Large scale transgenic and cluster deletion analysis of the HoxD complex separate an ancestral regulatory module from evolutionary innovations.

The ancestral role of the Hox gene family is specifying morphogenetic differences along the main body axis. In vertebrates, HoxD genes were also co-opted along with the emergence of novel structures such as limbs and genitalia. We propose that these functional recruitments relied on the appearance, or implementation, of regulatory sequences outside of the complex. Whereas transgenic human and murine HOXD clusters could function during axial patterning, in mice they were not expressed outside the trunk. Accordingly, deletion of the entire cluster abolished axial expression, whereas recently acquired regulatory controls were preserved.

Localized and transient transcription of Hox genes suggests a link between patterning and the segmentation clock.

During development, Hox gene transcription is activated in presomitic mesoderm with a time sequence that follows the order of the genes along the chromosome. Here, we show that Hoxd1 and other Hox genes display dynamic stripes of expression within presomitic mesoderm. The underlying transcriptional bursts may reflect the mechanism that coordinates Hox gene activation with somitogenesis. This mechanism appears to depend upon Notch signaling, as mice deficient for RBPJk, the effector of the Notch pathway, showed severely reduced Hoxd gene expression in presomitic mesoderm. These results suggest a molecular link between Hox gene activation and the segmentation clock. Such a linkage would efficiently keep in phase the production of novel segments with their morphological specification.

Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice.

We show here that the alpha, beta, and gamma isotypes of peroxisome proliferator-activated receptor (PPAR) are expressed in the mouse epidermis during fetal development and that they disappear progressively from the interfollicular epithelium after birth. Interestingly, PPARalpha and beta expression is reactivated in the adult epidermis after various stimuli, resulting in keratinocyte proliferation and differentiation such as tetradecanoylphorbol acetate topical application, hair plucking, or skin wound healing. Using PPARalpha, beta, and gamma mutant mice, we demonstrate that PPARalpha and beta are important for the rapid epithelialization of a skin wound and that each of them plays a specific role in this process. PPARalpha is mainly involved in the early inflammation phase of the healing, whereas PPARbeta is implicated in the control of keratinocyte proliferation. In addition and very interestingly, PPARbeta mutant primary keratinocytes show impaired adhesion and migration properties. Thus, the findings presented here reveal unpredicted roles for PPARalpha and beta in adult mouse epidermal repair.

Mechanisms of Hox gene colinearity: transposition of the anterior Hoxb1 gene into the posterior HoxD complex.

Transposition of Hoxd genes to a more posterior (5') location within the HoxD complex suggested that colinearity in the expression of these genes was due, in part, to the existence of a silencing mechanism originating at the 5' end of the cluster and extending towards the 3' direction. To assess the strength and specificity of this repression, as well as to challenge available models on colinearity, we inserted a Hoxb1/lacZ transgene within the posterior HoxD complex, thereby reconstructing a cluster with a copy of the most anterior gene inserted at the most posterior position. Analysis of Hoxb1 expression after ectopic relocation revealed that Hoxb1-specific activity in the fourth rhombomere was totally abolished. Treatment with retinoic acid, or subsequent relocations toward more 3' positions in the HoxD complex, did not release this silencing in hindbrain cells. In contrast, however, early and anterior transgene expression in the mesoderm was unexpectedly not suppressed. Furthermore, the transgene induced a transient ectopic activation of the neighboring Hoxd13 gene, without affecting other genes of the complex. Such a local and transient break in colinearity was also observed after transposition of the Hoxd9/lacZ reporter gene, indicating that it may be a general property of these transgenes when transposed at an ectopic location. These results are discussed in the context of existing models, which account for colinear activation of vertebrate Hox genes.

Hox genes in digit development and evolution.

Homeobox genes located in the 5' part of the HoxA and HoxD complexes are required for proliferation of skeletal progenitor cells of the vertebrate limb. Specific combinations of gene products determine the length of the upper arm (genes belonging to groups 9 and 10), the lower arm (groups 10, 11 and 12) and the digits (groups 11, 12 and 13). In these different domains, individual gene products quantitatively contribute to an overall protein dose, with predominant roles for groups 11 and 13. Quantitative reduction in the gene dose in each set results in truncations of the corresponding anatomical regions. The physical order of the genes in the HoxA and HoxD complexes, as well as a unidirectional sequence in gene activation, allow for completion of the process in a precise order, which in turn makes possible the sequential outgrowth of the respective primordia. While the skeletal patterns of upper and lower limb are relatively stable throughout the tetrapods, more variation is seen in the digits. Molecular analysis of the underlying regulatory processes promises further exciting insights into the genetic control of development, pathology and the course of evolution.

Genetic control of murine limb morphogenesis: relationships with human syndromes and evolutionary relevance.

Over the past ten years, the discovery and functional characterisation of murine Hox genes has led to a better understanding of some of the molecular mechanisms underlying limb development. It has also shed some light on the potential genetic events which have accompanied the fin-to-limb transition, an evolutionary step of critical importance which opened the way to the evolution of higher vertebrates. This convergence between developmental biology and the sciences of evolution is one of the synergistic interface that has been established recently thanks to the use of genetic engineering and transgenic animals. The increasing number of human genetic syndromes which are derived from mutations in developmental control genes remind us that many human genetic diseases are nothing else but alterations in our developmental programme. Here, we illustrate these various issues by discussing the function of Hox genes during limb development.

