Anterior-posterior differences in HoxD chromatin topology in limb development

A late phase of HoxD activation is crucial for the patterning and growth of distal structures across the anterior-posterior (A-P) limb axis of mammals. Polycomb complexes and chromatin compaction have been shown to regulate Hox loci along the main body axis in embryonic development, but the extent to which they have a role in limb-specific HoxD expression, an evolutionary adaptation defined by the activity of distal enhancer elements that drive expression of 5' Hoxd genes, has yet to be fully elucidated. We reveal two levels of chromatin topology that differentiate distal limb A-P HoxD activity. Using both immortalised cell lines derived from posterior and anterior regions of distal E10.5 mouse limb buds, and analysis in E10.5 dissected limb buds themselves, we show that there is a loss of polycomb-catalysed H3K27me3 histone modification and a chromatin decompaction over HoxD in the distal posterior limb compared with anterior. Moreover, we show that the global control region (GCR) long-range enhancer spatially colocalises with the 5' HoxD genomic region specifically in the distal posterior limb. This is consistent with the formation of a chromatin loop between 5' HoxD and the GCR regulatory module at the time and place of distal limb bud development when the GCR participates in initiating Hoxd gene quantitative collinearity and Hoxd13 expression. This is the first example of A-P differences in chromatin compaction and chromatin looping in the development of the mammalian secondary body axis (limb).     

Conserved expression domains for genes upstream and within the HoxA and HoxD clusters suggests a long-range enhancer existed before cluster duplication

The posterior HoxA and HoxD genes are essential in appendicular development. Studies have demonstrated that a "distal limb enhancer," remotely located upstream of the HoxD complex, is required to drive embryonic autopod expression of the posterior Hox genes as well as the two additional non-Hox genes in the region: Evx2 and Lnp. Our work demonstrates a similar mode of regulation for Hoxa13 and four upstream genes: Evx1, Hibadh, Tax1bp, and Jaz1. These genes all show embryonic (E11.5-E13.5) distal limb and genital bud expression, suggesting the existence of a nearby enhancer influencing the expression of a domain of genes. Comparative sequence analysis between homologous human and mouse genomic sequence upstream of Hoxa13 revealed a remote 2.25-kb conserved noncoding sequence (mmA13CNS) within the fourth intron of the Hibadh gene. mmA13CNS shares a common 131-bp core identity within a conserved noncoding sequence upstream of Hoxd13, which is located within the previously identified distal limb enhancer critical region. To test the function of this conserved sequence, we created mmA13CNS-Hsp86-lacZ transgenic mice. mmA13CNS directed a wide range of tissue expression, including the central nervous system, developing olfactory tissue, limb, and genital bud. Limb and genital bud expression directed by mmA13CNS is not identical to the patterns exhibited by Hoxa13/Evx1/Hibadh/Tax1bp1/Jaz1, suggesting that mmA13CNS is not sufficient to fully recapitulate their expression in those tissues. The Evx1- and Evx2-like central nervous system expression observed in these mice suggests that the long-range regulatory element(s) for the Hox cluster existed before the cluster duplication.     

The chick oligozeugodactyly (ozd) mutant lacks sonic hedgehog function in the limb

We have analyzed a new limb mutant in the chicken that we name oligozeugodactyly (ozd). The limbs of this mutant have a longitudinal postaxial defect, lacking the posterior element in the zeugopod (ulna/fibula) and all digits except digit 1 in the leg. Classical recombination experiments show that the limb mesoderm is the defective tissue layer in ozd limb buds. Molecular analysis revealed that the ozd limbs develop in the absence of Shh expression, while all other organs express Shh and develop normally. Neither Ptc1 nor Gli1 are detectable in mutant limb buds. However, Bmp2 and dHAND are expressed in the posterior wing and leg bud mesoderm, although at lower levels than in normal embryos. Activation of Hoxd11-13 occurs normally in ozd limbs but progressively declines with time. Phase III of expression is more affected than phase II, and expression is more severely affected in the more 5' genes. Interestingly, re-expression of Hoxd13 occurs at late stages in the distal mesoderm of ozd leg buds, correlating with formation of digit 1. Fgf8 and Fgf4 expression are initiated normally in the mutant AER but their expression is progressively downregulated in the anterior AER. Recombinant Shh protein or ZPA grafts restore normal pattern to ozd limbs; however, retinoic acid fails to induce Shh in ozd limb mesoderm. We conclude that Shh function is required for limb development distal to the elbow/knee joints, similar to the Shh(-/-) mouse. Accordingly we classify the limb skeletal elements as Shh dependent or independent, with the ulna/fibula and digits other than digit 1 in the leg being Shh dependent. Finally we propose that the ozd mutation is most likely a defect in a regulatory element that controls limb-specific expression of Shh.     

