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Abdominal-B homologs and limb development

The tetrapod limb consists of three distinct compartments, the stylopod (upper arm and thigh), zeugopod (lower arm and calf) and autopod (hand and foot). There is a broad correlation between the position of a compartment and its evolutionary history. The stylopod is the most ancient (possibly of Late Silurian origins) whereas the zeugopod and autopod are the most recent (being first encountered in Devonian sarcopterygians). Fins of Sarcopterygian fish have a zeugopod and an elaborate endoskeletal fin skeleton. Digits develop within the distal portion of this extensive endoskeleton. HoxD genes were probably not involved in the origins of body wall outgrowths in basal vertebrates because unpaired finds do not express these genes. These Hox genes were initially involved in specifiying region identity along the primary body axis, particularly in caudal segments. This function is similar to the role of Hox genes in Drosophila development. The same order of appearance of three limb segments in higher vertebrates is recapitulated during development. Early removal of the apical ectodermal ridge (AER) results in a limb with only a stylopod; the zeugopod and autopod are produced after successively later surgeries (Shubin, 1997).

The Abdominal-B related genes of the HoxD cluster are expressed in at least three distinct, independently regulated phases corresponding to the three physical limb compartments. In the first phase, two of these genes (HoxD-9 and HoxD-10) are expressed across the entire limb bud. This expression correlates with the time that the stylopod is specified. Subsequently, a second phase of expression is initiated in reponse to the secreted factor Sonic hedgehog (see Drosophila Hedgehog). Here Hox genes are expressed in a nested set centered around the Sonic-expressing cells, with HoxD-13 being expressed in the most restricted domain, and HoxD-12 and HoxD-11 each encompassing a broader domain. This pattern of expression coincides with the time of specification of the zeugopod. A third phase of expression is initiated later during limb development when these genes are all expressed across the majority of the distal portion of the limb bud. During this phase, the expreession of Hox genes still appears to be a consequence of the Sonic hedgehog signal, but the relative responsiveness of the different genes has changed so that HoxD-13 has the broadest expression domain and HoxD-12 and HoxD-11 are nested within it. HoxD-9 plays a pre-eminent role in phase I in the stylopod, HoxD-11 during phase II in the zeugopod, and HoxD-13 during phase III in the autopod. The expression of the dominant Hox genes in each phase is essential for the formation of the bones in each segment (Shubin, 1997).

The wrist (carpus) and ankle (tarsus) of most tetrapods, as well as the wrist of anurans, contains relatively small nodular skeletal elements. The anuran tarsus, however, comprises a pair of long bones, the proximal tarsals tibiale and fibulare, which resemble the lower leg bones, tibia and fibula (zeugopodium). An investigation asked whether the proximal tarsals of Xenopus are of zeugopodial character identity, i.e. whether they develop under the influence of the same genes that pattern the lower limb. Hoxa-11 expression in the forelimb bud was compared with that in the hind limb bud by whole-mount in situ hybridization. Hoxa-11 has been implicated in the development of the lower limb. In Xenopus three differences between Hoxa-11 expression are observed in fore- and hind limb buds: (1) Hoxa-11 expression is maintained until the hind limb bud reaches a larger size (2 mm) than that of the forelimb bud (1.5 mm); (2) Hoxa-11 expression is maintained over larger spatial domains than in the forelimb; and (3) Hoxa-11 expression has a pronounced posterior polarity in the hind limb, but not in the forelimb. Hind limb expression of Hoxa-11 can be understood as a heterochronic prolonging of the expression dynamic in the forelimb. The proximal tarsals start to develop within the expression domain of Hoxa-11, while in the forelimb the lower arm elements reach the distal expression limit of Hoxa-11. The gene expression data presented here support the notion of a zeugopodial identity of the proximal tarsal elements in Xenopus (Blanco, 1998).

Hox genes play a critical role in the development of the vertebrate axis and limbs, and previous studies have implicated them in the specification of positional identity, the control of growth, and the timing of differentiation. Axolotl limbs offer an opportunity to distinguish these alternatives because the sequence of skeletal differentiation is reversed along the anterior-posterior axis relative to that of other tetrapods. During early limb development, expression patterns of HoxD genes in axolotls resemble those in amniotes and anuran amphibians. At later stages, the anterior boundary of Hoxd-11 expression is conserved with respect to morphological landmarks, but there is no anterior-distal expansion of the posterior domain of Hoxd-11 expression similar to that observed in mice and chicks. Since axolotls do not form an expanded paddle-like handplate prior to digit differentiation, it is suggested that anterior expansion of expression in higher vertebrates is linked to the formation of the handplate, but is clearly not necessary for digit differentiation. The 5' HoxD genes are reexpressed during limb regeneration. The change in the expression pattern of Hoxd-11 during the course of regeneration is consistent with the hypothesis that the distal tip of the regenerate is specified first, followed by intercalation of intermediate levels of the pattern. Both Hoxd-8 and Hoxd-10 are expressed in non-regenerating wounds, but Hoxd-11 is specific for regeneration. It is also expressed in the posterior half of nerve-induced supernumerary outgrowths (Torok, 1998).

