Herault Y, Fraudeau N, Zakany J, Duboule D
Department of Zoology and Animal Biology, University of Geneva, Sciences III, Switzerland.
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.
Zakany J, Gerard M, Favier B, Duboule D
Department of Zoology and Animal Biology, University of Geneva, Sciences III, Switzerland.
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.
Zeller R, Duboule D
European Molecular Biology Laboratory, Heidelberg, Germany. Zeller@EMBL-Heidelberg.de
Molecular and developmental studies of limb pattern formation have recently gained widespread attention. The fact that vertebrate limbs are amenable to both genetic and embryological manipulations has established this model system as a valuable paradigm for studying vertebrate development. Limb buds are polarised along all three major axes and the establishment of the dorso-ventral (DV) polarity is dependent upon cues localised in the trunk, where a DV ectodermal interface is produced by confrontation of dorsal and ventral identities. By analogy to Drosophila imaginal disc development, this interface has been proposed to determine and position an ectodermal organising centre, the Apical Ectodermal Ridge (AER), controlling limb bud outgrowth. Recent fate mapping studies and studies of genes regulating DV limb polarity, AER formation and differentiation suggest, however, that DV patterning and AER induction, though coordinately regulated during limb bud outgrowth, may early on be more dissociated than expected.
Podlasek C A, Duboule D, Bushman W
Urology Department, Northwestern University Medical School, Chicago, Illinois, USA.
The role of the Hox gene Hoxd-13 in postnatal morphogenesis of the male accessory sex organs was examined by correlating the distribution and temporal regulation of expression in the accessory sex organs of postnatal mice with morphologic abnormalities of Hoxd-13-deficient transgenic mice. Previous studies of Hoxd-13 expression in the perinatal period have shown a broad domain of expression in the lower genitourinary tract, with expression in both mesenchyme and epithelium; focal expression was also noted in the epithelium of the nascent ducts of the developing prostate. Quantitative RT-PCR studies of Hoxd-13 expression in the 5 day mouse confirm widespread expression in the accessory sex organs developing from both the Wolffian duct and the urogenital sinus. Expression is down-regulated with age, and a detailed time course of expression in the developing prostate shows that the level of Hoxd-13 expression correlates with morphogenetic activity in the development of the prostate ductal system. Transgenic Hoxd-13-deficient mice display multiple abnormalities in the male accessory sex organs. The most severe abnormalities were observed in organs exhibiting ductal branching during postnatal development and included diminished mesenchymal folding in the seminal vesicles, decreased size and diminished ductal branching in the ventral and dorsal prostate, and agenesis of the bulbourethral gland. We conclude that Hoxd-13 expression in the postnatal period correlates with a period of intense morphogenetic activity in accessory sex organ development and that the function of Hoxd-13 is evidenced by morphologic abnormalities in accessory sex organs of the Hoxd-13-deficient mutant.
Herault Y, Duboule D
Departement de Zoologie et Biologie Animale, Faculte des Sciences, Universite de Geneve, Suisse.
Vertebrate limbs are an amazing example of successful adaptation to various environmental conditions. In higher vertebrates, forelimbs help to fly, swim, walk, dig or grasp, yet their basic structure (the sequence and spatial arrangement of bony elements) is always the same. This implies the existence of a unique developmental strategy for building a limb (a limb plan) that imposes early on a basic scheme, on the top of which subsequent species-specific customizations will occur. The description of such a universal limb plan, hence the idea that the genetic and developmental processes that generate this plan are very ancient, has been controversial for about a century. It is worth asking whether recent discoveries of important genes involved in these processes can bring novel arguments to the debate.
Herault Y, Kondo T, Zakany J, Duboule D
Departement de zoologie et biologie animale, faculte des Sciences, universite de Geneve-Science III, Suisse.
Herault Y, Hraba-Renevey S, van der Hoeven F, Duboule D
Department of Zoology and Animal Biology, University of Geneva, Sciences III, Switzerland.
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 (eve) gene, 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, we produced a null allele of Evx-2. Furthermore, and because Hoxd-13 function is prevalent over that of nearby Hoxd genes, we generated two different double mutant loci wherein both Evx-2 and Hoxd-13 were inactivated in cis. The analysis of these various genetic configurations revealed 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.
Beckers J, Gerard M, Duboule D
Department of Zoology and Animal Biology, University of Geneva, Sciences III, Quai Ernest Ansermet 30, Geneva 4, 1211, Switzerland. duboule@sc2a.unige.ch
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 which may be of importance for the regulation of Hoxd-11 expression. We have used transgenic mice 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.
Zakany J, Duboule D
Department of Zoology and Animal Biology, University of Geneva, Sciences III, Switzerland.
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.
Sordino P, Duboule D, Kondo T
Department of Zoology and Animal Biology, University of Geneva, Switzerland.
Vertebrate Hox genes are required for the establishment of regional identities along body axes. This gene family is strongly conserved among vertebrates, even in bony fish which display less complex ranges of axial morphologies. We have analysed the structural organization and expression of Abd-B related zebrafish HoxA cluster genes (Hoxa-9, Hoxa-10, Hoxa-11 and Hoxa-13) as well as of Evx-2, a gene closely linked to the HoxD complex. We show that the genomic organization of Hoxa genes in fish resembles that of tetrapods albeit intergenic distances are shorter. During development of the fish trunk, Hoxa genes are coordinately expressed, whereas in pectoral fins, they display transcript domains similar to those observed in developing tetrapod limbs. Likewise, the Evx-2 gene seems to respond to both Hox- and Evx-types of regulation. During fin development, this latter gene is expressed as the neighbouring Hox genes, in contrast to its expression in the central nervous system which does not comply with colinearity and extends up to anterior parts of the brain. These results are discussed in the context of the functional evolution of Hoxa versus Hoxd genes and their different roles in building up paired appendages.
Gerard M, Chen J Y, Gronemeyer H, Chambon P, Duboule D, Zakany J
Department of Zoology and Animal Biology, University of Geneva, Sciences III, Switzerland.
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.
Kondo T, Dolle P, Zakany J, Duboule D
Department of Zoology and Animal Biology, University of Geneva, Switzerland.
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.