Hox  gene  expression  in  mouse  and  corn  snake  embryonic  tails

Hox genes located in the 5’ region of the cluster are expressed posteriorly in the embryonic tail and have been proposed to impact on termination of body elongation via PSM shrinkage (Denans et al., 2015). We therefore collected tail tissue (post-cloacal) from E9.5 to E13.5 mouse embryos and from 2.5 dpo to 5.5 dpo corn snake embryos in order to assess gene expression by RNA-seq.

We first analyzed the expression in mouse embryonic tails and found that, as expected, the main source of variance between samples, i.e. the first axis in the principal component analysis, corresponded to the developmental stage (Figure 17A). Accordingly, sample order along this axis was a match to developmental stage order (Figure 17A). In addition, we observed that the expression dissimilarity between developmental stages increases with time. Taking E9.5 posterior trunk as a reference, we observe that E9.5 and E10.5 expression patterns are very similar, while later developmental stages have increasingly more expression divergence compared to E9.5 (Figure 17A and B). Interestingly, we found that Hox genes represented a big part of the differentially expressed genes (Figure 17B, blue circles).

Concerning the most variable genes between all samples, an almost equal distribution between increased and decreased expression levels over time was scored (Figure 17C). As expected, these genes were mainly enriched for gene ontology (GO) terms related with

embryonic development, mostly owing to the large amount of differentially expressed Hox genes (Figure 17D and E).

Figure 17 – RNA-seq analysis of mouse embryonic tails

A. (top) Principal component analysis performed using the 500 more variable genes across samples. Samples coming from different embryonic stages (E9.5-E13.5) are color-coded and duplicates differ by shape. (Bottom) Heatmap that plots the Euclidian distance between samples. Darker blue refers to smaller distances while light blue corresponds to

larger distances. B. MA-plot between E9.5 tail samples and all other samples. Each dot represents a gene and differentially expressed genes (p<0.1) are marked in red. Dots that refer to Hox genes are surrounded by blue circles. C. Heatmap of the 100 most variable genes across samples. The different samples are ordered and can be seen at the top of the graph. Duplicates are represented in purple and green. D. Gene ontology term enrichment of the 100 most variable genes across samples. E. Heatmap of the 20 most variable Hox genes across samples.

We then set out to compare transcription at the Hox loci in mouse and snake tails over a range of developmental stages. For this purpose two approaches were used. Our first approach consisted of mapping the RNA-seq reads to a reference genome. Data generated from mouse tissue was aligned with the mouse genome, while the corn snake derived data was aligned to the king cobra genome. The second method relied on de novo transcript assembly using the Trinity method (Grabherr et al., 2011).

Both methods showed differences either in levels or dynamics of gene expression between the two species (Figure 18 and Figure19). For instance, while genes in the mouse HoxA cluster are downregulated over developmental time, the snake Hoxa gene expression remains almost unchanged in the different samples (Figure 18A). In addition, the Hoxb1-4 genes show considerably higher levels of expression in the mouse tails than in the snake’s (Figure 18B). Since “posterior” (5’) Hox genes are more likely to have an impact on body elongation, we focused our analysis on this subset of genes. Interestingly, Hoxa13, Hoxb13 and Hoxd13 are expressed at lower levels in the snake than in the mouse posterior trunk (Figure 18 and Figure19). However, Hoxc13 and Hoxc12 genes are, on the contrary, expressed very strongly in the snake tail since very early stages (i.e. before the first caudal somite has been formed). This is in contrast to the mouse expression that is not very strong at this initial stage and progressively increases as the embryo gets older (Figure 18C).

Figure 18 – Gene expression profiles mapped on the Hox clusters

Hox gene transcription in mice and corn snakes across successive developmental stages.

Reads from mouse tail RNA-seq were mapped to the mouse genome while snake reads were mapped to the king cobra genome. Genes with distinct expression levels between the two species are highlighted in orange.

Figure 19 – Interspecies comparison of Hox13 gene expression

Average normalized RPKM values of two biological replicates of the different Hox13 genes in mouse (blue) and snake (red) embryo posterior trunk over developmental time:

E9.5 to E12.5 for the mouse and 2.5 dpo to 5.5 dpo for the snake.

Since we found important differences in expression of Hox13 genes in the snake embryonic tail, we set out to investigate how their expression could affect genes involved in axial elongation such as trunk Hox genes, Wnt and Fgf genes, as well as Cyp26a1 (“trunk genes”) that codes for a retinoic acid-degrading enzyme (Young et al., 2009). To establish a parallel between developmental stages between mouse and snake we counted the caudal somites at a given stage and calculated the percentage over total caudal somite number after somitogenesis completion (Figure 20A). We then analyzed the expression levels of Hox13 genes as well as Fgf8, Cyp26a1 and Cdx2 along developmental time. In order to more easily compare the dynamics of expression between genes, we first attributed to the maximum RPKM (reads per kilobase of transcript per million mapped reads) value for each gene the

value of 1 (Figure 20B). We find that in the mouse, Hoxc13 and Hoxb13 expression expectedly increases, while “trunk gene” expression steeply decreases over time. Hoxd13, on the other hand, reaches a peak of expression at E10.5. In the snake posterior trunk, however, both Hoxc13 and Hoxd13 are more highly expressed at early developmental stages and only Hoxb13 expression is progressively increased as Fgf8, Cyp26a1 and Cdx2 lower their expression levels.

In order to analyze these results in a more quantitative context, we plotted the RPKM absolute values of this group of genes over developmental time (Figure 20C). We found that, although the Hoxc13 gene was very highly expressed at the start of snake tail formation,

“trunk genes” were not significantly affected by these premature elevated levels of Hoxc13 expression in the corn snake posterior trunk (Figure 20C).

Figure 20 – Expression of genes involved in axial extension and termination

A. Close-up of the posterior extremity of corn snake embryos stained with DAPI. The red arrowhead indicates the positioning of the cloaca and genital buds. The developmental stage is depicted above and below are the percentages of tail somites formed at that stage relative to total amount of caudal somites at the end of somitogenesis B. and C. Plotted FPKM values for mouse (left) and for snake (right) samples over the different developmental stages. FPKM values are represented either relative to the highest expression of each gene (B) or in absolute values (C). Genes are color-coded as represented on the right of the plots.

Whole mount in situ hybridization of house snake embryos, a snake species largely comparable to the corn snake in terms of development, complemented our quantitative expression analysis with spatial information (Figure 21). We found that, indeed, at 2.5 dpo Hoxc13 is the only Hox13 gene with detectable gene expression in the snake tail. At this stage, Hoxa13, Hoxb13 and Hoxd13 expression was restricted to other tissues such as the hindgut or the cloaca. The Hoxa13 gene was the last Hox gene to be detectable and showed a

very posterior domain of expression in the house snake embryonic tail. On the other hand, Hoxb13 and Hoxc13 genes are activated earlier and their expression is scored throughout most of the post-cloacal region of the embryo (Figure 21). Hoxd13 represents an intermediate situation in terms both of timing of activation and expression domain (Figure 21).

Our gene expression analysis indicates that, although Hoxc13 is expressed unusually early and strongly in the snake tail bud, it does not result in the expected tail truncation or

“trunk gene” downregulation. It would therefore appear that the snake Hoxc13 gene has lost, at least in part, the ability to arrest body elongation by failing to repress genes that promote axial extension.

Figure 21 – Expression of Hox13 genes in the house snake tail bud

Whole-mount in situ hybridization of all Hox13 genes in house snake embryos across four developmental stages. The pictures are close-ups of the posterior extremity of the house snake stained embryos.