BAC transgenic analysis reveals enhancers sufficient for Hoxa13 and neighborhood gene expression in mouse embryonic distal limbs and genital bud.
ABSTRACT: We previously demonstrated that a approximately 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 beta-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.
Project description:BACKGROUND: Kangaroos and wallabies have specialised limbs that allow for their hopping mode of locomotion. The hindlimbs differentiate much later in development but become much larger than the forelimbs. The hindlimb autopod has only four digits, the fourth of which is greatly elongated, while digits two and three are syndactylous. We investigated the expression of two genes, HOXA13 and HOXD13, that are crucial for digit patterning in mice during formation of the limbs of the tammar wallaby. RESULTS: We describe the development of the tammar limbs at key stages before birth. There was marked heterochrony and the hindlimb developed more slowly than the forelimb. Both tammar HOXA13 and HOXD13 have two exons as in humans, mice and chickens. HOXA13 had an early and distal mRNA distribution in the tammar limb bud as in the mouse, but forelimb expression preceded that in the hindlimb. HOXD13 mRNA was expressed earlier in the forelimb than the hindlimb and was predominantly detected in the interdigital tissues of the forelimb. In contrast, the hindlimb had a more restricted expression pattern that appeared to be expressed at discrete points at both posterior and anterior margins of the limb bud, and was unlike expression seen in the mouse and the chicken. CONCLUSIONS: This is the first examination of HOXA and HOXD gene expression in a marsupial. The gene structure and predicted proteins were highly conserved with their eutherian orthologues. Interestingly, despite the morphological differences in hindlimb patterning, there were no modifications to the polyalanine tract of either HOXA13 or HOXD13 when compared to those of the mouse and bat but there was a marked difference between the tammar and the other mammals in the region of the first polyserine tract of HOXD13. There were also altered expression domains for both genes in the developing tammar limbs compared to the chicken and mouse. Together these findings suggest that the timing of HOX gene expression may contribute to the heterochrony of the forelimb and hindlimb and that alteration to HOX domains may influence phenotypic differences that lead to the development of marsupial syndactylous digits.
Project description:Vertebrates have four clusters of Hox genes (HoxA, HoxB, HoxC, and HoxD). A variety of expression and mutation studies indicate that posterior members of the HoxA and HoxD clusters play an important role in vertebrate limb development. In humans, mutations in HOXD13 have been associated with type II syndactyly or synpolydactyly, and, in HOXA13, with hand-foot-genital syndrome. We have investigated two unrelated children with a previously unreported pattern of severe developmental defects on the anterior-posterior (a-p) limb axis and in the genitalia, consisting of a single bone in the zeugopod, either monodactyly or oligodactyly in the autopod of all four limbs, and penoscrotal hypoplasia. Both children are heterozygous for a deletion that eliminates at least eight (HOXD3-HOXD13) of the nine genes in the HOXD cluster. We propose that the patients' phenotypes are due in part to haploinsufficiency for HOXD-cluster genes. This hypothesis is supported by the expression patterns of these genes in early vertebrate embryos. However, the involvement of additional genes in the region could explain the discordance, in severity, between these human phenotypes and the milder, non-polarized phenotypes present in mice hemizygous for HoxD cluster genes. These cases represent the first reported examples of deficiencies for an entire Hox cluster in vertebrates and suggest that the diploid dose of human HOXD genes is crucial for normal growth and patterning of the limbs along the anterior-posterior axis.
Project description:HoxA genes encode for important DNA-binding transcription factors that act during limb development, regulating primarily gene expression and, consequently, morphogenesis and skeletal differentiation. Within these genes, HoxA11 and HoxA13 were proposed to have played an essential role in the enigmatic evolutionary transition from fish fins to tetrapod limbs. Indeed, comparative gene expression analyses led to the suggestion that changes in their regulation might have been essential for the diversification of vertebrates' appendages. In this review, we highlight three potential modifications in the regulation and function of these genes that may have boosted appendage evolution: (1) the expansion of polyalanine repeats in the HoxA11 and HoxA13 proteins; (2) the origin of +a novel long-non-coding RNA with a possible inhibitory function on HoxA11; and (3) the acquisition of cis-regulatory elements modulating 5' HoxA transcription. We discuss the relevance of these mechanisms for appendage diversification reviewing the current state of the art and performing additional comparative analyses to characterize, in a phylogenetic framework, HoxA11 and HoxA13 expression, alanine composition within the encoded proteins, long-non-coding RNAs and cis-regulatory elements.
