The Genomik Origin of Hedgehog Spines

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Evolutionary processes have resulted in the most diverse adaptions to environmental challenges. How suchadaptations evolve and how they are encoded in the genome remains one of the great mysteries in biology. In this proposal, we will study a fascinating phenomenon in evolution, the development of spines in hedgehogs, in particular the four-toed hedgehog (Atelerix albiventris) for which we have a breeding colony. Our genomic analyses will be coupled with functional analyses, both on mice and hedgehogs. The project consists of five parts. (1) Our first aim is to compare the full-chromosome hedgehog genome we already produced to that of mouse and human to (i) search for loss and gain of genes and gene families, (ii) detect breaks of synteny, including copy number variations (deletions, duplications), and (iii) identify candidate genomic regions where Topologically Associating Domains (TAD) have been affected. We will then verify for the presence of these modifications in genomes of other hedgehogs and related species (mole and shrew) in order to identify those that are specific to the hedgehog lineage. (2) We will perform expression analysis from single cell (sc) data from dorsal and ventral skin of hedgehog and mouse embryos at two stages: (a) when the skin is undifferentiated and (b) when placodes, the precursor of hair/spines, form. From these data, we will generate an expression map of the mouse and hedgehog embryonic in skin. In parallel, we will identify active enhancer and promoter regions in the developing skin using ChIP-seq for chromatin modification and ATAC-seq. (3) In an integrated data analysis, we will merge the results from Aims 1 and 2 to identify changes in enhancer activity and/or genomic rearrangements at synteny breaks resulting in altered regulatory landscapes of genes differentially expressed in hedgehog dorsal skin. Comparisons will be performed within the hedgehog samples - pre-placode vs. placode skin and dorsal vs. ventral skin - as well by comparing with the mouse expression profiles. (4) We will carry out whole-mount in situ hybridizations on hedgehog and mouse embryos to determine the exact position and timing of expression of each candidate gene and compare the two species. Once the timing of expression of each candidate gene has been defined, we will proceed to functional testing in hedgehog and mouse ex-vivo skin cultures. (5) Finally, using CRISVar technology, we will be able to manipulate the mouse genome in a way that it recapitulates hedgehog-specific genomic rearrangements. We will also establish transgenesis in hedgehogs for the first time sing the iGONAD approach aiming to at least partially reverse the changes that led to the appearance of spines. The combined results of these aims will allow us to elucidate the genomic origin of hedgehog spines and advance our understanding of the evolutionary mechanisms responsible for the appearance of novel morphological characters.