Collinear regulations of vertebrate Hox gene clusters: structural constraints and evolutionary innovations

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Thesis

Reference

Collinear regulations of vertebrate Hox gene clusters: structural constraints and evolutionary innovations

TSCHOPP, Patrick

Abstract

The family of Hox gene transcription factors confers anterior-to-posterior positional information during embryonic development of a wide variety of animal species. In vertebrates, as in many invertebrates, these genes are found in clusters on the chromosome. A Hox gene's relative position within its cluster determines the time and place of gene activation during axial elongation in vertebrates. These phenomena are known as temporal and spatial collinearity, respectively. Across the animal kingdom, themes and variations of this strict correlation exist, as suggested by the recent genome analyses of various species and their embryonic Hox gene expression patterns. However, clearly Hox cluster architecture and gene position inside the complexes have important implications for the transcriptional regulation of this gene family in various developmental contexts.

TSCHOPP, Patrick. Collinear regulations of vertebrate Hox gene clusters: structural constraints and evolutionary innovations. Thèse de doctorat : Univ. Genève, 2011, no. Sc.

4297

URN : urn:nbn:ch:unige-149748

DOI : 10.13097/archive-ouverte/unige:14974

Available at:

http://archive-ouverte.unige.ch/unige:14974

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITE DE GENEVE FACULTE DES SCIENCES Départment de Zoologie et Professeur Denis Duboule Biologie Animale

Collinear Regulations of Vertebrate Hox Gene Clusters:

Structural Constraints and Evolutionary Innovations

THESE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Patrick Tschopp de

Ziefen (BL)

Thèse N° 4297

GENEVE

Atelier de reproduction Uni Mail 2011

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List of Figures and Tables

Figure 2.1. A split HoxD cluster reveals both positive and negative regulations for early expression in the developing trunk………31 Figure 2.2. Transgene scanning of the HoxD cluster……….33 Figure 2.3. Expression onset in the trunk depends on the respective distance to the telomeric extremity……….………34 Figure 2.4. Premature activation of posterior Hoxd genes follows the wild type progression in tissue specificity……….………….…35 Figure 2.5. Spatial collinearity is independent of the timing of transcriptional onset.

……….……….…………...37 Figure 2.6. Attenuated transcription for Hox genes moved towards the centromeric end of the cluster………...………...38 Figure 2.7. A two-phases model for the establishment of temporal and spatial collinearities of Hoxd genes in the trunk……….40 Figure 2.S1. Phenotypic alterations in the axial skeleton of mice with a split HoxD cluster……….………..43 Figure 3.1. A centromeric inversion, which separates a Hoxd11/lacZ transgene targeted right upstream of the HoxD cluster………49 Figure 3.2. Reduced number of skeletal elements in Inv mutant animals…………..50 Figure 3.3. Up-regulation of posterior Hoxd gene expression in the presomitic mesoderm of Inv mutant embryos………...……….…51 Figure 3.4. Zeugopodal elements are shortened in Inv animals……….….53 Figure 3.5. Expansion of zeugopodal expression domains of posterior Hoxd genes at later stages of limb development………...…………..54 Figure 3.S1. Schematic of the STRING approach………..59 Figure 3.S2. Phenotypic alterations in the distal limb of homozygous Inv animals...60 Figure 4.1. Probing the spinal cord’s transcriptional HoxD profile………65 Figure 4.2. Two transcriptional blocks define HoxD spatial collinearity in the spinal cord…...……….………...66 Figure 4.3. HoxD cluster deletion analysis reveals regulatory autonomy of the two transcriptional blocks………...………67 Figure 4.4. Cluster-internal deletions lead to ectopic expression of posterior Hoxd genes…….……….………...68 Figure 4.5. Ectopic HOXD10 function and its associated patterning phenotypes…..69 Figure 4.6. Whole-genome transcriptome analysis of the brachial Del(4-9) spinal cord……….………..71 Figure 4.7. A series of LacZ transgenes covering the HoxD complex………73 Figure 4.8. Hoxd genes’ transcriptional profiles in trans………74 Figure 4.9. Two transcriptional blocks set up HoxD spatial collinearity in the spinal cord……….………..76 Figure 4.S1. Spinal cord transcriptional activity in a 2Mb genomic region surrounding the murine HoxD cluster………..80 Figure 4.S2. Changes in Hoxd gene expression patterns after cluster-internal deletions……….………..81 Figure 4.S3. Generic transcriptional response of two targeted Hoxd transgenes at various positions inside the HoxD complex……….82

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Figure 4.S4. Spatial and temporal specificities of ectopic Hoxd10 expression in

Del(4-9) homozygous mutant embryos………83

Figure 4.S5. Hoxd10^high and Hoxc9^high cells tend to sort out from one another……..84

Figure 5.1. Functional diversification of vertebrate paralogous gene clusters via the evolution of specific global regulations……….……..87

Figure 5.2. Phenotypic rescue of Hoxd loss-of-function in limbs by Hoxc genes under the control of the HoxD regulatory landscape.……….88

Figure 5.3. Hoxc genes can functional substitute for Hoxd genes during digit development.……….………...90

Figure 6.1. Regulatory evolution at the murine HoxD cluster.………...97

Figure 7.1. Linear representation of the plasmids used for pronuclear injection of random Hoxdlac transgenes………...………103

Table I. Primer combinations for PCR-genotyping of new alleles………...105

Table II. Probes used for Southern blot-analyses……….105

Table III. Southern blot genotyping strategies for new alleles……….106

Table IV. Southern blot genotyping strategies for random transgenes……….106

Table V. Probes used for whole mount in situ hybridization………107

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Summary

The family of Hox gene transcription factors confers anterior-to-posterior positional information during embryonic development of a wide variety of animal species. In vertebrates, as in many invertebrates, these genes are found in clusters on the chromosome. A Hox gene’s relative position within its cluster determines the time and place of gene activation during axial elongation in vertebrates. These phenomena are known as temporal and spatial collinearity, respectively. Across the animal kingdom, themes and variations of this strict correlation exist, as suggested by the recent genome analyses of various species and their embryonic Hox gene expression patterns. However, clearly Hox cluster architecture and gene position inside the complexes have important implications for the transcriptional regulation of this gene family in various developmental contexts. Ever since the discovery of this conserved gene family, genetic tools have been and are being developed to experimentally address this intricate interrelationship between cluster structure and gene regulation.

