xv, 76 p. : ill. (some col.) Includes 4 video files. / Improved understanding of pharyngeal arch (PA) patterning and morphogenesis can reveal critical insights into the origins of craniofacial diseases, such as Fraser syndrome. PAs contain mesenchymal condensations, which give rise to most of the facial skeleton in vertebrates. Studies of Endothelin1 signaling reveal that the skeleton derived from the first two PAs are patterned into dorsal, intermediate, and ventral domains. Previous work has indicated that endothelin targets, including the Dlx genes, homeotically pattern dorsal versus ventral PA identity. I show that the Dlx gene family plays a vital role in PA intermediate-domain identity establishment. In WT fish, the PA intermediate domain is delineated by combined expression of all Dlx genes. Reduction of Dlx gene function results in loss of intermediate-domain identity. Conversely, ventral expansion of Dlx expression, seen in hand2 mutants, results in ventral expansion of intermediate-domain identity. Hence, PA intermediate-domain identity is defined by co-expression of Dlx genes. Epithelial-mesenchymal interactions play an important part in PA intermediate-domain morphogenesis. Zebrafish fras1 (epithelially expressed) and itga8 (mesenchymally expressed) mutants also show specific defects within intermediate-domain skeleton and epithelia. Facial phenotypes in fras1;itga8 double mutants look extremely similar to either single mutant, suggesting that fras1 and itga8 might participate in the same epithelial-mesenchymal interaction during PA intermediate-domain formation. Our developmental studies reveal that fras1 - and itga8 -dependent epithelial segmentation of the PA intermediate domain stabilizes developing skeletal elements. Lesions in human FRAS1 underlie many cases of Fraser syndrome, and this work provides an excellent developmental model for the craniofacial defects found in Fraser syndrome. Loss of either Dlx or fras1 function produces defects in the PA intermediate domain, yet seemingly during different developmental periods. Nonetheless, combined reduction of both Dlx and fras1 function synergistically increases skeletal defects, implying a molecular connection between early (Dlx -mediated) pattern formation and later (fras1 -mediated) pattern stabilization. Elucidation of the Dlx-fras1 interaction is an interesting topic which may unveil new molecules pertinent to Fraser syndrome. Supplemental movies highlighting skeletal and epithelial morphogenesis accompany this dissertation. / Committee in charge: Judith S. Eisen, Chairperson; Charles B. Kimmel, Advisor; John H. Postlethwait, Member; Chris Q. Doe, Member; Kennith E. Prehoda, Outside Member

The fitness function developed in this thesis directly links the physicochemical properties of an enzyme to evolutionary fates in a quantitative and predictive manner through a comparative study of empirical and simulated data. The success or failure of organisms during evolution is dictated by changes in molecular structure that give rise to changes in fitness revealed by evolutionary dynamics within a population. While the conceptual link between genotype, phenotype and fitness is clear, the ability to relate these in a quantitative manner remains difficult. I show here that predicting success during adaptation can depend critically upon enzyme kinetic and folding models. We used a 'weak link' method to favor mutations to an essential, but maladapted adenylate kinase gene within a microbial population that resulted in the identification of five mutants that arose nearly simultaneously and competed for success. The unique catalytic role of adenylate kinase in vivo is to maintain adenylate homeostasis by catalyzing the reaction: ATP + AMP [imaginary] ADP. The stabilizing substitutions retained this essential function and were shown to be necessary for viability at higher temperatures. Physicochemical characterization of these mutants demonstrated that, although steady-state enzyme activity is important, success within the population is critically dependent on resistance to denaturation and aggregation thus emphasizing the importance of proper folding in adaptation. In vitro activity is a product of critical catalytic and folding pathways, and hence is a valuable proxy for fitness. A fitness function relating in vitro measurements of enzyme activity and reversible and irreversible unfolding to growth rate must impose an activity threshold above which there is no added fitness benefit in order to reproduce in vivo evolutionary fates in an in silico population. The fitness function thereby links organismal adaptation to the properties of a single gene. Understanding the physical basis for adaptation of an organism is the first step in the development of approaches that can accurately model, and someday predict, the manner in which organisms would respond to new antibiotics and improve upon the current clinical regimens.

