Biology Theses and Dissertations

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Open Access
Molecular Architecture of the Octopus bimaculoides Central Nervous System
(University of Oregon, 2024-12-06) Songco, Jeremea; Niell, Cristopher
Interacting with our environments requires that we appropriately integrate sensory information and convert these inputs into a perception of our surroundings to generate basic and complex behaviors. Traditionally, model organisms, such as nematodes, flies, zebrafish, or even mice, have been used in the laboratory setting to investigate neural circuit formation and function. While these organisms have furthered our understanding of how different cell types wire up to drive complex behavior, there is much to be learned from exploring the brain of non-traditional organisms. Cephalopods have the largest brain among invertebrates and have a rich catalog of behaviors, including navigating complex underwater environments and rapid body-patterning known as camouflage. While seminal work during the 1960s revealed cellular properties of neurons using the giant squid axon, recent advancements in technology have permitted further characterization of cell types and circuits in a species that is unlike many of those used traditionally in the field of neuroscience. By investigating the brain of these animals, we can begin to understand fundamental mechanisms involved in the formation and function of complex neural circuits. Unlike model organisms, there are limited tools in genetic manipulation and the field has yet to produce a comprehensive brain atlas bridging anatomical, molecular, and functional properties of cell types in these animals. Therefore, my dissertation sought to develop key resources that will serve as a foundation for such studies once it becomes technically possible. I first contributed to the optimization and usage of functional imaging in an ex vivo preparation of the octopus brain in order to characterize response properties of visually responsive cells in the optic lobe, the main visual center which is a paired brain region that comprises 2/3 of the central nervous system of octopuses. We found evidence for retinotopic organization of responses to light (ON) and dark (OFF) spots, including spatial tuning properties that may be suggestive of environmental demands. To begin elucidating the diversity of unit responses we revealed in this initial study, I focused on developing a single-cell molecular atlas of the Octopus bimaculoides optic lobe by combining single cell RNA-sequencing (scRNA-seq) with multiplexed fluorescence in situ hybridization (FISH). We identified six classes of mature neuronal cell types in addition to a large population of immature neurons. Our FISH revealed sublaminar organization across the optic lobe, further characterizing the cell types that were initially identified in the 1960s based on morphology. An octopus’ ability to engage in a wide range of visually guided behaviors rests upon the various inputs and outputs the optic lobes have to other structures in the central nervous system. However, there has yet to be published a mapping of these structures as well as an in-depth understanding of the molecular landscape across the central nervous system. Therefore, I sought to develop the first brain-wide gene expression resource for cephalopods by characterizing all of the structures in this species through Hematoxylin & Eoisin (H&E) staining of serial sections of the brain, and I quantified expression for 40 genes, including functional and developmental determinants, across 20 identified brain regions. Together, this work reveals functional and molecular organization in the optic lobe as well as other brain regions, furthering our understanding of how a completely different organism can carry out complex behaviors. This dissertation includes previously published and unpublished co-authored material.
Embargo
Left-Right Patterning: Biophysical and Chemical Signaling Pathways that Contribute to Left-Right Axis Establishment
(University of Oregon, 2024-08-07) Luna-Arvizu, Gabriel; Grimes, Daniel
Cells sense their environment by detecting and responding to mechanical forces and chemical signals across their surface. Fluid flows, which can impart force and distribute chemicals, are critical for organ development, embryonic patterning, and contribute to host-microbe interactions and cancer metastasis. Despite the impacts on human health, flow signal sensation is understudied. We aim to elucidate how embryos break left-right (L-R) symmetry using flow-derived signals to understand cell communication. During development, beating cilia within structures called L-R organizers (LRO) generate an asymmetric flow that breaks symmetry. Cells sense flow, repressing the target gene dand5 on the left side only. The response to flow signals involves cilia yet mechanisms remain unclear. Our main hypothesis is that the Polycystin transmembrane protein Pkd1l1 complexes with cation channel Pkd2 to mediate flow signal sensation and establish L-R asymmetries. We found that pkd1l1 zebrafish mutants exhibited L-R defects in the heart, brain, pancreas, as well as displaying aberrant laterality-associated behaviors. Spatiotemporally, pkd1l1 and dand5 are co-expressed during flow stages. Global and cell-specific mutagenesis revealed Pkd1l1 functions cell-autonomously to maintain high levels of dand5, suggesting that flow normally represses Pkd1l1 on the left, leading to reduced dand5 on that side. Flow signals are thought to open Pkd2 channels, resulting in intraciliary Ca2+ signals. Mutation of pkd1l1 led to increased intraciliary Ca2+ signals, supporting a model in which Pkd1l1 represses Pkd2 channels until that repression is relieved by flow on the left.A second goal of this thesis was to discover novel causes of human laterality diseases. Recently, our team discovered DIXDC1 mutations in families exhibiting heterotaxia and situs inversus i.e. defective L-R symmetry breaking. Dixdc1 is thought to play a role in Wnt signaling, but had not previously been linked to L-R patterning. Using loss and gain of function approaches, we determined that perturbation of dixdc1a and dixdc1b, two zebrafish paralogs of DIXDC1, caused edema, abnormal body axis development, reduced forebrain and, importantly, severe heart laterality defects. Molecular evidence suggests dixdc1a and dixdc1b act upstream of dand5. Thus, we have uncovered a new role for the Wnt regulator Dixdc1 in L-R patterning across species. This further establishes an interplay between mechanical and chemical signals that orchestrate the breaking of symmetry during development. This dissertation includes previously published co-authored material.