The limb muscles, originating from the ventrolateral portion of the somites, exhibit position-specific morphological development through successive splitting and growth/differentiation of the muscle masses in a region-specific manner by interacting with the limb mesenchyme and the cartilage elements. The molecular mechanisms that provide positional cues to the muscle precursors are still unknown. The expression patterns of Hoxa-11 and Hoxa-13 are correlated with muscle patterning of the limb bud and muscular Hox genes are activated by signals from the limb mesenchyme. This study examines the regulatory mechanisms directing the unique expression patterns of Hoxa-11 and Hoxa-13 during limb muscle development. HOXA-11 protein is detected in both the myogenic cells and the zeugopodal mesenchymal cells of the limb bud. The earlier expression of HOXA-11 in both the myogenic precursor cells and the mesenchyme is dependent on the apical ectodermal ridge (AER), but later expression is independent of the AER. HOXA-11 expression in both myogenic precursor cells and mesenchyme is induced by fibroblast growth factor (FGF) signal, whereas hepatocyte growth factor/scatter factor (HGF/SF) maintains HOXA-11 expression in the myogenic precursor cells, but not in the mesenchyme. The distribution of HOXA-13 protein expression in the muscle masses is restricted to the posterior region. HOXA-13 expression in the autopodal mesenchyme is dependent on the AER but not on the polarizing region, whereas expression of HOXA-13 in the posterior muscle masses is dependent on the polarizing region but not on the AER. Administration of BMP-2 at the anterior margin of the limb bud induces ectopic HOXA-13 expression in the anterior region of the muscle masses followed by ectopic muscle formation close to the source of exogenous BMP-2. In addition, NOGGIN/CHORDIN, antagonists of BMP-2 and BMP-4, downregulate the expression of HOXA-13 in the posterior region of the muscle masses and inhibit posterior muscle development. These results suggested that HOXA-13 expression in the posterior muscle masses is activated by the posteriorizing signal from the posterior mesenchyme via BMP-2. On the contrary, the expression of HOXA-13 in the autopodal mesenchyme is affected by neither BMP-2 nor NOGGIN/CHORDIN. Thus, mesenchymal HOXA-13 expression is independent of BMP-2 from the polarizing region, but is under the control of as yet unidentified signals from the AER. These results show that expression of Hox genes is regulated differently in the limb muscle precursor and mesenchymal cells (Hashimoto, 1999).

Genes of the HoxD complex have a crucial role in the morphogenesis of vertebrate limbs. During development, their functional domains are colinear with their genomic positions within the HoxD cluster, such that Hoxd13 and Hoxd12 are necessary for digit development, whereas Hoxd11 and Hoxd10 are involved in making forearms. Mutational analyses of these genes have demonstrated their importance and illustrated the requirement for a precise control of their expression during early limb morphogenesis. To study the nature of this control, the posterior part of the HoxD complex was scanned with a targeted reporter transgene and the response of this foreign promoter to limb regulatory influences was analyzed. The results suggest that this regulation is achieved through the opposite effects of two enhancer elements that compete with one another in the process of interacting with promoters located nearby. The physical position of a given gene within this genomic interval, that controls potentially antagonistic regulatory events, might thus determine the gene's final expression pattern. One promoter, functioning for the digits, is thought to be located 5' of the HoxD13 locus, while the second, functioning for the forearm, is located in the vicinity of the HoxD10 and HoxD11 genes. This model provides a conceptual link between the morphology of the future limb and the genetic organization of the Hox gene cluster, a translation of a genomic context into a morphogenetic topology (Herault, 1999).

Development of paired appendages at appropriate levels along the primary body axis is a hallmark of the body plan of jawed vertebrates. Hox genes are good candidates for encoding position in lateral plate mesoderm along the body axis and thus for determining where limbs are formed. Local application of fibroblast growth factors (See Drosophila Branchless) to the anterior prospective flank of a chick embryo induces development of an ectopic wing, and FGF applied to posterior flank induces an ectopic leg. If particular combinations of Hox gene expression determine where wings and legs develop, then formation of additional limbs from flank should involve changes in Hox gene expression that reflect the type of limb induced. For example, induction of ectopic wing involves shifting the anterior boundary of Hoxd9 anteriorly from the more posterior flank- wing junction to the anterior limit of the wing bud. Hoxd9 in the flank behind the wing bud is subsequently downregulated. FGF beads in the posterior flank, which lead to ectopic legs, induce an anterior shift of the posterior boundary of the Hoxb9 domain and stronger Hoxc9 expression in the flank. These changes transform the normal flank pattern of Hox expression to a pattern normally found at the leg level. The same population of flank cells can be induced to form either a wing or a leg, and induction of these ectopic limbs is accompanied by specific changes in expression of three Hox genes in lateral plate mesoderm. This then reproduces, in the flank, expression patterns found at normal limb levels. Hox gene expression is reprogrammed in lateral plate mesoderm, but is unaffected in paraxial mesoderm. Independent regulation of Hox gene expression in lateral plate mesoderm may have been a key step in the evolution of paired appendages (Cohn, 1997).