<p>Evolutionary dynamics can be notoriously complex and difficult to analyze. In this dissertation I describe a population genetic regime where the dynamics are simple enough to allow a relatively complete and elegant treatment. Consider a haploid, asexual population, where each possible genotype has been assigned a fitness. When mutations enter a population sufficiently rarely, we can model the evolution of this population as a Markov chain where the population jumps from one genotype to another at the birth of each new mutant destined for fixation. Furthermore, if the mutation rates are assigned in such a manner that the Markov chain is reversible when all genotypes are assigned the same fitness, then it is still reversible when genotypes are assigned differing fitnesses. </p><p>The key insight is that this Markov chain can be analyzed using the spectral theory of finite-state, reversible Markov chains. I describe the spectral decomposition of the transition matrix and use it to build a general framework with which I address a variety of both classical and novel topics. These topics include a method for creating low-dimensional visualizations of fitness landscapes; a measure of how easy it is for the evolutionary process to `find' a specific genotype or phenotype; the index of dispersion of the molecular clock and its generalizations; a definition for the neighborhood of a genotype based on evolutionary dynamics; and the expected fitness and number of substitutions that have occurred given that a population has been evolving on the fitness landscape for a given period of time. I apply these various analyses to both a simple one-codon fitness landscape and to a large neutral network derived from computational RNA secondary structure predictions.</p> / Dissertation

<p>Despite its lack of septation, the tissue patterning of the arterial pole of the zebrafish is remarkably similar to the patterning of pulmonary and aortic arterial poles observed in mouse and chick. The secondary heart field (SHF) is a conserved developmental domain in avian and mammalian embryos that contributes myocardium and smooth muscle to the cardiac arterial pole. This field is part of the overall heart field, and its myocardial component has been fate mapped from the mesoderm to the heart in both mammals and birds. In this study I demonstrate that the population that gives rise to the arterial pole of the zebrafish can be traced from the epiblast, is a discrete part of the mesodermal heart field. This zebrafish SHF contributes myocardium after initial heart tube formation, giving rise to both smooth muscle and myocardium. I show that this field expresses Isl1, a transcription factor associated with the SHF in other species. I further show that differentiation, induced by Bmp signaling, occurs in this progenitor population as cells are added to the heart tube. Some molecular pathways required for SHF development in birds and mammals are conserved in teleosts, as Nkx2.5 and Nkx2.7 as well as Fgf8 regulate Bmp signaling in the zebrafish heart fields. Additionally, the transcription factor Tbx1 and the Sonic hedgehog pathway are necessary for normal development of the zebrafish arterial pole.</p> / Dissertation

In teleosts, the Yolk Syncytial Layer (YSL) is functionally similar to the anterior visceral endoderm found in mice and is required for morphogenesis of the overlying blastoderm. The YSL undergoes dramatic reorganization during early development through processes that mirror the morphogenetic movements of the blastoderm. The YSL and YSL nuclei (YSN) undergo epiboly, and during convergence and extension movements of the blastoderm, the YSN underneath the animal cap also converge and extend underneath the axial hypoblast. Our work with pollywog ( pwg ) maternal-effect mutants highlights the delicate control of the YSL during yolk morphogenesis, and provides novel insight into understanding which tissues of the embryo are affected by loss of a cohesive YSL. I found that pollywog encodes the zebrafish mitogen activated protein kinase kinase kinase 4 ( map3k4 ) gene and that it acts upstream of p38a MAPK in the YSL. I show that this pathway acts in the YSL along with a mixer gene family member, mix-type homeobox gene 1 ( mxtx1 ), to non-autonomously coordinate extracellular matrix deposition and morphogenetic movements in the overlying blastoderm. Our data describes an early and novel role for Map3k4, p38a and Mxtxl activity that is required for proper morphogenesis of the YSL and the blastoderm. In embryos lacking maternal Map3k4, the YSL undergoes a rapid and catastrophic retraction and the YSN lose their normal distribution around the yolk. The prechordal plate of pwg mutant embryos deflect laterally or plunge into the yolk, and the overall animalward extension of the prechordal plate is diminished. I also show that the anterior neural plate of pwg mutant embryos fail to converge dorsally to the same extent as in wild embryos. These data show that the p38 MAPK pathway is essential for maintaining normal yolk cell equilibrium during early development and that without proper cues from the YSL, the blastoderm cannot complete its morphogenetic movements. Incuded in this thesis is work highlighting the alpha-actinin gene family in zebrafish. alpha-actinins are actin microfilament crosslinking proteins. Vertebrate actinins fall into two classes: the broadly-expressed actinins 1 and 4 ( actn1 and actn4 ) and muscle-specific actinins, actn2 and actn3 . Members of this family have numerous roles, including regulation of cell adhesion, cell differentiation, directed cell motility, intracellular signaling and stabilization of f-actin at the sarcomeric Z-line in muscle. Here I identify five zebrafish actinin genes including two paralogs of ACTN3 . I describe the temporal and spatial expression patterns of these genes through embryonic development. All zebrafish actinin genes have unique expression profiles, indicating specialization of each gene. In particular the muscle actinins display preferential expression in different domains of axial, pharyngeal and cranial musculature. There is no identified avian actn3 and approximately 16% of humans are null for ACTN3 . Duplication of actn3 in the zebrafish indicates that variation in actn3 expression may promote physiological diversity in muscle function among vertebrates.