For some time now, mechanistic relationships between temporal and spatial collinearities have been suggested, such that the latter would be a consequence of the first. While this view has found support in the study of Hox gene regulation in the early limb bud, where a strong correlation between timing of activation and anterior to posterior extent of expression exists, the situation in the primary, rostral-to-caudal body axis appears to be more complex. Chapter 2 takes advantage of an extensive collection of genetic modifications at the murine HoxD complex to elucidate potential interdependencies of the two collinearities during trunk elongation, the ancestral place of action for this gene family.

During vertebrate evolution, Hox genes have been co-opted to pattern a variety of novel embryonic structures. Often, this diversification has been achieved by the acquisition of new expression specificities, such as for the limbs in the case of Hoxd genes. Chapter 3 addresses the question as to what extent the action of such newly evolved control elements could have been constrained by ancestral regulations, put in place to ensure correct gene activation in the primary body axis.

Since important aspects of spatial collinearity can be accomplished in the absence of gene clustering, temporal collinearity has been proposed as the main evolutionary constraint to keep Hox genes clustered. However, the preservation of Hox complexes to some degree in many animal species that do not display temporal collinearity, suggests a functional importance of gene clustering for spatial collinearity as well. In chapter 4, tiling array and transgenic analyses are used to investigate the potential molecular mechanisms underlying this apparent evolutionary constraint.

Two rounds of whole-genome duplications have paved the way for the astounding morphological diversification that has accompanied the transition from early chordates to vertebrates. The resulting genetic redundancy has been repeatedly exploited to expand gene functions, either by evolving protein structure or gene regulation. In the case of the murine HoxD cluster, the appearance of an extended regulatory landscape is thought to be at the evolutionary origin of digits. A genetic in vivo approach is developed in chapter 5, to exchange the entire upstream regulatory regions of two twin Hox complexes, HoxC and HoxD. The resulting allele is used to test whether the impact of such novel expression specificities is not only required, but also sufficient to give rise to evolutionary innovations like digits.

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Résumé en français

Les gènes Hox sont une famille de facteurs de transcription, qui confèrent l’information de position antéro-postérieur lors du développement embryonnaire de nombreuses espèces animales. Chez les vertébrés, ainsi que chez de nombreux invertébrés, ces gènes sont regroupés en clusters sur le chromosome. La position relative d’un gène Hox au sein du cluster détermine le moment et l’endroit d’activation de ce gène, lors de l’élongation axiale chez les vertébrés. Ces phénomènes sont appelés colinéarité temporelle et spatiale, respectivement. A travers le règne animal, plusieurs thèmes et variations de cette colinéarité stricte existent.

Ceci a été notamment suggéré par les récentes analyses génomiques de diverses espèces, et de leurs patterns d’expression des gènes Hox au niveau embryonnaire.

Cependant, l’architecture du cluster des gènes Hox, ainsi que la position des gènes à l’intérieur des complexes, ont d’importantes implications dans la régulation transcriptionnelle de cette famille de gènes, et ce dans de nombreux contextes développementaux. Depuis la découverte de cette famille de gènes très conservée, des outils génétiques ont et sont développés pour adresser expérimentalement cette relation complexe, entre la structure du cluster et sa régulation transcriptionnelle.

Il était généralement admis qu’il existait une relation causale entre les colinéarités temporelle et spatiale, telle que la dernière serait une conséquence de la première. Cette hypothèse était supportée par les études de la régulation des gènes Hox dans la formation des membres, où une forte corrélation entre le temps d’activation et l’extension antéro-postérieur existe. Toutefois, la situation au niveau de la formation de l’axe rostro-caudale du corps apparaît plus complexe. Dans le Chapitre 2, une importante collection de modifications génétiques dans le complexe murin des gènes HoxD est utilisée, afin d’élucider les interdépendances potentielles entre les deux colinéarités. Cette question est spécifiquement traitée au niveau de l’élongation du tronc, qui est la place ancestrale d’action des gènes de cette famille.

Lors de l’évolution des vertébrés, les gènes Hox ont été recrutés pour façonner une variété de nouvelles structures embryonnaires. Souvent, cette diversification a mené à l’acquisition de nouvelles spécificités d’expression, comme pour les membres dans le cas des gènes HoxD. Le Chapitre 3 adresse comment ces nouveaux éléments régulatrices auraient pu être contraints par des contrôles ancestraux, qui avaient été mis en place pour assurer une correcte activation génique, dans la formation de l’axe principal du corps.

Sachant que de nombreux aspects de la colinéarité spatiale peuvent être accomplis en l’absence d’un regroupement stricte des gènes Hox, la colinéarité temporelle a été proposée comme la contrainte principale pour maintenir les gènes Hox rassemblés sur le chromosome. Toutefois, la conservation partielle de complexes Hox chez certaines espèces animales qui ne montrent pas de colinéarité temporelle, suggère une importante fonction de la structure du cluster pour la colinéarité spatiale également. Des analyses de souris transgéniques et de ‘tiling array’ sont utilisés dans le Chapitre 4, afin d’étudier les mécanismes moléculaires potentiels sous-jacents à cette apparente contrainte évolutive.

Deux séries de duplications du génome entier ont ouvert la voie pour une diversification morphologique étonnante, qui a accompagné la transition des premiers chordés aux vertébrés. La redondance génétique résultante a été utilisée pour diversifier les fonctions des gènes, soit par l’évolution de la structure des protéines,

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soit par la modification de la régulation des gènes. Dans le cas du cluster HoxD, l’apparition d’un tel paysage régulatif a été proposée pour être à l’origine de l’évolution des doigts. Une approche génétique in vivo est développée dans le Chapitre 5, afin de pouvoir échanger des régions entières entre deux complexes Hox, notamment entre HoxC et HoxD. L’allèle résultant a été ensuite utilisé pour tester si de nouvelles spécificités d’expression ne sont pas seulement nécessaires, mais aussi suffisantes pour engendrer des innovations évolutives, telles que le développement des doigts.

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Chapter 1

General Introduction

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1.1 HOX GENE CLUSTERING AND COLLINEARITY

1.1.1. Hox genes and axial pattering

When looking in detail at the body plan of many bilaterian animals, a clear serially repeated, metameric organization becomes apparent in their primary body axis (Goodrich, 1913; De Robertis, 2008b; Couso, 2009). Different members of the familiy of Hox gene transcription factors play essential roles in specifying the identity of these segments at distinct anterior-to-posterior levels. The founding members were identified in the fruit fly Drosophila melanogaster, genetically mapping to spontaneous mutations that cause spectacular phenotypes by mis-specifying body segments. Such transformations in segment identity are also called homeotic mutations (Bateson, 1894), which lent their name to the shared protein motif of all Hox genes, the DNA-binding homeodomain (McGinnis et al., 1984). Hox genes are conserved throughout the animal kingdom, and genetic experiments using both gain- and loss-of-function approaches have firmly established their role also for vertebrate patterning (Krumlauf, 1994). Additionally, both spontaneous and engineered mutations affecting only the regulation of Hox genes have been shown to induce dramatic morphological changes, and hence their transcriptional control has to be tightly controlled. In numerous animal species, Hox genes are found clustered at a particular site in the genome, a type of organization that has important implications for their regulation as will be discussed later on.

