Thesis
Reference
Divergence in Hox gene regulation and the evolution of a snake-like body plan
GUERREIRO, Isabel
Abstract
While some aspects of vertebrate embryonic development are highly conserved, others are variable and determine the extraordinary morphological variability observed across species.
Hox genes are likely candidates in shaping the body plan of organisms over the course of evolution, owing to their instrumental functions in the patterning of developing structures.
Snakes, in particular, have a very elongated trunk and are limbless, representing one of the most extreme morphological adaptations in the vertebrate clade. In this study, we show that the evolution of the snake extreme body plan has been accompanied by major regulatory changes at the HoxD locus while respecting a very conserved global structural constraint.
GUERREIRO, Isabel. Divergence in Hox gene regulation and the evolution of a snake-like body plan. Thèse de doctorat : Univ. Genève, 2016, no. Sc. 4918
URN : urn:nbn:ch:unige-842528
DOI : 10.13097/archive-ouverte/unige:84252
Available at:
http://archive-ouverte.unige.ch/unige:84252
Disclaimer: layout of this document may differ from the published version.
UNIVERSITÉ DE GENÈVE Département de
Génétique et Évolution
FACULTÉ DES SCIENCES Professeur Denis Duboule
Divergence in Hox Gene Regulation and the Evolution of a Snake-like Body Plan
THÈSE
Présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
par
Isabel GUERREIRO
de
Lausanne (VD)
Thèse N° 4918
GENÈVE Atelier Repromail
2016
Table of contents
Summary ... 5
Résumé ... 7
Chapter 1 - Introduction ... 9
1.1 Hox genes in evolution and development ... 9
1.1.1 Hox gene clustering ... 9
1.1.2 Collinearity ... 12
1.1.3 The Hox code and posterior prevalence ... 12
1.2 Hox gene function ... 13
1.2.1 Hox genes and axial skeleton patterning ... 13
1.2.2 Hox function in secondary structures and organs ... 15
1.3 Hox gene regulation ... 18
1.3.1 Evolution of cis-‐regulatory regions ... 18
1.3.2 Repeat elements and the evolution of regulation ... 19
1.3.3 Hox gene regulation in the main body axis ... 20
1.3.4 Hox gene regulation in limbs ... 22
1.3.5 Hox gene regulation in genitals ... 24
1.3.6 Hox gene regulation in kidney and caecum ... 24
1.4 Evo-‐Devo of the snake ... 25
1.4.1 Hox gene expression in snakes ... 25
1.4.2 Transposable elements in squamate Hox clusters ... 26
1.5 Scope of the thesis ... 27
Chapter 2 - Results ... 29
2.1 The corn snake HoxD cluster and regulatory landscapes ... 29
2.1.1 HoxD cluster structure and spatial collinearity in the snake ... 33
2.2 Reorganization of snake mesodermal enhancers ... 35
2.2.1 Trunk mesoderm regulation in mouse and snakes ... 35
2.2.2 Mechanisms of Hoxd gene repression in the posterior trunk ... 39
2.3 Snake chromatin structure at the HoxD regulatory locus ... 41
2.3.1 Conservation of CTCF binding in the snake HoxD locus ... 42
2.3.2 Divergence of Hoxd gene regulation in the snake genital bud ... 44
2.3.3 Regulatory potential of conserved mouse enhancer sequences ... 47
2.4 Chromatin structure of the isolated HoxD cluster ... 50
2.5 Transcriptional analysis of snake embryonic samples ... 53
2.5.1 Hox gene expression in mouse and corn snake embryonic tails ... 53
2.5.2 Gene expression in the python vestigial hindlimbs ... 61
Chapter 3 - Discussion ... 67
3.1 The corn snake HoxD cluster structure and repeat content ... 67
3.2 Trunk mesodermal enhancers at the HoxD locus ... 67
3.2.1 Reallocation of snake mesodermal enhancers ... 68
3.2.2 Mechanisms of Hox gene repression in the posterior trunk ... 69
3.2.3 Towards a disorganized cluster? ... 69
3.3 A bimodal chromatin structure at the snake HoxD locus ... 71
3.3.1 Divergent regulatory strategy for genital bud patterning ... 72
3.3.2 Intrinsic bimodal organization of the human HoxD cluster ... 75
3.5 Differential Hox gene expression in mouse and snake tail buds ... 75
3.6 Gene expression in the python hindlimb bud ... 76
3.7 Concluding remarks ... 77
Chapter 4 - Materials and methods ... 79
4.1 BAC library construction, screening and sequencing ... 79
4.2 Sequence analysis and annotation ... 79
4.3 ChIP-‐sequencing ... 80
4.4 Mouse stocks ... 80
4.4.1 BAC transgenic lines ... 80
4.5 In situ hybridization and probe design ... 81
4.6 Enhancer trangenesis and lacZ staining ... 83
4.7 4C-‐sequencing ... 83
4.8 RNA extraction ... 84
4.9 RT-‐qPCR ... 85
4.10 RNA-‐seq data ... 85
Acknowledgements ... 89
References ... 91
List of figures and tables
Figure 1 - Corn snake HoxD cluster and surrounding regulatory landscapes ... 30
Figure 2 - Sequence conservation at the HoxD locus. ... 32
Figure 3 - The snake HoxD cluster. ... 34
Figure 4 – The regulatory potential of the vertebrate HoxD cluster ... 36
Figure 5 – Locating telomeric Hoxd trunk mesodermal enhancers ... 38
Figure 6 - Identification of a trunk mesoderm enhancer ... 39
Figure 7 – Mechanisms of maintenance of expression in the posterior trunk ... 40
Figure 8 – Hoxd gene interaction profiles in mouse and snake embryos ... 42
Figure 9 – CTCF binding site location and orientation at the HoxD locus ... 44
Figure 10 - Regulation of mouse and corn snake Hoxd genes in developing genitals. ... 46
Figure 11 - Interspecies comparison of the HoxD cluster regulatory potential ... 47
Figure 12 – Regulatory activity of mouse digit and GT enhancers in the snake ... 48
Figure 13 – Regulatory activity of mouse proximal limb enhancers in vertebrates ... 49
Figure 14 - HumanBAC integration sites ... 50
Figure 15 - Chromatin structure at the integration sites of HumanBAC lines ... 51
Figure 16 - Human HoxD gene expression in HumanBAC A and HumanBAC B ... 52
Figure 17 – RNA-seq analysis of mouse embryonic tails ... 54
Figure 18 – Gene expression profiles mapped on the Hox clusters ... 57
Figure 19 – Interspecies comparison of Hox13 gene expression ... 57
Figure 20 – Expression of genes involved in axial extension and termination ... 59
Figure 21 – Expression of Hox13 genes in the house snake tail bud ... 60
Figure 22 – RNA-seq analysis of python embryo hindlimbs, genitals and tail tip ... 62
Figure 23 – Python embryonic tissue RNA-seq mapping over genes involved in limb development ... 64
Figure 24 – RNA-seq profiles of python and lizard limbs and genitals ... 65
Figure 25 – Towards a disorganized cluster? ... 71
Figure 26 – Proposed model for the change in Hoxd gene regulation in snake genitalia ... 73
Table 1 - List of primers used to clone the probes for in situ hybridization ... 82
Table 2 - List of primers used for 4C-seq amplifications with snake tissues ... 84
Table 3 - List of snake and mouse primers used for qPCR ... 85
Summary
While some aspects of vertebrate embryonic development are highly conserved, others are variable and determine the extraordinary morphological variability observed across species. Hox genes are likely candidates in shaping the body plan of organisms over the course of evolution, owing to their instrumental functions in the patterning of developing structures. Snakes, in particular, have a very elongated trunk and are limbless, representing one of the most extreme morphological adaptations in the vertebrate clade. Initial studies have shown that the very extended body plan of snakes, in particular the corn snake, is due to a fast segmentation clock and a delayed shrinkage of the PSM (presomitic mesoderm). In chicken, this latter process has been shown to be associated with the activity of “posterior” Hox genes.
