Periodic Reporting for period 2 - Evoland (Evolution of regulatory landscapes at multiple timescales)
Reporting period: 2019-03-01 to 2020-08-31
The advent of next generation sequencing-based tools has made possible to identify genome-wide CREs and reveal how they are organized in the 3D genome. Here, we are integrating in a systematic and phylogenetically driven manner the contribution of CREs and their 3D organization to animal morphology at different evolutionary scales. This multidisciplinary approach allows to link evolution, regulatory information, genome 3D architecture and morphology. We are applying this strategy to study animal morphology along the evolution of deuterostome body plans, the generation of fin morphological diversity in vertebrates, and the recent phenotypic changes in fish adapted to cave environments.
Our work is already providing ground-breaking advances in our understanding of how coding and non-coding regulatory DNA are organized and integrated in the genome, and how this organization has shaped the evolution of genomes and impacted on body plan formation. Moreover, we are also using this integrative strategy to understand how alterations in cis-regulation and 3D chromatin architecture are associated to some human diseases, revealing the molecular base behind these pathologies.
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.
The comparison of some of this data has allowed demonstrating how cis-regulation has contributed to the evolution of body plan formation in the transition from invertebrate to vertebrates.
We contemplate, during the remaining of the project, to expand this analysis by including data from different echinoderm species and by comparing gene regulatory network that operate during development in different deuterostomes species.
On the other hand, the analysis of the skate genome, a basal vertebrate, will allow us to determine how vertebrate genomes have evolved. Moreover, our current epigenomic studies in different skate fins will likely unravel the molecular events that operate during development to generate the striking morphology of skate pectoral fins.
Finally, our epigenomic studies with Astyanax mexicanus, combined with functional analysis in zebrafish and the phylogenetic comparison between different fish species to detect accelerated regions in cavefish, will reveal critical CREs and genes associated to the morphological changes observed during adaptation of this specie to the cave life style.