1.1.2. Hox gene clustering: an evolutionary perspective

The clustered organization of Hox genes was first described in Drosophila melanogaster, where a total of eight Hox genes are distributed over two loci in the fly genome, namely the Antennapedia and the Bithorax complexes (Lewis, 1978;

Kaufman et al., 1980). This clustering of Hox genes into distinct genomic complexes was later on found conserved in higher vertebrates (Duboule and Dolle, 1989;

Graham et al., 1989). Ever thereafter, the discovery of Hox genes in new animal species was often automatically associated with the presence of a gene complex as well. However, the recent genome analyses of a variety of different non-classical model organisms has uncovered an unexpected diversity in the degree of clustering, as well as the number of Hox genes per species.

Early during animal evolution, before the split of cnidarians and bilaterians, a hypothetical ProtoHox cluster is thought to have contained between two to four genes, originating from tandem-duplications in cis of a single, ancestral ProtoHox gene (Garcia-Fernandez, 2005). This mode of gene amplification would also explain the clustered organization of this gene family to begin with, and why already in the cnidarian lineage Hox genes are found clustered (Chourrout et al., 2006; Ryan et al., 2007). The subsequent whole cluster duplication, along with a split of the resulting sister complexes, resulted in the generation of a classical Hox cluster and a ParaHox cluster (Brooke et al., 1998; Ferrier and Holland, 2001). Further gene duplications diversified the repertoire of each complex. Based on the Hox complement found nowadays in extant bilaterian phyla, it has been proposed that their last common ancestor must have possessed a Hox complement of at least eight Hox genes, as well

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as one Evx gene, likely organized in a single cluster (de Rosa et al., 1999; Garcia- Fernandez, 2005). From this putative evolutionary ground-state, however, a realm of different forms of Hox gene organizations have evolved, some of them far from this classical textbook depiction.

As mentioned above, the clustered organization of Hox genes was first described in Drosophila (Lewis, 1978), but only later on was it discovered that in fact two sub- cluster were present, likely stemming from a splitting event early in the dipteran lineage. In fact, splitting of the Hox complex had occurred at various positions in the order Diptera, likely due to different inversion and transposition events (Negre and Ruiz, 2007). In Drosophila melangaster the separation took place between the genes Antennapedia and Ultrabithorax, whereas in Drosophila virilis this had happened between Ultrabithorax and abdominal-A (Von Allmen et al., 1996; Lewis et al., 2003). An even greater degree of Hox disorganization has recently been reported for the urochordate Oikopleura dioica, as well as for the flatworm Schistosoma mansoni.

In both species, no clear clustering of Hox genes is present anymore, and the genes are found scattered throughout the genome (Seo et al., 2004; Pierce et al., 2005). In other lineages Hox clustering was maintained, although the ordering of their genes is not as strict as in the vertebrate clade. In the sea urchin Strongylocentrotus purpuratus, eleven Hox genes and one Evx gene are present on a single contig spanning 700kb. However, important genomic rearrangements must have occurred during the evolutionary history of this species, since gene order and transcriptional orientation are largely disturbed with respect to the vertebrate homologs. Probably this is due to an inversion event that translocated the anterior half of genes to the 5’

end of the cluster (Cameron et al., 2006).

Current evolutionary scenarios place a single Hox cluster at the advent of vertebrate evolution, a situation potentially reflected today by the cluster of the cephalochordate Amphioxus (Garcia-Fernandez and Holland, 1994). Gene duplication further expanded this species Abd-B-related gene repertoire such that the cluster now contains a total of 14 Hox genes, as well as two Evx genes at its 5’ end (Ferrier et al., 2000). Whether or not a Hox14 gene was part of the ancestral cluster is still debated, but its presence in both coelacanth and elephant shark could argue for the secondary loss of the gene in higher vertebrates (Powers and Amemiya, 2004; Venkatesh et al., 2007). This single, ancestral Hox complex was subsequently amplified in the vertebrate lineage to a total of four paralogous clusters commonly referred to as HoxA to HoxD, that likely originated from two rounds of whole genome duplications (Ravi et al., 2009). Intermediates of this process can already be found in the most primitive vertebrates, such as the sea lamprey Petromyzon marinus, member of the superclass Agnatha, where at least three clusters are present, depending on the evolutionary scenario favored (Force et al., 2002; Irvine et al., 2002). In fish, an additional round of whole genome duplication resulted in seven Hox clusters in crown teleostei, the HoxDb cluster in zebrafish having been reduced to single remaining microRNA (Amores et al., 1998; Woltering and Durston, 2006). Interestingly however, the total number of 48 Hox genes present is only slightly higher than in species with just four Hox complexes, arguing for an increased gene loss due to the greater level of genetic redundancy.

Today, in higher vertebrates such as mammals, a total of 39 Hox genes are distributed over four clusters, namely HoxA, HoxB, HoxC and HoxD (Garcia- Fernandez, 2005; Lemons and McGinnis, 2006). Genes of each complex are all transcribed from the same DNA strand, giving the clusters an intrinsic 5’ to 3’

polarity. Each clusters spans between 100 and 110 kilobases and is made up from 9 to

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to 11 Hox genes (Duboule, 2007). Based on sequence similarities, members of the different clusters can be classified in groups of paralogy, from 1 to 13, a gene order conserved in all vertebrate complexes. There is a considerable amount of functional redundancy amongst paralogous genes that allowed for a high degree of genetic flexibility, as shown by gene coding sequence swapping experiments (Greer et al., 2000). Paralogous genes have been lost repeatedly, such that only group 4, 9 and 13 are still present in all four modern vertebrate Hox clusters. This apparent evolutionary flexibility still seems to account for the ongoing modifications of each species Hox gene complement, even within vertebrates, as exemplified by the loss of Hoxb13 and Hoxd12 in Xenopus or the presence of Hoxc3 in squamates, amphibians and some species of fish (Hoegg and Meyer, 2005; Di-Poi et al., 2009). Remaining paralogs can thereafter be sub-functionalized, at least one maintaining the ancestral function, while others could acquire novel functions via e.g. the adoption of new expression specificities (see chapter 1.3.3, chapter 5 and Deschamps, 2007).