An examination of the Chiropteran HoxD locus from an evolutionary perspective

SUMMARY Duplications of Hox gene clusters have been suggested as a mechanism whereby new Hox functions can be developed while preserving critical ancestral roles. However, in tetrapods, particularly in mammals, there is great variability in limb structure morphologies that are known to be affected by Hox genes without further Hox cluster duplications. The lack of further duplications suggests that if Hox genes have played a direct role in the morphological elaboration of tetrapod limbs, the changes must have come about from Hox protein sequence changes or from changes regarding the amount, time, and place of Hox gene expression. To investigate whether such changes to Hox genes could play a role in limb elaboration, we examined the HoxD locus in bats, which have both highly elaborated fore‐ and hindlimbs. We found that while the Chiropteran HoxD13 protein was highly conserved, there was an expansion of HoxD13 expression in the posterior portion of the Chiropteran forelimb and into the leading edge of the wing membrane. We were also able to uncover a number of unique lineage‐specific sequence changes to a known HoxD limb enhancer, the Global Control Region (GCR). Further, mouse transgenic assays showed that the Chiropteran GCR has new limb enhancer activity domains beyond that reported for the Human GCR. These results suggest that modulation of Hox gene expression may be a mechanism for effecting morphological change in lineage‐specific manner while maintaining ancestral constraints and cluster integrity.     

Genetic Interactions Between Shox2 and Hox Genes During the Regional Growth and Development of the Mouse Limb

The growth and development of the vertebrate limb relies on homeobox genes of the Hox and Shox families, with their independent mutation often giving dose-dependent effects. Here we investigate whether Shox2 and Hox genes function together during mouse limb development by modulating their relative dosage and examining the limb for nonadditive effects on growth. Using double mRNA fluorescence in situ hybridization (FISH) in single embryos, we first show that Shox2 and Hox genes have associated spatial expression dynamics, with Shox2 expression restricted to the proximal limb along with Hoxd9 and Hoxa11 expression, juxtaposing the distal expression of Hoxa13 and Hoxd13. By generating mice with all possible dosage combinations of mutant Shox2 alleles and HoxA/D cluster deletions, we then show that their coordinated proximal limb expression is critical to generate normally proportioned limb segments. These epistatic interactions tune limb length, where Shox2 underexpression enhances, and Shox2 overexpression suppresses, Hox-mutant phenotypes. Disruption of either Shox2 or Hox genes leads to a similar reduction in Runx2 expression in the developing humerus, suggesting their concerted action drives cartilage maturation during normal development. While we furthermore provide evidence that Hox gene function influences Shox2 expression, this regulation is limited in extent and is unlikely on its own to be a major explanation for their genetic interaction. Given the similar effect of human SHOX mutations on regional limb growth, Shox and Hox genes may generally function as genetic interaction partners during the growth and development of the proximal vertebrate limb.     

Genomic regulation of Hox collinearity

During vertebrate limb development, Hoxd genes are transcribed in two temporal phases; an early wave controls growth and polarity up to the forearm and a late wave patterns the digits. In this issue of Developmental Cell, Tarchini and Duboule (2006) report that two opposite regulatory modules direct early collinear expression of Hoxd genes.     

BAC transgenic analysis reveals enhancers sufficient for Hoxa13 and neighborhood gene expression in mouse embryonic distal limbs and genital bud

SUMMARY We previously demonstrated that a ∼1 Mb domain of genes upstream of and including Hoxa13 is co-expressed in the developing mouse limbs and genitalia. A highly conserved non-coding sequence, mmA13CNS, was shown to be insufficient in transgenic mice to direct precise Hoxa13 -like expression in the limb buds or genital bud, although some LacZ expression from the transgene was reproducibly found in these tissues. In this report, we used β -globin minimal promoter LacZ recombinant BAC transgenes encompassing mmA13CNS to identify a single critical region involved in mouse Hoxa13 -like embryonic genital bud expression. By analyzing the expression patterns of these overlapping BAC clones in transgenic mice, we show that at least two sequences remote to the HoxA cluster are required collectively to drive Hoxa13 -like expression in developing distal limbs. Given that the paralogous posterior HoxD and neighboring genes have been shown to be under the influence of long-range distal limb and genital bud enhancers, we hypothesize that both long-range enhancers have one ancestral origin, which diverged in both sequence and function after the HoxA/D cluster duplication.     

