Conservation and Divergence of Regulatory Strategies at Hox Loci and the Origin of Tetrapod Digits

1The tetrapod limb is made out of a proximal-to-distal series of long bones, the stylopod, zeugopod in the arm, and the digits in the hand, the latter of which are separated from the former two by the mesopodium, an articulation based on an array of small roundish bones –. This skeletal organisation evolved during the Devonian as an adaptation to the buoyancy-lacking environment of the land,. The fossil record indicates that limbs evolved from fins via successive steps of distal elaboration, eventually resulting in the formation of the autopod as a tetrapod-specific evolutionary novelty, with fin radials or distal fin radials as putative evolutionary precursors of digits –.

2During mammalian limb development, the activity of both HoxA and HoxD gene clusters is essential and the absence of these two loci leads to rudimentary and truncated appendages. All long bones of the limb require the activation of Hox genes in different though partially overlapping combinations. Initially, Hoxd9 to Hoxd11 and Hoxa11 are expressed in the developing proximal limb (the presumptive arm). Subsequently, in a second phase of transcriptional activation, Hoxd9 to Hoxd13 as well as Hoxa13 are expressed in presumptive digits –. The existence of distinct regulatory modules for long bones on either side of the mesopodial articulation (the wrist and ankle), together with the separated evolutionary trajectories of these elements, has supported the view that tetrapod limbs are genetically organized following a specific bimodal pattern of proximal (arm and forearm) and distal (digits) long bones, which as such is not present in fish fins (refs. in ).

3The characterization of Hoxa and Hoxd expression patterns during fish fin bud development has re-enforced the view that changes in Hox genes' regulation were instrumental in the transformation of fins into limbs,,–. The exact nature of these changes, however, has remained controversial. The analysis of fin buds from various fish species lead to different conclusions regarding the existence in fishes of the late and distal phase of Hoxd gene expression, associated with the development of tetrapod digits (Figure 1A). This phase was indeed either considered as a tetrapod novelty,, implying an origin close to the stem of tetrapods, or alternatively as more ancestral and already present in fish –. This latter scenario suggests a deeper homology between fin radials and distal limb structures,,, which could potentially qualify distal fin radials as digit homologs. Regarding Hoxa genes, the expression of Hoxa11 and Hoxa13 is largely overlapping in fins,,,, instead of the mutually exclusive patterns observed in limbs, and is thus of little help in providing a proximo-distal (P-D) reference point.

4These analyses primarily rely on the comparison between gene expression domains whose interpretation is complicated when highly divergent structures are considered, such as fins and limbs, and hence whether or not any homology can be inferred from such expression analyses is unclear. The assessment of fish DNA sequences orthologous to tetrapod digit enhancers in transgenic mice indeed indicated their potential for regulating gene transcription in developing appendages,. However, these fish enhancers, related to tetrapod digit control sequences, appeared to drive transgene expression primarily in more proximal mouse limb territories rather than in digits. As an alternative to using gene expression to infer homologies, we looked at whether a comparison between the regulatory mechanisms underlying Hox gene transcription in both tetrapod and fish appendages could be more informative.

5The transcriptional regulation of the HoxD gene cluster during limb development becomes rather well understood. The successive proximal and distal waves of expression are controlled by distinct enhancer-containing regulatory landscapes, located in gene deserts on opposite sides of the gene cluster. A proximal landscape is located on the 3′ side of the gene cluster, whereas the distal (digit) landscape extends on the 5′ side –. These regulatory landscapes are regions of active enhancer-promoter interactions, as defined by chromosome conformation capture (4C), and their genomic extents and properties strongly suggest that they correspond to recently defined topological domains (Figure 1B) —that is, ca. 100 kb to megabase large chromatin domains, which provide a permissive environment for long-range enhancer—promoter interactions,. Hoxd9 to Hoxd11, which are located in the central part of the gene cluster, successively interact with either one of these 3′ and 5′-located regulatory landscapes depending upon which series of enhancers are active, thus switching their contacts from one landscape to the other at the time of the transition between cells with a proximal fate to cells forming the presumptive digits.