Hypodactyly (Hoxa13Hd) mice have a 50-bp deletion in the coding region of exon 1 of the Hoxa13 gene and have more severe limb defects than mice with an engineered deletion of the entire gene (Hoxa13-/-). Increased cell death isobserved in the autopod of Hoxa13Hd/Hd but not Hoxa13-/- limb buds. In addition, compound heterozygotes for one Hd allele and a Hoxa13- allele have a more severe limb phenotype than mice homozygous for the engineered null allele, suggesting a dominant-negative effect of the Hd mutation. The Hoxa13Hd deletion does not interfere with steady-state mRNA levels; however, its consequences on translation are unknown. In this paper, the Hoxa13 transcription initiation site has been characterized in limbs and the initiator methionine of HOXA13 has been determined. The Hoxa13Hd deletion results in a translational frame shift that leads to the loss of wild-type HOXA13 protein and the simultaneous production of a novel, stable protein in the limb buds of mutant mice. The mutant Hd protein (HOXA13Hd) consists of the first 25 amino acids of wild-type HOXA13 sequence, followed by 275 amino acids of an arginine- and lysine-rich, novel sequence that lacks the homeodomain. Like wild-type HOXA13, HOXA13Hd is localized to the nucleus in transfected COS-7 cells, perhaps mediated by the arginine- and lysine-rich peptide sequences created by the translational frame shift. To determine whether HOXA13Hd alters limb morphogenesis, the mutant mRNA was misexpressed throughout the developing limb bud using a Prx-1 promoter-Hd gene construct in transgenic mice. Three of 15 transgenic founder animals displayed reduction or absence of proximal and distal limb structures. It is proposed that the expression of HOXA13Hd plays a role in the profound failure of digit formation in Hoxa13Hd/Hd mice and explains the morphologic differences between these two Hoxa13 alleles. Several mechanisms could explain how HOXA13Hd leads to limb truncation in transgenic mice or to a more severe phenotype in Hypodactyly mutants. One possibility lies in the potential interference of HOXA13Hd with the function or expression of Hox genes in the limb bud. The Hoxa13Hd/Hd limb phenotype closely resembles that of mice with a complete absence of both Hoxa13 and Hoxd13: this raises the possibility that the Hoxa13Hd mutation, in addition to a possible effect on Hoxd13 transcription, might interfere with HOXD13 protein function. Another possibility may be that HOXA13Hd interferes with the critical epithelial-mesenchymal interactions between the apical ectodermal ridge (AER) and the underlying mesenchymal cells necessary to maintain both the AER and the proliferating mesenchyme (Post, 2000).

Targeted disruption of the Hoxd-10 gene, a 5' member of the mouse HoxD linkage group, produces mice with hindlimb-specific defects in gait and adduction. To determine the underlying causes of this locomotor defect, mutant mice were examined for skeletal, muscular and neural abnormalities. Mutant mice exhibit alterations in the vertebral column and in the bones of the hindlimb. Sacral vertebrae beginning at the level of S2 exhibit homeotic transformations to adopt the morphology of the next most anterior vertebra. In the hindlimb, there is an anterior shift in the position of the patella, an occasional production of an anterior sesamoid bone, and an outward rotation of the lower part of the leg, all of which contribute to the defects in locomotion. No major alterations in hindlimb musculature are observed, but defects in the nervous system are evident. There is a decrease in the number of spinal segments projecting nerve fibers through the sacral plexus to innervate the musculature of the hindlimb. Deletion of a hindlimb nerve is seen in some animals, and a shift is evident in the position of the lumbar lateral motor column. Though male fertility is affected in Hoxd-10 mutants, the testes are present in their normal locations and sperm production is normal. The effect of Hoxd-10 on fertility is therefore likely to be operational. Thus, the hindlimb locomotor defects in mutant males may interfere with their ability to mount and impregnate a female. These observations suggest a role for the Hoxd-10 gene in establishing regional identity within the spinal cord and imply that patterning of the spinal cord may have intrinsic components and is not completely imposed by the surrounding mesoderm (Carpenter, 1997).

In spite of recent breakthroughs in understanding limb patterning, the genetic factors determining the differences between the forelimb and the hindlimb have not been understood. The genes Pitx1 and Tbx4 encode transcription factors that are expressed throughout the developing hindlimb but not forelimb buds. Misexpression of Pitx1 in the chick wing bud induces distal expression of Tbx4, as well as HoxC10 and HoxC11, which are normally restricted to hindlimb expression domains. Wing buds in which Pitx1 is misexpressed develop into limbs with some morphological characteristics of hindlimbs: the flexure is altered to that normally observed in legs; the digits are more toe-like in their relative size and shape, and the muscle pattern is transformed to that of a leg (Logan, 1999).

