Spitz F, Gonzalez F, Peichel C, Vogt T F, Duboule D, Zakany J
Department of Zoology and Animal Biology, University of Geneva, Sciences III, 1211 Geneva 4, Switzerland.
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.
Michalik L, Desvergne B, Tan N S, Basu-Modak S, Escher P, Rieusset J, Peters J M, Kaya G, Gonzalez F J, Zakany J, Metzger D, Chambon P, Duboule D, Wahli W
Institut de Biologie Animale, Universite de Lausanne, Batiment de Biologie, CH-1015 Lausanne, Switzerland.
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.
Zakany J, Kmita M, Alarcon P, de la Pompa J L, Duboule D
Department of Zoology and Animal Biology, University of Geneva, Sciences III, Quai Ernest Ansermet 30, 1211 Geneva 4, Switzerland.
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.
Spitz F, Duboule D
Department of Zoology and Animal Biology, University of Geneva, Sciences III, 1211 Geneve 4, Switzerland.
Kmita M, Kondo T, Duboule D
Department of Zoology and Animal Biology, University of Geneva, Sciences III, Geneva, Switzerland.
Mammalian Hox genes are clustered at four genomic loci. During development, neighbouring genes are coordinately regulated by global enhancer sequences, which control multiple genes at once, as exemplified by the expression of series of contiguous Hoxd genes in either limbs or gut. The link between vertebrate Hox gene transcription and their clustered distribution is poorly understood. Experimental and comparative approaches have revealed that various mechanisms, such as gene clustering or global enhancer sequences, might have constrained this genomic organization and stabilized it throughout evolution. To understand what restricts the effect of a particular enhancer to a precise set of genes, we generated a loxP/Cre-mediated targeted inversion within the HoxD cluster. Mice carrying the inversion showed a reciprocal re-assignment of the limb versus gut regulatory specificities, suggesting the presence of a silencer element with a unidirectional property. This polar silencer appears to limit the number of genes that respond to one type of regulation and thus indicates how separate regulatory domains may be implemented within intricate gene clusters.
Duboule D
Kmita M, van Der Hoeven F, Zakany J, Krumlauf R, Duboule D
Department of Zoology and Animal Biology, University of Geneva, Sciences III, 1211 Geneva 4, Switzerland.
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.
Vogt T F, Duboule D
Department of Molecular Biology, Princeton University, New Jersey 08544, USA.
Zakany J, Duboule D
Department of Zoology and Animal Biology, University of Geneva, Sciences III, Switzerland.
Zakany J, Duboule D
Department of Zoology and Animal Biology, University of Geneva, Sciences III, Quai Ernest Ansermet 30, CH-1211 Geneva 4, Switzerland.
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.
Kondo T, Duboule D
Department of Zoology and Animal Biology, University of Geneva, Sciences III, Switzerland.
Vertebrate Hox genes are activated in a spatiotemporal sequence that reflects their clustered organization. While this colinear relationship is a property of most metazoans with an anterior to posterior polarity, the underlying molecular mechanisms are unknown. Previous work suggested that Hox genes were made progressively available for transcription in the course of gastrulation, implying the existence of an element capable of initiating a repressive conformation, subsequently relieved from the clusters sequentially. We searched for this element by combining a genomic walk with successive transgene insertions upstream of the HoxD complex followed by a series of deletions. The largest deficiency induced posterior homeotic transformations coincidentally with an earlier activation of Hoxd genes. These data suggest that a regulatory element located upstream of the complex is necessary for setting up the early pattern of Hox gene colinear activation.
Gerard M, Zakany J, Duboule D
Department of Zoology and Animal Biology, University of Geneva, Sciences III, Switzerland.
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.