When looking at the overall organization of vertebrate Hox clusters as compared to their invertebrate counterparts, the degree of organization seems to be by far the highest (reviewed in Duboule, 2007). Their size is considerably more compacted than in other species, and no non-Hox genes can be found interspersed in the complex, as is for example the case in both Drosophila and sea urchin. All Hox genes are in the same transcriptional orientation and literally no repetitive sequences can be found inside the clusters, arguing for strong selective pressure to exclude the invasion by mobile genetic elements at the base of vertebrate evolution (Fried et al., 2004;

Amemiya et al., 2008). An exception from this strict exclusion of repetitive elements has recently been reported for both squamates and caecilieans (Di-Poi et al., 2009;

Mannaert et al., 2010). Species of the two orders can show extensive deviations from the “classical” vertebrate body plan. Changes in Hox gene expression, potentially originating from this altered Hox complex organization, have been put forward as a possible explanation for this observed morphological diversity (Di-Poi et al., 2010).

Nevertheless, given the apparent flexibility of Hox gene clustering throughout evolution, it is actually quite surprising to see vertebrates having the highest degree of order in their Hox complexes, whereas a considerable amount of disorganization has been found in all other bilaterians investigated so far. Since in species with a split Hox cluster, genes of all groups of paralogy can be found in their sub-clusters following the same order as in their vertebrate counterparts, it is likely that a single, continuous Hox complex existed at the base of the bilaterian radiation (Garcia-Fernandez, 2005;

Ryan et al., 2007). With respect to the ancestral state of Hox clustering, two potential scenarios have been put forward to discuss the apparent paradox surrounding the increased organization of vertebrate Hox genes: Either the tightly structured form of Hox clustering found nowadays in vertebrates represents the evolutionary ground- state that has deteriorated repeatedly during the evolution of all other lineages, or, alternatively, a loosely organized, single Hox complex was specifically consolidated and compacted in the case of vertebrates (Duboule, 2007). To support the latter scenario, an increased regulatory complexity at vertebrate Hox loci has been put forward as a potential evolutionary constraint to compact the clusters, including both newly acquired expression specificities, as well as the ancient collinear transcription mechanism(s).

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13 1.1.3 Collinearity

One of the most fascinating aspects of Hox gene regulation is the phenomenon of collinearity (Kmita and Duboule, 2003). First discovered in Drosophila, it describes the correspondence of gene order within the complex to the anterior-to-posterior (AP) distribution of their transcripts, as well as their domains of action, along the main body axis (Lewis, 1978). Subsequently, the same “spatial collinearity” was found to occur in vertebrates, where genes of all four clusters show expression territories along the AP axis according to the relative position they occupy in their respective complexes (Gaunt et al., 1988; Duboule and Dolle, 1989; Graham et al., 1989).

Accordingly, genes at one extreme of the cluster, the 3’ end, are expressed in more anterior structures than their counterparts at the 5’ end, leading to the denomination of

“anterior genes” and “posterior genes”, respectively. During vertebrate embryogenesis, Hox genes show a collinear distribution of their transcripts in a variety of tissues along the primary body axis, such as e.g. in the paraxial mesoderm, the developing digestive system or the spinal cord (Gaunt et al., 1988; Graham et al., 1989; Yokouchi et al., 1995; see also Chapter 4). Additionally, similar correspondences of gene order and staggered expression domains are also found in secondary body axes, such as the external genitalia or the limb (Dolle et al., 1989;

Dolle et al., 1991; Haack and Gruss, 1993; Nelson et al., 1996).

Whatever the underlying regulatory mechanism, such coordinated response in gene activity to the genomic organization provides a tempting way of explaining as to what evolutionary constraints might have kept these genes clustered. Spatial collinear Hox transcript distribution in the primary body axis has been reported in all bilaterian species investigated so far, however, this does not always depend on a strictly clustered organization of the genes themselves (e.g. C. elegans; Wang et al., 1993). In the most extreme cases such trans-collinearity, i.e. the collinear distribution according to groups of paralogy rather then gene position in an actual cluster, can occur with all Hox genes randomly distributed throughout the genome (Seo et al., 2004; Duboule, 2007). Obviously, such apparent lack of interdependence of clustering and spatial collinearity is at odds with claims that the phenomenon might represent a major constraint on keeping Hox gene linked together. A similar paradoxical situation had already been described for randomly integrated Hox transgenes in the mouse. There again, relatively short genomic sequences surrounding single Hox genes were able to reproduce the major aspects of the endogenous gene’s spatial transcript distribution, even when placed outside of its originally clustered context (Puschel et al., 1991;

Whiting et al., 1991).

However, the slight differences in the temporal dynamics of transgene activation, as compared to the endogenous genes, have led to the notion that the presence of a cluster could potentially be required for the temporal fine-tuning during the activation process. Indeed, apart from the spatial distribution, the temporal progression of Hox gene activation during vertebrate embryogenesis also follows the gene order inside the complexes, a phenomenon not found in Drosophila. This “temporal collinearity”

is observed in all vertebrates, and can be found in both primary and secondary body axes (Dolle et al., 1989; Deschamps and van Nes, 2005). In the primary body axis, the trunk, Hox genes become sequentially activated in a 3’ to 5’ fashion during a time window spanning approximately embryonic day 7.5 (E7.5) to E9. Likewise, after the appearance of the limb bud at around E9, a similar wave of activation is observed for

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Hoxd genes in the secondary body axis, as well as for the 5’-located Hoxa genes (Dolle et al., 1989; Haack and Gruss, 1993).

Due to the progressive growth dynamics of both structures, a causal link has been proposed between the two types of collinearities; temporal collinearity acting as a molecular clock that would fix the rostral limit of expression, and hence spatial collinearity, by timing the activation process of each gene to the moment the respective segment would form (Duboule, 1994). During early limb patterning, indeed, heterochronies in the temporal activation of Hoxd genes translate into concomitant changes in spatial transcript distribution (Tarchini and Duboule, 2006).

The situation in the primary body axis, however, is strikingly different, as no coherent impact upon spatial collinearity could be documented when changing the onset of Hoxd gene expression, thereby also questioning the evolutionary relationship of the two types of collinearities (see Chapter 2 and Tschopp et al., 2009).