Synpolydactyly, an inherited human abnormality of the hands and feet, is caused by expansions of a polyalanine stretch in the amino-terminal region of HOXD13. The homozygous phenotype includes the transformation of metacarpal and metatarsal bones to short carpal- and tarsal-like bones. The mutations identify the polyalanine stretch outside of the DNA binding domain of HOXD13 as a region necessary for proper protein function (Maragaki, 1996).

Vertebrate gene members of the HoxD complex are essential for proper development of the appendicular skeletons. Inactivation of these genes induces severe alterations in the size and number of bony elements. Evx-2, a gene related to the Drosophila even-skipped, is located close to Hoxd-13 and is expressed in limbs like the neighbouring Hoxd genes. To investigate whether this tight linkage reflects a functional similarity, a null allele of Evx-2 was produced. Furthermore, and because Hoxd-13 function is prevalent over that of nearby Hoxd genes, two different double mutant loci were generated wherein both Evx-2 and Hoxd-13 were inactivated in cis. The analysis of these various genetic configurations reveals the important function of Evx-2 during the development of the autopod as well as its genetic interaction with Hoxd-13. These results show that, in limbs, Evx-2 functions like a Hoxd gene. A potential evolutionary scenario is discussed, in which Evx-2 was recruited by the HoxD complex in conjunction with the emergence of digits in an ancestral tetrapod (Hérault, 1996).

Ulnaless (Ul), an X-ray-induced dominant mutation in mice, severely disrupts development of forearms and forelegs. The mutation maps onto chromosome 2, tightly linked to the HoxD complex. In particular, 5'-located (posterior) Hoxd genes are involved in limb development; combined mutations within these genes result in severe alterations in appendicular skeleton. Strong reductions and malformations are observed in both the radius and ulna of mutants. Several engineered alleles of the HoxD complex were used to genetically assess the potential linkage between Ul and HoxD. Ulnaless is allelic to Hoxd genes. Important modifications in the expression patterns of the posterior Hoxd-12 and Hoxd-13 genes at the Ul locus suggest that Ul is a regulatory mutation that interferes with a control mechanism shared by multiple genes to coordinate Hoxd function during limb morphogenesis (Hèraul, 1997).

Vertebrate Hoxd genes are essential determinants of limb morphogenesis. In order to understand the genetic control of their complex expression patterns, a combined approach was used involving interspecies sequence alignments in parallel with transgenic analyses, followed by in vivo mutagenesis. A regulatory element has been identified that is located in the vicinity of the Hoxd-12 gene. While this element is well conserved in tetrapods, little sequence similarity is found when compared to the cognate fish DNA. The regulatory potential of this region XI (RXI) was first assayed in the context of a Hoxd-12/lacZ reporter transgene and shown to direct reporter gene expression in posterior limb buds. A deletion of this region was generated by targeted mutagenesis in ES cells and introduced into mice. Analyses of animals homozygous for the HoxDRXI mutant allele reveals the function of this region in controlling Hoxd-12 expression in the presumptive posterior zeugopod where it genetically interacts with Hoxa-11. Downregulation of Hoxd-12 expression is also detected in the trunk suggesting that RXI may mediate a rather general function in the activation of Hoxd-12. These results support a model whereby global as well as local regulatory influences are necessary to build up the complex expression patterns of Hoxd genes during limb development (Hèrault, 1998).

The semi-dominant mouse mutation Ulnaless alters patterning of the appendicular but not the axial skeleton. Ulnaless mutant forelimbs and hindlimbs have severe reductions of the proximal limb and less severe reductions of the distal limb. Genetic and physical mapping has failed to separate the Ulnaless locus from the HoxD gene cluster. The Ulnaless limb phenotypes are not recapitulated by targeted mutations in any single HoxD gene, suggesting that Ulnaless may be a gain-of-function mutation in a coding sequence or a regulatory mutation. Deregulation of 5' HoxD gene expression is observed in Ulnaless limb buds. There is ectopic expression of Hoxd-13 and Hoxd-12 in the proximal limb and reduction of Hoxd-13, Hoxd-12 and Hoxd-11 expression in the distal limb. Skeletal reductions in the proximal limb may be a consequence of posterior prevalence, whereby proximal misexpression of Hoxd-13 and Hoxd-12 results in the transcriptional and/or functional inactivation of Hox group 11 genes. The Ulnaless digit phenotypes are attributed to a reduction in the distal expression of Hoxd-13, Hoxd-12, Hoxd-11 and Hoxa-13. In addition, Hoxd-13 expression is reduced in the genital bud, consistent with the observed alterations of the Ulnaless penian bone. No alterations of HoxD expression or skeletal phenotypes were observed in the Ulnaless primary axis. It is propose that the Ulnaless mutation alters a cis-acting element that regulates HoxD expression specifically in the appendicular axes of the embryo (Peichel, 1997).

