Decision letter: Reorganisation of Hoxd regulatory landscapes during the evolution of a snake-like body plan

Abstract

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Within land vertebrate species, snakes display extreme variations in their body plan, characterized by the absence of limbs and an elongated morphology. Such a particular interpretation of the basic vertebrate body architecture has often been associated with changes in the function or regulation of Hox genes. Here, we use an interspecies comparative approach to investigate different regulatory aspects at the snake HoxD locus. We report that, unlike in other vertebrates, snake mesoderm-specific enhancers are mostly located within the HoxD cluster itself rather than outside. In addition, despite both the absence of limbs and an altered Hoxd gene regulation in external genitalia, the limb-associated bimodal HoxD chromatin structure is maintained at the snake locus. Finally, we show that snake and mouse orthologous enhancer sequences can display distinct expression specificities. These results show that vertebrate morphological evolution likely involved extensive reorganisation at Hox loci, yet within a generally conserved regulatory framework. https://doi.org/10.7554/eLife.16087.001 eLife digest Animals with a backbone can look remarkably different from one another, like fish and birds, for example. Nevertheless, these animals – which are also known as vertebrates – have many genes in common that shape their bodies during development. These genes include a family called the Hox genes, which control how an animal’s body parts develop from its head to its tail and are needed to shape the animal’s limbs. Hox genes are found clustered in groups within a vertebrate’s DNA, and large regions of DNA on either side of a Hox cluster can, in some cases, physically interact with the Hox genes to regulate their expression. So how do the same genes produce different body shapes? Different vertebrates regulate where and when their Hox genes are switched off and on in different ways. As such, it is likely that differences in gene regulation, rather than in the genes themselves, lead embryos to develop into the distinct shapes seen across the animal kingdom. Snakes – for example – evolved from a lizard-like ancestor into elongated limbless animals as they have adapted to a burrowing lifestyle. However, it was not known if changes in how Hox genes are regulated have played a role in shaping the distinct body plan of snakes. Guerreiro et al. have now compared how Hox genes are regulated in snakes, mice and other vertebrates, focusing on corn snakes and one particular cluster of Hox genes called the HoxD cluster. The comparison revealed that these Hox genes are regulated differently in developing snakes than in other vertebrate embryos. This is particularly the case for tissues that show the most differences when compared with other animals (such as the torso and genitals) or that are absent (such as the limbs). Although Hoxd genes are activated at different times and places in snakes than in other vertebrates, snake Hox genes appear to be regulated using the same general mechanisms as mouse Hox genes. Guerreiro et al. suggest that changes to Hoxd gene regulation have contributed to the evolution of the snake’s shape and have most likely influenced the body shapes of other vertebrates as well. However, the findings also suggest that these gene regulatory changes have been constrained by an existing regulatory mechanism that has been maintained throughout evolution. It remains for future work to address whether these changes in Hox gene regulation are a cause or a consequence of the snake’s extreme body shape, or indeed a combination of the two. https://doi.org/10.7554/eLife.16087.002 Introduction Even though vertebrate species can display different morphologies, they all contain a strikingly similar repertoire of transcription factors and signalling molecules. In particular, genes with critical functions during embryonic development are often largely pleiotropic and highly conserved across species (for references, see Kirschner et al., 2005; Duboule and Wilkins, 1998). This universality of genetic and genomic principles has changed the evolutionary paradigm from the question of the nature of similarities to that of how distinct traits could evolve using such related developmental pathways (Carroll, 2008; De Robertis, 2008). Initially, Hox genes, as well as their structural and functional organization into genomic clusters were found well conserved across bilateria (Duboule and Dolle, 1989; Akam, 1989; Garcia-Fernandez and Holland, 1994; Graham et al., 1989; McGinnis et al., 1984). In addition, their mis-expression led to changes in the identity of both insect and vertebrate segments, illustrating their crucial role in the patterning of animal structures, even though the structures they specify are of very different