Embargo
Landscapes of (Co)Evolution: A Tale of Two Signals
(University of Oregon, 2024-08-07) Caudill, Victoria; Bohannan, Brendan
Evolution between species and within specific environments has resulted in a diverse array of traits and genetic variation. These are the result of interactions that have occurred across space and throughout time. As evolution progresses, it generates signals and patterns that can be used to unravel mysteries of the past and provide insight into future possibilities. By examining the signals left behind by the process of evolution, I have gained valuable insights onto what has occurred in the past. In chapter II, I use spatial simulations to explore the (co)evolutionary trajectories of levels of toxin resistance and toxin production in the predator-prey Thamnophis garter snake – Taricha newt system. Specifically, I examine how possi- ble genetic architectures of the toxin and resistance traits affect the coevolutionary dynamics by manipulating both mutation rate and effect size of mutations across many simulations. I find that coevolutionary dynamics alone were not sufficient in our simulations to produce the striking mosaic of levels of toxicity and resistance observed in nature. Instead, simulations with ecological heterogeneity (in trait cost- liness or interaction rate) did produce such patterns. In chapter III, I examined landscapes of genetic variation in cichlids, a species complex that has recently ra- diated. I used the phylogenetic relationship and population genetic measurements (mean nucleotide diversity and divergence) to describe large-scale variation across the genome. These patterns are likely caused by complex effects of inversions, in- trogression, and linked selection. Together, these findings contribute to building a strong foundation for understanding the evolutionary signals of natural selection and how its’ impacts vary along the genome. This dissertation includes previously published and unpublished co-authored material.This dissertation includes previously published and unpublished co-authored material.
Open Access
Spatiotemporal Motifs of Cerebral Theta Oscillations in Freely Moving Mice
(University of Oregon, 2024-08-07) Sattler, Nicholas; Wehr, Michael
Mice are the most commonly used mammal in systems neuroscience, yet the ability to implement large-scale neural recordings and behavioral monitoring in freely moving conditions has remained methodologically challenging. Due to these difficulties, traditional methods resort to heavily restraining mice in “head-fixed conditions” to achieve stable recordings of neural activity and behavioral monitoring. Such practices, however, drastically restrict the flexibility of both their behavior and the neural activity intended to be studied in the first place. In freely moving conditions, movement-related activity across the cerebral cortex has been shown to be organized by theta oscillations. While there has been significant progress in the ability to simultaneously record from large numbers of neurons using calcium imaging and electrophysiological probes, our understanding of theta oscillations on global cortical function has similarly been stymied by these method’s shortcomings in temporal resolution and spatial scale respectively.This dissertation aims to address these issues through the development and integration of new methodologies. In Chapter II, I present a novel head-mounted camera system for monitoring freely moving mice. The advantages of this system allow large-scale electrophysiological recordings to be paired with multi-camera headsets, enabling the ability to simultaneously record the fine movements of the eyes, ears, whiskers, nostrils, and visual field in freely moving conditions. In Chapter III, I utilize these techniques to identify a distinct behavioral pattern of the nostrils and ears during freely moving mouse behavior. Additionally, through the development and integration of cortex-wide electrocorticography —a method of electrophysiology that records across large areas of the cortex at high spatiotemporal resolution— I also identify distinct cortex-wide spatiotemporal motifs of cerebral theta oscillations prominent during natural behavior. These novel findings open up new avenues of investigation for understanding how the brain integrates information into the production of ongoing behavior in natural conditions. This dissertation includes previously published and unpublished co-authored material. This dissertation also includes seven supplementary videos related to the material described in Chapter II.