Genes of the HoxD complex related to the Drosophila Abd-B gene are involved in the morphogenesis of vertebrate paired appendages. Hoxd-11, for instance, is necessary in combination with other Hox genes for the proper development of different parts of the tetrapod limbs. Sequence comparisons between the mouse, chicken, and zebrafish Hoxd-11 loci have revealed the conservation of several blocks of DNA sequence that may be of importance for the regulation of Hoxd-11 expression. Transgenic mice have been used to show that one of these conserved elements specifically drives expression in a proximal-posterior part of developing forelimbs. Production of mice transgenic for a full fish Hoxd-11 construct as well as for mouse-fish Hoxd-11 chimeric constructs shows that the fish counterpart of this sequence is able to elicit expression in mouse forelimbs as well, though in a slightly different domain. However, this fish element requires the presence of the mouse promoter and does not work in its own context. These results are discussed in light of both the control of Hoxd gene expression during limb development and the use of a comparative interspecies approach to understand the regulation of genes involved in vertebrate development (Beckers, 1996).

Hox genes are important regulators of limb pattern in vertebrate development. Misexpression of Hox genes in chicks using retroviral vectors provides an opportunity to analyze gain-of-function phenotypes and to assess their modes of action. The misexpression phenotype for Hoxd-13 is described and compared to the misexpression phenotype of Hoxd-11. Both genes are AbdominalB homologs. Hoxd-13 misexpression in the hindlimb results in a shortening of the long bones, including the femur, the tibia, the fibula and the tarsometatarsals. Mutations in an alanine repeat region in the N-terminus of Hoxd-13 have recently been implicated in human synpolydactyly. N-terminal truncations of Hoxd-13 lacking this repeat were constructed and were found to produce a similar, although slightly milder, misexpression phenotype than the full-length Hoxd-13 (Goff, 1997).

The stage of bone development regulated by Hox genes has not previously been examined. The changes in bone lengths caused by Hoxd-13 misexpression are late phenotypes that first become apparent during the growth phase of the bones. Hox genes can pattern the limb skeleton by regulating the rates of cell division in the proliferative zone of growing cartilage. Hoxd-11, in contrast to Hoxd-13, acts both at the initial cartilage condensation phase in the foot and during the later growth phase in the lower leg. Ectopic Hoxd-13 appears to act in a dominant negative manner in regions where it is not normally expressed. A model is proposed in which all Hox genes are growth promoters, regulating the expression of the same target genes, with some Hox genes being more effective promoters of growth than other Hox genes. According to this model, the overall rate of growth in a given region is the result of the combined action of all of the Hox genes expressed in that region competing for the same target genes (Goff, 1997).

The limb muscle precursor cells migrate from the somites to congregate in the dorsal and ventral muscle masses of each limb bud. Complex muscle patterns are formed by successive splitting of the muscle masses and their subsequent growth and differentiation in a region-specific manner. Hox genes, known as key regulator genes for cartilage pattern formation in the limb bud, are expressed in the limb muscle precursor cells. At stage 18, HOXA-11 protein is expressed in the premyoblasts in the limb bud, but not in the somitic cells or migrating premyogenic cells in the trunk. By stage 24, HOXA-11 expression begins to decrease from the posterior halves of the muscle masses. HOXA-13 is expressed strongly in the myoblasts of the posterior part of the limb dorsal/ventral muscle masses and weakly in a few myoblasts of the anterior part of the dorsal muscle mass. Transplantation of the lateral plate of the presumptive wing bud to the flank induces migration of premyoblasts from somites to the graft. Under these conditions, HOXA-11 expression is induced in the migrating premyoblasts of the ectopic limb buds. Application of retinoic acid at the anterior margin of the limb bud causes duplication of the autopodal cartilage and transformation of the radius to the ulna; at the same time, it induces duplication of the muscle pattern along the anteroposterior axis. Under these conditions, HOXA-13 is also induced in the anterior region of the ventral muscles in the zeugopod. These results suggest that Hoxa-11 and Hoxa-13 expression in the migrating premyoblasts is under the control of the limb mesenchyme and the polarizing signal(s). In addition, these results indicate that these Hox genes are involved in muscle patterning in the limb buds (Yamamoto, 1998).

The five most 5' HoxD genes, which are related to the Drosophila Abd-B gene, play an important role in patterning axial and appendicular skeletal elements and the nervous system of developing vertebrate embryos. Three of these genes, Hoxd11, Hoxd12, and Hoxd13, act synergistically to pattern the hindlimb autopod. This study examined the combined effects of two additional 5' HoxD genes: Hoxd9 and Hoxd10. Both of these genes are expressed posteriorly in overlapping domains in the developing neural tube and axial mesoderm as well as in developing limbs. Locomotor behavior in animals carrying a double mutation in these two genes is altered; these alterations include changes in gait, mobility, and adduction. Morphological analysis shows alterations in axial and appendicular skeletal structure, hindlimb peripheral nerve organization and projection, and distal hindlimb musculature. These morphological alterations are likely to provide the substrate for the observed alterations in locomotor behavior. The alterations observed in double-mutant mice are distinct from the phenotypes observed in mice carrying single mutations in either gene, but exhibit most of the features of both individual phenotypes. This suggests that the combined activity of two adjacent Hox genes provides more patterning information than activity of each gene alone. These observations support the idea that adjacent Hox genes with overlapping expression patterns may interact functionally to provide patterning information to the same regions of developing mouse embryos (de la Cruz, 1999).