nature in various taxa (e.g. (Maeda, 2006; Lewis, 1978; Krumlauf, 1994). Tetrapods generally have four clusters of Hox genes (HoxA, HoxB, HoxC and HoxD), originating from genome duplications early in the vertebrate lineage (see e.g. Lemons and McGinnis, 2006) and located on different chromosomes, unlike fishes or some jawless vertebrates, which have more (Prince et al., 1998; Mehta et al., 2013; Amores et al., 1998). In addition, all vertebrate Hox clusters described thus far implement a particular type of regulatory process referred to as collinearity, whereby Hox genes are expressed sequentially in both time and space following their topological organization within each genomic cluster (Gaunt et al., 1988; Izpisua-Belmonte et al., 1991). This regulatory property is first observed during axial extension and, subsequently, in some structures such as the limbs (see (Deschamps, 2007; Deschamps and van Nes, 2005). In this latter case, and while the detailed underlying mechanism may be distinct from that at work in the major body axis (Kmita and Duboule, 2003), the general principle remains the same and was likely co-opted in the course of tetrapod evolution (Spitz et al., 2001), through the emergence of global enhancers located at remote positions on both sides of the cluster (Lonfat et al., 2014). These complex regulations were extensively studied in the mouse, in particular at the HoxD locus, by using various targeted approaches in vivo. The HoxD cluster is surrounded by two gene deserts of approximately 1 Mb (megabase) in size, each one containing distinct sets of enhancers capable of activating specific sub-groups of target Hoxd genes depending on their location within the cluster. Each of these two gene deserts can be superimposed to a Topologically Associating Domain (TAD), i.e. a chromatin domain where DNA-DNA interactions in cis are privileged, for example between promoters and enhancers, and determined through chromosome conformation capture technologies (Dixon et al., 2012; Nora et al., 2012). The centromeric gene desert can activate the transcription of the Hoxd9 to Hoxd13 genes, whereas the telomeric gene desert, which is further subdivided into two sub-TADs (Andrey et al., 2013) controls the expression of Hoxd1 to Hoxd11 (see [Lonfat and Duboule, 2015]). This bimodal regulation allows for the selected expression of Hoxd gene sub-groups in a series of secondary embryonic structures. During limb development, for instance, the telomeric TAD initially controls all genes from Hoxd3 to Hoxd11 in the proximal part of the limb bud, whereas more posterior genes such as Hoxd13 or Hoxd12 are controlled subsequently in the most distal aspect of the incipient limb by enhancers located within the centromeric TAD (Andrey et al., 2013). This latter regulatory landscape also controls transcription of the same posterior genes during the outgrowth of external genitalia (Lonfat et al., 2014). Since snakes are limbless animals and they display highly specialized and divergent external genitals (Tschopp et al., 2014), the existence of such a bimodal type of regulation at the snake HoxD locus was uncertain. Therefore, we set out to investigate Hox gene regulation in snakes. While these animals cannot yet be considered as genuine model systems (Guerreiro and Duboule, 2014; Milinkovitch and Tzika, 2007), recent advances in their genomic analyses make their study increasingly interesting in an Evo-Devo context (Castoe et al., 2013; Gilbert et al., 2014; Ullate-Agote et al., 2014; Vonk et al., 2013). These analyses revealed that snakes, regardless of their extreme morphologies, have a tetrapod-like complement of Hox genes with only a few exceptions (Vonk et al., 2013; Di-Poï et al., 2010). Consequently, the serpentiform body plan may have evolved either along with changes in time and space of Hox gene expression or with a different interpretation of Hox protein functions (see [Di-Poï et al., 2010; Woltering et al., 2009]). The analysis of Hox gene expression in the developing corn snake (Pantherophis guttatus) revealed a surprisingly well conserved collinear mRNA distribution along the anterior-posterior axis. However, the rather strict correlation between morphological landmarks and the anterior borders of Hox transcript domains, usually seen in mammals and birds, was not always present in snakes (Burke et al., 1995; Woltering et al., 2009) (see also Head and Polly [2015]). It was thus concluded that some Hox proteins had likely changed (part of) their functionality. In addition, the fact that the most posterior Hox genes were poorly expressed in the extending tailbud was tentatively associated to the unusually large number of segments (Di-Poï et al., 2010), together with an increased pace in segmentation (Gomez et al., 2008). In this work, we used a combination of experimental approaches to try and elucidate the nature of the differences in Hoxd gene regulation between