Open Access
Reciprocal Subsidies: Phytodetrital Influence on the Reproduction of Methanotrophic Mussels, and the Contribution of Mussel Larvae to Planktonic Food Webs in the Gulf of Mexico and Western Atlantic
(University of Oregon, 2024-08-07) Plowman, Caitlin; Young, Craig
There is growing evidence that chemosynthesis-based ecosystems in the deep sea influence the benthic and pelagic habitats around them, though the extent of these connections is unknown. Bathymodiolin mussels are among the dominant fauna at deep-sea chemosynthetic systems such as methane seeps and hydrothermal vents. Despite their symbiotic bacteria, many species in the subfamily have shown evidence of periodic reproduction driven by seasonal changes in surface productivity. In this dissertation, I explore the reproductive patterns of bathymodiolin mussels from methane seeps in the Gulf of Mexico and Northwestern Atlantic and how they contribute to connections between surface waters and the deep sea. In Chapter II, I use gonad histology to examine how the reproductive phenology changes with the season, ocean basin, and depth for three species of bathymodiolin mussels, Gigantidas childressi, Bathymodiolus brooksi, and Bathymodiolus heckerae. I find inter- and intra-specific variations across a bathymetric gradient and between the two ocean basins. The results further support that surface productivity plays a role in bathymodiolin reproduction. In Chapter III, I use carbon and nitrogen stable isotopes to investigate phytodetritus as a potential source of supplemental nutrition for reproduction. I also compare the phenology to satellite-derived measurements of surface productivity. Many of the sampled populations of bathymodiolin mussels incorporate phytodetrital nutrition, but it is not a necessary subsidy for reproduction. The onset of gametogenesis occurs near a peak in surface productivity, and spawning occurs during a change in surface productivity that is shortly followed by a peak. Thus, surface productivity may be an environmental cue that helps synchronize the gametogenic phenology of these mussels. While Chapters II and III examine how the water column affects the reproduction of bathymodiolin mussels, Chapter IV examines how spawned mussel eggs influence the water column. For Gigantidas childressi from three seep sites, I used histology to estimate individual fecundity, bomb calorimetry to measure the energy contained in an egg, and video of the sites to determine the size of the mussel beds. All metrics vary among the sites; changes in fecundity correspond to changes in phenology, while the nutrient density of eggs increased with depth. Each factor affects the total energy released through spawned eggs, but the estimated values are on par with the photosynthetic production of oligotrophic surface waters. Most of the spawned eggs are eaten and thus may represent a critical trophic subsidy for the surrounding ecosystems. The energy these mussels use to create gametes comes mostly from chemosynthetic sources. Seasonal pulses of phytodetritus may then trigger the spawning of these eggs, releasing much of that energy into the pelagic food web. This research highlights the intimate reciprocal connection between processes in the surface ocean and the deep seafloor. This dissertation includes previously unpublished co-authored material.
Open Access
INTERCONNECTED GENOMIC LANDSCAPES OF SEQUENCE VARIATION, MEIOTIC RECOMBINATION, AND GERMLINE CHROMATIN IN C. ELEGANS
(University of Oregon, 2024-08-07) Bush, Zachary; Libuda, Diana
Meiosis is a specialized cell division used by sexually reproducing organisms to generate haploid gametes, such as sperm and eggs. During meiosis, cells must repair DNA damage and accurately segregate parental copies of each chromosome into daughter cells. Although there is potential for new DNA mutations and chromosome rearrangements in each meiotic division, meiotic cells preferentially use high-fidelity mechanisms of DNA repair such as crossovers to ensure faithful genome inheritance. Crossovers serve critical functions in repairing DNA damage and promote accurate chromosome segregation, but they also introduce genetic diversity in progeny. In the nematode Caenorhabditis elegans, like many species, there is sex-specific regulation of crossing over, but the mechanisms that lead to sexual dimorphisms in this process remain unclear. To investigate sex-specific regulation of crossing over, I leveraged the density of genetic variation in the Bristol and Hawaiian populations of C. elegans to generate high-resolution maps of crossovers in sperm and egg cells, respectively. In Chapter 2, I completed whole-genome assembly of the Bristol and Hawaiian strains of C. elegans and comprehensively detailed their genetic variation at multiple scales and complexities. I found while many genetic variants are small, such as single nucleotide polymorphisms (SNPs) and insertion/deletions (<50bp), most of the variation between these two populations is comprised of large (>50bp) sequence gains, losses, and rearrangements. Further, I demonstrate the role of specific chromosome structures in influencing where SNPs, indels, and rearrangements accumulate in the genome. In Chapter 3, I defined genomic variations between different laboratory lineages of the Bristol and Hawaiian strains to demonstrate the degree of genetic drift and genomic structural variations accumulating in laboratory model organisms. In chapter 4, I developed a method that leverages the SNPs identified in Chapter 2 to map crossovers with sub-kilobase precision C. elegans sperm and eggs, respectively. I found that the crossover distribution and rate is sexually dimorphic, as well as demonstrating that the chromosomal structures associated with different states of germline gene expression are differentially associated with crossing over in developing eggs versus sperm. By determining the genomic features associated with crossover sites in each sex, I have illuminated the potential mechanisms that lead to sexually dimorphic distributions of crossing over. Taken together, the work in this dissertation fills critical gaps in our knowledge of how specific chromosome structures influence mechanisms that promote genomic integrity for inheritance by the next generation. This dissertation includes previously unpublished co-authored material.
