Developmental genetic basis of stickleback evolved tooth gain

An abundance of morphological diversity is seen across nature yet we know little of the mechanistic underpinnings of these evolved changes. Developmental patterning is achieved by relatively few signaling pathways, which have been modified throughout evolution to give rise to the various forms we see today. Despite extensive studies characterizing mutant phenotypes under laboratory settings, we still know little about the developmental and genetic mechanisms underlying evolved phenotypes in nature. Outstanding questions include: How have developmental mechanisms been modified? When the same phenotype evolves, are the genetic bases the same or different? And in cases with continuously regenerating morphology, how are these phenotypes maintained over time and what signaling pathways play a role? In this dissertation I address these questions using the teeth of the threespine stickleback fish, Gasterosteus aculeatus, as a model./nTeeth have long served as a model system to study basic questions about vertebrate organogenesis, morphogenesis, and evolution. In most non-mammalian vertebrates, teeth regenerate throughout adult life. New model systems that undergo continuous tooth replacement are sorely needed to complement developmental studies of tooth formation in mice, which do not replace their teeth. Fish have evolved a tremendous amount of diversity in dental patterning in both their oral and pharyngeal dentitions, offering numerous opportunities to study how morphology develops, regenerates, and evolves in different lineages./nThreespine stickleback fish have emerged as a new system to study how morphology evolves, and provide a particularly powerful system to study the development and evolution of dental morphology. Sticklebacks have undergone an adaptive radiation, with oceanic marine populations repeatedly colonizing and rapidly adapting to freshwater lakes and creeks throughout the northern hemisphere. Colonization of freshwater environments is accompanied by a variety of changes to the head skeleton, many of which are likely adaptive for the major shift in diet from small zooplankton in the ocean to larger prey in freshwater. Natural variation in dental patterning exists between stickleback fish populations providing an opportunity to dissect the developmental genetic basis of tooth formation and replacement. Marine and freshwater sticklebacks can be intercrossed and their F1 hybrids are fertile, allowing forward genetic mapping of genomic regions controlling evolved differences./nIn chapter 1, I introduce the oral and pharyngeal dentition in sticklebacks and provide morphological, histological, and molecular evidence for homology of oral and pharyngeal teeth. Next, using a dense developmental time-course of lab-reared animals, the temporal and spatial sequence of early tooth formation for the ventral pharyngeal dentition is described. This sequence is highly stereotypical allowing the characterization of the first tooth replacement event and providing a guide for future phenotyping. Finally, the early sequence of tooth development is compared to that described in other fish, revealing that major changes to how dental morphology arises and regenerates have evolved across different fish lineages./nIn chapter 2, I focus on how the variation in dental patterning manifests during development. Previous work had identified a freshwater population with increased tooth number arising late in development, however the mechanism of how teeth were gained over time remained elusive. Here, using a vital dye pulse-chase method, we showed increased tooth number results from an increased tooth replacement rate. We also identified a second freshwater population which has convergently evolved tooth gain allowing us to study whether the developmental and genetic bases underlying this phenotype are the same or different. Despite the similar evolved phenotype of more teeth and an accelerated adult replacement rate, the timing of tooth number divergence and the spatial patterns of newly formed teeth are different in the two freshwater populations, suggesting distinct developmental mechanisms underlie the evolved changes. Using genome-wide linkage mapping in marine-freshwater F2 genetic crosses, we found largely non-overlapping genomic regions controlling tooth patterning in the two high-toothed populations. This work represents one of the first demonstrations of distinct developmental genetic bases underlying evolved changes in morphology in vertebrates./nIn chapter 3, I test the role of BMP signaling on stickleback tooth formation and replacement. Although distinct genomic regions underlying evolved tooth gain in two freshwater populations were identified, the reoccurrence of BMP pathway members (ex. Bmp6, Msxe, Bmp7a) appearing as candidates in loci underlying evolved tooth gain suggests the hypothesis that changes in different components of the BMP signaling pathway underlies convergent evolution of tooth gain. Using the small molecule BMP signaling inhibitor LDN-193189, we showed BMP signaling plays both positive and negative roles in tooth development and replacement. BMP knockdown results in failure to initiate late forming primary positions while premature replacement occurs at early, established tooth positions. Notably, this accelerated tooth replacement is independent of tooth shedding and may be a mechanism used to evolved gains in tooth number. Late in development at a stage after individual tooth positions can be tracked, BMP knockdown in marine fish increased the number of newly forming teeth, likely due to increased tooth replacement. Collectively these data suggest that during stickleback tooth formation and replacement, BMPs positively regulate tooth development while negatively regulating tooth replacement and suggest this pathway has been modified during freshwater adaptation to achieve evolved tooth gain./nIn chapter 4, I provide a protocol demonstrating how to dissect and flat-mount the internal branchial skeleton. By mounting this complex three-dimensional skeleton into largely two-dimensions, one can easily phenotype a variety of internal traits including pharyngeal tooth patterning. This method is a fast and relatively inexpensive way to study variation of trophic traits and is also applicable to a wide variety of fish species. In sticklebacks, we have used this method to visualize and precisely measure skeletal morphology in genetic crosses to map genomic regions controlling craniofacial patterning./nTogether this dissertation makes significant progress toward understanding the developmental genetic basis of evolved tooth gain in stickleback fish. These results have broad implications for understanding the repeatability of evolution, mechanisms of evolved gain traits, the process and signaling pathways of tooth replacement, and how signaling pathways can be modulated to produce morphological variation.

Advisor: Miller, Craig T