Detection of targeted GFP-Hox gene fusions during mouse embryogenesis

The ability to use a vital cell marker to study mouse embryogenesis will open new avenues of experimental research. Recently, the use of transgenic mice, containing multiple copies of the jellyfish gene encoding the green fluorescent protein (GFP), has begun to realize this potential. Here, we show that the fluorescent signals produced by single-copy, targeted GFP in-frame fusions with two different murine Hox genes, Hoxa1 and Hoxc13, are readily detectable by using confocal microscopy. Since Hoxa1 is expressed early and Hoxc13 is expressed late in mouse embryogenesis, this study shows that single-copy GFP gene fusions can be used through most of mouse embryogenesis. Previously, targeted lacZ gene fusions have been very useful for analyzing mouse mutants. Use of GFP gene fusions extends the benefits of targeted lacZ gene fusions by providing the additional utility of a vital marker. Our analysis of the Hoxc13GFPneo embryos reveals GFP expression in each of the sites expected from analysis of Hoxc13lacZneo embryos. Similarly, Hoxa1GFPneo expression was detected in all of the sites predicted from RNA in situ analysis. GFP expression in the foregut pocket of Hoxa1GFPneo embryos suggests a role for Hoxa1 in foregut-mediated differentiation of the cardiogenic mesoderm.

Expression of the Hox gene complex in the indirect development of a sea urchin

Hox complex genes control spatial patterning mechanisms in the development of arthropod and vertebrate body plans. Hox genes are all expressed during embryogenesis in these groups, which are all directly developing organisms in that embryogenesis leads at once to formation of major elements of the respective adult body plans. In the maximally indirect development of a large variety of invertebrates, the process of embryogenesis leads only to a free-living, bilaterally organized feeding larva. Maximal indirect development is exemplified in sea urchins. The 5-fold radially symmetric adult body plan of the sea urchin is generated long after embryogenesis is complete, by a separate process occurring within imaginal tissues set aside in the larva. The single Hox gene complex of Strongylocentrotus purpuratus contains 10 genes, and expression of eight of these genes was measured by quantitative methods during both embryonic and larval developmental stages and also in adult tissues. Only two of these genes are used significantly during the entire process of embryogenesis per se, although all are copiously expressed during the stages when the adult body plan is forming in the imaginal rudiment. They are also all expressed in various combinations in adult tissues. Thus, development of a microscopic, free-living organism of bilaterian grade, the larva, does not appear to require expression of the Hox gene cluster as such, whereas development of the adult body plan does. These observations reflect on mechanisms by which bilaterian metazoans might have arisen in Precambrian evolution.

Organization of the Hox gene cluster in the grasshopper, Schistocerca gregaria

The conserved organization of the Hox genes throughout the animal kingdom has become one of the major paradigms of evolutionary developmental biology. We have examined the organization of the Hox genes of the grasshopper, Schistocerca gregaria. We find that the grasshopper Hox cluster is over 700 kb long, and is not split into equivalents of the Antennapedia complex and the bithorax complex of Drosophila melanogaster. SgDax and probably also Sgzen, the grasshopper homologues of fushi-tarazu (ftz) and Zerknüllt (zen), respectively, are also in the cluster, showing that the non-homeotic Antp-class genes, “accessory genes,” are an ancient feature of insect Hox clusters.

Hox cluster genomics in the horn shark, Heterodontus francisci

Reconstructing the evolutionary history of Hox cluster origins will lead to insights into the developmental and evolutionary significance of Hox gene clusters in vertebrate phylogeny and to their role in the origins of various vertebrate body plans. We have isolated two Hox clusters from the horn shark, Heterodontus francisci. These have been sequenced and compared with one another and with other chordate Hox clusters. The results show that one of the horn shark clusters (HoxM) is orthologous to the mammalian HoxA cluster and shows a structural similarity to the amphioxus cluster, whereas the other shark cluster (HoxN) is orthologous to the mammalian HoxD cluster based on cluster organization and a comparison with noncoding and Hox gene-coding sequences. The persistence of an identifiable HoxA cluster over an 800-million-year divergence time demonstrates that the Hox gene clusters are highly integrated and structured genetic entities. The data presented herein identify many noncoding sequence motifs conserved over 800 million years that may function as genetic control motifs essential to the developmental process.