snakes and mice at comparable stages of their early development. We find that, even though the structural organization of the corn snake HoxD cluster resembles that of tetrapods, the extreme body plan observed in snakes is associated with an extensive regulatory restructuring. In snakes, mesodermal enhancers are mostly located inside the cluster itself, whereas other vertebrates make use of long-range regulations located at remote positions. In addition, we show that despite the loss of limbs, the bimodal chromatin organisation at the Hoxd locus found in tetrapods is conserved in the snake lineage. However, we find that the regulation of snake Hoxd genes during the development of the external genitalia is different from that of other tetrapods, even though the general logic is conserved. In this latter case, the change in enhancer activity from a limb to an external genital specificity seems to have occurred. Altogether, we conclude that Hoxd gene regulation in the snake is in many ways distinct from the situation in mammals. We discuss the possible causative nature of these changes in the evolutionary transformation towards a serpentiform body plan. Results To analyse the regulation of the snake HoxD cluster, we initially had to complement the available genomic information (Ullate-Agote et al., 2014) with high coverage sequencing of the gene cluster itself, along with the two flanking gene deserts. We screened a corn snake custom-made BAC library using as probes DNA sequences conserved from mammals to the anole lizard, present within this large DNA interval. A scaffold was built out of 13 overlapping BACs, which were selected for sequencing and from which a 1.3 Mb large DNA sequence of high quality was obtained (Figure 1—figure supplement 1A). The structural analysis of the corn snake HoxD cluster revealed that, as for other species of snakes whose genomes were recently released (Vonk et al., 2013; Castoe et al., 2013), all Hoxd genes but Hoxd12 are present and share the same transcriptional orientation within the cluster. When compared to the mouse, the corn snake cluster is about 1.5 fold larger (Figure 1A) (Vonk et al., 2013), likely due to a higher repeat content (Di-Poï et al., 2009) and consistent with the structures of the HoxD clusters of both the king cobra and the python (Castoe et al., 2013; Vonk et al., 2013). Figure 1 with 2 supplements see all Download asset Open asset The snake HoxD cluster. (A) Schematic representation at the same scale of the mouse (top) and corn snake (bottom) HoxD clusters. Exons are represented by black rectangles. (B) Whole-mount in situ hybridization of corn snake embryos at 8.5 dpo (days post oviposition) showing expression of Hoxd4, Hoxd9, Hoxd11 and Hoxd13. Numbers define the somite number where the most anterior levels of expression are detected. The black arrowhead points to the neural tube whereas the white arrowhead shows mesoderm. A single black arrowhead indicates that the neural and mesodermal boundaries coincided. (C) Detection of both H3K9me3 and H3K27me3 histone modifications by ChIP-seq in corn snake brain (top and middle tracks) and of H3K27me3 marks in the posterior trunk of 0.5–2.5 dpo snake embryos (bottom track). Blue is for brain and orange for posterior trunk, as schematized on the left. The black peaks in the top track represent artifactual signals also present in the input chromatin mapping. https://doi.org/10.7554/eLife.16087.003 We plotted the sizes of the mammalian, reptile, bird and fish HoxD clusters against the size of their respective genome. When reptiles were excluded from the linear regression analysis, an R2 value of 0.43 was scored, indicating significant correlation. However, when the corn snake, king cobra and Burmese python cluster sizes were added to the analysis, the R2 value was reduced to 0.027 (Figure 1—figure supplement 1B). Even though snake Hox clusters show a size larger than what would be expected based on their genome size, the green anole lizard cluster is, from the vertebrate species analysed, the one with the lowest level of correlation with genome size (R2=0.0012). When we performed the same correlation analysis for the regulatory gene deserts that surround the HoxD cluster, high values of R2 were scored both by excluding and including squamate values. However, the size of the squamate 3’ gene desert clearly showed a better correlation with genome size than the 5’ gene desert (Figure 1—figure supplement 1B). Because the increased size of the cluster in Squamata correlated with the presence of a high number of transposable elements (Di-Poï et al., 2009, 2010), we investigated the number and type of repeats present in the HoxD locus. We found that the corn snake cluster contains more than twice as many repeats as the mouse counterpart. In addition, while the mouse HoxD cluster is mainly composed of SINEs (short interspersed elements), the corn snake cluster is composed