Control of colinearity in AbdB genes of the mouse HoxD complex.

During development, vertebrate Hox genes are activated in a temporal and spatial sequence colinear with the position of the genes within their clusters. To investigate the mechanistic basis of this phenomenon, we used the ES cell technology and the loxP/Cre system to engineer a conditional fusion of the 5' exon of Hoxd-13 with the 3' exon of Hoxd-12. This hybrid transcription unit was regulated like Hoxd-11, with expression limits in the trunk, limbs, intestinal, and urogenital systems more anterior than those expected for either Hoxd-13 or Hoxd-12. An in vivo interspecies replacement by the fish homologous DNA fragment showed that anteriorization was not due to a distance effect, thus suggesting the presence of a regulatory element between Hoxd-13 and Hoxd-12 that may contribute to the establishment, early on, of a repressive state over these two genes.

Ulnaless (Ul), a regulatory mutation inducing both loss-of-function and gain-of-function of posterior Hoxd genes.

Ulnaless (Ul), an X-ray-induced dominant mutation in mice, severely disrupts development of forearms and forelegs. The mutation maps on chromosome 2, tightly linked to the HoxD complex, a cluster of regulatory genes required for proper morphogenesis. In particular, 5'-located (posterior) Hoxd genes are involved in limb development and combined mutations within these genes result in severe alterations in appendicular skeleton. We have used several engineered alleles of the HoxD complex to genetically assess the potential linkage between these two loci. We present evidence indicating that 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.

Interspecies exchange of a Hoxd enhancer in vivo induces premature transcription and anterior shift of the sacrum.

The precise activation, in space and time, of vertebrate Hox genes is an essential requirement for normal morphogenesis. In order to assess for the functional potential of evolutionary conserved Hox regulatory sequences, a phylogenetically conserved bipartite regulatory element necessary for proper spatial and temporal activation of the Hoxd-11 gene was replaced by its fish counterpart in the HoxD complex of mice, using an ES cell-based targeted exchange. Fetuses carrying this replacement activated Hoxd-11 transcription prematurely, which led to a rostral shift of its expression boundary and a consequent anterior transposition of the sacrum. These results demonstrate the high phylogenetic conservation of regulatory mechanisms acting over vertebrate Hox complexes and suggest that minor time difference (heterochronies) in Hox gene activation may have contributed to important morphological variations in the course of evolution.

Deletion of a HoxD enhancer induces transcriptional heterochrony leading to transposition of the sacrum.

A phylogenetically conserved transcriptional enhancer necessary for the activation of Hoxd-11 was deleted from the HoxD complex of mice by targeted mutagenesis. While genetic and expression analyses demonstrated the role of this regulatory element in the activation of Hoxd-11 during early somitogenesis, the function of this gene in developing limbs and the urogenital system was not affected, suggesting that Hox transcriptional controls are different in different axial structures. In the trunk of mutant embryos, transcriptional activation of Hoxd-11 and Hoxd-10 was severely delayed, but subsequently resumed with appropriate spatial distributions. The resulting caudal transposition of the sacrum indicates that proper vertebral specification requires a precise temporal control of Hox gene expression, in addition to spatial regulation. A slight time delay in expression (transcriptional heterochrony) cannot be compensated for at a later developmental stage, eventually leading to morphological alterations.

Developmental regulation and asymmetric expression of the gene encoding Cx43 gap junctions in the mouse limb bud.

The Gja1 gene encoding the gap junction connexin 43 (Cx43) is dynamically regulated during limb morphogenesis. Transcript expression is found in many regions of the limb bud known to be important in regulating limb growth and patterning. In the newly emerged limb bud, Gja1 transcripts are first expressed in the ventrodistal margin of the ectoderm, and later transcript expression is localized to the apical ectodermal ridge (AER). Interestingly, transcript expression in the ventrodistal ectoderm is initiated left/right asymmetrically, with some strain backgrounds showing reverse sidedness in the fore vs. hindlimb buds. In legless, a mouse mutant exhibiting both limb and left/right patterning defects, Gja1 transcripts could not be detected in this region. However, in the i.v./i.v. embryo, a mutant with randomization of body situs the same pattern of Gja1 asymmetry was found in the limb ectoderm regardless of body situs. This suggests that Gja1 transcript expression is not directly linked to signaling pathways involved in specification of the left/right axis. In addition to transcript expression in the apical ectodermal ridge, Gja1 transcripts were also found at high levels in the ventral ectoderm. In the limb bud mesenchyme, Gja1 transcripts were distributed in a posterior distal gradient, coincident with tissue known to have polarizing activity. With limb outgrowth and the initiation of limb mesenchyme condensation. Gja1 transcripts were localized in the presumptive progress zone, and in the condensing mesenchyme. In more proximal regions of the limb where mesenchyme differentiation has been initiated, Gja1 transcripts were expressed only in the outer mesenchymal cells comprising the presumptive perichondrium. Further analysis of transgenic mice ectopically expressing Wnt-1 in the limb mesenchyme revealed alterations in the pattern of Gja1 transcript expression in conjunction with the perturbation of limb mesenchyme condensation and differentiation. Together, these findings indicate that Cx43 gap junctions may mediate cell-cell interactions important in cell signaling processes involved in limb growth and patterning.