Expression of a Hox gene, Cnox-2, and the division of labor in a colonial hydroid

We report the isolation and expression of the Hox gene, Cnox-2, in Hydractinia symbiolongicarpus, a hydrozoan displaying division of labor. We found different patterns of aboral-to-oral Cnox-2 expression among polyp polymorphs, and we show that experimental conversion of one polyp type to another is accompanied by concordant alteration in Cnox-2 expression. Our results are consistent with the suggestion that polyp polymorphism, characteristic of hydractiniid hydroids, arose via evolutionary modification of proportioning of head to body column.

A conserved mode of head segmentation in arthropods revealed by the expression pattern of Hox genes in a spider

Chelicerates constitute a basic arthropod group with fossil representatives from as early as the Cambrian period. Embryonic development and the subdivision of the segmented body region into a prosoma and an opisthosoma are very similar in all extant chelicerates. The mode of head segmentation, however, has long been controversial. Although all other arthropod groups show a subdivision of the head region into six segments, the chelicerates are thought to have the first antennal segment missing. To examine this problem on a molecular level, we have compared the expression pattern of Hox genes in the spider Cupiennius salei with the pattern known from insects. Surprisingly, we find that the anterior expression borders of the Hox genes are in the same register and the same relative segmental position as in Drosophila. This contradicts the view that the homologue of the first antennal segment is absent in the spider. Instead, our data suggest that the cheliceral segment is homologous to the first antennal segment and the pedipalpal segment is homologous to the second antennal (or intercalary) segment in arthropods. Our finding implies that chelicerates, myriapods, crustaceans, and insects share a single mode of head segmentation, reinforcing the argument for a monophyletic origin of the arthropods.

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.

The conserved role of Krox-20 in directing Hox gene expression during vertebrate hindbrain segmentation.

Transient segmentation in the hindbrain is a fundamental morphogenetic phenomenon in the vertebrate embryo, and the restricted expression of subsets of Hox genes in the developing rhombomeric units and their derivatives is linked with regional specification. Here we show that patterning of the vertebrate hindbrain involves the direct upregulation of the chicken and pufferfish group 2 paralogous genes, Hoxb-2 and Hoxa-2, in rhombomeres 3 and 5 (r3 and r5) by the zinc finger gene Krox-20. We identified evolutionarily conserved r3/r5 enhancers that contain high affinity Krox-20. binding sites capable of mediating transactivation by Krox-20. In addition to conservation of binding sites critical for Krox-20 activity in the chicken Hoxa-2 and pufferfish Hoxb-2 genes, the r3/r5 enhancers are also characterized by the presence of a number of identical motifs likely to be involved in cooperative interactions with Krox-20 during the process of hindbrain patterning in vertebrates.

Class 3 Hox genes in insects and the origin of zen.

We have cloned, from a beetle and a locust, genes that are homologous to the class 3 Hox genes of vertebrates. Outside the homeobox they share sequence motifs with the Drosophila zerknüllt (zen) and z2 genes, and like zen, are expressed only in extraembryonic membranes. We conclude that the zen genes of Drosophila derive from a Hox class 3 sequence that formed part of the common ancestral Hox cluster, but that in insects this (Hox) gene has lost its role in patterning the anterio-posterior axis of the embryo, and acquired a new function. In the lineage leading to Drosophila, the zen genes have diverged particularly rapidly.

A structural model for a homeotic protein-extradenticle-DNA complex accounts for the choice of HOX protein in the heterodimer.

The genes of the homeotic complex (HOX) encode DNA binding homeodomain proteins that control developmental fates by differentially regulating the transcription of downstream target genes. Despite their unique in vivo functions, disparate HOX proteins often bind to very similar DNA sequences in vitro. Thus, a critical question is how HOX proteins select the correct sets of target genes in vivo. The homeodomain proteins encoded by the Drosophila extradenticle gene and its mammalian homologues, the pbx genes, contribute to HOX specificity by cooperatively binding to DNA with HOX proteins. For example, the HOX protein labial cooperatively binds with extradenticle protein to a 20-bp oligonucleotide that is sufficient to direct a labial-like expression pattern in Drosophila embryos. Here we have analyzed the protein-DNA interactions that are important for forming the labial-extradenticle-DNA complex. The data suggest a model in which labial and extradenticle, separated by only 4 bp, bind this DNA as a heterodimer in a head-to-tail orientation. We have confirmed several aspects of this model by characterizing extradenticle-HOX binding to mutant oligonucleotides. Most importantly, mutations in base pairs predicted to contact the HOX N-terminal arm resulted in a change in HOX preference in the heterodimer, from labial to Ultrabithorax. These results demonstrate that extradenticle prefers to bind cooperatively with different HOX proteins depending on subtle differences in the heterodimer binding site.