of different types of transposable elements including LINEs (long interspersed elements) and DNA transposons (Figure 1—figure supplement 1C). The 5’ and 3’ gene deserts that surround the cluster contain a similar amount of repeats in the two species. Both gene deserts in mice include a wider range of repeat element types than in the cluster itself, yet SINEs remain the most represented transposable elements, whereas snake gene deserts mostly included LINEs (Figure 1—figure supplement 1C). Our deep DNA sequence of the entire corn snake HoxD locus, including both flanking gene deserts, allowed a global conservation analysis to be performed between non-coding regions amongst different vertebrate species. Surprisingly, we found that the pattern of conservation of the snake HoxD genomic landscape is almost identical to that of the chicken, when compared to the mouse sequence (Figure 1—figure supplement 2). The snake HoxD cluster Because the silencing of transposable elements is often paralleled by the modification of histone H3 at lysine 9 (H3K9me3 (Kidwell and Lisch, 1997; Martens et al., 2005; Friedli and Trono, 2015), heterochromatin-like islands within the snake Hox clusters may be associated with severe modifications in gene regulation (Di-Poï et al., 2009; Woltering et al., 2009). H3K9me3 modifications are normally not found at Hox loci in tetrapods, which like many other genomic loci containing genes of importance for development, are also poor in transposons (Simons et al., 2007). Therefore, we performed a ChIP-seq experiment with an antibody against this histone modification on micro-dissected embryonic snake brain (Figure 1), a tissue that we routinely use as a negative control for Hox gene expression (Figure 1B). No particular H3K9me3 enrichment was scored over the length of the HoxD cluster and the closest located peak was identified in an intron of the Lunapark gene, i.e. at a position unlikely to have any critical impact on Hoxd gene expression (Figure 1C). In tetrapods, the proper collinear regulation of Hox gene transcription was associated with the progressive removal of H3K27me3 coverage (Soshnikova and Duboule, 2009), a histone modification deposited by the Polycomb complex PRC2 (Margueron and Reinberg, 2011). We checked if this repressive system would operate similarly during the elongation of the snake body axis by performing an H3K27me3 ChIP-seq, either in the embryonic brain, or in a part of the posterior trunk excluding the post-cloacal region (Figure 1C). In the absence of Hox gene transcription (brain), the entire cluster was decorated with H3K27me3 marks, forming a dense domain of Polycomb repression as seen previously in other species. In contrast, the posterior trunk tissue displayed an H3K27me3 coverage specifically over the 5’ part of the gene cluster, containing the most ‘posterior’ Hoxd genes (Figure 1C). In parallel, whole-mount in situ hybridization (WISH) to assess Hoxd gene expression revealed a clear correlation between the domain of active Hoxd genes and the absence of the H3K27me3 mark (Figure 1B). From these experiments, we concluded that both spatial collinearity and the associated dynamics of chromatin structure accompanying progressive gene activation are implemented in snakes as in any other vertebrate species studied thus far. Regulatory potential of the HoxD cluster in vertebrates In tetrapods, regulatory elements controlling Hox gene expression are found at various positions. The mouse HoxD cluster for instance contains regulatory elements, which are mainly involved in driving Hox gene expression along the anterior-posterior body axis during gastrulation, whereas remote enhancers located outside of the cluster itself regulate transcription in other organs or structures (Spitz et al., 2001; Lonfat and Duboule, 2015). Therefore, to try and identify snake-specific differences in the modes of regulations, we compared the regulatory potential of the snake HoxD cluster with that of other vertebrates by using a BAC transgenic approach in mice, whereby BACs containing HoxD clusters of either human, mouse, chicken, snake and zebrafish were randomly integrated in the mouse genome (Figure 2A). The expression of Hoxd4 was monitored by in situ hybridization with species-specific probes and, under these experimental conditions, all mammalian transgenic BACs showed transcript patterns restricted to the dorsal part of the main embryonic body axis (Figure 2B), resembling the pattern obtained when a single copy Hoxd4/LacZ transgene was used (Tschopp et al., 2012). Interestingly, however, this pattern represented only a subset of the full Hoxd4 expression pattern as seen either by WISH on control embryos (Figure 2B), or on previous reporter Hoxd4/lacZ transgenes likely integrated as tandem repeats (Zhang et al., 1997). Indeed, expression was scored mostly in the neural tube, yet not in the ventral mesodermal tissues of the upper