Nevertheless, what seems to be clear is that the occurrence of temporal collinearity, no matter in which species or embryonic context, requires the presence of an intact genomic cluster. Animals outside the vertebrate clade reported to display temporal collinearity all possess a single, intact Hox complex, and hence temporal collinearity has been proposed as the main evolutionary constraint in keeping these genes clustered (Monteiro and Ferrier, 2006). An intriguing possibility is that the need to activate Hox genes in a temporal progression could be intrinsically linked to a species mode of development. Vertebrates elongate their primary body axis by a step- wise addition of body segments in a posterior growth zone, and premature activation of Hox genes can lead to posterior transformations of skeletal elements or, even more dramatically, severe truncations of the animal (see chapter 3 and Gerard et al., 1996;

Young et al., 2009). Accordingly, species that show drastically different modes of development might have lost such constraint on the temporal orchestration of gene activation, and consequently the need to maintain their Hox genes clustered.

Examples include a fixed cell lineage, as found in C. elegans and the urochordate Oikopleura mentioned above, or the rapid early development in long germ band insects such as Drosophila, where the structural constraints imposed by temporal collinearity might even turn out to be detrimental to the process (Ferrier and Holland, 2002; Negre et al., 2005; Duboule, 2007).

For any evolutionary novel form of global gene regulation, it is likely that it will also favor the maintenance of a clustered organization of its targets, once put in place (Deschamps, 2007). At the same time, this type of regulation also implies a graded, and hence collinear response based on gene order in the complex as the most parsimonious solution for the resulting transcriptional output. Such type of secondary collinearities can be observed e.g. for Hoxd genes in the developing digits and the external genitalia in the phenomenon of “quantitative collinearity”. Hoxd13, the gene lying closest to the enhancer sequences responsible for gene activation in the autopod and the genital tubercle, shows the highest levels of steady-state mRNAs, with Hoxd genes lying further inside the complex being transcribed with progressively decreasing efficiency (Dolle et al., 1989; Dolle et al., 1991). Gene order inside the cluster, and not the nature of each gene’s promoter, is the key determining factor for this transcriptional response, as genomic rearrangements in the posterior HoxD complex can easily relocate the enhancer output (Kmita et al., 2002a). A mechanistic explanation involving initial enhancer contacting outside the complex and subsequent promoter scanning has recently been put forward, based on a combination of experimental, quantitative data-sets and mathematical modeling (Montavon et al., 2008).

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15 1.2 HOX GENE FUNCTION

1.2.1 Homeotic transformations

As outlined above, one of the hallmarks of Hox gene function is their ability to give rise to so-called homeotic transformations, thereby changing the identity of body segments. In both invertebrates and vertebrates, loss-of-function of a given Hox gene can lead to anterior transformations, i.e. a posterior segment usually patterned by the affected Hox gene adopts a more anterior fate. Vice versa, the ectopic expression of a Hox gene at more rostral levels than normal can impose a posterior fate upon these anterior segments (Akam, 1987; Krumlauf, 1994). Originally identified by a set of spontaneously occurring mutations in Drosophila, the systematic use of molecular genetic tools has proven the near-universal application of these principles during invertebrate and vertebrate embryogenesis. Along with the advent of gene targeting techniques in murine embryonic stem cells, each individual of the 39 Hox genes has been genetically inactivated, either by itself or in combination. In parallel, Hox gene overexpression by classical transgenesis or changes in their expression patterns due to engineered regulatory mutations, have generated a wealth of information regarding their function, some of which will be reviewed in the following paragraphs.

1.2.2 Hox gene function in the primary body axis of vertebrates

The segmented nature of the vertebrate bodyplan is most apparent in its internal skeletons, made up of the vertebral column in the primary body axis. During embryogenesis, the precursors of the individual vertebrae are laid down as a series of seemingly homogenous structures called the somites. Their identity and later anatomical appearance are to a large extent determined by the combinatorial action of HOX proteins they express (the “Hox code”; Kessel and Gruss, 1991). Changes in Hox gene expression patterns during evolution coincide with the shift of anatomical boundaries in the skeleton of different species, giving further support to their instrumental role in vertebrae patterning (Burke et al., 1995). Gene action follows a spatial collinear distribution, such that inactivation of genes on one extremity of the cluster affect more anterior structures than genes lying on the opposite, posterior side of the complex. Targeted mutations in group 3 and 4 genes lead to various alterations in the cervical part of the axial skeletons, causing anterior transformations of cervical vertebrae C1, C2 and C3 (Condie and Capecchi, 1993; Ramirez-Solis et al., 1993;

Horan et al., 1995), and can also impose such patterning function when expressed ectopically (Lufkin et al., 1992). Genes of the middle groups of paralogy can affect the thoracic skeleton (Le Mouellic et al., 1992), whereas mutations in genes belonging to the posterior, Abd-B-related class transform the lumbo-sacral and caudal regions (Davis and Capecchi, 1994; Fromental-Ramain et al., 1996a; Godwin and Capecchi, 1998). Although Hox genes are usually expressed from their anterior boundary onwards throughout the body axis to the posterior end, the phenotypic consequences of gene inactivations are usually confined to the anterior limits of expression. At more caudal levels, posterior genes that are expressed in overlapping domains dominantly impose their patterning function over their anterior counterparts, an effect termed

“posterior prevalence” (Duboule and Morata, 1994). This dominant function of posterior genes can also help to explain the posterior transformation phenotypes

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observed in various gain-of-function alleles, by overexpressing posterior Hox genes using either heterologous promoters in classical transgenes or targeted regulatory mutations at endogenous gene loci (Gerard et al., 1996; Carapuco et al., 2005).

Another important aspect when discussing Hox gene function in the primary body axis is the great degree of functional redundancy amongst members of the same group of paralogy. Genes of all four complexes are expressed in largely similar patterns and paralogous HOX proteins often display an almost equivalent function as shown by swapping experiments, with the small differences between paralogs attributed to subtle changes in expression specificities (Greer et al., 2000; Tvrdik and Capecchi, 2006). The resulting buffering capacity of this redundant system is probably most impressively illustrated by an allelic series removing all six alleles of the three paralogous Hox10 genes, namely Hoxa10, Hoxc10 and Hoxd10. While in the absence of five of the six alleles a largely normal axial skeleton is formed, removing the last remaining Hox10 copy leads to the complete anterior transformation of the lumbar region into a thoracic fate, all vertebra now bearing ribs (Wellik and Capecchi, 2003).