6In contrast, genes that are situated at either extremity of the cluster always interact within their neighbor landscape and will not switch their contacts. Hoxd13 for instance will only interact with 5′-located enhancers and, as a consequence, will be transcribed only in the distal limb territory. Therefore, a bimodal chromatin organization of the HoxD locus prefigures the bimodal expression of Hoxd genes with their proximal and/or distal specificities, leading to the tetrapod P-D limb axis. The expression patterns of Hoxa genes suggested that this collinear property of Hoxd genes may also apply to their Hoxa paralogs and each gene cluster on its own is capable of specifying a complete limb P-D axis, as demonstrated by the full deletion of either HoxA or HoxD,,. However, whether or not these functional similarities reflect a conservation of regulatory strategies remained elusive. Here, we report that this mode of regulation is globally conserved between both gene clusters, suggesting that its emergence predated the origin of tetrapods. In addition, we looked at the situation in fishes and analyzed the regulatory potential of their Hox clusters in the context of transgenic mice.

7We first evaluated whether the bimodal regulatory strategy observed at the HoxD cluster was particular to this locus or, in contrast, was shared with HoxA during limb development, in which case such regulatory modalities would likely predate the duplication of Hox clusters and hence the emergence of tetrapods. We looked at the expression patterns of Hoxa genes to see how well they adhere to the proximal-to-distal restrictions observed for Hoxd genes in budding limbs,,. Although a weak expression of Hoxa4 was scored proximal to the digits in E12.5 limbs, strong expression of Hoxa9 and Hoxa10 was detected both in developing digits and in a more proximal domain, corresponding to the presumptive forearm. In contrast, both Hoxa13 and the Hoxa11 antisense transcript (Hoxa11as) accumulate only in the distal, presumptive digit domain and in the future wrist (Figure 2A). A noticeable difference with Hoxd genes was observed, however, as Hoxa11 transcripts are absent from this distal domain, while present in the proximal territory, suggesting that Hoxa11 may escape the distal regulation imposed on the Hoxa9 to Hoxa13 genes, unlike in the case of HoxD, where Hoxd9 to Hoxd13 genes are coregulated in digits.

8It has, however, recently been shown that Hox13 group genes repress Hoxa11 in the distal limb. In the absence of Hoxa13 function, the expression of Hoxa11 shifts into the distal limb bud to partially overlap with the expression of the inactivated Hoxa13 transcript, as detected by using a 3′UTR probe (Figure 2B, compare left and central panels). When doses of Hoxa13 and Hoxd13 functions were progressively removed, the distal extension was strengthened and Hoxa11 transcripts were found almost throughout the entire developing autopod (Figure 2B, right panels), much like its antisense Hoxa11as transcript in the wild-type condition. Although this result indicates that both Hoxa13 and Hoxd13 products repress Hoxa11 expression, it also demonstrates that the Hoxa11 promoter can readily respond to the distal regulation, much like Hoxa13. Therefore, it appears that the HoxA cluster, like HoxD, is the target of a distal limb (digit) global enhancer, which can regulate at least two distinct promoters. On the other hand, Hoxa4, Hoxa9, Hoxa10, and Hoxa11 are transcribed in a wider territory, including the proximal limb region, suggesting that as for the HoxD cluster, Hoxa genes are regulated by two distinct regulatory modules during limb budding and patterning.

9This regulatory dichotomy may correlate with another similarity between the HoxA and the HoxD cluster—that is, the fact that HoxA too is located at the junction between two topological domains. These data were, however, obtained in ES cells and thus we further characterized the three-dimensional chromatin dynamics of the HoxA cluster during limb development, in comparison with forebrain cells where all Hox genes are inactive, at least at this stage. We implemented circular chromosome conformation capture and deep sequencing (4C-seq),, using Hoxa4, Hoxa9, Hoxa11, and Hoxa13 as baits in E12.5 dissected presumptive digits, proximal limb, and forebrain cells. The distribution of contacts over an 8 Mb DNA interval (Figure S1A), as judged by the number of sequence reads, shows that ca. 90% of the interactions are concentrated within the regions corresponding to four topological domains as determined in ES cells and located on either side of the cluster (Figure S1B, shaded area from −2 to +2), with particularly strong contacts with the regions corresponding to the two flanking domains (Figure S1B, from −1 to +1). This observation was strongly reminiscent of the situation described for the HoxD cluster (Figure S1B, bottom and ).