trunk, i.e. above the level of hindlimbs. To better determine which mesodermal components had their Hox gene expression affected in the isolated human BAC line, we performed a Hoxd4 WISH in a sectioned embryo. We found that, at least at this stage of development, the human Hoxd4 gene was expressed only in the neural tube (Figure 2—figure supplement 1A). Figure 2 with 3 supplements see all Download asset Open asset Location of Hoxd trunk mesodermal enhancers. (A) Schematic representation, at the same scale, of the mouse, human, chicken, corn snake and zebrafish BAC clones used to generate the transgenic mouse lines. Exons are represented by black rectangles. (B) Lateral view of whole-mount in situ hybridizations of Hoxd4 using E11.5 mouse embryos transgenic either for the mouse, the human, the chicken, the zebrafish or the corn snake BAC. (C) Schemes illustrating the various deletion stocks (top) and whole-mount in situ hybridization of E12.5 mouse embryos with the Hoxd4 probe in corresponding deleted mutant embryos (bottom). LoxP sites are indicated as red triangles, the HoxD cluster is represented by a black rectangle and other genes are shown with grey rectangles. vm indicates expression in the ventral mesoderm and white asterisks represent the absence of expression in this tissue. (D) ChIP-seq analysis over the mouse and snake HoxD loci of H3K27acetylation using anterior trunk mesodermal tissue of E11.5 mouse embryos and 5.5 dpo corn snake embryos (left). Green boxes under each ChIP-seq mapping represent peaks called by the MACS algorithm (Zhang et al., 2008). On the right, a graphical representation is shown of the percentage of conserved regions between the mouse and corn snake HoxD loci, which are enriched for H3K27ac in each species. https://doi.org/10.7554/eLife.16087.006 We then investigated the expression of Hoxd4 from either the chicken or the zebrafish BAC transgenic lines and found a similar expression pattern, again mostly limited to the neural tube as well as the dorsal-most part of the somites (Figure 2B). Altogether, these results suggested that enhancers controlling the robust expression of Hoxd genes in various mesodermal derivatives are, for the most part, located outside of the cluster itself. Alternatively, some mesodermal enhancers could be located inside the HoxD cluster, yet they may require additional sequences located at remote positions to properly impact upon the transcription of target genes in physiological conditions. Consequently, we searched for the location(s) of such enhancers outside the HoxD cluster by using a set of targeted deletions flanking the locus on either side of it. Reshuffling mesodermal enhancers We first analysed the expression of Hoxd4 in mouse embryos lacking the centromeric TAD, which contains strong enhancers with digit and genital specificities (Lonfat et al., 2014). Mutant embryos carrying this HoxDDel(Atf2-Nsi) deletion (Montavon et al., 2011) showed a domain of Hoxd4 expression comparable to control embryos (Figure 2C; Del(Atf2-Nsi). In contrast, embryos carrying the HoxDDel(Attp-Sb3) deletion of the opposite TAD, located telomeric to the HoxD cluster, which contains various enhancer sequences (Andrey et al., 2013; Delpretti et al., 2012), displayed reduced amounts of mRNA steady state levels in the ventral mesoderm (Figure 2C; Del(Attp-Sb3). Consistently, the repositioning of potential telomeric enhancers several megabases far from the target genes, through the HoxDInv(Attp-CD44) inversion, displayed no clear expression in the ventral mesoderm of the thoraco-lumbar region (Figure 2—figure supplement 2). These results indicate that the telomeric gene desert contains most of the enhancers necessary for Hox gene expression in ventral mesoderm. However, unlike what was observed in the Human BAC line, Hoxd4 expression in the HoxDDel(Attp-Sb3) and HoxDInv(Attp-CD44) mutant lines was also scored in the dorsal-most part of the somites and not exclusively in the neural tube (Figure 2—figure supplement 1A and 2). To more precisely localize potential mesodermal enhancers within the deleted DNA interval, we used four additional mutant stocks carrying smaller deletions. Both the HoxDDel(Sb2-Sb3) and the HoxDDel(Sb2-65) mutant alleles (Andrey et al., 2013) resulted in expression patterns for Hoxd4 similar to that obtained with the Del(Attp-Sb3) deletion of the entire gene desert (Figure 2C; Del(Sb2-Sb3), Del(Sb2-65), i.e. lacking any detectable expression in ventral mesoderm. In contrast, such mesodermal expression was scored in the smaller HoxDDel(65-Sb3) and HoxDDel(Attp-Sb2) deletion alleles (Figure 2—figure supplement 2; Del(65-Sb3), Del(Attp-Sb2). This set of analyses indicated the presence of mesodermal enhancer(s) within a segment of the telomeric gene desert. In addition, the distribution of H3K27ac modifications in the mouse trunk mesodermal tissue, a histone mark associated with putative active enhancers and