This study demonstrates severe malformations of the appendicular skeleton in mice that overexpres Hoxc11. Consistent with the endogenous expression pattern, the most conspicuous defect in Hoxc11 overexpressing neonates is aplasia/hypoplasia of the fibula. This is preceded at day 15.5 of embryonic development by marked reduction of chondrocyte proliferation, lack of PTHR expressing prehypertrophic cells, and the absence of hypertrophic and calcifying chondrocytes. Combined with the lack of an overt phenotype in the majority of Hoxc11 overexpressing embryos at day 13.5, the data suggest inhibition of chondrocyte differentiation during the elongation phase of the fibula bone as a primary effect of elevated Hoxc11 expression. This interpretation is further corroborated by Hoxc11 reporter gene expression in the joint areas at embryonic day 15.5, suggesting an involvement of the periarticular perichondrium in generating the mutant phenotype (Papenbrock, 2000).

The most 5' mouse Hoxa and Hoxd genes, which occupy positions 9-13 and which are related to the Drosophila AbdB gene, are all active in patterning developing limbs. Inactivation of individual genes produces alterations in skeletal elements of both forelimb and hindlimb; inactivation of some of these genes also alters hindlimb innervation. Simultaneous inactivation of paralogous or nonparalogous Hoxa and Hoxd genes produces more widespread alterations, suggesting that combinatorial interactions between these genes are required for proper limb patterning. The effects of simultaneous inactivation of Hoxa10 and Hoxd10 on mouse hindlimb skeletal and nervous system development has been examined. These paralogous genes are expressed at lumbar and sacral levels of the developing neural tube and surrounding axial mesoderm as well as in developing forelimb and hindlimb buds. Double-mutant animals demonstrate impaired locomotor behavior and altered development of posterior vertebrae and hindlimb skeletal elements. Alterations in hindlimb innervation have also been observed, including truncations and deletions of the tibial and peroneal nerves. Animals carrying fewer mutant alleles show similar, but less extreme phenotypes. These observations suggest that Hoxa10 and Hoxd10 coordinately regulate skeletal development and innervation of the hindlimb (Wahba, 2001).

Polyalanine expansion in the human HOXD13 gene induces synpolydactyly (SPD), an inherited congenital limb malformation. A mouse model was isolated, which shows a spontaneous alanine expansion due to a 21-bp duplication at the corresponding place in the mouse gene. This mutation (synpolydactyly homolog, spdh), when homozygous, causes malformations in mice similar to those seen in affected human patients. The genetics of this condition has been studied, using several engineered Hoxd alleles, and by looking at the expression of Hox and other marker genes. The mutated SPDH protein induces a gain-of-function phenotype, likely by behaving as a dominant negative over other Hox genes. The mutation, however, seems to act independently from Hoxa13 and doesn't appear to affect Hox gene expression, except for a slight reduction of the HOXD13 protein itself. Developmental studies indicate that the morphological effect is mostly due to a severe retardation in the growth and ossification of the bony elements, in agreement with a general impairment in the function of posterior Hoxd genes (Bruneau, 2001).

Mesenchymal patterning is an active process whereby genetic commands coordinate cell adhesion, sorting and condensation, and thereby direct the formation of morphological structures. In mice that lack the Hoxa13 gene, the mesenchymal condensations that form the autopod skeletal elements are poorly resolved, resulting in missing digit, carpal and tarsal elements. In addition, mesenchymal and endothelial cell layers of the umbilical arteries (UAs) are disorganized, resulting in their stenosis and in embryonic death. To further investigate the role of Hoxa13 in these phenotypes, a loss-of-function allele was generated in which the GFP gene was targeted into the Hoxa13 locus. This allele allows FACS isolation of mesenchymal cells from Hoxa13 heterozygous and mutant homozygous limb buds. Hoxa13GFP expressing mesenchymal cells from Hoxa13 mutant homozygous embryos are defective in forming chondrogenic condensations in vitro. Analysis of pro-adhesion molecules in the autopod of Hoxa13 mutants reveals a marked reduction in EphA7 expression in affected digits, as well as in micromass cell cultures prepared from mutant mesenchymal cells. Finally, antibody blocking of the EphA7 extracellular domain severely inhibits the capacity of Hoxa13GFP heterozygous cells to condense and form chondrogenic nodules in vitro, which is consistent with the hypothesis that reduction in EphA7 expression affects the capacity of Hoxa13–/– mesenchymal cells to form chondrogenic condensations in vivo and in vitro. EphA7 and EphA4 expression are also decreased in the mesenchymal and endothelial cells that form the umbilical arteries in Hoxa13 mutant homozygous embryos. These results suggest that an important role for Hoxa13 during limb and UA development is to regulate genes whose products are required for mesenchymal cell adhesion, sorting and boundary formation (Stadler, 2001).