10Within the domains of high interactions (i.e., the shaded areas in Figure S1B), the occurrence of contacts was quantified and no difference was observed in the distribution of interactions for either Hoxa4 or Hoxa13, when either proximal or distal limb bud samples were used. Hoxa4 establishes interactions primarily with the 3′ neighborhood of the gene cluster in both distal (Figure 3; 72%) and proximal (71%) samples, whereas Hoxa13 mostly contacts the opposite, 5′-located landscape in the same two samples (Figure 3; 66% and 63%, respectively) (the 3′ and 5′ orientation of the cluster is given following the direction of transcription of Hox genes). In contrast, both Hoxa9 and Hoxa11 increase their interactions with the 5′-located landscape in digit cells, when compared to proximal limb cells (Figure 3A,B; from 26% to 39% and from 39% to 53%, respectively). This shift in contacts observed with more centrally located Hoxa genes (Hoxa9, Hoxa11) is comparable to the situation described for the HoxD cluster (Figure 3D,E, Figure S2, and,). The increase in interactions between these genes and the 5′ landscape in distal cells suggests that several Hoxa genes located at the 5′ extremity of the cluster are coordinately regulated in the presumptive digit domain. These results and the analogy with the HoxD cluster are in line with the presence of functional enhancer sequences in the 5′ landscape, capable of activating transcription with distal limb specificity (Figure S3),.

11Because the HoxA cluster appears to respond to a bimodal regulation that shares several features with that reported for Hoxd genes, we propose that this operational mode is a core component of both Hoxa and Hoxd gene regulation during limb proximal to distal patterning and thus likely predates the emergence of tetrapods. The data obtained from the forebrain samples show that this partitioning between 5′ and 3′ regulatory landscapes is only partially tissue-specific and largely independent from the transcriptional activity, as noted for a large proportion of topological domains (Figure 3C,D,E and Figure S2),.

12Because this chromatin partitioning at and around the tetrapod HoxA and HoxD clusters is associated with distal and proximal regulatory capacities, we looked at its presence in fish Hox clusters as a potential indication that distinct regulations may also be at work during fin development. Data obtained from limb tissues, brain, and ES cells all show this biased distribution in interactions corresponding to the existence of two flanking topological domains, indicating that such a structural organization exists in both expressing and nonexpressing tissues. Consequently, we used whole zebrafish embryos at day 5 postfertilization (dpf) to visualize the interaction profiles of related fish Hox clusters, instead of dissected fin bud tissues, which would have met our technical limitations due to their small size and the amount of tissue required for 4C analysis. Teleosts underwent an additional genome duplication and have up to eight Hox cluster loci –, of which HoxAa, HoxAb, and HoxDa are the most relevant for fin development,. Accordingly, we used viewpoints in Hoxa4a, Hoxa9a, Hoxa11a, Hoxa13a, Hoxa2b, Hoxa11b, Hoxa13b, Hoxd4a, Hoxd10a, Hoxd11a, and Hoxd13a for 4C experiments (Figure 4 and Figure S4).

13We observed that fish Hox13 genes also display a clear bias in their interactions toward their 5′ flanking neighborhood (Figure 4). For example, Hoxa13a, Hoxa13b and Hoxd13a show 67%, 79% and 66%, respectively, of their total contacts with their 5′ landscapes. In contrast, only 27% of the contacts established by Hoxa4a and 26% of the contacts established by Hoxd4a were scored over their 5′ landscapes, these latter genes interacting mostly with the 3′ neighborhood of the gene clusters (73% and 74% respectively). Therefore, as in the mouse, genes located at either end of the clusters establish preferential contacts with either their 3′ or 5′ neighboring landscape. In contrast, interactions involving Hoxa9a, Hoxa11a, Hoxa11b, Hoxd10a, or Hoxd11a—that is, genes located at more central positions—are rather equally distributed between the two landscapes on either side of the cluster (Figure 4). We thus concluded that the chromatin partitioning observed in tetrapods at the HoxA and HoxD loci is also present in fishes. These results suggest that the structural component of the mechanism underlying the distinct proximal and distal phases of Hox gene expression predates the evolution of tetrapod limbs. Therefore, a resemblance greater than anticipated may exist between distal fins and limb structures, as recently proposed,,.

