glands, adrenal glands, thymus, pancreas, skin, otic vesicles, glandular stomach, and hair follicles, and agenesis of the lungs, anterior pituitary, thyroid, teeth, and limbs. Detailed analysis of limb development revealed an essential role for FgfR2-IIIb in maintaining the AER. Its absence does not prevent expression of Fgf8, Fgf10, Bmp4, and Msx1, but does prevent induction of Shh and Fgf4, indicating that these genes are downstream targets of FgfR2-IIIb activation. In the absence of FgfR2-IIIb, extensive apoptosis of the limb bud ectoderm and mesenchyme occurs between E10 and E10.5, providing evidence that Fgfs act primarily as survival factors. It is proposed that FgfR2-IIIb is not required for limb bud initiation, but is essential for its maintenance and growth (Revest, 2001).

Uncoupling Sonic hedgehog control of pattern and expansion of the developing limb bud

Sonic hedgehog (Shh), which regulates proliferation in many contexts, functions as a limb morphogen to specify a distinct pattern of digits. How Shh's effects on cell number relate to its role in specifying digit identity is unclear. Deleting the mouse Shh gene at different times using a conditional Cre line, it was found that Shh functions to control limb development in two phases: a very transient, early patterning phase regulating digit identity, and an extended growth-promoting phase during which the digit precursor mesenchyme expands and becomes recruited into condensing digit primordia. This analysis reveals an unexpected alternating anterior-posterior sequence of normal mammalian digit formation. The progressive loss of digits upon successively earlier Shh removal mirrors this alternating sequence and highlights Shh's role in cell expansion to produce the normal digit complement (Zhu, 2008).

The finding that loss of Shh during the 'expansion' phase reduces digit number without altering identities of remaining digits supports the view that digit identity is in fact determined at a very early stage, in so far as Shh requirement is concerned. However, there is also evidence that Shh may act in part through secondary signals and that interdigit signals downstream of Shh function to regulate digit identity at late stages, thus relaying the patterning function of Shh beyond the time period of direct Shh activity. The data do not contradict a contribution of late signals acting downstream of Shh, but rather suggest that the signal relay is set in motion during the earliest phases of Shh action. Indeed, the action of late signals regulating identity would provide one mechanism for maintaining normal digit identities when reduced cell numbers become allocated into fewer condensations following later Shh removal. Alternatively, normal digit identities could be maintained in the face of reduced cell number if early digit condensation precursors were expanded at different times. However, there is currently no evidence for differential spatial proliferation zones in the limb bud as required by this model (Zhu, 2008).

Crosstalk between the fibroblast growth factor (FGF) and Sonic Hedgehog (Shh) pathways is critical for proper patterning and growth of the developing limb bud. This study shows that FGF-dependent activation of the ETS transcription factors Etv4 and Etv5 contributes to proximal-distal limb outgrowth. Surprisingly, blockage of Etv activity in early distal mesenchyme also resulted in ectopic, anterior expansion of Shh, leading to a polydactylous phenotype. These data indicate an unexpected function for an FGF/Etv pathway in anterior-posterior patterning. FGF activity in the limb is not only responsible for maintaining posterior-specific Shh expression, but it also acts via Etvs to prevent inappropriate anterior expansion of Shh. This study identifies another level of genetic interaction between the orthogonal axes during limb development (Mao, 2009).

These data support a role for an FGF/Etv pathway in repressing Shh expression in the anterior mesenchyme, thereby contributing to the posterior restriction of a Shh-producing organizing center. Based on the temporal pattern of ectopic Shh expression seen in the limb bud after EtvEnR misexpression, it is speculated that endogenous Etv4/5 acts to repress Shh in two distinct domains, one extending from the ZPA across the distal limb bud, and a second, later domain in the anterior of the limb bud. The later timing of ectopic anterior Shh expression is consistent with the fact that in other mutants exhibiting ectopic anterior Shh activity, the anterior domain is observed at a later time in limb development than the normal domain at the posterior margin. Both of these functions of Etv4/5, in the distal and anterior limb, must occur before ~E10.5. Thus, there is an early time window, prior to Shh activation, when all distal mesenchyme is competent to activate Shh but broad activation is blocked by Fgf/Etv4/5 action. This broad competence is then lost shortly thereafter. Whether the loss of competence reflects a change in the intrinsic properties of mesenchyme, or a redundant mechanism for restricting Shh expression to the posterior margin, is unclear (Mao, 2009).