In the central nervous system of vertebrates, only the eight rhombomeres (r1 to r8) of the hindbrain show a clearly segmented morphology apparent from the outside, even though only transiently during embryogenesis. Indeed, Hox genes play important roles in specifying and maintaining this segmented structure. Genetic inactivation of Hoxa1 leads to a loss of r4 and a concomitant reduction of both r4 and r6 (Lufkin et al., 1991; Carpenter et al., 1993), while in Hoxa2 and Hoxa2/Hoxb2 double mutants incorrect boundaries between r1 and r4 are formed (Gavalas et al., 1997; Davenne et al., 1999). Additionally, cell-type specification and neuronal migration defects have been reported in the hindbrain of Hoxb1 mutant mice (Studer et al., 1996).

The spinal cord, on the other hand, appears morphological largely unsegmented from the outside along its rostral-to-caudal axis. At the cellular and molecular level, however, different neuronal populations occupy specific anterior-posterior as well as dorso-ventral positions, thereby determining functionally distinct columns of motor- and interneurons (Landmesser, 1978; Hollyday, 1980). In the developing vertebrate spinal cord, two opposing morphogen gradients emanating from the neighboring axial mesodermal tissue, namely retinoids at the anterior and FGFs and Gdf11 at the posterior end, have been shown initiate the overlapping HoxB and HoxC expression patterns that coincide with the columnar sub-divisons (Ensini et al., 1998; Liu et al., 2001; Bel-Vialar et al., 2002). The functional importance of Hox genes in specifying these columns has been shown in vivo using both overexpression approaches in chicken and genetic studies in mice. Ectopic expression of Hox6, Hox9 and Hox10 proteins in the chicken neural tube is sufficient to induce the columnar fates normally associated with their endogenous expression domains, as judged by columnar marker gene expression and motor neuron connectivity (Dasen et al., 2003; Shah et al., 2004;

see chapter 4). Additionally, cross-repressive interactions amongst Hox genes have been proposed to refine their initial expression boundaries (Dasen et al., 2003). In mice, the genetic inactivation of group 10 genes leads to defects in patterning and nerve projections of the lateral motor column (LMC) characteristic at lumbar levels (Carpenter et al., 1997; Wahba et al., 2001; Tarchini et al., 2005), whereas the absence of a single Hox9 protein, namely Hoxc9, leads to an extension of an LMC fate throughout the entire thoracic region (Jung et al., 2010). For anterior Hox genes potentially determining LMCs at brachial levels, no clear alterations in columnar specification has been reported for single gene inactivations, likely reflecting a higher degree of redundancy at these anatomical positions (Jung et al., 2010). Inside these

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motor neuron columns, different combinations of Hox genes are expressed in discrete pools that they help to functionally specify, thereby generating an anatomical correct and diversified map of neuronal connections innervating the different muscles of the limb (Dasen et al., 2005). At the thoracic levels, the median and hypaxial motor columns (MMC and HMC) show greatly reduced neuronal diversity, probably reflecting the simplified task to innervate the body wall when compared to limbs.

Based on the observation that the genetic inactivation of an essential Hox co-factor, FoxP1, reverts all LMC neurons to an HMC-like ground-state, it was proposed that the co-option of Hox proteins into discrete pools was an essential step for the diversified appendage innervation during tetrapod evolution (Dasen et al., 2008;

Rousso et al., 2008).

Hox genes are also important to pattern a variety of internal organs. The combined inactivation of Hoxa11, Hoxc11 and Hoxd11 leads to a complete agenesis of the kidney (Wellik et al., 2002). This is phenocopied by the ectopic expression of Hoxd13, likely overruling the patterning activity of group 11 genes in a posterior prevalent manner (Kmita et al., 2000). Members of the HoxD complex have also been shown to pattern the digestive tract, following the rules of spatial collinearity: while the in cis deletion of Hoxd4 to Hoxd13 affects the formation of the smooth muscle layer at the ileocaecal sphincter, inactivation of Hoxd12 and Hoxd13 only leads to malformations at more posterior levels, namely the anal sphincter (Kondo et al., 1996;

Zakany and Duboule, 1999). Ectopic expression of posterior genes can lead to agenesis of the caecum by down-regulating the activity of Fgf10 and Pitx1 (Zacchetti et al., 2007).

1.2.3 Hox genes and evolutionary novelties

Apart from their presumably ancestral function in the main body axis, Hox genes of different clusters have been repeatedly co-opted to pattern novel structures during the course of vertebrate evolution. The genetic flexibility gained by the presence of up to four paralogous genes has been exploited by assigning them with different functions, often through the acquisition of new expression specificities (see chapter 1.3 and chapter 5; Deschamps, 2007). Posterior HoxC genes are expressed in hair follicles, and the loss of functional Hoxc13 leads to the lack of external hair (Godwin and Capecchi, 1998; Shang et al., 2002). Hoxb8 has been reported to be expressed in microglia cells, a link with the hematopoietic system that is further reinforced by the frequent overexpression of Hox genes in various types of leukemias (Grier et al., 2005; Chen et al., 2010). Hoxa10, Hoxa11 and Hoxd11 are expressed in adult uterine horns, the former two being essential for uterine receptivity (Hsieh-Li et al., 1995;

Satokata et al., 1995; Beckers and Duboule, 1998; Taylor, 2000)

More important for this thesis, Hox genes have also been recruited to pattern the secondary body axes that emerged during the course of tetrapod evolution. Genes of the HoxA and HoxD clusters are expressed in discrete patterns during the formation of both the fore- and the hindlimb (reviewed in Zakany and Duboule, 2007), while HoxC genes are expressed in either one of the structures (Nelson et al., 1996). In the presumptive forearm, Hoxd transcripts are present in anterior-to-posterior staggered domains, while posterior Hoxa genes display a proximal-to-distal distribution of their transcripts (Dolle et al., 1989; Yokouchi et al., 1991). In the most distal part of the limb, the developing autopod, Hoxd13 to Hoxd9 are expressed in a quantitative collinear manner (see above and Montavon et al., 2008), while of the HoxA cluster,

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only Hoxa13 is transcribed. The absence of these expression patterns in the fins of teleost fish led to the proposal that the acquisition of a second, distal phase of Hox expression could have been a causative factor for the neomorphic emergence of digits in higher tetrapods (Sordino et al., 1995; van der Hoeven et al., 1996a; see also chapter 5). Single and combined gene inactivation experiments have demonstrated the importance of Hox genes for the patterning and growth of the appendicular skeleton.