14The bias of fish Hox13 genes to contact their immediate 5′ environment suggested that, similar to their tetrapod counterparts, they might be used as the distal contribution of a bimodal regulatory strategy. We investigated the potential presence in these fish landscapes of enhancers driving limb-specific expression. Accordingly, we generated transgenic mice with fish Hox clusters including their entire 5′ flanking regions. We selected Pufferfish (Tetraodon nigroviridis) BACs because, due to the compressed genome of this species, they permit the transgenic analysis of entire syntenic regions.

15Mice transgenic for the fish HoxAa 5′ landscape showed expression of Hoxa11a, Hoxa13a, and Evx1 in hindlimb buds, but with a proximal-only specificity, whereas no distal expression was observed despite the presence of the 5′ flanking genomic region (Figure 5A).

16Likewise, the fish Hoxa10b, Hoxa11b, and Hoxa13b genes were strongly expressed in mouse limb buds transgenic for the fish HoxAb 5′ landscape. Here again, however, the expression domain matched a proximal zone and transcription was not scored in developing digits (Figure 5B). Because the pufferfish HoxAb BAC contains both the 5′ and 3′ neighborhoods, we implemented 4C-seq on transgenic mouse limbs and could confirm that strong interactions occurred between both Hoxa13b and Hoxa11b with the 5′ flanking region, despite the transcriptional outcome, which was restricted to a proximal domain (Figure S5). Also, the HIBADHb, TAX1BP1b, and JAZF1b genes, located next to Hoxa13b, were co-expressed with HoxAb genes, further illustrating that a global regulation is associated with this 5′ landscape (Figure 5B), as is the case for the mouse locus where these genes are co-expressed along with Hoxa13.

17Of note, the onset of the fish Hoxa13b expression in transgenic limb occurs in distal limb bud cells located underneath the apical ectodermal ridge (AER; Figure 6A, arrowhead), similar to the “late” expression pattern of this gene during fin bud development. In mice, however, the expression territory of this fish transgene does not follow the distal extension of the bud and thus remains at a more proximal position (Figure 6A, arrows). This result illustrates the difficulty of using relative parameters such as “proximal” or “distal” when assigning homology between fins and limbs (Figure 6B). Altogether, the fish regions syntenic to the mouse HoxA cluster failed to elicit expression in presumptive digit cells during limb budding. Instead, when introduced into mice, fish HoxA genes were all transcribed in proximal limb domains.

18We also analyzed the HoxDa cluster by using two BACs extending either 5′ or 3′ from the fish cluster. The 5′ BAC covers a region of the fish genome syntenic with the digit regulatory landscape, upstream of the mouse HoxD cluster, whereas the 3′ BAC is syntenic with the proximal limb regulatory landscape. In both cases, when introduced into transgenic mice, the fish Hoxda genes were expressed in a restricted domain, always located in the proximal limb bud, whereas no transcript was detected distally (Figure 5C). In this context, the fish genes were expressed according to their relative proximity to the flanking landscapes; Hoxd4a, Hoxd9a, and Hoxd10a were indeed preferentially transcribed whenever their closely located 3′ landscape was included, whereas Hoxd11a, Hoxd12a, and Evx2 were preferentially expressed when the opposite 5′ landscape was present. However, the same proximal specificity was observed in both cases, suggesting that regulatory domains exist on both sides of the fish HoxDa cluster, which are able to control fish Hoxda gene transcription in a proximal domain of the mouse limb bud, rather than in the digits (Figure 5C). These results are in agreement with the capacity of zebrafish, skate, and coelacanth sequences orthologous to mouse HoxD “digit enhancers” to drive expression essentially in proximal, rather than distal, domains of murine transgenic limb buds.