Targeted mutation of the Hoxd11 gene causes reduced male fertility, vertebral transformation, carpal bone fusions, and reductions in digit length. A duplication of the Hoxd11 gene was created with the expectation that the consequences of restricted overexpression in the appropriate cells would provide further insight into the function of the Hoxd11 gene product. Genetic assays have demonstrated that two tandem copies of Hoxd11 are functionally indistinguishable from the normal two copies of the gene on separate chromosomes with respect to formation of the axial and appendicular skeleton. Extra copies of Hoxd11 cause an increase in the lengths of some bones of the forelimb autopod and a decrease in the number of lumbar vertebrae. Further, analysis of the Hoxd11 duplication demonstrated that the Hoxd11 protein can perform some functions supplied by its paralog Hoxa11. For example, the defects in forelimb bones are corrected when extra copies of Hoxd11 are present in the Hoxa11 homozygous mutant background. Thus, it appears that Hoxd11 can quantitatively compensate for the absence of Hoxa11 protein, and therefore Hoxa11 and Hoxd11 are functionally equivalent in the zeugopod. However, extra copies of Hoxd11 did not improve male or female fertility in Hoxa11 mutants. Interestingly, the insertion of an additional Hoxd11 locus into the HoxD complex does not appear to affect the expression patterns of the neighboring Hoxd10, -d12, or -d13 genes (Boulet, 2002).

The 5' members of the Hoxa and Hoxd gene clusters play major roles in vertebrate limb development. One such gene, HOXD13, is mutated in the human limb malformation syndrome synpolydactyly. Both polyalanine tract expansions and frameshifting deletions in HOXD13 cause similar forms of this condition, but it remains unclear whether other kinds of HOXD13 mutations could produce different phenotypes. A six-generation family is described in which a novel combination of brachydactyly and central polydactyly co-segregates with a missense mutation that substitutes leucine for isoleucine at position 47 of the HOXD13 homeodomain. The HOXD13(I47L) mutant protein is compared, both in vitro and in vivo, to the wild-type protein and to an artificial HOXD13 mutant, HOXD13(IQN), which is completely unable to bind DNA. The mutation causes neither a dominant-negative effect nor a gain of function, but instead impairs DNA binding at some sites bound by wild-type HOXD13. Using retrovirus-mediated misexpression in developing chick limbs, it has been shown that wild-type HOXD13 can upregulate chick EphA7 in the autopod, but that HOXD13(I47L) cannot. In the zeugopod, however, HOXD13(I47L) produces striking changes in tibial morphology and ectopic cartilages, which are never produced by HOXD13(IQN), consistent with a selective rather than generalized loss of function. Thus, a mutant HOX protein that recognizes only a subset of sites recognized by the wild-type protein causes a novel human malformation, pointing to a hitherto undescribed mechanism by which missense mutations in transcription factors can generate unexpected phenotypes. Intriguingly, both HOXD13(I47L) and HOXD13(IQN) produce more severe shortening in proximal limb regions than does wild-type HOXD13, suggesting that functional suppression of anterior Hox genes by more posterior ones does not require DNA binding and is mediated by protein:protein interactions (Caronia, 2003).

Mutations in the 5' or posterior murine Hox genes (paralogous groups 9-13) markedly affect the formation of the stylopod (i.e. humerus or femur), zeugopod (i.e. radius and ulna in the forelimb, or tibia and fibula in the hindlimb) and autopod (i.e. the carpals and tarsals, metacarpals and metatarsals, and digits) of both forelimbs and hindlimbs. Targeted disruption of Hoxa11 and Hoxd11 or Hoxa10, Hoxc10 and Hoxd10 result in gross mispatterning of the radius and ulna or the femur, respectively. Similarly, in mice with disruptions of both Hoxa13 and Hoxd13, development of the forelimb and hindlimb autopod is severely curtailed. Although these examples clearly illustrate the major roles played by the posterior Hox genes, little is known regarding the stage or stages at which Hox transcription factors intersect with the limb development program to ensure proper patterning of the principle elements of the limb. Moreover, the cellular and/or molecular bases for the developmental defects observed in these mutant mice have not been described. In this study, it was shown that malformation of the forelimb zeugopod in Hoxa11/Hoxd11 double mutants is a consequence of interruption at multiple steps during the formation of the radius and ulna. In particular, reductions in the levels of Fgf8 and Fgf10 expression may be related to the observed delay in forelimb bud outgrowth that, in turn, leads to the formation of smaller mesenchymal condensations. However, the most significant defect appears to be the failure to form normal growth plates at the proximal and distal ends of the zeugopod bones. As a consequence, growth and maturation of these bones is highly disorganized, resulting in the creation of amorphous bony elements, rather than a normal radius and ulna (Boulet, 2004).