The patterning actions of different genes along the proximal-to-distal axis are again collinear with their position in the complex. Mutations in group 9 genes lead to alterations in the stylopod, group 11 genes affect the zeugopod and finally group 13 genes are required for proper autopod development (Dolle et al., 1993; Davis and Capecchi, 1994; Fromental-Ramain et al., 1996a). At all of the three anatomical positions, functional redundancy between paralogous Hoxa and Hoxd is apparent, as shown by the increased severity of phenotypes in compound mutants (Davis et al., 1995; Fromental-Ramain et al., 1996a; Fromental-Ramain et al., 1996b). In addition to their patterning function, Hox genes play also important roles in the growth dynamics of the limb (Goff and Tabin, 1997). In mice, ectopic expression of group 13 Hox genes at more proximal locations, due to engineered or induced regulatory mutations, can have deleterious effects upon the growth of the affected structure, likely by suppressing the function of group 11 genes (van der Hoeven et al., 1996b;

Herault et al., 1997; Peichel et al., 1997; see also chapter 3). This posterior prevalence of group 13 genes seems also to be utilized in a wild-type situation, e.g. by keeping in check the action Hoxd11 to prevent supernummary digit condensations in the autopod (Kondo et al., 1998; Kmita et al., 2002a). Moreover, limbs lacking all HoxA and HoxD functions are arrested early during development, as their posterior genes are essential for the initial activation of Shh in the zone of polarizing activity that in turn is required for further limb bud outgrowth to occur (Kmita et al., 2005; Tarchini et al., 2006).

Intriguingly, genes of both HoxA and HoxD complexes are also expressed in the developing external genitalia, and deletions of posterior genes cause defects in the formation of the penile bone (Kondo et al., 1997; Zakany et al., 1997a). The similar regulatory mechanism underlying these novel expression domains in both limbs and external genitalia have led to the notion that both of them could have been co-opted together, as an essential adaptation to a terrestrial life style (Zakany et al., 1997a;

Spitz et al., 2003; Montavon et al., 2008).

1.3 HOX GENE REGULATION

1.3.1 Genetic approaches to study Hox gene regulation

The transcriptional regulation of any developmental gene is faced by a variety of very complex challenges. Genes need to be activated both temporally and spatially in a highly coordinated and reproducible manner to guarantee proper development (Ohler and Wassarman, 2010). Different strategies have evolved to cope with such requirements, however, the situation of clustered genes, as found for the Hox complexes, presents an additional layer of complication. While it certainly simplifies the task of a concerted regulation of all members in a given complex, the dense array of promoters and enhancers found in and around Hox clusters calls for a tightly controlled orchestration to ensure the differential transcription required at the single

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gene level. Over the years a wide range of genetic approaches, some of them specifically developed to tackle the problem of Hox gene regulation, have been employed to try and decipher the complex interplay between the clustered organization of these genes and their regulatory elements.

Following the first efforts studying Hox gene expression by means of in situ hybridization, the question quickly arose as to how these patterns were generated at the transcriptional level, i.e. what kind of regulatory elements were responsible for them. The development of tagged reporter transgenes was therefore a suitable strategy to scan genomic fragments surrounding Hox genes for their enhancer activities, once taken outside the context of the cluster. Indeed, many of the larger transgenes are able to reproduce important aspects of the spatial transcript distribution of the Hox gene they surround, and enhancer-bashing assays helped to fine-map the responsible DNA sequences (Zakany et al., 1988; Whiting et al., 1991; Charite et al., 1995). While these randomly integrated transgenes where mainly able to reproduce expression patterns in the trunk, they mostly failed to do so in evolutionary novel domains such as the limb or the genital buds mentioned above (Renucci et al., 1992; Gerard et al., 1993). Such expression specificities were only recovered when the same transgenes were targeted into the context of their endogenous cluster, implying an important role played by the enlarged cluster environment (van der Hoeven et al., 1996b; Spitz et al., 2001).

Therefore, experimental set-ups had to be devised to both modify and scan this intricate relation of genomic cluster structure and its resulting transcriptional output.

From a cluster-centric point of view, loxP sites (e.g. left behind from selection cassettes used for the targeted mutagenesis of regulatory elements) were “recycled” in an approach termed “targeted meiotic recombination” (TAMERE; Herault et al., 1998b). If such loxP sites are on homologous chromosomes at different position, trans-allelic recombination can be induced by Cre-expression during homologous pairing in meiotic prophase. The resulting recombined clusters are either shortened or enlarged by the fragment the loxP sites were originally spanning. This then allows evaluating the impact of cluster size, both distance- and promoter-wise, on gene regulation.

To up-scale the original transgenic approaches to the level of whole clusters and large genomic fragments surrounding them, BAC-transgenesis, both tagged and un- tagged, has been successfully applied to scan larger pieces of DNA sequence for their regulatory potential (Spitz et al., 2001; Spitz et al., 2003; Lehoczky and Innis, 2008).

The finding that remote, global enhancers are responsible for many, if not all, evolutionary novel expression domains of Hox genes, led to the notion of “regulatory landscapes”. Transcription units residing within such landscapes would show a similar expression pattern due to these global enhancers, without a clear functional relevance necessarily attached to all genes displaying it (bystander effects; Spitz and Duboule, 2008).

To modify such extended genomic regions, refined ES cell-based methods had previously been developed (Ramirez-Solis et al., 1995; Smith et al., 1995). However, as for the TAMERE-approach mentioned above, the desire to avoid tedious ES cell culture work led the development of a purely genetic in vivo approach to alter the architecture of entire regulatory landscapes. LoxP sites located in trans on homologous chromosomes at great distance to one another can easily be brought in cis, by simple meiotic crossover. Once on the same chromosome, expression of a Cre- transgene can induce inversions or deletions between the two loxP sites, depending on their orientation relative to each other. This sequential targeted recombination- induced genomic (STRING) approach allows for the targeted disruption of extended

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regulatory landscapes by simple means of mouse breeding (Spitz et al., 2005).

Moreover, a modified TAMERE approach using loxP sites located on non- homologous chromosomes became recently possible thanks to the strong Hprt-Cre line. This has been successfully used to generate site-directed chromosomal translocations in vivo, opening the possibility to exchange and re-shuffle entire regulatory landscapes between different loci in the genome (PANTHERE; see chapter 5 and Wu et al., 2007).

From the various approaches mentioned above, a wealth of data has been amassed over the years, some of which will be discussed in the following paragraphs.

It depicts a complex interplay between the structure of Hox clusters, the regulatory elements present within and outside of them, and the neighboring genomic regions, all contributing towards a coordinated transcriptional output.