19Our results show that, similar to the HoxD gene cluster, the HoxA cluster establishes preferential contacts with the two flanking genomic neighborhoods, such that Hoxa13 strongly interacts with the telomeric DNA (i.e., with its 5′ side), whereas Hoxa4 contacts its centromeric (i.e., on the 3′ side) landscape. The existence of such a structural bias in both gene clusters suggests that the ancestral gene cluster, before its duplication at the root of the vertebrate taxon, already displayed such a general bimodal chromatin structure. This may indicate the presence of a generic regulatory constraint imposed to these gene clusters, such as the necessity to functionally separate, in space and time, the most posterior genes from their anterior neighbors, the former proteins being generally dominant over the latter.

20This split of both HoxA and HoxD clusters into two chromatin domains precisely matches the results obtained by using Hi-C on ES cell material. Interestingly, however, the same dataset reveals that neither HoxB nor HoxC seem to display this feature, suggesting it may have been lost subsequently. This might relate to the fact that these latter two gene clusters are truncated either for their anterior (HoxC) or posterior (HoxB) genes.

21Our 4C experiments also confirmed that many interactions were present in all the tissues assayed, regardless of their transcriptional activity, as previously observed,. In addition, the general extent of our interaction domains precisely coincided with the topological domains as defined by using the Hi-C dataset of Dixon et al., further suggesting that many of those interactions associated with such topological domains are constitutive in nature. For example, the strong 3′ HoxA interacting peak observed at the border between topological domains −2 and −1 (Figure 3, Chr6: 51,120,000) was present in all tissues investigated. This peak colocalizes with both proximal limb enhancers (elements 406 and 407 and human element 1465) and branchial arch enhancers (elements 402 to 406), as reported in a genome-wide enhancer screen.

22It may be that such a constitutive contact anchors the Hox cluster at the vicinity of tissue-specific enhancers, thus working as a priming mechanism for enhancer–promoter interactions. In this context, the presence of constitutive contacts with anchoring points rather than with the actual enhancers themselves might reflect the fact that Hox genes are regulated by multiple tissue-specific enhancers in time and space. The presence of a constitutive, poised regulatory architecture may have evolved at these loci to facilitate the successive implementation of multiple regulations, by providing a stable framework to be complemented by tissue-specific factors.

23The problem raised by the fin-to-limb transition shows that developmental expression patterns cannot always be used to infer homologies between distinct species. Because both Hoxd13 and Hoxa13—that is, the two tetrapod genes essential for digit development—display this strong regulatory tropism towards their upstream genomic neighborhoods, we looked at whether the related fish Hox genes would display the same behavior and found that fish Hox gene clusters have the same conformational organization. This observation suggested a level of conservation between the regulation of these genes in both tetrapods and fishes higher than anticipated. However, the existence of such separated chromatin domains including the fish Hox13 genes and their flanking genomic sequences does not lead to a clear partitioning of regulatory activities, at least when introduced into transgenic mice. All fish regulatory landscapes assayed, taken from either sides of the clusters, indeed elicited comparable proximal expression in mouse limbs and were thus unable to respond to those signals, triggering the emergence of the digital plate in mouse.

24The existence in fishes, of regulatory landscapes showing proximal specificities in transgenic murine limbs, may be related to the fact that fins can display elaborate proximal-to-distal patterns, as illustrated by combinations of radials and distal radials. Both zebrafish and paddlefish, as well as shark Hox, genes appear to be activated in a partially heterotopic manner consistent with the presence of these distinct fin segments,,, and hence such P-D patterns may result from biphasic regulations emanating from opposite regulatory landscapes. In this view, it is conceivable that the regulatory balance between these two landscapes in fishes contributes to the wide variety of P-D patterns observed in the fins of various species (Figure 7). Given the inability of fish regulatory sequences to activate transcription in the mouse digital plate, however, this P-D division would not be homologous to the regulatory partitioning observed between the arm and the hand in tetrapods.