Identification of planarian homeobox sequences indicates the antiquity of most Hox/homeotic gene subclasses.

The homeotic gene complex (HOM-C) is a cluster of genes involved in the anteroposterior axial patterning of animal embryos. It is composed of homeobox genes belonging to the Hox/HOM superclass. Originally discovered in Drosophila, Hox/HOM genes have been identified in organisms as distantly related as arthropods, vertebrates, nematodes, and cnidarians. Data obtained in parallel from the organization of the complex, the domains of gene expression during embryogenesis, and phylogenetic relationships allow the subdivision of the Hox/HOM superclass into five classes (lab, pb/Hox3, Dfd, Antp, and Abd-B) that appeared early during metazoan evolution. We describe a search for homologues of these genes in platyhelminths, triploblast metazoans emerging as an outgroup to the great coelomate ensemble. A degenerate PCR screening for Hox/HOM homeoboxes in three species of triclad planarians has revealed 10 types of Antennapedia-like genes. The homeobox-containing sequences of these PCR fragments allowed the amplification of the homeobox-coding exons for five of these genes in the species Polycelis nigra. A phylogenetic analysis shows that two genes are clear orthologues of Drosophila labial, four others are members of a Dfd/Antp superclass, and a seventh gene, although more difficult to classify with certainty, may be related to the pb/Hox3 class. Together with previously identified Hox/HOM genes in other flatworms, our analyses demonstrate the existence of an elaborate family of Hox/HOM genes in the ancestor of all triploblast animals.

Gain of function mutations for paralogous Hox genes: implications for the evolution of Hox gene function.

To investigate the functions of paralogous Hox genes, we compared the phenotypic consequences of altering the embryonic patterns of expression of Hoxb-8 and Hoxc-8 in transgenic mice. A comparison of the phenotypic consequences of altered expression of the two paralogs in the axial skeletons of newborns revealed an array of common transformations as well as morphological changes unique to each gene. Divergence of function of the two paralogs was clearly evident in costal derivatives, where increased expression of the two genes affected opposite ends of the ribs. Many of the morphological consequences of expanding the mesodermal domain and magnitude of expression of either gene were atavistic, inducing the transformation of axial skeletal structures from a modern to an earlier evolutionary form. We propose that regional specialization of the vertebral column has been driven by regionalization of Hox gene function and that a major aspect of this evolutionary progression may have been restriction of Hox gene expression.

Sexage et phylogénie, à partir des gènes CHD (-Z et -W) et COX-1, des oiseaux de proie du Québec et de perroquets d’attrait vétérinaire

Connaître le sexe d’un oiseau est important pour divers domaines notamment pour les vétérinaires, les écologistes ainsi que pour les éleveurs d’oiseaux qui veulent former des couples qui serviront à la reproduction. Plusieurs espèces d’oiseaux, juvéniles et adultes, n’ont pas de dimorphisme sexuel. L’utilisation de l’ADN est une façon rapide de déterminer le sexe à partir d’un échantillon de sang, de muscle, de plumes ou de fèces. Par contre, la méthode devrait être validée pour chaque espèce et idéalement, standardisée. Le premier objectif de cette étude est de développer une méthode de sexage par séquençage des oiseaux à partir des séquences du gène CHD, en utilisant les oiseaux de proie et les perroquets vus en clinique au Québec. Un deuxième objectif est de faire l’identification de l’espèce à sexer, à partir du gène mitochondrial COX-1 et aussi à partir des séquences CHD-Z et CHD-W, utilisés pour le sexage. Un troisième objectif est d’évaluer les séquences sorties (CHD-Z, CHD-W et COX-1) en vue d’une étude phylogénique. Une extraction d’ADN a été effectuée chez 27 espèces de perroquets, 34 espèces d’oiseaux de proie, une corneille (Corvus brachyrhynchos) et un poulet (Gallus gallus). Une amplification par PCR a été exécutée pour les exons partiels 23 et 24 du gène CHD. Le séquençage de cet amplicon permettait de savoir s’il s’agissait d’un mâle (séquence simple CHD-Z) ou d’une femelle (séquences CHD-Z et CHD-W qui se chevauchent). Afin d’avoir des séquences CHD-W distinctes, un sous-clonage a été fait chez les femelles de chaque espèce. De cette manière, les séquences partielles du gène CHD, Z et W, ont été trouvées pour les espèces échantillonnées. Une étude phylogénique a été effectuée avec les séquences de COX-1, CHD-Z et CHD-W grâce au site « Clustal-Omega ». La méthode de sexage des oiseaux par séquençage du gène CHD est standard et efficace. Le gène COX-1 permet une meilleure identification des espèces parentes et le gène CHD-Z est le plus utile pour étudier la phylogénie profonde.