Open Access
Evolution of Innate Immune Protein Complexes, Toll-like Receptor 4 and Calprotectin, in Early Vertebrates and Zebrafish
(University of Oregon, 2024-08-07) Orlandi, Kona; Harms, Michael
The innate immune system is our first line of defense against pathogens as well as our interface with our commensal microbiota. Toll-like receptor 4 (TLR4) and calprotectin are two innate immune proteins that are tightly associated with inflammatory disorders. Zebrafish (Danio rerio) has been successfully used to model the human innate immune system, but TLR4 and calprotectin models have not been developed because of their significant divergence in humans and zebrafish. Here, we set out to reveal the evolutionary and functional relationships between human and zebrafish TLR4 and calprotectin. We used phylogenetic analyses to define the evolutionary relationships between homologous proteins and characterized their immune functions in cell-based assays. We found that an antagonist of human TLR4 is a potent agonist for zebrafish TLR4, but when tested in live fish there was no difference in immune stimulation. We further investigated the evolutionary origin of this change in ligand specificity and determine that TLR4 in the cyprinid order of fish likely convergently evolved sensitivity to LPS. Our characterization of zebrafish proteins homologous to human calprotectin also suggest that the zebrafish proteins do not share functional similarities to calprotectin during the immune response. We conclude that although humans and zebrafish share many immune system characteristics, the TLR4 and calprotectin immune responses are not directly comparable. This dissertation includes previously published and unpublished co-authored material. Supplement files are multiple sequence alignments and phylogenetic trees for TLR4 and MD-2.
Open Access
The Role of Cilia Motility in Axial Morphogenesis
(University of Oregon, 2024-08-07) Irons, Zoe; Grimes, Daniel
The spine is the core of the vertebrate body axis, providing structural support and playing a role in establishing body shape. The linearity of the spine is essential for proper function. Scoliosis, or curvature of the spine, is present in 3-4% of the human population and can cause chronic pain as well as compression of the lungs and heart in severe cases. In this dissertation, I used zebrafish to study the role of cilia motility in morphogenesis of the body axis and the development of scoliosis. Previous work has established cilia motility as essential for zebrafish embryos to uncurl their bodies from around the yolk during the first 24 hours post fertilization. In Chapter I, I found that the protein Daw1 regulates the timely onset of cilia motility, and that embryos are able to remodel their body axes up to 3 days post fertilization. In Chapter II, I investigated factors functioning downstream of cilia motility that control body and spine morphogenesis. The Reissner fiber assembles downstream of cilia motility. Using zebrafish lacking Daw1, I showed that this fiber also assembles in response to a delayed cilia motility cue. The expression of the Urotensin-like peptides, Urp1 and Urp2, is increased as a result of cilia motility in the central canal. By generating mutants lacking both peptides, I showed that these peptides are dispensable for early body morphogenesis, but critical for the maintenance of spinal straightness in adulthood. This work revealed new components of spinal morphology that could be playing roles in human scoliosis. Lastly, in Chapter IV I investigated the mechanism stops further morphogenetic tissue movements once a linear body axis is generated. This occurs in embryos lacking Pkd2, a calcium ion channel known to play a role in flow sensory pathways. Using epistatic tests, I showed that pkd2 acts in a pathway independent of cilia motility and fluid flow to prevent axis over-straightening. Collectively, this dissertation advances our understanding of the role of cilia motility, fluid flow, and downstream factors in body axis straightening and the maintenance of spinal straightness. This work contains both unpublished and published co-authored materials.