On the other hand, mice that lack the HoxA and HoxD clusters, only develop the very proximal part of the limb. These facts indicate that Hox genes could have played an important role in the evolution of a snake-like body plan.
In this study, we used an interspecies comparative approach where different aspects of Hoxd gene regulation were assessed. We find that, although spatial collinearity and associated epigenetic mark dynamics are conserved in the corn snake, other regulatory modalities have been largely restructured. We used a BAC transgenesis approach where clusters from different vertebrate species were randomly integrated in the mouse genome in order to assess the regulatory potential contained within these sequences. We found that, while the majority of mesodermal enhancers in the vertebrate species assayed appear to be mostly located outside of the cluster, the corn snake cluster is sufficient to drive normal Hoxd gene expression in most of the mouse trunk.
The profile of HoxD locus interactions with its genomic landscapes obtained from mouse and snake genital bud, revealed important regulatory differences between the two species in this tissue. However, despite the profound differences observed in morphology and regulation of Hoxd genes, the bimodal chromatin structure at the snake HoxD locus is maintained. In addition, highly conserved sequences that in the mouse have a role in driving Hox gene expression in limbs and genitals, were found to be conserved in the snake.
However, in what concerns “limb enhancers”, this conservation was restricted to the sequence level, since most snake counterparts were unable to drive reporter gene expression in the mouse limb. Interestingly, the snake version of a limb-only regulatory sequence in the mouse appears to have been coopted to a genital-only function.
A comparative transcriptomic analysis of the posterior trunk of mouse and snake embryos over a range of developmental time revealed that a highly expressed Hox13 gene, Hoxc13, seems to have lost its function in repressing genes involved in trunk elongation in the
snake. Consequently, the delay in shrinking of the PSM is likely to be due to the loss of axial termination activity of this gene.
In addition, we have produced a transcriptome dataset of the python vestigial hindlimb buds that will bring important contributions to understanding the molecular mechanisms that underlie limb loss. We found that Hoxd genes are not expressed in this tissue, while 5’ Hoxa genes are highly expressed. This includes Hoxa13, whose expression is usually restricted to the distal region of the limb bud.
Altogether, our results show that the evolution of the snake extreme body plan has been accompanied by major regulatory changes at the HoxD locus while respecting a global structural constraint.
Résumé
Chez les vertébrés, certains aspects du développement embryonnaire sont très conservés, tandis que d'autres présentent de fortes divergences. Ces variations sont tenues pour responsables de l'extraordinaire diversité morphologique présente parmi les espèces de ce clade. Les gènes Hox sont impliqués dans la formation de l'axe corporel des organismes.
Au cours de l'évolution, ils ont acquis de multiples fonctions dans la formation des structures en développement. Le tronc très allongé des serpents et leurs membres inexistants représentent des exemples très particuliers d'adaptations morphologiques extrêmes.
Certaines études ont montré que le corps filiforme du serpent des blés est dû à une horloge de segmentation rapide ainsi qu'à un délai dans le rétrécissement du mésoderme présomitique lors de la somitogenèse. Chez le poulet, ce processus de diminution de la zone présomitique dépend de l'activité de gènes Hox postérieurs (situés en 5' du groupe). D'autre part, les souris auxquelles on a retiré les groupes de gènes HoxA et HoxD développent uniquement la partie très proximale de leur membre.
Dans cette étude, nous avons fait appel à une approche comparative inter-espèces où différents aspects de la régulation des gènes Hoxd ont été évalués. Bien que la colinéarité spatiale et les marqueurs épigénétiques qui y sont associés soient conservés dans le serpent des blés, nous avons découvert que certains mécanismes de régulation ont été largement restructurés. Dans le but d'évaluer le potentiel régulateur intrinsèque aux groupes Hox de différentes espèces de vertébrés, nous avons eu recours à la transgénèse par BAC, afin d'intégrer ces séquences au hasard dans le génome de souris. Malgré le fait que la plupart des éléments régulateurs des gènes Hoxd dans le mésoderme qui ont été étudiés semblent être localisés à l'extérieur du groupe, nous avons trouvé que la région homologue à HoxD chez le serpent des blés est suffisante pour promouvoir une expression normale de ces gènes dans presque tout le tronc.
Les profils d'interactions longue-distance du locus HoxD avec son environnement génomique obtenus à partir du tubercule génital d'embryons de souris et de serpent ont révélé d'importantes divergences de régulation entre les deux espèces dans ce tissu. Cependant, malgré les profondes différences observées tant dans leur morphologie que dans la régulation de leurs gènes Hoxd, la structure bimodale de la chromatine au niveau de ce locus est maintenue chez le serpent comme chez la souris. De plus, certaines séquences particulièrement conservées, qui dans la souris ont le rôle de guider l'expression des gènes Hox dans les membres et les organes génitaux, se trouvent être présentes à l'identique chez le serpent. Toutefois, en ce qui concerne les éléments régulateurs actifs dans les membres, la conservation se limite au niveau de la séquence, étant donné que la plupart des régions homologues provenant du serpent sont incapables d'activer l'expression du gène rapporteur
dans ces structures. Curieusement, la "version serpent" d'une séquence particulière active le gène rapporteur dans le tubercule génital uniquement, alors qu'elle est connue chez la souris pour être un élément régulateur spécifique des membres.
Une analyse transcriptomique comparative du tronc postérieur d'embryons de souris et de serpents à différents stades de développement a révélé qu'Hoxc13, un gène Hox très fortement exprimé dans ce tissu, semble avoir perdu chez le serpent sa fonction de répresseur des gènes impliqués dans l'élongation du tronc. Par conséquent, le raccourcissement du mésoderme présomitique, qui se produit de manière différée dans cette espèce, est vraisemblablement dû à la perte d'activité de terminaison axiale de ce gène.
De plus, nous avons produit un ensemble de données à partir des membres postérieurs vestigiaux du python, ce qui contribuera certainement à la compréhension des mécanismes moléculaires à l'origine de la disparition des membres chez les serpents. Nous avons trouvé que les gènes Hoxd ne sont pas exprimés dans cette zone, tandis que les gènes Hoxa situés en 5' du groupe HoxA le sont fortement. Ceci inclut Hoxa13, dont l'expression est normalement restreinte à la région distale du bourgeon de membre.
Dans leur ensemble, nos résultats montrent que l'évolution extrême de l'axe corporel des serpents a été accompagnée par des changements majeurs de régulation au niveau du locus HoxD tout en respectant une certaine contrainte structurelle globale.