Modularisation épistatique des loci à trait quantitatif associés à la pression artérielle et identification de gènes candidats pour l’hypertension

Problématique: L’hypertension artérielle essentielle, facteur de risque majeur dans le développement des maladies cardiovasculaires, est un trait multigénique complexe dont les connaissances sur le déterminisme génétique nécessitent d’être approfondies. De nombreux loci à trait quantitatif (QTLs); soit des gènes responsables de faire varier la pression artérielle (PA), ont été identifiés chez l’humain et le modèle animal. Cependant, le mystère plane encore sur la façon dont ces gènes fonctionnent ensemble pour réguler la PA. Hypothèse et objectif: Plutôt qu’une addition de QTLs ayant chacun une action infinitésimale sur la PA, une interaction épistatique entre les gènes serait responsable du phénotype hypertendu. Ainsi, l’étude de cette épistasie entre les gènes impliqués, directement ou indirectement, dans l’homéostasie de la PA nous permettrait d’explorer de nouvelles voies de régulation moléculaire en cause dans cette maladie. Méthodes: Via la réalisation de souches congéniques de rats, où un segment chromosomique provenant d’une souche receveuse hypertendue (Dahl Salt Sensitive, SS/Jr) est remplacé par son homologue provenant d’une souche donneuse normotendue (Lewis, LEW), des QTLs peuvent être mis en évidence. Dans ce contexte, la combinaison de QTLs via la création de doubles ou multiples congéniques constitue la première démonstration fonctionnelle des interactions intergéniques. Résultats: Vingt-sept combinaisons au total nous ont menés à l’appréciation d’une modularisation des QTLs. Ces derniers ont été catégorisés selon deux principaux modules épistatiques (EMs) où les QTLs appartenant à un même EM sont épistatiques entre eux et participent à une même voie régulatrice. Les EMs/cascades agissent alors en parallèle pour réguler la PA. Grâce à l’existence de QTLs ayant des effets opposés sur la PA, nous avons pu établir l’ordre hiérarchique entre trois paires de QTLs. Cependant, lorsque cette suite régulatrice ne peut être déterminée, d’autres approches sont nécessaires. Nos travaux nous ont mené à l’identification d’un QTL situé sur le chromosome 16 du rat (C16QTL), appartenant au EM1 et qui révélerait une nouvelle voie de l’homéostasie de la PA. Le gène retinoblastoma-associated protein 140 (Rap140)/family with sequence similarity 208 member A (Fam208a), présentant une mutation non synonyme entre SS/Jr et LEW est le gène candidat le plus plausible pour représenter C16QTL. Celui-ci code pour un facteur de transcription et semblerait influencer l’expression de Solute carrier family 7 (cationic amino acid transporter, y+ system) member 12 (Slc7a12), spécifiquement et significativement sous exprimé dans les reins de la souche congénique portant C16QTL par rapport à la souche SS/Jr. Rap140/Fam208a agirait comme un inhibiteur de la transcription de Slc7a12 menant à une diminution de la pression chez Lewis. Conclusions: L’architecture complexe de la régulation de la PA se dévoile mettant en scène de nouveaux acteurs, pour la plupart inconnus pour leur implication dans la PA. L’étude de la nouvelle voie de signalisation Rap140/Fam208a - Slc7a12 nous permettra d’approfondir nos connaissances quant à l’homéostasie de la pression artérielle et de l’hypertension chez SS/Jr. À long terme, de nouveaux traitements anti-hypertenseurs, ciblant plus d’une voie de régulation à la fois, pourraient voir le jour.