25The bimodal limb bauplan is characterized by a clear morphological separation between the long bones of the arm and the forearm, on the one side, and of the hand, on the other side. This separation is controlled by opposite regulatory landscapes and gives rise to the presence in between of a nodular articulation critical for the function of the limbs and not observed in any fish fins: the mesopodium –. Because the development of long bones requires high doses of HOX products whereas a lower dose is associated with carpal-like small bones, it was proposed that the mesopodium results from the offset between the proximal and distal Hox expression territories, made possible by the existence of distinct regulatory landscapes. In this context, a potential scenario emerges for the fin-to-limb transition whereby two partially overlapping expression domains in fins progressively segregated to generate entirely distinct expression territories.

26This model hypothesizes the evolution of a bimodal “proximal–proximal” patterning system in fins (without mesopodium) into a bimodal “proximal–distal” system in limbs, including an articulation and thus postulates the transformation of a regulatory landscape from a “proximal” to a “distal” specificity. The mechanisms underlying this “regulatory homeosis” are elusive, but modifications in the structure and function of the AER, a source of growth factors in the tip of growing limb buds, may have been instrumental. During fin development, the cessation of endoskeletal expansion coincides with the transformation of the AER into the apical ectodermal fold (AEF) from which the exoskeletal fin rays will develop. Various models have predicted that the folding of the ectodermal layer plays a key role in the termination of fin distal growth, possibly due to the interruption of AER-derived proliferative signals by its dense extracellular matrix,,–. In this view, the abrogation of ectodermal folding in tetrapods may have lead to a prolonged exposure to AER signals, leading to increased Hox expression and extended distal growth, thus resulting in the formation of the autopod.

27However, we show here that this model cannot fully account for our transgenic results, as expression of fish Hox genes is not observed distal to the mesopodium, even in the absence of ectodermal folding. We conclude that there is an intrinsic inability of fish enhancers to respond to the distal limb regulatory program in the mouse. Consequently, the absence of a clear distal expression territory in fin buds separated from (but concomitant with) the proximal expression domains is likely not caused by a mere physical obstacle induced by the folding of the ectodermal layer. Rather, an ancestral fish 5′ regulatory landscape may have evolved to better respond to distal ectodermal signals, and the reinforced transcription of Hox genes distally might have promoted supplementary growth by delaying ectodermal folding. This situation is illustrated by the effect of overexpressing Hoxd13a in zebrafish fins, which leads to increased distal growth at the expense of the AEF. Alternatively, the capacity for ancestral fish enhancers to respond to the appropriate distal signals may have existed from early on but be repressed, in which case tetrapod loci may have simply overcome this repression, for instance, through the loss of repressor binding sequences.

28It has been pointed out that chondrichthyans or actinopterygians could be more informative regarding the fin-to-limb transition than extant teleosts,, which may have lost primitive characters. Yet fins of all these species consist of both radials and distal radials and exhibit similar HoxA and HoxD expression patterns,,–,,. Furthermore, our results with transgenic pufferfish sequences are consistent with the patterns found when enhancers isolated from more primitive fishes were used. In all cases indeed,, these enhancers did not elicit expression patterns as distal as one would have expected for bona fide digit enhancers,.

29In conclusion, although digits are likely formed through the action of tetrapod-specific Hox enhancers, the underlying regulatory circuitry relies upon an ancestral framework already implemented in fish, illustrating the retrofitting of preexisting genomic infrastructure. In this context, the question regarding the homology between fin radials and digits may receive different answers depending on which level is considered within the regulatory hierarchy. Fish have the necessary genes and higher order regulatory architecture to form digits and likely implement the 5′ regulatory landscape to pattern distal fin radials. Accordingly, digits could be considered as a specialized type of distal radials as both structures rely on a unique ancestral regulatory strategy. However, the fish 5′ regulatory landscapes are unable to specify a distinct digit territory and, as such, this regulatory feature defines a clear tetrapod synapomorphy. Therefore, a qualification of distal radials as digits (senso “classical” homology, that is, with a common ancestral structure) is not supported by our results.

30All animal experiments were performed according to Swiss regulations under license no. 1008/3482/0 (to D.D.).