Chapter 1 - Introduction
1.1 Hox genes in evolution and development
A great variety of species has arisen over the course of evolution by acquiring distinct morphological traits that become specified during embryonic development. Although the coupling of evolution and development (Evo-Devo) has long intrigued biologists, it was only with the advent of molecular genetics that this discipline truly emerged with the finding that key developmental molecules are conserved throughout a variety of different phylogenetic clades. These discoveries first started with the realization that Hox genes were present in distinct animal phyla that undergo very different developmental programs (Moczek et al., 2015).
Hox genes, formerly termed homeotic selector genes, were first described in the fruit fly (Drosophila melanogaster) by Ed Lewis as genes whose misexpression resulted in the change of segment identity of a segment into another. These phenotypes, termed homeotic transformations, revealed the crucial role of homeotic genes in fruit fly patterning.
Furthermore, it was proposed that Hox genes could have been implicated in the evolution of Drosophila from its four-winged ancestor (Lewis, 1978).
All homeotic genes in the fruit fly were found to contain a DNA sequence named homeobox. This sequence is 183 base pair-long and codes for a helix-turn-helix 61 amino acid motif (homeodomain) with DNA binding ability. Subsequently, homeobox-containing genes were discovered in the genome of many other animal species, including vertebrates (McGinnis et al., 1984). These discoveries were followed by the realization that many important players in embryonic development were conserved throughout evolution. With this knowledge, new interdisciplinary approaches have arisen that made it possible to tackle biological questions in a variety of different fields.
1.1.1 Hox gene clustering
Hox genes code for transcription factors that have been found in most metazoans including cnidarians and all bilaterians (Lemons and McGinnis, 2006). An ancestral ProtoHox cluster containing few genes is thought to have arisen before the split between cnidarians and bilaterians over 1000 million years ago. After analyzing Hox genes in cnidarian species, it was proposed that this ProtoHox cluster contained only two anteriorly expressed genes. The cluster would have been duplicated, originating identical Hox and
ParaHox clusters, which subsequently diverged and expanded by tandem duplications in cis (Garcia-Fernandez, 2005). The sequencing of Hox genes in different bilaterian species has motivated the hypothesis that a minimum of seven genes would have been present in the ancestral bilaterian cluster (de Rosa et al., 1999). However, this initial cluster has evolved differently in different species acquiring distinct organizations and Hox gene number.
In the fruit fly Drosophila melanogaster, where Hox genes were first identified, a set of eight genes located on chromosome 3 is split in two complexes separated by eight megabases: Antennapedia (ANT-C) and Bithorax (BX-C) (Lewis, 1978). Later, vertebrate Hox genes were also found to be organized in a cluster, which led to the erroneous assumption that the presence of Hox genes in a species was necessarily associated with a cluster-like organization (Duboule, 2007). Furthermore, clustered Hox genes were thought to be essential for the correct spatial expression of Hox genes and by consequence to be under a strong functional constraint (Lewis, 1978). However, the increasing data on Hox gene repertoire and organization of different animals has shown that this is not always the case. For example, Nematostella vectensis and Eleutheria dichotoma, two cnidarians, have shown to have so-called atomized Hox clusters (Duboule, 2007) that are very unorganized, with Hox genes isolated or in small groups scattered in the genome (Kamm et al., 2006).
Interestingly, species closely related to Drosophila melanogaster have their two Hox complexes split at a different location. More basal insect and diptera species, however, have an intact cluster indicating this to be the insect ancestral condition (Ferrier and Minguillon, 2003). However, although this cluster is present at a single locus in the insect genome, the intergenic regions are large and the transcription orientation is not always the same (Duboule, 2007).
In what concerns echinoderms, the sea urchin presents a very interesting Hox gene organization. It would appear that Hox genes, although organized in one single locus, have rearranged their order in the cluster in comparison with the ancestral condition (Cameron et al., 2006). Echinoderms are the sister group of hemichordates but are less diverged and show more similarities with chordates. The analysis of Hox gene organization in two species of this phylum has revealed a cluster composed of 12 genes in the correct order. The 10 first genes have the same transcriptional orientation as the chordate counterparts, while the two more posterior genes are inverted (Freeman et al., 2012).
Echinoderms and Hemichordates compose together the group of Ambulacraria which is a sister group of Chordates. Within the Chordate phylum Cephalocordates (Amphioxus) represent the most basal subphylum (Putnam et al., 2008). The Amphioxus Hox cluster contains 15 Hox genes in the same locus displaying the same transcriptional orientation and has not suffered any rearrangements. While the recently discovered 15th Hox gene has only been discovered in the Amphioxus genome (Putnam et al., 2008), Hox14 paralogs have been
found in vertebrate species such as the lamprey, cartilageneous fishes or coelacanth. This gene is thus unlikely to result from an amphioxus-specific duplication. However, Hox14 appears to have a distinct function from other Hox genes and thus to be under a more relaxed selective pressure, which could explain why it is missing in most species of the vertebrate lineage (Kuraku et al., 2008).
Urochordates, the sister group of vertebrates, have a dramatically different Hox gene organization. Ascidians, for example, have been shown to have nine Hox genes distributed in two different chromosomes with a highly rearranged organization and large intergenic regions containing unrelated genes (Ikuta et al., 2004). Another Urochordate, Oikopleura dioica presents an even greater level of disorganization (Seo et al., 2004).
Early in the vertebrate evolution, two events of genome duplication occurred that originated four Hox clusters. As a consequence, over the course of vertebrate evolution, several events of gene loss occurred and, to date, no extant vertebrate has been shown to possess a complete Hox cluster (McClintock et al., 2001; Wagner, 1998).
Within the vertebrate clade, there are two superclasses: Agnatha (jawless vertebrates like lampreys and hagfish) and Gnathostomata (jawed vertebrates). Agnathan Hox clusters are large and have a high repeat content, superior to the percentage of repeats in the genome (Mehta et al., 2013). The Japanese lamprey has six clusters instead of four most likely due to segmental duplications (Mehta et al., 2013; Smith and Keinath, 2015).
In Gnathostomes most lineages have four Hox clusters, except for some fish groups that suffered additional large-scale duplications resulting in a higher number of clusters (seven in zebrafish, for example). In comparison to their Agnathan counterparts, gnathostome Hox clusters tend to be more compact and lower in repeat content (Mehta et al., 2013).
In particular, all mammalian species analyzed so far have 39 Hox genes organized in four clusters, HoxA, HoxB, HoxC and HoxD that are located in four different chromosomes.
All Hox genes are ordered and have the same transcriptional orientation within each cluster of about 100 kilobases in size. Based on homology, thirteen paralogous groups have been identified although only groups 4, 9 and 13 are represented in all clusters (Lemons and McGinnis, 2006).
The increasing genomic information supports a model whereby an ancestral bilaterian cluster would have suffered extensive changes over the course of evolution to the point of Hox genes being scattered in the genome surrounded by unrelated non-Hox genes in an atomized conformation. It is therefore surprising to find gnathostomes as the only bilaterians to have countered this trend by consolidating their clusters. The increase in cluster and Hox gene number would be expected to confer a higher flexibility permitting genes to be rearranged without resulting in adverse effects. This unique consolidation observed in gnathostomes is therefore likely to have presented an important advantage to the lineage. The
implementation of global long-range enhancers that require the close proximity of genes for a coordinate regulation to take place has been proposed as an explanation (Duboule, 2007).