Anterior-posterior (A-P) patterning of the vertebrate limb is controlled by sonic hedgehog (SHH) signaling, and the precise restriction of Shh expression to the posterior limb bud is essential for its polarizing effect. Fibroblast growth factor (FGF) signaling, a key control of proximal-distal (P-D) limb outgrowth, is known to promote Shh expression in the posterior limb bud. This study shows that conditional knockout of the FGF-activated transcription factor genes Etv4 and Etv5 in mouse leads to ectopic Shh expression in the anterior limb bud and a preaxial polydactyly (PPD) skeletal phenotype. These unexpected results suggest that ETV4 and ETV5 act downstream of FGF signaling to inhibit Shh expression in the anterior limb bud. This finding elucidates a novel aspect of the mechanism coordinating limb development along the A-P and P-D axes (Zhang, 2009).

Normal patterning of tissues and organs requires the tight restriction of signaling molecules to well-defined organizing centers. In the limb bud, one of the main signaling centers is the zone of polarizing activity (ZPA) that controls growth and patterning through the production of sonic hedgehog (SHH). The appropriate temporal and spatial expression of Shh is crucial for normal limb bud patterning, because modifications, even if subtle, have important phenotypic consequences. However, although there is a lot of information about the factors that activate and maintain Shh expression, much less is known about the mechanisms that restrict its expression to the ZPA. This study shows that BMP activity negatively regulates Shh transcription and that a BMP-Shh negative-feedback loop serves to confine Shh expression. BMP-dependent downregulation of Shh is achieved by interfering with the FGF and Wnt signaling activities that maintain Shh expression. FGF induction of Shh requires protein synthesis and is mediated by the ERK1/2 MAPK transduction pathway. BMP gene expression in the posterior limb bud mesoderm is positively regulated by FGF signaling and finely regulated by an auto-regulatory loop. These study emphasizes the intricacy of the crosstalk between the major signaling pathways in the posterior limb bud (Bastida, 2009).

The basic helix-loop-helix transcription factor Twist1 is essential for normal limb development. Twist1^-/- embryos die at midgestation. However, studies on early limb buds found that Twist1^-/- mutant limb mesenchyme has an impaired response to FGF signaling from the apical ectodermal ridge, which disrupts the feedback loop between the mesenchyme and AER, and reduces and shifts anteriorly Shh expression in the zone of polarizing activity. This study combined Twist1 null, hypomorph and conditional alleles to generate a Twist1 allelic series that survives to birth. As Twist1 activity is reduced, limb skeletal defects progress from preaxial polydactyly to girdle reduction combined with hypoplasia, aplasia or mirror symmetry of all limb segments. With reduced Twist1 activity there is striking and progressive upregulation of ectopic Shh expression in the anterior of the limb, combined with an anterior shift in the posterior Shh domain, which is expressed at normal intensity, and loss of the posterior AER. Consequently limb outgrowth is initially impaired, before an ectopic anterior Shh domain expands the AER, promoting additional growth and repatterning. Reducing the dosage of FGF targets of the Etv gene family, which are known repressors of Shh expression in anterior limb mesenchyme, strongly enhances the anterior skeletal phenotype. Conversely this and other phenotypes are suppressed by reducing the dosage of the Twist1 antagonist Hand2. These data support a model whereby multiple Twist1 activity thresholds contribute to early limb bud patterning, and suggest how particular combinations of skeletal defects result from differing amounts of Twist1 activity (Krawchuk, 2011).

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).

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