Project description:Hand-foot-genital syndrome (HFGS) is a rare, dominantly inherited condition affecting the distal limbs and genitourinary tract. A nonsense mutation in the homeobox of HOXA13 has been identified in one affected family, making HFGS the second human syndrome shown to be caused by a HOX gene mutation. We have therefore examined HOXA13 in two new and four previously reported families with features of HFGS. In families 1, 2, and 3, nonsense mutations truncating the encoded protein N-terminal to or within the homeodomain produce typical limb and genitourinary abnormalities; in family 4, an expansion of an N-terminal polyalanine tract produces a similar phenotype; in family 5, a missense mutation, which alters an invariant domain, produces an exceptionally severe limb phenotype; and in family 6, in which limb abnormalities were atypical, no HOXA13 mutation could be detected. Mutations in HOXA13 can therefore cause more-severe limb abnormalities than previously suspected and may act by more than one mechanism.
Project description:HoxA genes exhibit central roles during development and causal mutations have been found in several human syndromes including limb malformation. Despite their importance, information on how these genes are regulated is lacking. Here, we report on the first identification of bona fide transcriptional enhancers controlling HoxA genes in developing limbs and show that these enhancers are grouped into distinct topological domains at the sub-megabase scale (sub-TADs). We provide evidence that target genes and regulatory elements physically interact with each other through contacts between sub-TADs rather than by the formation of discreet "DNA loops". Interestingly, there is no obvious relationship between the functional domains of the enhancers within the limb and how they are partitioned among the topological domains, suggesting that sub-TAD formation does not rely on enhancer activity. Moreover, we show that suppressing the transcriptional activity of enhancers does not abrogate their contacts with HoxA genes. Based on these data, we propose a model whereby chromatin architecture defines the functional landscapes of enhancers. From an evolutionary standpoint, our data points to the convergent evolution of HoxA and HoxD regulation in the fin-to-limb transition, one of the major morphological innovations in vertebrates.
Project description:The amniote phallus and limbs differ dramatically in their morphologies but share patterns of signaling and gene expression in early development. Thus far, the extent to which genital and limb transcriptional networks also share cis-regulatory elements has remained unexplored. We show that many limb enhancers are retained in snake genomes, suggesting that these elements may function in non-limb tissues. Consistent with this, our analysis of cis-regulatory activity in mice and Anolis lizards reveals that patterns of enhancer activity in embryonic limbs and genitalia overlap heavily. In mice, deletion of HLEB, an enhancer of Tbx4, produces defects in hindlimbs and genitalia, establishing the importance of this limb-genital enhancer for development of these different appendages. Further analyses demonstrate that the HLEB of snakes has lost hindlimb enhancer function while retaining genital activity. Our findings identify roles for Tbx4 in genital development and highlight deep similarities in cis-regulatory activity between limbs and genitalia.
Project description:To elucidate the role of Hox genes in limb cartilage development, we identified the target genes of HOXA11 and HOXA13 by ChIP-Seq. The ChIP DNA fragment contained evolutionarily conserved sequences and multiple highly conserved HOX binding sites. A substantial portion of the HOXA11 ChIP fragment overlapped with the HOXA13 ChIP fragment indicating that both factors share common targets. Deletion of the target regions neighboring Bmp2 or Tshz2 reduced their expression in the autopod suggesting that they function as the limb bud-specific enhancers. We identified the Hox downstream genes as exhibiting expression changes in the Hoxa13 knock out (KO) and Hoxd11-13 deletion double mutant (Hox13 dKO) autopod by Genechip analysis. The Hox downstream genes neighboring the ChIP fragment were defined as the direct targets of Hox. We analyzed the spatial expression pattern of the Hox target genes that encode two different categories of transcription factors during autopod development and Hox13dKO limb bud. (a) Bcl11a, encoding a repressor of cartilage differentiation, was expressed in the E11.5 autopod and was substantially reduced in the Hox13dKO. (b) The transcription factors Aff3, Bnc2, Nfib and Runx1t1 were expressed in the zeugopodal cartilage but not in the autopod due to the repressive or relatively weak transcriptional activity of Hox13 at E11.5. Interestingly, the expression of these genes was later observed in the autopodal cartilage at E12.5. These results indicate that Hox13 transiently suspends the cartilage differentiation in the autopodal anlage via multiple pathways until establishing the paddle-shaped structure required to generate five digits.