In humans and mice, loss of HOXA13 function causes defects in the growth and patterning of the digits and interdigital tissues. Analysis of Hoxa13 expression reveals a pattern of localization overlapping with sites of reduced Bmp2 and Bmp7 expression in Hoxa13 mutant limbs. Biochemical analyses identified a novel series of Bmp2 and Bmp7 enhancer regions that directly interact with the HOXA13 DNA-binding domain and activate gene expression in the presence of HOXA13. Immunoprecipitation of HOXA13-Bmp2 and HOXA13-Bmp7 enhancer complexes from the developing autopod confirm that endogenous HOXA13 associates with these regions. Exogenous application of BMP2 or BMP7 partially rescues the Hoxa13 mutant limb phenotype, suggesting that decreased BMP signaling contributes to the malformations present in these tissues. Together, these results provide conclusive evidence that HOXA13 regulates Bmp2 and Bmp7 expression, providing a mechanistic link between HOXA13, its target genes and the specific developmental processes affected by loss of HOXA13 function (Knosp, 2004).

Pattern formation along the proximal-distal (PD) axis in the developing limb bud serves as a good model for learning how cell fate and regionalization of domains, which are essential processes in morphogenesis during development, are specified by positional information. Detailed fate maps for the limb bud of the chick embryo were constructed in order to gain insights into how cell fate for future structures along the PD axis is specified and subdivided. In the limb, there is a single long cartilage in the most proximal region (stylopod) followed by two long cartilage elements (the zeugopod), and the most distal structures of the limb are carpals/tarsals and digits (the autopod). The fate map revealed that there is a large overlap between the prospective autopod and zeugopod in the distal limb bud at an early stage (stage 19), whereas a limb bud at this stage has already regionalized the proximal compartments for the prospective stylopod and zeugopod. A clearer boundary of cell fate specifying the prospective autopod and zeugopod could be seen at stage 23, but cell mixing was still detectable inside the prospective autopod region at this stage. Detailed analysis of HOXA11 AND HOXA13 expression at single cell resolution suggested that the cell mixing is not due to separation of some different cell populations existing in a mosaic. These findings suggest that a mixable unregionalized cell population is maintained in the distal area of the limb bud, while the proximal region starts to be regionalized at the early stage of limb development (Sato, 2007).

A combination of activation and repression by a colinear hox code controls forelimb-restricted expression of tbx5 and reveals hox protein specificity

Tight control over gene expression is essential for precision in embryonic development and acquisition of the regulatory elements responsible is the predominant driver for evolution of new structures. Tbx5 and Tbx4, two genes expressed in forelimb and hindlimb-forming regions respectively, play crucial roles in the initiation of limb outgrowth. Evolution of regulatory elements that activate Tbx5 in rostral lateral plate mesoderm (LPM) was essential for the acquisition of forelimbs in vertebrates. This study identified such a regulatory element for Tbx5 and demonstrated Hox genes are essential, direct regulators. While the importance of Hox genes in regulating embryonic development is clear, Hox targets and the ways in which each protein executes its specific function are not known. This study reveals how nested Hox expression along the rostro-caudal axis restricts Tbx5 expression to forelimb. Hoxc9, which is expressed in caudal LPM where Tbx5 is not expressed, can form a repressive complex on the Tbx5 forelimb regulatory element. This repressive capacity is limited to Hox proteins expressed in caudal LPM and carried out by two separate protein domains in Hoxc9. Forelimb-restricted expression of Tbx5 and ultimately forelimb formation is therefore achieved through co-option of two characteristics of Hox genes; their colinear expression along the body axis and the functional specificity of different paralogs. Active complexes can be formed by Hox PG proteins present throughout the rostral-caudal LPM while restriction of Tbx5 expression is achieved by superimposing a dominant repressive (Hoxc9) complex that determines the caudal boundary of Tbx5 expression. These results reveal the regulatory mechanism that ensures emergence of the forelimbs at the correct position along the body. Acquisition of this regulatory element would have been critical for the evolution of limbs in vertebrates and modulation of the factors that were identified can be molecular drivers of the diversity in limb morphology (Nishimoto, 2014).

Intrinsic properties of limb bud cells can be differentially reset

An intrinsic timing mechanism specifies the positional values of the zeugopod (i.e. radius/ulna) and then autopod (i.e. wrist/digits) segments during limb development. This study addressed if this timing mechanism ensures that patterning events occur only once by grafting GFP-expressing autopod progenitor cells to the earlier host signaling environment of zeugopod progenitor cells. Early and late autopod progenitors fated for the wrist and phalanges, respectively, both contribute to the entire host autopod indicating that the autopod positional value is irreversibly determined as revealed by Hoxa13 (see Drosophila AbdominaB) expression. Evidence is provided that Hoxa13 provides an autopod-specific positional value that correctly allocates cells into the autopod, most likely through the control of cell-surface properties as shown by cell-cell sorting analyses. However, only the earlier autopod cells can adopt the host proliferation rate to permit normal morphogenesis. Therefore, these findings reveal that the ability of embryonic cells to differentially reset their intrinsic behaviors confers robustness to limb morphogenesis. It is speculated that this plasticity could be maintained beyond embryogenesis in limbs with regenerative capacity (Saiz-Lopez, 2017).

Table of contents

Abdominal-B: Biological Overview | Promoter Structure | Transcriptional Regulation | Targets of activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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