1.1.2 Collinearity
One of the most fascinating aspects of Hox gene expression first reported in Drosophila melanogaster by Ed Lewis was that the position of Hox genes in the cluster reflected the location of their gene expression domains in the developing embryo (Lewis, 1978). This phenomenon, termed spatial collinearity, was subsequently found to be conserved in mice as well (Duboule and Dollé, 1989; Graham et al.). Therefore, genes located at the 3’
end of the cluster such as Hox1 genes are expressed in more anterior domains of the embryo, while genes located in the 5’ end of the cluster are expressed in the embryonic tail. Spatial collinearity has since been found both in vertebrate and invertebrate species regardless of the organization level of the cluster (Monteiro and Ferrier, 2006).
In species that have a progressive axis formation and segmentation, such as vertebrates and short-germ band insects, 3’ Hox genes are not only expressed more anteriorly but they are also activated earlier in development, whereas 5’ located genes start to be expressed later (Kmita and Duboule, 2003). This phenomenon, termed temporal collinearity appears to require a cluster-like Hox gene organization (Tschopp et al., 2009). Details on the regulation mechanisms behind collinearity are detailed in section 1.3.3.
1.1.3 The Hox code and posterior prevalence
The collinear activation of Hox genes in nested overlapping domains has generated the hypothesis that the combination of Hox genes in a given anterior-posterior (AP) region of the embryo would determine its fate – the Hox code (Kessel and Gruss, 1991). Loss and gain of function experiments are however inconsistent with a combinatorial activity of all Hox transcription factors expressed in a tissue. For example, Hox gene overexpression doesn’t result in homeotic transformations where more posterior genes were expressed (Duboule and Morata, 1994). These observations led to the hypothesis that Hox genes act hierarchically, with more posterior genes overruling anterior gene activity. This system is referred to as
“posterior prevalence”. In some cases, however, a combined activity of different paralogous groups has been shown to take place, for example in mouse rib cage patterning (McIntyre et al., 2007).
1.2 Hox gene function
1.2.1 Hox genes and axial skeleton patterning
Somite formation and differentiation
In vertebrates Hox genes have an instrumental role in patterning the axial skeleton.
Vertebrae arise from somites, paired structures sequentially produced from the unsegmented paraxial mesoderm in a rostral to caudal direction that flank the neural tube (Hirsinger et al., 2000). During the somitogenesis process, cells in the presomitic mesoderm (PSM) continue to divide to provide tissue for somites to form. Therefore, the final number of somites results from the coordination between somitogenesis and axial extension (Gomez et al., 2008).
Somitogenesis relies on two molecular mechanisms: the segmentation clock and the determination front. The molecular oscillator or segmentation clock is activated at somite formation sending a wave of gene expression through the presomitic mesoderm. These cycling genes are members of FGF, Wnt and Notch pathways (Bénazéraf and Pourquié, 2013). Interestingly, it would appear that the molecular oscillator mechanism is employed also in short germ-band insect segmentation (Sarrazin et al., 2012).
The wavefront process on the other hand relies on the FGF and Wnt caudal to rostral gradient in the PSM. Once a threshold of activity is reached, the cells located in this region of the PSM (the determination front) become responsive to the segmentation clock signal and initiate segmentation (Morimoto et al., 2005).
Following segmentation, somites are patterned in response to signals originating from neighbouring tissues. The ventral part of the somite de-epithelializes in response to sonic hedgehog (Shh) signal from the notochord and floor plate and forms the sclerotome. The dorsal part of the somite, on the other hand, is exposed to Wnt secretion from the dorsal neural tube and ectoderm and differentiates to form the dermomyotome. Later, dermomyotome cells delaminate and migrate to form the myotome. The sclerotome gives rise to the axial skeleton while the myotome and the dermomyotome give rise to skeletal muscle and dorsal dermis (Brent and Tabin, 2002). Although all somites initially look very similar to each other, they originate distinct vertebrae morphologies depending on their anterior- posterior location in the body axis. This specification of vertebral identities has been shown to be determined by Hox gene activity (Casaca et al., 2014).
Hox genes and the axial skeleton
The mouse vertebral spine is composed of seven cervical vertebrae, thirteen rib- bearing thoracic vertebrae, six lumbar vertebrae, four sacral vertebrae and approximately 28 caudal vertebrae. This axial formula varies extensively in different vertebrate species. Snakes represent one of the most extreme cases and can have more than 300 total vertebrae. The corn snake, for example, has been shown to have a total of 296 vertebrae from which three cervical, more than 200 thoracic, 4 cloacal (vertebrae with small forked ribs) and 70 caudal (Gomez et al., 2008).
The detailed study of Hox gene expression in chicken and mouse embryos showed a correlation between the anterior limits of Hox gene expression and the boundaries of distinct axial skeleton regions (Burke et al., 1995). Loss and gain of function experiments have since revealed this correlation to be of a causal nature.
The fact that vertebrates have four or more clusters results in a high level of functional redundancy between the paralogous group members. For example, swapping experiments with the Hoxa3 and Hoxd3 genes showed that these genes were for the most part functionally interchangeable (Greer et al., 2000). For this reason, initial loss of function mutations of single Hox genes in mice resulted in very mild, if any, phenotypic defects.
Mutations of entire paralog groups were therefore often necessary to determine the nature of Hox gene activity in axial skeleton patterning (Wellik and Capecchi, 2003). This is well exemplified by the loss of function of the entire Hox10 paralog group, which results in ectopic rib formation in lumbar and sacral vertebrae. Accordingly, the overexpression of Hoxa10 in the PSM resulted in completely ribless embryos. These experiments revealed the rib-repressing role of Hox10 genes, essential for establishing the thoracolumbar transition (Carapuço et al., 2005; Wellik and Capecchi, 2003).
The knockout of paralogous group 11 results in an anteriorization of sacral vertebrae that assume a lumbar identity. Hox11 genes were therefore proposed to partially repress Hox10 rib-repressing activity and specify sacral vertebrae (Wellik and Capecchi, 2003).
Hox5-9 loss of function experiments revealed collinear and partially overlapping skeletal defects in the thoracic vertebrae showing the importance of these paralogous groups in rib cage patterning (McIntyre et al., 2007). Later, Hox6 genes were shown to be able to induce rib formation through overexpression in the PSM that resulted in ectopic rib formation throughout the length of the axial skeleton (Vinagre et al., 2010).
On the other hand, loss of function experiments of Hox3 to Hox5 genes have revealed the function of these genes in cervical vertebrae specification (Condie and Capecchi, 1994;
Horan et al., 1995; McIntyre et al., 2007).
Hox function in axial elongation
Hox genes have been implicated not only in specifying morphologically distinct regions along the axial skeleton but also in defining the length of the main body axis.
