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DEVELOPMENTAL AND EVOLUTIONARY COMPARATIVE ANALYSIS OF A REGULATORY LANDSCAPE IN MAMMALS AND BIRDS

Modifications in gene regulation during development are considered to be a driving force in the evolution of organisms. Part of these changes involve rapidly evolving cis-regulatory elements (CREs), which interact with their target genes through higher-order 3D chromatin structures. However, how such 3D architectures and variations in CREs contribute to transcriptional evolvability remains elusive. During vertebrate evolution, Hox genes were redeployed in different organs in a class-specific manner, while maintaining the same basic function in organizing the primary body axis. Since a large part of the relevant enhancers are located in a conserved regulatory landscape, this gene cluster represents an interesting paradigm to study the emergence of regulatory innovations. Here, we analysed Hoxd gene regulation in both murine vibrissae and chicken feather primordia, two mammalian- and avian-specific skin appendages which express different subsets of Hoxd genes, and compared their regulatory modalities with the regulations at work during the elongation of the posterior trunk, a mechanism highly conserved in amniotes. We show that in the former two structures, distinct subsets of Hoxd genes are contacted by different lineage-specific enhancers, likely as a result of using an ancestral chromatin topology as an evolutionary playground, whereas the regulations implemented in the mouse and chicken embryonic trunk partially rely on conserved CREs. Nevertheless, a high proportion of these non-coding sequences active in the trunk appear to have functionally diverged between the two species, suggesting that transcriptional robustness is maintained despite a considerable divergence in CREs’ sequence, an observation supported by a genome-wide comparative approach.


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Changes in the spatial and temporal regulation of genes critical for developmental processes have 55 greatly contributed to the evolution of animal morphologies (see for example (Carroll, 2008;Rebeiz et al., 56 2015). The expression of such genes is generally modulated by combinations of cis-regulatory elements 57 (CREs), short DNA sequences enriched for transcription factor binding sites, which tend to evolve more 58 rapidly than the genes they control (Edwards et al., 2013;Long et al., 2016;Spitz and Furlong, 2012). CREs 59 can be spread over large distances around the gene(s) they regulate, which gives to the spatial organization of 60 chromatin a particular importance. Indeed, the existence of such 'regulatory landscapes' (Spitz et al., 2003) 61 implies that enhancer-promoter interactions involve spatial proximity, which is generally thought to occur 62 through large chromatin loops (Rao et al., 2014).

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We initially analyzed the expression profiles of Hoxd genes in mouse VPs and chicken FPs (Fig. 1).

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Skin appendage development starts with the interaction of the skin ectoderm with its underlying mesoderm, 130 resulting in the thickening of the epithelium into a placode and in the condensation of dermal cells into a papilla 131 (Mikkola, 2007;Sawyer and Knapp, 2003;Widelitz and Chuong, 1999). VP development starts at early

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Whole-mount in situ hybridization (WISH) revealed strong Hoxd1 expression in the dermal papillae 138 of the VPs ( Fig. 1B; Fig. S1). Furthermore, a comparative analysis of VPs from single E12.5 embryos revealed 139 that Hoxd1 transcripts accumulate slightly after ectodermal placode formation, marked by the expression of 140 Shh (Chiang et al., 1999) (Fig. S1). Hoxd3 and Hoxd4 are also expressed in VPs, yet at much lower levels than 141 Hoxd1 (Fig. 1B, Fig. S1). Faint levels of Hoxd8 mRNAs were sporadically detected in the VPs, while Hoxd9 142 and more 5'-located Hoxd mRNAs were never observed in these structures (Fig. 1B, Fig. S1). In situ 143 hybridization on cryostat sections confirmed that Hoxd1 transcripts accumulate in the dermal mesenchyme of 144 VPs, but not in the ectoderm-derived portion of the follicle (Fig. S1). Therefore, Hoxd1-Hoxd4 are

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Hox genes are expressed in the paraxial and lateral mesoderm of the main embryonic axis, as well as 156 in the neural tube, and display a progressively more restricted expression domain depending on their position uniformly transcribed in the mouse paraxial and lateral mesoderm as well as in the neural tube, Hoxd1 161 displayed a biphasic expression pattern. Transcripts accumulated in the embryonic tailbud, yet they rapidly 162 disappeared from the presomitic mesoderm and remained undetected in lateral mesoderm progenitors or in the 163 neural tube (Fig. 1F). Instead, Hoxd1 was specifically re-activated during somite condensation, coupling the 164 transcription of this gene to the segmentation clock (Dale and Pourquié, 2000;Zákány et al., 2001). Similar to 165 the mouse pattern, the chicken HOXD1 expression also differed from its neighbouring paralogs, even though 166 its transcripts remained detectable in the formed somites all along the main body axis (Fig. 1G). These

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In order to see whether these differences and similarities in regulatory specificities could be associated

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In the mouse VPs, the MAR mapped within the D1 region ( Fig. 3A, top, red-filled rectangle), in 219 agreement with the stronger expression of Hoxd1 compared to its neighbouring paralogs ( Fig. 1B; Fig. S1).

