Evolution—the great tinkerer—has produced the astounding diversity of form within and between existing species. It is a fundamental goal of evolutionary biology to understand the origin of such diversity. What types of genes underlie evolved changes in morphology? Are certain types of mutations (notably changes within regulatory regions) more likely to be used to produce adaptive changes in form? When distinct populations evolve similar morphological changes, are the underlying genetic bases changes to the same genes, the same genetic pathways, or largely independent? Are changes in form modular, or are their concerted changes to multiple developmentally similar organs? The ever cheapening cost of sequencing, coupled the availability of high-quality reference genomes, allows high-throughput approaches to identifying the loci of evolution. The emergence of a robust genome engineering system, CRISPR/Cas9, allows for efficient and direct testing of a gene's phenotype. Combining both of these techniques with a model system with naturally evolved phenotypic variation, the threespine stickleback, allows for systems-level answers to the many evolutionary questions.
Chapter one outlines the field of evolutionary developmental biology. It proposes two alternative viewpoints for thinking about the evolution of form. The first is the view of the `Modern Synthesis', linking Mendelian inheritance with Darwinian natural selection, which explains evolution as the change in allele frequencies over time. The second views evolution through the lens of deep homology, focusing on changes to developmental programs over time, even across related organs within the same animal. It then introduces key concepts within evolutionary and developmental biology, including cis-regulation of gene expression, and gene regulatory networks. It then provides examples of evolution reusing similar gene regulatory networks, including Hox genes, Pax6 dependent eye initiation, and ectodermal placode development. Teeth use highly conserved signaling pathways, during both their initiation and replacement. Threespine sticklebacks Gasterosteus aculeatus have repeatedly adapted following a shift from marine to freshwater environments, with many independently derived populations sharing common morphological traits, including a gain in tooth number. The following chapters investigate this gain in tooth number in multiple distinct populations of sticklebacks.
Chapter two describes the discovery and mapping of a spontaneous stickleback albino mutation, named casper. casper is a sex-linked recessive mutation that results in oculocutaneous albinism, defective swim bladders, and blood clotting defects. Bulked segregant mapping of casper mutants revealed a strong genetic signal on chromosome 19, the stickleback X chromosome, proximal to the gene Hps5. casper mutants had a unique insertion of a G in the 6th exon on Hps5. As mutants in the human orthologue of Hps5 resulted in similar albino and blood clotting phenotypes, Hps5 is a strong candidate underlying the casper phenotype. Further supporting this model, genome editing of Hps5 phenocopied casper. Lastly, we show that casper is an excellent tool for visualizing the activity of fluorescent transgenes at late developmental stages due to the near-translucent nature of the mutant animals.
Chapter three details the fine mapping of a quantitative trail locus (QTL) on chromosome 21 controlling increases in tooth number in a Canadian freshwater stickleback population. Recombinant mapping reduced the QTL-containing region to an 884kb window. Repeated QTL mapping experiments showed the presence of this QTL on multiple, but not all, wild derived chromosomes from the Canadian population. Comparative genome sequencing revealed the perfect correlation with genetic data of ten variants, spanning 4.4kb, all within the 4 th intron of the gene Bmp6. Transgenic analysis of this intronic region uncovered its role as a robust tooth enhancer. TALEN induced mutations in Bmp6 revealed required roles for the gene in stickleback tooth development. Finally, comparative RNA-seq between Bmp6 wild-type and mutant dental tissue showed a loss of mouse hair stem cell genes in Bmp6 mutant fish teeth, suggesting deep homology of the regeneration of these two organs.
Chapter four investigates the evolved changes in gene expression that accompany evolved increases in tooth number in two distinct freshwater populations. Independently derived stickleback populations from California and Canada have both evolved increases in tooth number, and previous work suggested that these populations used distinct genetic changes during their shared morphological changes. RNA-seq analysis of dental tissue from both freshwater populations compared to marine revealed a gain in critical regulators of tooth development in both freshwater populations. These evolved changes in gene expression can be partitioned in cis changes (mutations within regulatory elements of a gene) and trans changes (changes to the overall regulatory environment) using phased RNA-seq data from marine-freshwater F1 hybrids. Many genes show evidence for stabilizing selection of expression levels, with cis and trans changes in opposing directions (Abstract shortened by ProQuest.).
|Advisor:||Miller, Craig T.|
|Commitee:||Eisen, Michael B., Nachman, Michael W., Rokhsar, Daniel S.|
|School:||University of California, Berkeley|
|Department:||Molecular & Cell Biology|
|School Location:||United States -- California|
|Source:||DAI-B 80/01(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Genetics, Bioinformatics|
|Keywords:||Bioinformatics, Developmental biology, Evolution, Genetics, Genome editing, Genomics|
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