This process of axis elongation arrest is associated with FGF and Wnt signaling inhibition that, in turn, results in the downregulation of T/Brachyury and Cyp26A1, a retinoic acid-degrading enzyme. The resulting increased levels of retinoic acid promote differentiation and death of progenitor cells, which lead to PSM shrinkage (Gomez et al., 2008; Olivera- Martinez et al., 2012; Young et al., 2009).
Hoxb13 or Hoxc13 inactivation result in a longer tail as a result of both cell division increase and cell death decrease in mutant embryos (Economides et al., 2003). Accordingly, gain of function experiments of Hoxa13, Hoxb13 and Hoxc13 all result in shorter truncated tails, reminiscent of the mutant phenotypes obtained from the knockout of Cdx genes. At the molecular level Wnt signaling and retinoic acid degradation are downregulated similarly to what is observed during the process of axial termination in wild type mice. These results point to an essential role of Hox13 genes to arrest axial elongation. On the other hand, the rescue of the truncated Cdx mutant phenotype by a Hox8 transgene showed that Cdx and Hox genes expressed in the embryonic trunk promote axial extension by maintaining Wnt signaling and degrading RA. However, genes belonging to paralogous group 13 seem to exert a dominant activity over trunk Hox genes successively blocking their activity from trunk to tail transition until full axial extension arrest (Young et al., 2009).
In chicken it has recently been shown that posterior Hox genes repress Wnt and FGF pathways with increasing strength as genes are collinearly activated. As a consequence mesodermal cell ingression is reduced and the PSM shrinks (Denans et al., 2015).
1.2.2 Hox function in secondary structures and organs
The duplication of Hox clusters early in vertebrate evolution has allowed for Hox genes to be co-opted for additional functions in the patterning of secondary structures such as limbs and external genitalia, as well as organs like the cecum or the metanephric kidneys (Dollé et al., 1989; Dollé et al., 1991; Patterson et al., 2001; Pitera et al.; Zacchetti et al., 2007).
Hox function in limbs
Limbs originate from the lateral plate mesoderm and depend on two signaling centers: the apical ectodermal ridge (AER), necessary for limb outgrowth and a zone of
polarizing activity (ZPA). Interestingly, Hox gene misexpression interferes with these structures that are indispensable in limb development (Zakany and Duboule, 2007).
Hoxa and Hoxd genes are expressed in the early limb following collinear temporal activation, reminiscent of the sequential activation of Hox genes in the main body axis (Tarchini and Duboule, 2006). During limb development, Hoxa and Hoxd genes are expressed in two phases. The first wave of expression patterns the arm (stylopod) and forearm (zeugopod) and follows a collinear spatiotemporal dynamic, reminiscent of the sequential activation of Hox genes in the main body axis. In contrast, the second wave of expression, that patterns the hands and feet (autopod), mostly relies on the expression of 5’ Hoxd genes as well as Hoxa13. Instead of employing a collinear mechanism like in the embryonic trunk and early limb, digit patterning is achieved through a mechanism of quantitative collinearity.
Posterior Hoxd genes are expressed in increasing amounts from Hoxd9 to Hoxd13 with Hoxd13 and Hoxa13 being the only Hox genes expressed in the more anterior digit (the thumb) (Montavon et al., 2008). Interestingly, the observation of increasing levels of transcription with respect to topological organization in the cluster was originally described in the context of the genital bud (Dollé et al., 1991)
As Hoxd genes become expressed in the early limb bud following a temporal order of activation, genes that lie in the 5’ end of the cluster (Hoxd10-Hoxd13) get restricted to the posterior and distal area of the developing limb bud. A deletion that produces a small cluster composed only of Hoxd11-Hoxd13 resulted in limbs with no AP polarity. This was shown to be the result of Sonic hedgehog (Shh) ectopic activation in the anterior side of the limb bud.
Therefore, overexpression of 5’ Hox genes in the early limb bud greatly affects the anterior- posterior (AP) polarity of the autopod (Zákány et al., 2004). Conversely, the complete absence of both HoxA and HoxD clusters is accompanied by a loss of Shh expression in the limb bud (Kmita et al., 2005). These clusters have also been shown to be necessary to activate Gremlin1 (Grem1), a gene involved in the induction of Fgf expression at the AER that is essential for limb growth (Sheth et al., 2013).
Shh is secreted in a posterior to anterior gradient by the zone of polarizing activity (ZPA), a signaling center located in the posterior mesenchyme of the limb bud that is essential for correct AP patterning of the distal limb (Zeller et al., 2009). In turn, Shh signaling promotes the activating function of the Gli1 and Gli2 transcription factors and prevents the processing of Gli3 to a repressive form (Gli3R). Therefore, inverse gradients of Shh signaling and Gli3R are established along the anterior-posterior axis of the developing limb bud (Wang et al., 2000). Limbs of Gli3 and Shh double mutants lack digit identity and are polydactylous. Such a phenotype could be explained by the disruption of a Turing-type mechanism, suggested to have a key role in digit patterning (Sheth et al., 2012). Hox genes emerged as candidates for modulating this system since mutations in 5’ Hox genes, namely
Hoxd13 and Hoxd12, have resulted in polydactyly (Kmita et al., 2002). In addition, these posterior Hox genes have been shown to both regulate and be regulated by the Shh-Gli3 system (Zákány et al., 2004). The limb analysis of mouse embryos carrying mutations for Gli3, Hoxd10-13 and Hoxa13 showed that the decreasing amount of posterior Hox products was correlated with a higher degree of polydactyly. A model was proposed whereby distal Hox genes modulate the Turing-like mechanism behind digit patterning (Sheth et al., 2012).
From an evolutionary standpoint, it would appear that this mechanism was present already in the patterning of chondrichthyan and basal actinopterygian fins. A change in the expression domains of posterior Hox genes, Shh and Gli3 would have allowed the evolution of the current tetrapod limb morphology (Sheth et al., 2012).
The importance of Hox gene expression in limbs is well illustrated by the Hoxa and Hoxd gene deletion phenotypes. For redundancy reasons the compound mutants of Hoxa and Hoxd genes from the same paralogous group are the most informative. Mice lacking both the HoxA and HoxD clusters have no limbs and only maintain a small part of the stylopod (Kmita et al., 2005). When both Hoxa11 and Hoxd11 are absent, the zeugopods are very reduced and the knockout of both Hoxa13 and Hoxd13 results in mice with no digits (Davis et al., 1995;
Fromental-Ramain et al., 1996). Accordingly, the Hox10 triple mutant resulted in defects and reduction of the stylopod (Wellik and Capecchi, 2003).
Hox function in genitals
Although external genitalia and limbs have distinct form and function, the initial phase of genital tubercle (GT) development is in many ways reminiscent of limb bud development (Cohn, 2011). In particular, Hox gene expression in developing genital buds is very similar to that of autopods. Indeed, Hoxd9-Hoxd13 and Hoxa13 genes are expressed following the same quantitative collinearity needed for correct digit patterning (Montavon et al., 2008). The progressive removal of posterior Hoxd genes and Hoxa13 is accompanied by an increase in mutant phenotype severity, ending with complete agenesis of both digits and external genitalia in the most severe case (Kondo et al., 1997; Zákány et al., 1997). The hand- foot-genital syndrome, caused by a mutation in the Hoxa13 gene that leads to the generation of a truncated Hoxa13 protein, is an example of a natural occurring human disorder that affects both structures (Mortlock and Innis, 1997).
