title: Eda, gene pathways and alternative splicing: the genetic basis of adaptive evolution in threespine stickleback
Creators: name: Rodríguez Ramírez, Carlos Ernesto
Publisher: name: Universität Bern
Year: 2024
Subjects: subject: 570 Life sciences; biology

description: Understanding the genetic basis of adaptation is one of the main goals of evolutionary biology. This entails understanding which and how many loci underlie adaptive phenotypes (the genetic architecture), but also entails understanding how the underlying genes and mutations work and through which mechanisms the create these adaptive phenotypes (the molecular mechanisms). While much progress has been made in the last decades in discovering the genetic architecture of phenotypic variation in the wild, understanding the molecular mechanisms linking genotype and phenotype remains challenging. Understanding this link however is essential to understand why certain genes are involved in adaptive evolution and others not. Answering these questions is essential not only to understand how individuals have adapted to their current environments, but also to one day be able to predict how they might adapt to new environments.
A model that is at the forefront of research in this field is the threespine stickleback (Gasterosteus aculeatus), a teleost fish widespread throughout the oceans of the Northern Hemisphere. After the Last Glacial Age, threespine stickleback repeatedly and independently adapted and colonized freshwater habitats across its distribution range. This interesting evolutionary history coupled with rich ecological knowledge and a wealth of genetic tools make threespine stickleback a great system to study the genetic basis of adaptation. One of the most characteristic traits found in freshwater sticklebacks is the repeated loss of defensive lateral plates. This phenotype has been mapped to a large effect gene, Ectodysplasin A (Eda), which is strongly under selection in freshwater populations. Interestingly, Eda is a pleiotropic gene, and besides the lateral plates it also causes changes in the patterning of the sensory lateral line and schooling behaviour. However, despite years of research on Eda, not much is known yet about the molecular mechanisms through which Eda causes these phenotypic effects.
In Chapter 1 of this thesis, I describe my work in looking for the causative mutation of the phenotypic effects of Eda, which remains unknown to this day. In this study my collaborators and I test a candidate 16 bp deletion in the first intron of Eda with a CRISPR-Cas9 manipulation experiment. Unfortunately, our results show that our candidate has no effect on the number of lateral plates. We conclude that the causative region of Eda needs to be narrowed down further before more candidate mutations are tested. These results also serve as a reminder of how seemingly strong candidate mutations might still turn out to have no effect on the phenotype under study.
In Chapter 2, I describe my work in exploring the downstream molecular effects of the Eda haplotype. This is a 16 kb region of the genome that includes Eda plus two neighbouring genes (Tnfsf13b and Garp), which are linked in wild populations of stickleback. Using RNAseq I found that the Eda haplotype causes downstream effects in hundreds of genes in skin. These include genes involved in bone and neuronal development pathways, making them strong candidates to be the mediators of the phenotypic effects of Eda on the lateral plates and the lateral line. I also find evidence of an effect of the haplotype in immune genes and an immune organ (head kidney), which could be related to the presence of Tnfsf13b and/or Garp. Finally, I find that the haplotype effect on downstream genes happens not only through changes in gene expression levels, but also by changes in alternative splicing patterns. Interestingly, these two mechanisms affect mostly non-overlapping sets of genes that also present differences in their average expression and pleiotropy levels.
In Chapter 3 of this thesis, I described the results of a study inspired by our previous results on alternatively spliced genes. In this study I explore the role of alternative splicing in the marine-freshwater divergence of threespine stickleback. By comparing publicly available RNAseq data of marine and freshwater populations, I find over one hundred differentially spliced genes (DSGs) between the ecotypes. I also find that these genes are enriched in regions of the genome associated to phenotypic divergence and in regions of the genome with signatures of divergent selection between ecotypes. Furthermore, I find that different types of splicing seem to contribute differently to the divergence, with splicing of mutually exclusive exons being the most common in our DSGs and having the strongest signatures of genetic divergence. Taken together the results of this chapter suggest that alternative splicing might mediate some of the adaptive divergence between marine and freshwater sticklebacks.
I finalize this thesis with a general discussion, where I discuss how the results of this thesis give us interesting insights into the molecular mechanism mediating adaptive evolution in threespine stickleback and highlight the value and the need to keep studying these questions in our field.