Identification of the molecular changes underlying head morphology variation in closely related Drosophila species
by Montserrat Torres Oliva
Date of Examination:2016-05-23
Date of issue:2016-09-09
Advisor:Dr. Nico Posnien
Referee:Dr. Nico Posnien
Referee:Prof. Dr. Martin Göpfert
Persistent Address:
http://dx.doi.org/10.53846/goediss-5851
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Abstract
English
The great diversity of adult morphologies that we can observe in nature is the product of millions of years of evolution of the underlying developmental programs. The genes that code for the transcription factors and signaling molecules that govern these processes are remarkably conserved across great phylogenetic distances. Thus, it is thought that gene expression divergence is the main driver of morphological evolution. The possibility to study genome-wide patterns of gene expression based on high-throughput transcriptome sequencing (RNA-seq) can provide unprecedented new insights into how the mechanisms that regulate gene expression have evolved to give rise to such outstanding variety in phenotypes. Insects show a striking morphological diversity, especially in the size and shape of their head and eyes. To understand what parts of the gene regulatory networks that govern head and eye development can evolve to generate morphological differences without disturbing the fundamental developmental programs, a deeper knowledge of these networks is necessary. In the fruit fly Drosophila melanogaster, many transcription factors that govern compound eye development are known. However, few target genes of these regulators have been identified, and still little is known about the development of the other organs and cell types that are also part of the fly head. Here I have performed developmental transcriptomics on three key stages of D. melanogaster head development in order to obtain a more detailed description of these processes and all the implicated genes. Most interestingly, by gene co-expression analyses I found that the well-known transcription factor Hunchback may play a central role during late eye-antennal imaginal disc development. And indeed, subsequent functional analyses revealed a critical role of Hunchback in the development of a subtype of retinal glia cells that is involved in axon guidance and the formation of an intact blood-brain barrier. This finding and the additional identification of other transcription factors and target genes that I could validate, certify that genome-wide developmental gene co-expression analysis is a powerful tool to increase our knowledge on gene regulatory networks governing developmental processes. Recent studies have identified significant differences in the size of the heads and compound eyes in the three closely related Drosophila species D. melanogaster, D. simulans and D. mauritiana. D. melanogaster has a wider face and smaller eyes than its sibling species, while D. mauritiana has the biggest eyes and a much narrower face. Therefore, these three species represent a good model to identify the nodes of the developmental networks that present divergent expression levels that could give rise to adult morphological differences. Although genomic references are available for these species, the comparability of these resources varied greatly. In order to perform an unbiased inter-species analysis of differential gene expression, I first developed a pipeline to reciprocally re-annotate their genomes. A rigorous benchmarking of this new pipeline in comparison to previously available methods showed that my strategy increased the number of genes that I could compare and it resulted in the most unbiased results. Additionally, this analysis represents the first comprehensive evaluation of existing statistical methods in the context of inter-specific expression divergence. The unbiased references allowed me to reliably perform a comprehensive transcriptomics analysis to identify all differentially expressed genes between D. melanogaster, D. mauritiana and D. simulans during key stages of head and eye development. By studying allele-specific expression of the viable F1 hybrids, I could identify the regulatory mechanisms underlying the divergent gene expression between these species. Interestingly, I have found that most gene expression differences in developing tissues are due to changes in the upstream regulatory genes, what is known as variation in trans. These results are different to what has been previously reported in adult Drosophila tissues and could indicate that different stages of an organism’s life are subject to different evolutionary mechanisms influencing gene expression divergence. Finally, it has been shown that the compound eyes of D. mauritiana are bigger than D. simulans eyes due to differences in facet size. I have combined available quantitative trait loci data with my genome-wide differential gene expression data to identify the genetic basis of these observed morphological differences. This unbiased strategy in combination with functional tests in D. melanogaster has led to the identification of a single gene, namely ocelliless, as being the most likely candidate for its regulatory region to have evolved to give rise to the observed morphological differences in eye size. In conclusion, I could identify new regulatory interactions underlying Drosophila head formation. Additionally, I revealed some of the potential molecular changes that may have given rise to morphological diversity. All in all, this work shows how comprehensive transcriptomics analyses can greatly contribute to a better understanding of both developmental and evolutionary processes.
Keywords: eye development; Drosophila; evolution; transcriptomics