1.3.2 Initiation, establishment and maintenance of Hox gene expression in the primary body axis.

In the primary body axis, Hox gene regulation can be roughly sub-divided into three phases, namely the initiation of expression during early gastrulation, the establishment of the spatial expression patterns specific for each gene and their subsequent maintenance throughout embryogenesis (Deschamps et al., 1999).

Hox genes start to be expressed at the late streak stage, in cells of the posterior streak region contributing to extraembryonic tissues. More posterior Hox genes become expressed at progressively later time-points (temporal collinearity), even though the induction wave to prime cells for Hox expression precedes the actual activation (Forlani et al., 2003). Once activated, expression expands to more rostral levels into cells contributing to the elongating embryonic axis, in a way that does not correlate with cell lineage movements (Deschamps and Wijgerde, 1993). The definitive Hox codes are therefore not fixed at the node, but only later at more anterior levels. A number of TRANS-acting factors has been identified as potential upstream activators in both mesodermal and neurectodermal derivatives. Early caudal to rostral gradients of Wnt and Fgf signaling, as well as little later on retinoic acid (RA) have been implicated in the modulation of Hox gene expression (Kessel and Gruss, 1991;

Pownall et al., 1998; Niederreither et al., 1999; Dubrulle et al., 2001; Aulehla et al., 2003; Forlani et al., 2003). However, whether these pathways regulate the transcription of Hox genes directly or indirectly is less clear. Cdx genes, members of the ParaHox family and homologs of the Drosophila gene caudal, have emerged as potential mediators of the afore-mentioned signaling pathways (reviewed in Deschamps and van Nes, 2005). The early expression of the three paralogs, Cdx1, Cdx2 and Cdx4 follows closely the pattern of Hox genes at these timepoints (Gamer and Wright, 1993; Meyer and Gruss, 1993; Beck et al., 1995). Mutations in Cdx genes affect vertebral patterning in agreement with their impact on the establishment of Hox codes from cervical to caudal levels (van den Akker et al., 2002). Moreover, Cdx genes are known to regulate Hox genes directly via Cdx binding sites (Subramanian et al., 1995; Charite et al., 1998). At least for the effects of exogenous FGF it has been shown that its activating function on 5’ Hox genes is directly mediated by Cdx (Isaacs et al., 1998). Fgfs are also implicated in Hox regulation during somite formation, just as is the Notch pathway in establishing a cycling expression of anterior Hoxd genes in the pre-somitic mesoderm (Dubrulle et al., 2001; Zakany et al., 2001).

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In parallel to these molecular interactions of signaling pathways and transcription factors, higher order chromatin dynamics accompany the sequential activation of Hox genes during early embryogenesis as well ES cell in vitro differentiation (reviewed in Sproul et al., 2005; Soshnikova and Duboule, 2009a). During this time-window, activating and repressive histone marks, such as trimethylation at lysine 4 or acetylation at lysine 9 as well as trimethylation at lysine 27 of histone H3, follow inverse temporal collinear changes in their distribution from 3’ to 5’ of the cluster (Chambeyron and Bickmore, 2004; Soshnikova and Duboule, 2009b). These changes precede the actual gene activation, however they do not rely on a in cis spreading mechanism sensu stricto, since a split HoxD cluster still shows largely correct distribution of chromatin marks (Soshnikova and Duboule, 2009b). Mutations in the enzymatic complexes that deposit these marks, namely the Polycomb group genes, confirm a functional role for these chromatin signatures, as changes therein induce heterochronies in Hox gene activation (Bel-Vialar et al., 2000). Moreover, the overall organization of the DNA seems to change during the activation process, as both decondensation and repositioning of activated genes outside of their chromosome territories have been reported for HoxB and HoxD clusters (Chambeyron et al., 2005;

Morey et al., 2007).

Once the genes are transcriptionally activated, correct rostral expression limits have to be established (spatial collinearity). As vertebrates extend their body axis progressively by a posterior growth zone (Selleck and Stern, 1992; reviewed in Wilson et al., 2009), it has been speculated that this temporal progression of Hox gene activation along with axis elongation could as well directly determine the establishment of the late Hox expression boundaries (Duboule, 1994). Indeed, in chicken, the overexpression of posterior Hox genes into epiblast cells has been reported to delay their ingression through the streak and thereby determining the final axial position they contribute to (Iimura and Pourquie, 2006). However, as in mice engineered heterochronies in gene activation do not systematically lead to changes in the anterior limits of expression, a slightly more complex picture including multiple, superimposed regulatory strategies emerges (van der Hoeven et al., 1996b; Kondo and Duboule, 1999; Tschopp et al., 2009). In accordance with these results, the expression patterns of several Hox genes are known to be highly dynamic during this establishment phase. The final rostral limits of expression of e.g. Hoxb8 or Hoxa7 and Hoxa10 are only established long after the initial activation took place (Deschamps and Wijgerde, 1993; Gaunt et al., 1999). This anterior expansion of expression depends on cell-to-cell signaling rather than on cell movements, as shown by lineage tracing experiments (Forlani et al., 2003). Amongst the signals that can influence Hox expression boundaries is RA. Group 1 to 4 genes are known to possess retinoic acid response elements (RARE) in their proximity, important for setting the final expression limits in both neurectoderm and paraxial mesoderm (e.g. Dupe et al., 1997). Genetic inactivation of retinoic acid receptors (RAR) binding to RAREs leads to anterior transformations and changing RA availability in vivo alters Hox expression patterns (Kessel and Gruss, 1991; Lohnes et al., 1994; Niederreither et al., 1999). In the hindbrain, cross-regulation between Hox genes as well as the transcription factors Krox-20 and kreisler have been shown to establish the final expression patterns of anterior genes (Sham et al., 1993; Cordes and Barsh, 1994; Gould et al., 1997).

Once the correct expression boundaries have been established, members of the Polycomb group (PcG) and Trithorax group (TrxG) proteins play essential roles in preserving them. Originally identified in Drosophila, where mutations in them give rise to homeotic transformations, they compose a family of histone modifying

Collinear regulations of vertebrate Hox gene clusters: structural constraints and evolutionary innovations

Thesis

Reference

Collinear regulations of vertebrate Hox gene clusters: structural constraints and evolutionary innovations

Collinear Regulations of Vertebrate Hox Gene Clusters:

Structural Constraints and Evolutionary Innovations

Table of Contents

List of Figures and Tables

Summary

Résumé en français

Chapter 1

General Introduction