Regulation of number and size of digits by posterior Hox genes: a dose-dependent mechanism with potential evolutionary implications.

The proper development of digits, in tetrapods, requires the activity of several genes of the HoxA and HoxD homeobox gene complexes. By using a variety of loss-of-function alleles involving the five Hox genes that have been described to affect digit patterning, we report here that the group 11, 12, and 13 genes control both the size and number of murine digits in a dose-dependent fashion, rather than through a Hox code involving differential qualitative functions. A similar dose-response is observed in the morphogenesis of the penian bone, the baculum, which further suggests that digits and external genitalia share this genetic control mechanism. A progressive reduction in the dose of Hox gene products led first to ectrodactyly, then to olygodactyly and adactyly. Interestingly, this transition between the pentadactyl to the adactyl formula went through a step of polydactyly. We propose that in the distal appendage of polydactylous short-digited ancestral tetrapods, such as Acanthostega, the HoxA complex was predominantly active. Subsequent recruitment of the HoxD complex contributed to both reductions in digit number and increase in digit length. Thus, transition through a polydactylous limb before reaching and stabilizing the pentadactyl pattern may have relied, at least in part, on asynchronous and independent changes in the regulation of HoxA and HoxD gene complexes.

Synpolydactyly in mice with a targeted deficiency in the HoxD complex.

The morphogenesis of mammalian digits requires the function of several genes of the HoxD complex during development of limb buds. Using embryonic stem (ES) cells and a site-specific recombination system (loxP/Cre), we have induced a deficiency that eliminates the products of the Hoxd-13, Hoxd-12 and Hoxd-11 genes simultaneously. A Hoxd-11/lacz reporter gene replaced the deleted region in order to monitor the effect of this triple inactivation at the cellular level. Mice homozygous for this deficiency showed small digit primordia, a disorganized cartilage pattern and impaired skeletal mass. These alterations are similar to the defects seen in a human synpolydactyly, suggesting that this syndrome, which is associated with a subtle mutation in HOXD13 (ref. 8), may involve the loss of function of several Hoxd genes. These results indicate the existence of a functional hierarchy among these genes and provide us with an animal model to study human digit malformations.

In vivo targeted mutagenesis of a regulatory element required for positioning the Hoxd-11 and Hoxd-10 expression boundaries.

Vertebrate Hox genes are required for the proper organization of structures along the rostrocaudal axis. Hoxd-11 is expressed in the posterior part of the embryo, up to the level of prevertebra 27, and its expression boundary is reproduced by a Hoxd-11/lacZ transgene. Expression of this transgene anterior to prevertebra 27 is prevented by the silencing activity of a cis-acting element, region IX. Using transgenic mice, we show that Hoxd-11 repression by region IX is necessary to position the sacrum properly. This silencing activity depends on phylogenetically conserved sequences able to bind in vitro retinoic acid receptors and COUP-TFs. ES cells were used to generate mice carrying a subtle mutation that abolishes binding of nuclear receptors to region IX. Mutant mice display an anterior shift of their lumbosacral transition inherited as a codominant trait. In mutant embryos, expression of both Hoxd-11 and Hoxd-10 mRNAs in the prevertebral column is anteriorized. These results illustrate the sharing, in cis, of a single regulatory element in order to establish the expression boundaries of two neighboring Hoxd genes.

Function of posterior HoxD genes in the morphogenesis of the anal sphincter.

Vertebrate 5'-located HoxD genes are expressed in the most caudal part of the digestive tract and their potential functions during gut development have been assessed by gene disruptions. We have inserted reporter lacZ sequences within the Hoxd-12 gene and analysed the morphology of the gut in these mice as well as in Hoxd-13 mutant animals. When homozygous, both mutations induce an important disorganization of the anorectal region. In particular, severe alterations of the smooth muscle layers of the rectum led to defective morphogenesis of the internal anal sphincter. Similarly, Hoxd-12 and Hoxd-13 functionally overlap during digit development. The function of these genes in the morphogenesis of the digestive system as well as their functional evolution are discussed.

Gene transpositions in the HoxD complex reveal a hierarchy of regulatory controls.

Vertebrate Hox genes are activated following a temporal sequence that reflects their linear order in the clusters. We introduced two Hoxd transcription units, labeled with lacZ, to an ectopic 5' position in the HoxD complex. Early expression of the relocated genes was delayed and resembled that of the neighboring Hoxd-13. At later stages, locus-dependent expression in distal limbs and the genital eminence was observed, indicating that common regulatory mechanisms are used for several genes. These experiments also illustrated that neighboring genes can share the same cis-acting sequence and that moving genes around in the complex induces novel regulatory interferences. These results suggest that high order regulation controls the activation of Hox genes and highlight three important constraints responsible for the conservation of Hox gene clustering.