In the first objective, to unravel the contribution of CREs to the evolution of deuterostome body plans, we have de novo sequenced the genome of the European amphioxus and have generated multiple epigenomic and transcriptomic data. This information and a similar dataset generated in zebrafish and other species, allowed us to study the impact of the regulatory genome in the transition from invertebrates to vertebrates. We found that the genomic duplication that took place in the transition between invertebrates and vertebrates was accompanied by an increase in genomic regulatory regions. This new regulatory information allowed many duplicated genes to acquire more specific expression patterns, contributing to an increase of cellular complexity and the appearance of new tissues and structures characteristic of vertebrates. The indicated work was published in Nature (Maelétaz et al., Nature 2018).
We have also explored the evolutionary contribution of an enhancer critical for the expression of the Sonic Hedgehog (Shh) gene and its function in fin and limb development. Using deletion studies with the CRISPR/Cas9 technology, we found that this enhancer and its target gene Shh are also essential for dorsal fin development. This indicates that the regulatory network that operates in vertebrates paired appendages was co-opted from the dorsal fins of ancestral fishes. This work was published in Nature Genetics (Letelier et al., Nature Genet 2018).
To try to address how signaling pathways have changed during evolution, we have perturbed different pathways in amphioxus and zebrafish and compared the effect of this signaling modulation in gene expression and cis-regulation. We have observed a clear increase in the number of developmental genes controlled by these pathways in the transition from invertebrates to vertebrates. This impact can be explained through the incorporation of new cis-regulatory elements in their genomic regulatory landscapes. Many of these developmental genes correspond to regulators of other pathways, pointing out to an increase of interconnection between these pathways in vertebrates. We have just submitted the manuscript of this work for publication.
In the second objective, we have first generated a high quality de novo genome for the skate Leucoraja erinacea. Using this genome as a reference, we have generated a large epigenomic dataset that included: gene expression, open chromatin and 3D chromatin structure information in skate fins. By integrating all this data, and combining it with functional studies in mouse and zebrafish embryos, we are trying to unravel the regulatory mechanism behind the unique shape of pectoral skate fins. Although this work is still in progress, we are testing a candidate mechanism that could explain how the striking morphology of skate pectoral fins is generated during evolution.
For the third objective of this project, we have generated a large epigenomic dataset (RNA-seq, ATAC-seq and HiChIP) in cavefish and surface populations of Astyanax mexicanus. By integrating and comparing this dataset, we are trying to unravel the regulatory mechanism behind cave adaptation. In our analysis we have found specific CREs associated to differentially expressed genes in surface and cavefish. Interestingly, we found that many CREs that are downregulated in cavefish are associated with developmental genes critical for body plan formation. We are now using transgenic assays in zebrafish to test the regulatory activity of some of these regions. We are also trying now to identify accelerated regions in both populations to try to point critical genes and regulatory elements associated to cave adaptation.