<p>The problem is two fold: how does natural selection operate on systems of interacting genes and how does natural selection operate in natural populations. To address the first problem, I have conducted a theoretical investigation into the evolution of control and the distribution of mutations in a simple system of interacting genes, a linear metabolic pathway. I found that control is distributed unevenly between enzymes, with upstream enzymes possessing the greatest control and accumulating the most beneficial mutations during adaptive evolution. To address the second problem, I investigated the evolution of copper tolerance in the common yellow monkeyflower, Mimulus guttatus. I genetically mapped a major locus controlling copper tolerance, Tol1. A Dobzhansky-Muller incompatibility was hypothesized to also be controlled by Tol1, however, we have demonstrated that it maps to another, tightly linked locus, Nec1. Finally, we investigated the parallel evolution of copper tolerance in multiple new discovered mine populations. We found that copper tolerance has evolved in parallel multiple times via at least two distinct physiological mechanisms. In four mine populations, there was a strong signal of selection at markers linked to Tol1, implying that copper tolerance has evolved via the same genetic mechanisms in these populations.</p> / Dissertation

<p>Gene regulatory networks (GRNs) provide a systems-level orchestration of an organism’s genome encoded anatomy. As biological networks are revealed, they continue to answer many questions including knowledge of how GRNs control morphogenetic movements and how GRNs evolve. Morphogenesis is a complex orchestration of movements by cells that are specified early in development. </p><p> The activation of an upstream GRN is crucial in order to orchestrate downstream morphogenetic events. In the sea urchin, activation of the endomesoderm GRN occurs after the asymmetric 4th cleavage. Embryonic asymmetric cell divisions often are accompanied by differential segregation of fate-determinants into one of two daughter cells. That asymmetric cleavage of the sea urchin micromeres leads to a differential animal-vegetal (A/V) nuclear accumulation of cell fate determinants, β-Catenin and SoxB1. Β-Catenin protein is localized into the nuclei of micromeres and activates the endomesoderm gene regulatory network, while SoxB1 is excluded from micromeres and enters the nucleus of the macromeres, the large progeny of the unequal 4th cleavage. Although nuclear localization of β-Catenin and SoxB1 shows dependence on the asymmetric cleavage, the mechanics behind the asymmetrical division has not been demonstrated. In Chapter 3, we show that micromere formation requires the small RhoGTPase, Cdc42 by directing the apical/basal orientation of the mitotic spindle at the apical cortex. By attenuating or augmenting sea urchin Cdc42 function, micromere divisions became defective and failed to correctly localize asymmetrically distributed determinants. As a consequence, cell fates were altered and multiple A/V axes were produced resulting in a “Siamese-twinning” phenotype that occurred with increasing frequency depending on the quantitative level of perturbation. Our findings show that Cdc42 plays a pivotal role in the asymmetric division of the micromeres, endomesoderm fate-determinant segregation, and A/V axis formation.</p><p> This dissertation also characterizes, at high resolution, the repertoire of cellular movements contributing to three different morphogenetic processes of sea urchin development: the elongation of gut, the formation of the primary mouth, and the migration of the small micromeres (the presumptive primordial germ cells) in the sea urchin, Lytechinus variegatus. Descriptive studies of the cellular processes during the different morphogenetic movements allow us to begin investigating their molecular control. </p><p>In Chapter 4, we dissected the series of complex events that coordinate gut and mouth morphogenesis. Until now, it was thought that lateral rearrangement of endoderm cells by convergent extension was the main contributor to sea urchin archenteron elongation and that cell divisions were minimal during elongation. We performed cell transplantations to live image and analyze a subset of labeled endoderm cells at high-resolution in the optically clear sea urchin embryo. We found that the endomesoderm cells that initially invaginate into the sea urchin blastocoel remained contiguous throughout extension, so that, if convergent extension were present, it was not a major contributor