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In the latter line, the entire HoxD cluster is deleted, such as to be able to see the effect of the HoxD Del(attP-SB2)lac

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To assess whether the D1 region is also necessary and sufficient for the specific Hoxd1 expression

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Instead, ß-galactosidase activity was not detected neither in tgN EC2 , nor in tgN EC3 transgenic embryos (Fig.   277 S4B). We next tested these candidate enhancers at E9.5 to evaluate their regulatory potential in forming 278 somites. EC2, which was the only candidate sequence with significant H3K27ac enrichment in posterior trunk 279 cells, displayed reproducible activity in forming somites (Fig. 4H), unlike tgN EC1 or tgN EC3 (Fig. S4C). We 280 concluded that the only VP enhancer sequence identified was not active during somite formation, unlike a 281 somite enhancer located nearby (2.7 kb), within the D1 region.

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The fact that two independent tissue-specific regulations rely on enhancers located in a close proximity

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To take this analysis genome-wide, we compared the H3K27ac profiles across different mouse and

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H3K27ac ChIP-seq datasets. pCREs were separated into putative enhancers and promoters, hereafter referred 304 to as 'enhancers' and 'promoters' for simplicity, with 'enhancers' mapping more than 2 kb away from a gene 305 transcription start site and 'promoters' mapping less than 2 kb away. pCREs which were H3K27ac-positive in 306 more than one of the analyzed tissues were categorized as 'pleiotropic' while those being active in only one 307 tissue were named 'specific' for the sake of classification. In both mouse and chicken, most promoters were 308 pleiotropic while the majority of enhancers were specific (Fig. 5A)

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The intersection between the same tissue in mouse and in chicken represents elements that have both 327 a conserved sequence and regulatory function (Fig. 5C, overlaps circled in black). This intersection is smaller 328 than any difference between two sets, which indicates that DNA sequence similarity between two species can 329 be maintained amongst regulatory sequences despite divergences in their regulatory functions. We could 330 observe that CNEs often display enhancer activities in a chicken tissue that is different from the mouse tissue, 331 supporting this conclusion (Fig. 5C, overlaps circled in white). As a consequence, the regulatory activity of a 332 given sequence conserved between these two species cannot be used to predict its function(s) in the two 333 species.

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Altogether, we show that both the global genomic organisation of the HoxD locus and specific domains 335 of preferential enhancer-promoter interactions are highly conserved between mouse and chicken. However, 336 the regulatory activities of individual CREs are largely species-specific and such sequences are systematically 337 found within those segments of regulatory landscapes that preferentially interact with the promoters of the 338 most highly transcribed genes. We also show that the lineage-specific evolution of regulatory activities could 339 either generate new regulatory relationships or, conversely, maintain ancestral transcription patterns.           Table S1 and were backcrossed continuously to Bl6/CBA mixed animals and 470 maintained as heterozygous stocks. Chick embryos from a White Leghorn strain were incubated at 37.5°C and 471 staged according to Hamburger and Hamilton (1951).

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The different CRE candidate sequences were amplified using PCR-specific primers (see Table S2) and the 474 Expand High Fidelity PCR system (Roche). Amplified fragments were gel-purified and cloned into the pSK-   Table S2. Digoxigenin (Dig) labeled probes were synthesized in vitro by 484 linearizing the respective plasmids using specific restriction enzymes and transcribed in vitro either with 485 T7/T3, or Sp6 RNA polymerase (Table S3) and the Dig-RNA labeling mix (Roche). The probes were purified 486 using the RNA easy mini kit. WISH experiments were performed as described in (Woltering et al., 2009).

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Probes are listed in (Table S3). In situ experiments in sections were performed according to (Sockanathan and

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exons and sequence conservation using bedtools intersect. pCREs were quantified using R (v3.6.1) and the bar 544 plots of Figure 5A were done with the ggplot2 package (v3.

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Bottom: Profiles of chicken HH18 embryonic posterior trunk cells (green) and of HH18 brain cells.