promoters, was clearly enriched in the telomeric gene desert when compared to the centromeric counterpart (Figure 2D, top) with 18 significant peaks telomeric to the cluster versus only 7 located in the centromeric gene desert. We thus concluded that most trunk mesodermal enhancers acting over Hoxd4 and presumably affecting other Hoxd genes, are located in the telomeric gene desert. Because the regulatory sequences located in the telomeric TAD were described to globally drive concomitant expression of several genes located in the central part of the gene cluster rather than individual Hoxd genes (Delpretti et al., 2013; Andrey et al., 2013), we also analysed the expression of both Hoxd3 and Hoxd8 in the absence of the telomeric gene desert. Similar to Hoxd4, the expression of these two other Hoxd genes was lost in the ventral mesoderm (Figure 2—figure supplement 1B) thus suggesting that the telomeric gene desert contains sequences necessary for the expression of multiple Hoxd genes in the ventral mesoderm of the upper trunk. Reorganization of mesodermal enhancers in the snake HoxD locus Next, we analysed the transgenic line carrying the snake HoxD cluster and found that, in this case, expression of Hoxd4 in the trunk was not dorsally restricted as observed in all other vertebrate BACs assayed thus far. The expression pattern in the main body axis was in fact reminiscent of the endogenous mouse Hoxd4 expression, with equally strong signals in both the neural tube and mesodermal derivatives (Figure 2B). Therefore, in contrast to other vertebrate species, enhancers located within the snake cluster appear sufficient to drive Hoxd gene expression in the ventral mesoderm. In order to assess if this increase in regulatory potential within the cluster was correlated with a reduction of long-range regulatory elements in the surrounding gene deserts we performed a comparative analysis of H3K27ac profiles between snake and mouse trunk tissues dissected from similar body parts. A global assessment of the profiles suggested that there were relatively less enriched sequences outside of the snake HoxD cluster than outside its mouse counterpart (Figure 2D, bottom). In order to be able to directly compare the ChIP-seq datasets in mouse and snake, we identified 27 DNA regions conserved between the two species and located within the telomeric desert and scored their enrichments with acetylation of H3K27. While 40% of these conserved sequences were acetylated in the mouse sample, only 22% of them were significantly decorated by this chromatin mark in the snake tissue (Figure 2D, right). Overall, these results indicate that the enhancers required to control snake Hoxd gene expression in the trunk mesoderm are, at least for the most part positioned within the cluster rather than in the telomeric gene desert. These DNA segments acetylated in snakes were found clustered in two regions of the gene desert as demonstrated by peak calling, whereas the mouse acetylated DNA regions span a larger portion of the gene desert (Figure 2D). Of note, one of the acetylated peaks in the mouse was scored over a region conserved in mammals, birds and amphibians, but not in snakes (Figure 2—figure supplement 3A and B). To confirm the enhancer activity of this sequence (MSS), we cloned the mouse version upstream of a LacZ reporter gene. As expected, MSS was able to drive expression in the trunk mesoderm from the forelimb to more posterior parts of the embryo (Figure 2—figure supplement 3C). Bimodal regulation in the snake HoxD regulatory locus At the mouse HoxD locus, a bimodal regulatory strategy associated with particular chromatin conformations was reported to control Hox gene expression in a variety of organs and structures. Such global controls involve separate sets of target Hoxd genes, which are thus re-activated after the major body axis is laid down (Andrey et al., 2013; Spitz et al., 2001). Most of these structures, however, are either missing in snakes, such as the limbs or the intestinal cecum or whenever present, they are nevertheless substantially different from their mammalian counterparts. Because mouse Hoxd genes contact such remote enhancer sequences via long-range interactions included within two opposite TADs (Montavon et al., 2011), we set out to see whether such a bimodal type of regulatory topology would also exist in snakes, even in the absence of many of the related functionalities. We thus used whole mouse and snake embryos of similar size to characterize the interaction profile of Hoxd genes with their surrounding regulatory landscapes. We used the 4C-seq version of chromosome conformation capture (Dekker et al., 2002; de Laat and Dekker, 2012) with four different Hoxd genes as viewpoints to assess their potential interaction tropism with either one of the flanking gene deserts. As observed in the mouse, significant interactions between the snake viewpoints and the centromeri