to elongation. We also found a prevalence of cell divisions throughout archenteron elongation that increased the number of cells within the gut linearly over time; however, we showed that the proliferation did not contribute to growth, and their spindle orientations were randomized during divisions and therefore did not selectively contribute to the final gut length. When cell divisions were inhibited, we saw no difference in the ability of the cells within the gut to migrate in order to elongate. Also in Chapter 4, we describe our observations of the cell biological processes underlying primary mouth formation at the end of gastrulation. Using time-lapse microscopy, photo-convertible Kaede, and an assay of the basement membrane remodeling, we describe a sequential orchestration of events that leads to the fusion of the oral ectoderm and the foregut endoderm. Our work characterizes, at higher resolution than previously recorded, the temporal sequence and repertoire of the cellular movements contributing to the length of the sea urchin larval gut and tissue fusion with the larval primary mouth.</p><p> In Chapter 5, the migration of the small micromeres to the coelomic pouches in the sea urchin embryo provides an exceptional model for understanding the genomic regulatory control of morphogenesis. An assay using the robust homing potential of these cells reveals a “coherent feed-forward” transcriptional subcircuit composed of Pax6, Six3, Eya, and Dach1 that is responsible for the directed homing mechanism of these multipotent progenitors. The linkages of that circuit are strikingly similar to a circuit involved in retinal specification in Drosophila suggesting that systems-level tasks can be highly conserved even though the tasks drive unrelated processes in different animals.</p><p> The sea urchin gene regulatory network (GRN) describes the cell fate specification of the developing embryo; however, the GRN does not describe specific cell biological events driving the three distinct sequences of cell movements. Our ability to connect the GRN to the morphogenetic events of gastrulation, primary mouth formation, and small micromere migration will provide a framework for characterizing these remarkable sequences of cell movements in the simplest of deuterostome models at an unprecedented scale.</p> / Dissertation

abstract: Gene-centric theories of evolution by natural selection have been popularized and remain generally accepted in both scientific and public paradigms. While gene-centrism is certainly parsimonious, its explanations fall short of describing two patterns of evolutionary and social phenomena: the evolution of sex and the evolution of social altruism. I review and analyze current theories on the evolution of sex. I then introduce the conflict presented to gene-centric evolution by social phenomena such as altruism and caste sterility in eusocial insects. I review gene-centric models of inclusive fitness and kin selection proposed by Hamilton and Maynard Smith. Based their assumptions, that relatedness should be equal between sterile workers and reproductives, I present several empirical examples that conflict with their models. Following that, I introduce a unique system of genetic caste determination (GCD) observed in hybrid populations of two sister-species of seed harvester ants, Pogonomyrmex rugosus and Pogonomyrmex barbatus. I review the evidence for GCD in those species, followed by a critique of the current gene-centric models used to explain it. In chapter two I present my own theoretical model that is both simple and extricable in nature to explain the origin, evolution, and maintenance of GCD in Pogonomyrmex. Furthermore, I use that model to fill in the gaps left behind by the contributing authors of the other GCD models. As both populations in my study system formed from inter-specific hybridization, I review modern discussions of heterosis (also called hybrid vigor) and use those to help explain the ecological competitiveness of GCD. I empirically address the inbreeding depression the lineages of GCD must overcome in order to remain ecologically stable, demonstrating that as a result of their unique system of caste determination, GCD lineages have elevated recombination frequencies. I summarize and conclude with an argument for why GCD evolved under selective mechanisms which cannot be considered gene-centric, providing evidence that natural selection can effectively operate on non-heritable genotypes appearing in groups and other social contexts. / Dissertation/Thesis / M.S. Biology 2012