Even though there is a strong resemblance between genital bud development and limb development, important distinctions between the morphogenesis processes of the two structures have been pointed out. An important difference, for example, is the fact that while the limb and genital bud both have a mesodermal and ectodermal component, only the genital bud has an additional endodermal component (Cohn, 2011).
Hox function in other structures
Hox genes also play an important role in kidney organogenesis. Double mutants for Hoxa11 and Hoxd11 resulted in abnormal kidneys and triple mutants for all Hox11 genes result in kidney agenesis (Patterson et al., 2001; Wellik et al., 2002). Ectopic expression of Hoxd13 in the developing metanephric blastema originated mice with no kidneys, likely through downregulation of Hox11 genes (Kmita et al., 2000). Abnormal kidneys were also observed upon Hoxb8 and Hoxb7 overexpression (Argào et al., 1995; Charité et al., 1994).
Rearrangements within the Hoxd cluster showed that posterior gene gain of function results in hypoplasia of the kidney. On the other hand, Hoxd4-Hoxd9 gene deletion resulted in microcystic defects and increased apoptosis in the kidneys (Di-Poï et al., 2007).
The correct patterning and function of the gastrointestinal tract is disturbed by Hox gene misexpression in a regional manner (Boulet and Capecchi, 1996; Kondo et al., 1996). In the caecum Hoxd genes were found to be expressed in a non-collinear spatial pattern but more posterior genes such Hoxd13 and Hoxd12 are completely absent. In fact, mutant mice in which Hoxd12 is ectopically expressed in this tissue do not form a caecum (Zacchetti et al., 2007).
The deletion of Hoxc13 has led to the surprising finding that this gene is essential for hair development. Indeed Hoxc13 mutant animals lack vibrissae and have brittle hair, which eventually leads to alopecia. Accordingly, the integration of a reporter gene in the Hoxc13 locus revealed expression in nails, vibrissae, hair follicles and the filiform papillae of the tongue. Although other Hox genes have been reported to be expressed in the skin, they appear to show a collinear type of expression, which is not the case for Hoxc13 (Duboule, 1998;
Godwin and Capecchi, 1998).
1.3 Hox gene regulation
1.3.1 Evolution of cis-regulatory regions
Vertebrates all develop from one single fertilized cell to a complex multicellular organism and comprise a wide range of morphologies. Interestingly, different vertebrate species acquire distinct forms that are for the most part specified during embryonic development using the same key developmental genes. Therefore, morphological variation is
most probably stemming from either mutations that affect transcription factors and signaling molecules directly (trans) or mutations in the sequences that regulate expression of a gene located in the same chromosome (cis) (Carroll, 2008; Wittkopp and Kalay, 2012).
Mutations in transcription factor sequence are often disadvantageous or deleterious and are therefore under stronger selective pressure. However, gene expression can be driven by a variety of different tissue or time-specific enhancers. Mutations in these regulatory sequences are less likely to be negatively selected and could in some cases represent an evolutionary advantage (Wittkopp and Kalay, 2012).
Conservation of non-coding sequences has long been used as a method to identify enhancer elements (Alonso et al., 2009). This approach has led to the discovery of highly conserved elements that have either maintained or changed the regulatory specificity compared to the homologous sequence in other species (Maeso et al., 2013; Royo et al., 2011).
However, recent advances in high-throughput technologies have made it possible to obtain genome-wide analysis of gene transcription, chromatin structure and transcription factor binding in different tissues and cell types, mostly of mammalian species. These studies have clearly demonstrated that cis-regulatory sequences have evolved very rapidly and can show distinct transcription factor occupancy despite sequence conservation (Odom et al., 2007; Schmidt et al., 2010; Villar et al., 2015; Yue et al., 2014). However, the comparison between mouse and human transcription factor binding complemented with assessment of open chromatin regions has revealed that pleiotropic regulatory regions are associated with higher conservation of transcription factor occupancy (Cheng et al., 2014). It has also been proposed that when embryonic tissues are used there is less variation in transcription factor occupancy perhaps because developmental enhancers are under higher selective pressure (Sakabe and Nobrega, 2013).
1.3.2 Repeat elements and the evolution of regulation
Transposable element (TE) derived content makes up at least half of the mouse and human genomes (Lander et al., 2001; Waterston et al., 2002). These segments of DNA were first found in maize and are characterized by their ability to “jump” and replicate within genomes (McClintock, 1956). TEs are classified in different families by mode of transposition and sequence similarity. Class I TEs (retroelements) transpose through an RNA intermediate that is then reverse transcribed back to DNA and inserted back in the genome.
Within this class are the long terminal repeat retrotransposons (LTRs) and long and short interspersed elements (LINES and SINES). Class II elements, on the other hand, are DNA
transposons and do not use an RNA intermediate. Instead they transpose through a “cut and paste” mechanism or replicate from DNA to DNA into the genome using a “copy and paste”
mechanism (Feschotte and Pritham, 2007; Finnegan, 1992).
TE insertion or excision from the genome can, in some cases, result in phenotypic consequences. However, TE-mediated regulatory changes can take a long time to evolve into a new type of regulation. For this reason it is not always easy to identify if novel regulatory mechanisms have arisen from TEs since these elements might have degraded beyond recognition (Kidwell and Lisch, 1997).
The insertion of repeats in exons is often deleterious and thus it is normally under negative selection. In rare cases, however, these types of mutations can result in phenotypic diversity (Rubin et al., 1982). On the other hand, TE-mediated mutations in regulatory regions are more likely to affect gene expression only in particular tissues or change the timing of activation and thus have a reduced deleterious effect while creating the opportunity for genetic and phenotypic variation (Kidwell and Lisch, 1997). TEs can also disrupt the host genome by inducing ectopic recombination or chromosomal insertions and translocations (Kidwell and Lisch, 2001).
In many cases TEs are targeted for silencing through RNAi-based mechanisms and heterochromatin formation, which could result in the silencing of nearby regions as a side effect (Feschotte and Pritham, 2007). More recently, evidence has accumulated indicating that transposable elements play a role in the fast evolutionary pace of enhancer elements by providing new transcription factor binding sites (Bourque et al., 2008; Kunarso et al., 2010;
Schmidt et al.).
1.3.3 Hox gene regulation in the main body axis
Initiation, establishment and maintenance of Hox gene expression
Hox gene expression is initially activated in the primitive streak during gastrulation and subsequently spreads anteriorly at specific timings (Deschamps and van Nes, 2005;
Forlani et al., 2003). FGF and Wnt signaling, essential for gastrulation movements, play an important role in the regulation of Hox gene expression in the forming mesoderm (Dubrulle et al., 2001; Forlani et al., 2003; Kessel and Gruss, 1991). Retinoic acid (RA) as well as segmentation genes such as components of the Notch signaling pathway also appear to modulate Hox gene expression insuring that the correct rostral boundaries are established (Cordes et al., 2004; Kessel and Gruss, 1991).