314C libraries were constructed as described before. Mouse libraries consisted of 52 dissected E12.5 proximal forelimb buds, distal forelimb buds, or forebrains. Zebrafish libraries consisted of approximately 300 5 dpf embryos from the TU strain. Transgenic mouse libraries containing the Tetraodon HoxAb (C0AA043AG01) BAC contained 48 E12 proximal and distal hindlimb buds. The sequencing data for these samples were combined and processed as a whole limb sample. For the mouse baits used for 4C, the primary restriction enzyme used was NlaIII (New England Biolabs, R0125L), and the secondary restriction enzyme was DpnII (New England Biolabs, R0543M). For the zebrafish baits, the primary restriction enzyme was DpnII (New England Biolabs, R0543M), and the secondary enzyme was TaqαI (New England Biolabs, R0149M). In the latter case, DNA was cut for 8 h at 65°C. For the Tetraodon baits, assessed in transgenic mice, the primary restriction enzyme used was DpnII (New England Biolabs, R0543M), and the secondary enzyme was NlaIII (New England Biolabs, R0125L). For each viewpoint, between 1.3 and 2.6 µg of the 4C library was amplified using 16 individual PCR reactions with inverse primers containing Illumina Solexa adapter sequences (Table S2). Multiplexed samples were sequenced on the Illumina HiSeq system using 100 bp single-end reads according to the manufacturer's specifications. 4C-seq reads were sorted, aligned, and translated to restriction fragments using the 4C-seq pipeline of the BBCF HTSstation (available at http://htsstation.epfl.ch ). Mouse samples were mapped to the ENSEMBL Mouse assembly NCBIM37 (mm9) and zebrafish samples were mapped to the ENSEMBL Zebrafish assembly Zv9. Transgenic Tetraodon samples in mouse were mapped to a custom genome containing the Tetraodon BAC (C0AA043AG01) and ENSEMBL Mouse assembly NCBIM37 (mm9), thus minimizing the chance of mapping nonspecific reads. The directionality of signal was calculated on 4C-seq patterns over the regions mentioned in Table S1A. Data are summarized in Table S1B. In the figures, smoothed 4C-seq patterns (running mean, window size 11) are visualized except in Figure S4B, which shows unprocessed data. Topological domains shown to complement the 4C-seq experiments are ES cell HiC data take from (http://chromosome.sdsc.edu/mouse/hi-c/database.php). Mouse domains selected for 4C-seq analyses correspond to two topological domains located centromeric and telomeric of the clusters (i.e., four domains in total) and are described in Figure S1. The zebrafish regions were selected on basis of synteny with the mouse domains analyzed. Similar experiments involving the HoxD cluster as shown in Figure S2A,B were previously reported. The experiments shown here were, however, repeated together with the analysis of Hoxa genes in order to compare datasets produced under the exact same conditions.

32BAC constructs were identified using the Genoscope Tetraodon genome browser (http://www.genoscope.cns.fr/externe/tetranew/). BAC numbers and genomic positions (TETRAODON8) correspond to HoxAa, C0AB048AA04 (Chr21:2,888,799–3,037,908); HoxAb, C0AA043AG01 (Chr8:6,699,347–6,844,622); HoxDa 3′, C0AB015CD05 (Chr2:13,313,310–13,458,212); HoxDa 5′, C0AB043BH04 (Chr2:13,417,090–13,578,446). BAC clones were obtained from Genoscope, France. A PISceI meganuclease site was introduced into the vector backbones using standard EL250 cell-based recombineering technology. BAC DNA was prepared using a Nucleobond Midiprep Kit, linearized with PISceI (New England Biolabs, R0696L), incubated with SDS according to the manufacturer's instructions, 2× chloroform-phenol purified, ethanol precipitated, and dialyzed against microinjection buffer containing protamines [70 µM spermidine (Sigma, S2626), 30 µM spermine (Sigma, S3256)]. Constructs were microinjected using standard protocols for pronuclear injection. BAC lines were genotyped using primer pairs every 5 to 10 kb in combination with deep sequencing using 4C-seq and mapping of the reads on the BAC sequence to confirm its integrity. A HoxAb transgenic line was mapped using embryonic hindlimb samples (Figure S5), and adult mouse ear samples of HoxDa 3′ and 5′ BAC lines were processed using 4C-seq specifically for the purpose of integrity mapping using a viewpoint located in Hoxd11a (unpublished data). BAC diagrams in Figure 5 represent the regions that were found to be integrated using PCR (all lines) or 4C-seq data (HoxAb, HoxDa 3′, and HoxDa 5′) in the transgenic lines presented.