abstract: How fast is evolution? In this dissertation I document a profound change that occurred around the middle of the 20th century in the way that ecologists conceptualized the temporal and spatial scales of adaptive evolution, through the lens of British plant ecologist Anthony David Bradshaw (1926–2008). In the early 1960s, one prominent ecologist distinguished what he called “ecological time”—around ten generations—from “evolutionary time”— around half of a million years. For most ecologists working in the first half of the 20th century, evolution by natural selection was indeed a slow and plodding process, tangible in its products but not in its processes, and inconsequential for explaining most ecological phenomena. During the 1960s, however, many ecologists began to see evolution as potentially rapid and observable. Natural selection moved from the distant past—a remote explanans for both extant biological diversity and paleontological phenomena—to a measurable, quantifiable mechanism molding populations in real time. The idea that adaptive evolution could be rapid and highly localized was a significant enabling condition for the emergence of ecological genetics in the second half of the 20th century. Most of what historians know about that conceptual shift and the rise of ecological genetics centers on the work of Oxford zoologist E. B. Ford and his students on polymorphism in Lepidotera, especially industrial melanism in Biston betularia. I argue that ecological genetics in Britain was not the brainchild of an infamous patriarch (Ford), but rather the outgrowth of a long tradition of pastureland research at plant breeding stations in Scotland and Wales, part of a discipline known as “genecology” or “experimental taxonomy.” Bradshaw’s investigative activities between 1948 and 1968 were an outgrowth of the specific brand of plant genecology practiced at the Welsh and Scottish Plant Breeding stations. Bradshaw generated evidence that plant populations with negligible reproductive isolation—separated by just a few meters—could diverge and adapt to contrasting environmental conditions in just a few generations. In Bradshaw’s research one can observe the crystallization of a new concept of rapid adaptive evolution, and the methodological and conceptual transformation of genecology into ecological genetics. / Dissertation/Thesis / Doctoral Dissertation Biology 2015

abstract: Early weaning, slow somatic and dental growth, and late age at reproduction are all part of a suite of energetic trade-offs that have shaped human evolution. A similar suite of energetic trade-offs has shaped the evolution of the indriid-palaeopropithecid clade, though members of this clade exhibit extremely fast dental development and nearly vestigial deciduous teeth. The development and functional occlusion of the primary postcanine dentition (i.e., deciduous premolars and molars) coincides with several life history parameters in great apes and indriids. This dissertation explored great ape dental macrowear, molar development in indriids, and molar size in lemurs with a broader goal of improving reconstructions of life history profiles in extinct primates. To this aim, macrowear and dental development were analyzed in apes and lemurs, respectively. Occlusal casts (six great ape species; N=278) were scanned to track mandibular fourth deciduous premolar (dp4) macrowear. Utilizing dental topographic analyses, changes in occlusal gradient and terrain were quantified. A subset of the great ape data (four species; n=199) was analyzed to test if differences in dp4 wear correlate with age at weaning. Using dental histology, molar development was reconstructed for Indri indri (n=1) and Avahi laniger (n=1). Life history and molar size data were collected from the literature. The results of this dissertation demonstrate that most great apes exhibited evidence of topographic maintenance, suggesting dp4s wear in a manner that maintain functional efficiency during growth and development; however, the manner in which maintenance is achieved (e.g., preservation of relief or complexity) is species specific. Dp4 macrowear is not correlated with age at weaning in great apes and is probably unreliable to reconstruct age at weaning in hominins. The pace of molar development in members of the indriid- palaeopropithecid clade did not correlate with body or brain size, an association present in several other primates. Associations of molar size with age at weaning suggest that expanding other developmental models (e.g., the inhibitory cascade) to life history is worth consideration. The broad variation in macrowear, dental development, and size highlights how the primary dentition may correlate with different life history parameters depending on the species and ecological setting, an important consideration when using teeth to reconstruct life history profiles. / Dissertation/Thesis / Doctoral Dissertation Anthropology 2016