Hox gene expression in neurectoderm is modulated independently from mesodermal expression. Normal Hox gene expression in the hindbrain is dependent on promoter sensitivity to RA signaling (Gavalas and Krumlauf, 2000). More posterior neural tissue has a distinct embryonic origin and relies on RA and FGF opposing actions. RA is produced by neighboring mesoderm and commits cells to a neural fate, while FGF maintains a posterior stem cell zone. This process is essential for a concerted formation of mesodermal and neurectodermal tissue along the main AP axis (del Corral and Storey, 2004). Therefore RA and FGF regulate Hox gene expression in both mesoderm and neurectoderm although different rostral boundaries of expression in the two tissues indicate that different regulatory specificities are involved (del Corral and Storey, 2004; Deschamps and van Nes, 2005).
While Fgf, Wnt and RA have been shown to modulate Hox gene expression, it is not clear if this is achieved through direct or indirect regulation. Cdx genes, on the other hand, have been shown to directly regulate Hox gene expression in the developing AP axis in a dose-dependent manner and are likely to relay positional information provided by Fgf, Wnt and RA (Deschamps and van Nes, 2005).
Once Hox active or inactive states of expression have been established, they are maintained by Polycomb (PcG) and trithorax (trxG) group proteins. In fruit flies, PcG mutants were shown to result in posterior homeotic transformations caused by the anteriorization of homeotic gene expression (Lewis, 1978). Later, trxG genes were identified for suppressing PcG mutant phenotypes. The antagonist action of these protein groups has now long been recognized for their role in maintaining cell identity (Ringrose and Paro, 2004). Hox genes are kept in a silent state by the action of PcG, while trxG ensures that Hox genes remain transcriptionally active. The silencing activity of PcG is achieved by two Polycomb complexes: PRC1 and PRC2. The PRC2 complex contains a component with methyltransferase activity that catalyzes methylation of Lysine 27 (K27) in histone H3 (H3).
This histone modification recruits PRC1 that has a chromatin compaction activity and contains an ubiquitin E3 ligase named RING1B. RING1B catalyzes the monoubiquitylation of histone H2A which results in Hox gene silencing (Schuettengruber and Cavalli, 2009). On the other hand, trxG proteins catalyze the trimethylation H3 at lysine 4, a histone mark usually associated with active gene expression (Schuettengruber et al., 2007). Therefore, PcG and trxG are essential for maintaining Hox gene expression spatially restricted throughout development.
Regulation of spatial and temporal collinearity
In the past it has been hypothesized that, in vertebrates, the ordered and nested expression along the anterior-posterior axis (spatial collinearity) was a read-out of the
sequential activation of Hox genes with respect to their relative order in the chromosome (temporal collinearity) (Duboule, 1994). Currently, evidence has surfaced that counter this intuitive explanation and dissociate the temporal and spatial aspects of collinearity. In the first place, as previously mentioned (see section 1.1.2) although temporal collinearity occurs only in animals with a relatively organized Hox cluster, spatial collinearity is a more widespread phenomenon and has been reported even in species with scattered atomized clusters (Seo et al., 2004). Secondly, single Hox transgenes randomly integrated in the mouse genome have been show to resemble endogenous spatial expression (Oosterveen et al., 2003). In addition, a series of deletions within the HoxD cluster that changed Hox gene activation timing was shown to have, for the most part, no effect on their final domain of expression. It was also demonstrated that, although sequences inside the cluster played a role in temporal collinearity, other sequences located in the surrounding 5’ (centromeric) and 3’ (telomeric) gene deserts provided negative and positive regulatory influences, respectively, in order for the activation to occur in the correct timing (Tschopp and Duboule, 2011).
The action of PcG and trxG has been shown to have an important role in establishing chromatin compartments during Hox gene collinear expression in time and space (Noordermeer et al., 2014; Noordermeer et al., 2011; Soshnikova and Duboule, 2009). At very early stages the H3K4me3 mark only covers the 3’-most Hox genes that are active in the developing embryo. The remaining genes that are yet to be expressed are instead covered by H3K27me3. As the embryo develops, the activation of more 5’ genes in the tail bud is accompanied by a progressive removal of the H3K27me3 mark that is replaced by H3K4me3 (Soshnikova and Duboule, 2009). Recently it has been shown that this change in epigenetic status is reflected at the level of chromatin 3D conformation, keeping inactive genes isolated from active genes. A similar epigenetic mechanism is associated to spatial collinearity. In the forebrain where no Hox genes are expressed, the entire cluster is covered by H3K27me3 and compacted in one single structure. In contrast, more posterior tissues show a bimodal organization where the H3K4me3 domain is restricted to the Hox genes that are expressed, while inactive genes lie in a separate compartment covered by H3K27me3 (Noordermeer et al., 2011). Although these histone changes are tightly related to the collinear expression essential for correct axial skeleton patterning, the mechanisms behind the recruitment of PcG and trxG are not well understood (Beisel and Paro, 2011).
1.3.4 Hox gene regulation in limbs
Neofunctionalization of Hox genes is thought to have occurred by acquiring enhancers that conferred additional expression specificities. Perhaps as a way to avoid
interference with more ancestral modes of regulation, these novel enhancers have often been found to lie outside of the Hox cluster, at least as far as HoxA and HoxD clusters are concerned (Berlivet et al., 2013; Duboule, 2007; Montavon et al., 2011). While both clusters are surrounded by long-range regulatory regions, the HoxD regulatory landscapes have the particularity of being flanked by two 1 megabase (Mb)-long gene deserts that appear to mostly contain regulatory sequences (Andrey et al., 2013; Montavon et al., 2011). The random integration of a human HoxD cluster devoid of the surrounding genomic context results in expression limited to the main body axis. Conversely, the deletion of the cluster yields reporter gene expression in the limb bud, showing that Hoxd gene regulation in the limb originates from sequences located outside the cluster (Spitz et al., 2001).
The two waves of Hox gene expression in developing limbs (see section 1.2.3) have been shown to rely on distinct regulatory modalities. By complementing chromosome capture techniques with disruption of Hox regulatory landscapes and detection of histone marks, a full picture of the complex regulation behind limb patterning starts to emerge. The regulatory mechanisms behind limb Hoxd gene expression, in particular, have been extensively studied (Andrey et al., 2013; Montavon et al., 2011).
Even though the regulation of Hox gene expression in the limb has long been attributed to sequences located outside of the cluster, it is only recently that the full extent of these regulatory landscapes has been revealed (Andrey et al., 2013; Montavon et al., 2011).
Indeed, only the deletion of the entire centromeric or telomeric gene desert resulted in the complete abrogation of gene expression either in distal or proximal limb, respectively.
Chromosomal conformation capture experiments and histone 3 lysine 27 acetylation (H3K27ac) enrichment, a histone mark for putative active promoters and enhancers, revealed limb enhancer candidates spread across the length of the two landscapes. LacZ reporter experiments confirmed the limb regulatory activity of some of these sequences (Andrey et al., 2013; Montavon et al., 2011).
Hi-C techniques have shown that the two regulatory gene deserts correspond to two topological associating domains (TADs) with the boundary located in the 5’ region of the cluster. Interestingly, it has been found that the change between early and late regulation of Hox genes in the limb bud relies on the switch between the centromeric and telomeric TADs.