Hox gene expression in limbs: colinearity by opposite regulatory controls

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, we have scanned the posterior part of the HoxD complex with a targeted reporter transgene and analyzed the response of this foreign promoter to limb regulatory influences. The results suggest that this regulation is achieved through the opposite effects of two enhancer elements which would compete with each other for interacting with nearby-located promoters. The physical position of a given gene within this genomic interval of opposite regulations might thus determine its final expression pattern. 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.     

Function and regulation of Alx4 in limb development: complex genetic interactions with Gli3 and Shh

The role of the aristaless-related homeobox gene Alx4 in antero-posterior (AP-) patterning of the developing vertebrate limb has remained somewhat elusive. Polydactyly of Alx4 mutant mice is known to be accompanied by ectopic anterior expression of genes like Shh, Fgf4 and 5'Hoxd. We reported previously that polydactyly in Alx4 mutant mice requires SHH signaling, but we now show that in early Alx4-/- limb buds the anterior ectopic expression of Fgf4 and Hoxd13, and therefore disruption of AP-patterning, occurs independently of SHH signaling. To better understand how Alx4 functions in the pathways that regulate AP-patterning, we also studied genomic regulatory sequences that are capable of directing expression of a reporter gene in a pattern corresponding to endogenous Alx4 expression in anterior limb bud mesenchyme. We observed, as expected for authentic Alx4 expression, expansion of reporter construct expression in a Shh-/- background. Total lack of reporter expression in a Gli3-/- background confirms the existence of Gli3-dependent and -independent Alx4 expression in the limb bud. Apparently, these two modules of Alx4 expression are linked to dissimilar functions.     

Control of vertebrate limb outgrowth by the proximal factor Meis2 and distal antagonism of BMPs by Gremlin

The mechanisms controlling growth and patterning along the proximal-distal axis of the vertebrate limb are yet to be understood. We show that restriction of expression of the homeobox gene Meis2 to proximal regions of the limb bud is essential for limb development, since ectopic Meis2 severely disrupts limb outgrowth. We also uncover an antagonistic relationship between the secreted factors Gremlin and BMPs required to maintain the Shh/FGF loop that regulates distal outgrowth. These proximal and distal factors have coordinated activities: Meis2 can repress distal genes, and Bmps and Hoxd genes restrict Meis2 expression to the proximal limb bud. Moreover, combinations of BMPs and AER factors are sufficient to distalize proximal limb cells. Our results unveil a novel set of proximal-distal regulatory interactions that establish and maintain outgrowth of the vertebrate limb.     

Developmental genetic basis for the evolution of pelvic fin loss in the pufferfish Takifugu rubripes

Paired appendages were a key developmental innovation among vertebrates and they eventually evolved into limbs. Ancient developmental control systems for paired fins and limbs are broadly conserved among gnathostome vertebrates. Some lineages including whales, some salamanders, snakes, and many ray-fin fish, independently lost the pectoral, pelvic, or both appendages over evolutionary time. When different taxa independently evolve similar developmental morphologies, do they use the same molecular genetic mechanisms? To determine the developmental genetic basis for the evolution of pelvis loss in the pufferfish Takifugu rubripes (fugu), we isolated fugu orthologs of genes thought to be essential for limb development in tetrapods, including limb positioning (Hoxc6, Hoxd9), limb bud initiation (Pitx1, Tbx4, Tbx5), and limb bud outgrowth (Shh, Fgf10), and studied their expression patterns during fugu development. Results showed that bud outgrowth and initiation fail to occur in fugu, and that pelvis loss is associated with altered expression of Hoxd9a, which we show to be a marker for pelvic fin position in three-spine stickleback Gasterosteus aculeatus. These results rule out changes in appendage outgrowth and initiation genes as the earliest developmental defect in pufferfish pelvic fin loss and suggest that altered Hoxd9a expression in the lateral mesoderm may account for pelvis loss in fugu. This mechanism appears to be different from the mechanism for pelvic loss in stickleback, showing that different taxa can evolve similar phenotypes by different mechanisms.