33The Hoxa13 and Hoxd13 mouse knockout lines were previously described,. In situ hybridization was performed as described with a temperature of prehybridization, hybridization, and posthybridization steps increased to 68.5°C. For Tetraodon probes, the SSC concentration in the hybridization mix was lowered to 1.3× to increase specificity (for the Evx2 probe, 0.5×SSC was used), and the posthybridization SSC washes were done using 4×30 minutes 2×SSC-T. In all experiments using transgenic Tetraodon probes, wild-type embryos were coprocessed for each probe and stage to monitor specificity of the probes (unpublished data). Except in brain vesicles, susceptible to probe trapping, nonspecific signal was never observed using the conditions described above.

34Probes were amplified using PCR from BAC DNA or limb cDNA and cloned into pGEMTE easy vector systems (Promega A1360). Primer sequences are given in Table S3. DIG-labeled RNA probes were synthesized using Sp6 or T7 polymerase (Promega). Probes for Hoxd11 and Hoxa13 were described previously,. The probe used to detect the Hoxa11 sense transcript was kindly provided by Dr. C. Fromental-Ramain and corresponds to a ScaI-HpaI fragment in the 3′ UTR of Hoxa11 (mm10: Chr6:52,242,847–52,243,385).

35In situ hybridization images were acquired using Leica Application Suite software v3.3.1 in combination with a Leica DFC300FX camera and Leica MZFLIII microscope. Images were edited in Adobe Photoshop software using the brightness/contrast function. Limb buds shown in Figure 1B stained for Hoxa13 are right side limb buds coming from the same embryos as the left side limb buds stained for Hoxa11 and are mirrored for purpose of comparison. Similarly, the Hoxa4 sample in Figure 1A is a mirror image right side forelimb bud. In Figure 1A the same wild-type forelimb specimen is used as in Figure 1B to illustrate the wild-type expression of Hoxa11.

36Unprocessed 4C-seq data for mouse and zebrafish samples are available from the Gene Expression Omnibus repository under accession number GSE47644. Ensembl IDs for genes used in this study are as follows: Mouse (Mus musculus), Hoxa4; ENSMUSG00000000942| Hoxa9; ENSMUSG00000038227| Hoxa10; ENSMUSG00000000938| Hoxa11; ENSMUSG00000038210| Hoxa11as; ENSMUSG00000086427| Hoxa13; ENSMUSG00000038203| Hoxd4; ENSMUST00000111980| Hoxd11; ENSMUSG00000042499| Hoxd13; ENSMUSG00000001819|; Zebrafish (Danio rerio), Hoxa4a; ENSDARG00000057724| Hoxa9a; ENSDARG00000096510| Hoxa11a; ENSDARG00000009045| Hoxa13a; ENSDARG00000007609| Hoxa2b; ENSDARG00000023031| Hoxa9b; ENSDARG00000007009| Hoxa13b; ENSDARG00000036254| Hoxd4a; ENSDARG00000059276| Hoxd10a; ENSDARG00000057859| Hoxd11a; ENSDARG00000059267| Hoxd13a; ENSDARG00000059256|; Pufferfish (Tetraodon nigroviridis), Hoxa11a; ENSTNIG00000001767| Hoxa13a; ENSTNIG00000009207| Evx1; ENSTNIG00000000875| Hoxa10b; ENSTNIG00000001780| Hoxa11b; ENSTNIG00000000494| Hoxa13b; ENSTNIG00000001781| HIBADHb; ENSTNIG00000018428| TAX1BP1b; ENSTNIG00000018429| JAZF1b; ENSTNIG00000018430| Hoxd4a; ENSTNIG00000001765| Hoxd9a; ENSTNIG00000016957| Hoxd10a; ENSTNIG00000001775| Hoxd11a; ENSTNIG00000001776| Hoxd12a; ENSTNIG00000001777| Evx2; ENSTNIG00000001817|.