This switch is mainly seen for Hox9-11 genes that are expressed both in proximal and distal limb. When the early limb bud starts to develop this subset of genes interacts mostly with the telomeric TAD while later, as the autopod forms, the pattern of contacts changes and there is a significant increase of interactions with the centromeric TAD (Andrey et al., 2013). The Hoxa genes that together with Hoxd genes pattern the limb were also shown to employ a bimodal type of regulation with long-range regulatory sequences. Interestingly, zebrafish HoxA and HoxD clusters were shown to also possess a bimodal chromatin structure
(Woltering et al., 2014). The invertebrate amphioxus Hox cluster, however, was found to be located in a single structural domain (Acemel et al., 2016). The bimodal chromatin structure found at the HoxA and HoxD loci would therefore represent a vertebrate evolutionary novelty that has predated tetrapod evolution.
1.3.5 Hox gene regulation in genitals
As previously stated (see section 1.2.3), genital and digit specification is achieved by the activity of the same subset of Hox genes. In addition to this, regulatory elements located centromeric to the HoxD cluster were shown to drive reporter gene expression both in the genital tubercle and in limbs (Gonzalez et al., 2007; Spitz et al., 2003). Recently, it has been shown that Hoxd gene regulation in limbs and genitals is achieved by sequences located in the same TAD, centromeric to the cluster. In addition, even though most enhancers activate Hoxd expression in both appendages, it appears that a small number of regulatory elements are either limb or genital-specific. A similar mechanism of enhancer and topology sharing to activate gene expression in genitals and limbs was also found in the HoxA regulatory landscapes (Lonfat et al., 2014).
These experiments led to the proposal that pre-existing contacts would have facilitated the acquisition of new enhancer functions by acquiring binding sites for tissue- specific transcription factors. Such “genomic niches” might have been in place before the divergence of the HoxA and HoxD clusters and could have provided a fertile ground for new regulatory elements to emerge (Lonfat and Duboule, 2015). The fact that such an organization is seen in teleost fishes that have no limbs and no external genitalia supports this evolutionary argument (Woltering et al., 2014).
Recently, the genome-wide analysis of H3K27ac coverage in different tissues revealed that enhancer sharing between genitals and limbs is not a feature exclusive of the Hox landscapes. Instead, more than 1500 regions in limb and genital tissues were found to be H3K27ac-rich in both appendage types (Infante et al., 2015).
1.3.6 Hox gene regulation in kidney and caecum
Mutant strains of different deletions within the HoxD cluster revealed that posterior and anterior Hox genes are involved in the development of either mesenchymal or epithelial components of the early kidney, respectively. Regulation by long-range enhancers was proposed to be necessary for correct Hoxd gene expression in this tissue. Although distinct
regulatory sequences are likely to be involved in activating the two groups of genes in their respective compartments, it would appear that both mesenchymal and epithelial enhancers are located in the telomeric gene desert (Di-Poï et al., 2007).
Hoxd gene regulation in the caecum was also found to be regulated by several regulatory elements located throughout the telomeric gene desert. Interestingly, orthologous sequences from a species that does not have a caecum, the hedgehog, were still able to drive Hox gene expression in the caecum of the mouse in a transgenic context (Delpretti et al., 2013).
1.4 Evo-Devo of the snake
The snake elongated and limbless morphology has emerged as an adaptation to the burrowing life style adopted by an ancestral lizard (Vidal and Hedges, 2004). The resulting divergent morphology of this vertebrate is of great interest to understand the underlying mechanisms of some important aspects of embryonic development. Indeed, partial or complete limb loss in snakes would bring insight into limb field determination and limb development and would help to elucidate how limb loss has evolved multiple times over the course of evolution. One study has approached these questions by using pythons, a species with vestigial hindlimbs that develop into copulatory spurs in the adult. Interestingly, python limb buds are devoid of an apical ectodermal ridge, a signaling center instrumental for limb growth, and do not express Shh (Cohn and Tickle, 1999).
Another striking feature of the snake body plan is the very elongated body plan composed of a large number of vertebrae that originate from somites. The mechanisms behind such an extreme phenotype can bring important insight into the processes of axial extension, segmentation and main body axis patterning. The study of somitogenesis in corn snake embryos showed that the large amount of somites mainly results from both a faster segmentation clock and a delay in the shrinkage of the presomitic mesoderm, compared to other vertebrates (Gomez et al., 2008).
1.4.1 Hox gene expression in snakes
Owing to their important patterning function both in the main body axis and limbs, Hox genes have been proposed to play a role in the generation of snake-like body plans (Woltering, 2012). The sequencing of the king cobra and python genomes have revealed that, despite their extreme body plan, most Hox genes are present with the exception of Hoxd12
and have maintained a cluster-like organization (Castoe et al., 2013; Vonk et al., 2013).
Detailed expression analysis in corn snake embryos surprisingly showed that spatial collinearity was respected in such a way that 3’ Hox genes were expressed more anteriorly than 5’ Hox genes. However, expression of paralogous groups 7 to 9 showed no apparent correlation with morphological boundaries (Woltering et al., 2009). In addition, Hoxa13 and Hoxd13 that belong to the paralog group involved in axial termination were found to be expressed transiently in the embryonic tail at very low and transient levels. Surprisingly, Hoxa10 and Hoxc10 genes, which in mammals code for transcription factors with rib- repressing activity, were found to be expressed in somites that would originate rib-bearing vertebrae (Di-Poï et al., 2010; Woltering et al., 2009). This expression pattern was initially attributed to loss of function of the Hoxa10 and Hoxc10 proteins. However, when overexpressed in the mouse presomitic mesoderm, the snake Hoxa10 gene was able to repress rib formation. In this case, a target gene enhancer was shown to have a polymorphism in the Hox binding site, thereby preventing Hox10 transcription factor binding and further rib- supressing processes to take place (Guerreiro et al., 2013).
1.4.2 Transposable elements in squamate Hox clusters
Squamates are a diverse order of reptiles that comprise all lizards and snakes. The first squamate genome to be sequenced was from the green anole lizard (Anolis carolinensis).
The analysis of the lizard Hox clusters revealed them to have incorporated a large amount of transposable element content in comparison with the Hox clusters of other vertebrates. As a result, Anolis carolinensis Hox clusters were found to be 1.5 to 2.5 fold larger. Interestingly, the increase in size appeared to be larger in the cluster itself compared to the surrounding genomic regions (Di-Poï et al., 2009). The sequencing of the posterior part of other squamate Hox clusters showed that the unusual high repeat content observed was not exclusive to the green anole lizard but rather appeared to be present in all squamates (Di-Poï et al., 2010).
Interestingly, the Hox clusters of all other jawed vertebrates investigated to date are mostly devoid of repeat content, likely due to the restraints imposed by the action of long range global enhancers (Duboule, 2007). This led to the hypothesis that the unusually large number of TE content present in squamate Hox clusters may have led to Hox gene misexpression promoting morphological diversity in squamates (Di-Poï et al., 2009; Di-Poï et al., 2010).
The complete Anolis genome sequence revealed the presence of a large variety of TEs that showed traces of recent evolution and activity (Alfoldi et al., 2011). Accordingly, the recent sequencing of the Burmese python, king cobra, corn snake and other snake genomes has also shown the presence of diverse types of repeat elements (Castoe et al., 2013; Ullate-