Project description:While the vertebrate body plan is highly conserved amongst all species of this taxon, extreme variations thereof can be documented in snakes, which display both an absence of limbs and an unusually elongated trunk. As Hox genes are strong candidates both for the making and the evolution of this body plan, their comparative study in such a morphologically diverged group is informative regarding their potential causative importance in these processes. In this work we use an interspecies comparative approach where different aspects of regulation at the HoxD locus are investigated. We find that although spatial collinearity and associated epigenetic mark dynamics are conserved in the corn snake, other regulatory modalities have been largely restructured. A BAC transgenic approach indeed revealed that, while the majority of mesodermal enhancers in vertebrates appear to be mostly located outside of the cluster, the corn snake contains most mesodermal trunk enhancers within the HoxD cluster. We also find that, despite the absence of limbs and an altered Hoxd gene regulation in external genitalia, the bimodal chromatin structure at the corn snake HoxD locus is maintained. The analysis of particular enhancer sequences initially defined in the mouse and further isolated at the snake orthologous locus showed differences in their specificities for the limb and genital bud expression. Of particular interest, a snake counterpart of a mouse limb-only enhancer sequence evolved into a genital-only enhancer. Such a regulatory exaptation suggests that enhancer versatility may have been an important factor to accompany the transition towards the snake body plan. These results show that vertebrate morphological evolution is likely to have been associated with extensive reorganization at the HoxD regulatory landscapes while respecting a very conserved general regulatory framework. Overall design: 4C-seq analysis of mouse and snake whole embryo and genital bud tissue using viewpoints contained within the cluster
Project description:The fin-to-limb transition represents one of the major vertebrate morphological innovations associated with the transition from aquatic to terrestrial life and is an attractive model for gaining insights into the mechanisms of morphological diversity between species. One of the characteristic features of limbs is the presence of digits at their extremities. Although most tetrapods have limbs with five digits (pentadactyl limbs), palaeontological data indicate that digits emerged in lobed fins of early tetrapods, which were polydactylous. How the transition to pentadactyl limbs occurred remains unclear. Here we show that the mutually exclusive expression of the mouse genes Hoxa11 and Hoxa13, which were previously proposed to be involved in the origin of the tetrapod limb, is required for the pentadactyl state. We further demonstrate that the exclusion of Hoxa11 from the Hoxa13 domain relies on an enhancer that drives antisense transcription at the Hoxa11 locus after activation by HOXA13 and HOXD13. Finally, we show that the enhancer that drives antisense transcription of the mouse Hoxa11 gene is absent in zebrafish, which, together with the largely overlapping expression of hoxa11 and hoxa13 genes reported in fish, suggests that this enhancer emerged in the course of the fin-to-limb transition. On the basis of the polydactyly that we observed after expression of Hoxa11 in distal limbs, we propose that the evolution of Hoxa11 regulation contributed to the transition from polydactyl limbs in stem-group tetrapods to pentadactyl limbs in extant tetrapods.
Project description:There is no obvious morphological counterpart of the autopod (wrist/ankle and digits) in living fishes. Comparative molecular data may provide insight into understanding both the homology of elements and the evolutionary developmental mechanisms behind the fin to limb transition. In mouse limbs the autopod is built by a "late" phase of Hoxd and Hoxa gene expression, orchestrated by a set of enhancers located at the 5' end of each cluster. Despite a detailed mechanistic understanding of mouse limb development, interpretation of Hox expression patterns and their regulation in fish has spawned multiple hypotheses as to the origin and function of "autopod" enhancers throughout evolution. Using phylogenetic footprinting, epigenetic profiling, and transgenic reporters, we have identified and functionally characterized hoxD and hoxA enhancers in the genomes of zebrafish and the spotted gar, Lepisosteus oculatus, a fish lacking the whole genome duplication of teleosts. Gar and zebrafish "autopod" enhancers drive expression in the distal portion of developing zebrafish pectoral fins, and respond to the same functional cues as their murine orthologs. Moreover, gar enhancers drive reporter gene expression in both the wrist and digits of mouse embryos in patterns that are nearly indistinguishable from their murine counterparts. These functional genomic data support the hypothesis that the distal radials of bony fish are homologous to the wrist and/or digits of tetrapods.