Trunk  mesoderm  regulation  in  mouse  and  snakes

In order to confirm the involvement of sequences outside of the cluster in the regulation of the mouse Hoxd gene expression in the trunk, we analyzed homozygous embryos for the deletion of the entire HoxD cluster (Del(d1-d13)d11lacZ). This line has a Hoxd11-lacZ knock-in gene that replaces the cluster and acts as a reporter for the regulatory activity of the neighboring regulatory gene deserts (Spitz et al., 2001) (Figure 4C). As expected, in situ hybridization of Hoxd11 in embryos of this line revealed expression in the trunk mesoderm (Figure 4D). In addition, two mouse lines containing BACs that span parts of the telomeric (3’) gene desert (Delpretti et al., 2013) (Figure 4C): Tel1 and Tel2, showed reporter gene expression in the trunk mesoderm (Figure 4D).

Figure 4 – The regulatory potential of the vertebrate HoxD cluster

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. Scheme illustrating the deletion of the HoxD cluster as well as the location of the two telomeric BACs where a lacZ gene has been inserted: Tel1 and Tel2. D. Whole-mount in situ hybridization of an E12.5 embryo for Hoxd11 in the deletion line and beta-galactosidase staining of E11.5 and E12.5 of the Tel1 and Tel2 BAC lines, respectively. E. ChIP-seq analysis over the mouse and snake HoxD loci of H3K27 acetylation 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 called peaks 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.

LoxP sites are indicated as red triangles, the HoxD cluster is represented by a black rectangle and other genes are shown with grey rectangles.

We complemented these results by analyzing the expression of mouse embryos that lacked either the centromeric gene desert (Del(Atf2-Nsi) (Montavon et al., 2011)) or most of the telomeric gene desert (Del(Attp-Sb3) (Andrey et al., 2013)). Since the Del(Attp-Sb3) allele does not completely delete the telomeric regulatory landscape, we also used the Inv(Attp-CD44) (Andrey et al., 2013) line that brings the telomeric desert more than 25 Mb away from the HoxD cluster (Figure 5A). While Del(Atf2-Nsi) embryos showed comparable trunk

expression to the control, Del(Attp-Sb3) and Inv(Attp-CD44) mutants had reduced expression in mesodermal expression of the upper trunk (Figure 5A). We looked in further detail into the regulatory potential of the telomeric gene desert by using mouse lines with smaller deletions:

Del(Sb2-Sb3), Del(Sb2-65), Del(65-Sb3) and Del(Attp-Sb2) (Andrey et al., 2013). The deletions Sb2-Sb3 and Sb2-65 resulted in a similar expression pattern as the one obtained with the telomeric gene desert deletion (Del(Attp-Sb3)) (Figure 5A). On the other hand, Hoxd4 expression in embryos carrying the smallest deletions: Del(65-Sb3) and Del(Attp-Sb2), was scored in the correct domain of expression (Figure 5B). Accordingly, the analysis of H3K27ac distribution, a histone mark associated with putative active enhancers and promoters, in the mouse trunk mesodermal tissue showed more enrichment in the telomeric gene desert than in the centromeric gene desert (Figure 4E, top). Together these results confirm the existence of mesodermal enhancers outside of the HoxD cluster and show that this regulatory activity is for the most part contained in the telomeric gene desert of the mouse.

We then examined transgenic mouse embryos carrying the integration of the snake HoxD cluster. Contrary to the expression results obtained in other BAC transgenics, the snake Hoxd4 expression pattern was not merely restricted to the neural tube. In fact the main body axis expression pattern was reminiscent of the endogenous mouse Hoxd4 expression with both neural tube and mesodermal expression (Figure 4B, SnakeBAC). It would therefore appear that, in contrast with the other vertebrate species analyzed, most of the necessary enhancers to drive expression in the snake trunk mesoderm lie within the HoxD cluster. To confirm these observations we performed an H3K27ac ChIP-seq using anterior trunk mesoderm from 5.5 dpo (days post oviposition) snake embryos (roughly equivalent to E12.5 mouse embryos) and compared it to the mouse acetylation profile. As expected, the snake gene deserts showed very weak coverage over either the 5’ or 3’ gene desert in comparison with the mouse enrichment in these genomic regions (Figure 4E). In order to be able to directly compare the ChIP-seq results between mouse and snake, we analyzed 27 regions in the 3’ gene desert conserved in both species and counted the number of enriched sequences.

While the mouse H3K27ac ChIP-seq showed enrichment for about 40% of these conserved sequences, in the snake only 22% of them were acetylated (Figure 4E, right). These snake conserved regions were found to be clustered in only two regions of the gene desert as demonstrated by peak calling, while mouse acetylated regions are scattered through the telomeric gene desert (Figure 4E, under each profile).

Figure 5 – Locating telomeric Hoxd trunk mesodermal enhancers

A. Schemes illustrating the deletion stocks that result in downregulation of Hoxd4 in the upper trunk mesoderm (top) and whole-mount in situ hybridization of E12.5 mouse embryos with the Hoxd4 probe in the corresponding deleted mutant embryos (bottom) B.

Schemes illustrating the deletion mouse stocks that showed similar Hoxd4 expression as the control (top) and corresponding whole-mount in situ hybridization of E12.5 mouse embryos with the Hoxd4 probe (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.

As an example of the loss of mesodermal regulatory capacity in the snake HoxD telomeric gene desert, we focused on a peak that was acetylated in the mouse and that is widely conserved in vertebrates (including lizard and frog), but not in snakes (Figure 6A and B). In order to confirm the enhancer activity of this region, we cloned this mouse mesodermal sequence (MSS) upstream of a lacZ reporter gene. Indeed, this 2.5 kb region was able to drive reporter gene expression both in the mouse upper trunk and posterior trunk mesoderm (Figure 6C). We therefore validated the function of a mouse mesodermal enhancer located in the telomeric gene desert that contains a sequence that is not conserved in snakes.

Figure 6 - Identification of a trunk mesoderm enhancer

A. ChIP-seq mapping over the HoxD cluster and the flanking 3’-located, telomeric gene desert of H3K27 acetylation in dissected mouse upper trunk tissue. Green boxes under the mapping represent peaks identified by the MACS software. The MSS peak is identified by an asterisk. The HoxD cluster is represented by a black rectangle and the grey box represents the Mtx2 gene. B. Conservation plots of the MSS sequence (left). The plots show conservation from 50% to 100%, with a pink color when conservation is above 75%. On the right, the MSS enhancer activity is shown as assessed by lacZ reporter assay at E12.5.