Open Access
Cellular mechanisms underlying how early life stressors disrupt respiratory control
(University of Oregon, 2024-08-07) Beyeler, Sarah; Huxtable, Adrianne
Sufficient breathing is essential to maintaining homeostatic blood gases, yet stressors early in life undermine neural circuits controlling breathing in neonates, leading to potentially life-threatening respiratory insufficiency. Further, since respiratory control circuitry continues to develop postnatally, many early life stressors have lasting negative consequences for adult breathing and can increase risk for developing adult ventilatory control disorders, such as sleep apnea. For instance, neonatal inflammation acutely disrupted neonatal central respiratory activity and caused lasting disruption in adult respiratory control by abolishing respiratory motor plasticity, which disrupts the ability of the neural networks controlling breathing to learn and adapt. Yet, identifying mechanisms mediating this loss of adult plasticity and the extent of impairments to the neural control of breathing beyond plasticity are still necessary to understand how neonatal inflammation contributes to adult ventilatory insufficiencies and the risk for developing ventilatory control disorders. Additionally, infants exposed to maternal opioids have diverse negative health consequences, including respiratory distress. However, many factors contribute to these negative health outcomes, confounding the ability to understand how maternal opioids directly impact the neonatal respiratory system. Thus, this dissertation focuses on (1) identifying lasting disruptions in adult breathing after neonatal inflammation, (2) cellular mechanisms contributing to these impairments in adults after neonatal inflammation and (3) mechanisms underlying how maternal opioids impairs neonatal respiratory circuits. I will begin by providing an overview of respiratory control circuitry and development, introducing microglia and their role in neuroinflammatory signaling, and why it is important to understand how early life stressors disrupt respiratory control in Chapter I. Our laboratory previously identified that neonatal inflammation caused lasting abolishment of adult respiratory motor plasticity, where loss of plasticity has the potential to disrupt the ability of the neural networks controlling breathing to learn and adapt. Chapter II of this dissertation extends this work by identifying medullary microglia (primary immune cells in the brain) as a key cell type likely contributing to this loss of adult plasticity. Further, we determined neonatal inflammation sex-dependently impaired respiratory control beyond plasticity, increasing our understanding of how neonatal inflammation may contribute to adult ventilatory insufficiencies and the risk for developing ventilatory control disorders. We identified that neonatal inflammation caused lasting augmentation of adult male hypercapnic responses, consistent with males being at increased risk for sleep apnea. Chapter III of this dissertation focuses on understanding how a subsequent adult inflammatory challenge affects breathing in adults after neonatal inflammation to determine their risk for breathing disruptions during illness and disease. This research significantly advances our understanding of how impairments in microglial inflammatory responses may contribute to adult breathing vulnerability after neonatal inflammation. Finally, Chapter IV of this dissertation investigated mechanisms underlying how neonatal breathing is impaired by maternal opioids, another early life stressor. We determined maternal opioids directly impaired neonatal respiratory control networks, likely contributing to neonatal breathing deficits after maternal opioids, which we identified previously. Together this dissertation significantly advances our understanding of cellular mechanisms underlying how two early life stressors, neonatal inflammation and maternal opioids, disrupt respiratory control. Such an understanding is necessary to develop novel therapeutic strategies to support breathing at all ages. This dissertation includes previously published, co-authored material.
Open Access
EXPLORING THE CATALYTIC ACTIVITY OF THE [BIG+] PRION AND DYNAMICS OF TRNA T-LOOP MODIFICATIONS IN SACCHAROMYCES CEREVISIAE
(University of Oregon, 2024-08-07) Shaw, Ethan; Garcia, David
RNA modifications affect stability, structure, and interactions with other molecules. They are important for the optimal functioning of RNA, and without them RNA can be degraded more easily and translation can become less efficient. The majority of known chemical modifications on RNA have been found on tRNAs. The modifications are so tightly packed together that many methods used to detect modifications on other RNAs are ineffective on tRNAs. In this work, I used a new and developing technology to detect RNA modifications - nanopore sequencing. Using direct RNA sequencing of tRNAs to detect modifications, I investigated 1) the catalytic activity of the yeast prion [BIG+] and 2) the incorporation of m5U54, Ψ55, and m1A58 in the T-loop of tRNAs. [BIG+] causes yeast cells to proliferate more quickly with a shorter lifespan allowing them to thrive in nutritionally rich environments. These phenotypes could be caused by altered translation in these cells. Specifically, global translation is increased and certain genes are translated more quickly. However, the mechanism behind these changes is poorly understood. Here, I explore the catalytic activity of [BIG+]. [BIG+] arises from the protein Pus4, a pseudouridine synthase. I hypothesized that a change in the levels of pseudouridylation in [BIG+] cells could be driving the changes in translation. I observed that [BIG+] has retained its catalytic activity and a change in pseudouridylation is unlikely to explain why cells with [BIG+] proliferate more quickly. Additionally, I co-developed one of the first direct RNA-sequencing methods to comprehensively detect Ψ55 and m1A58 in the T-loop of tRNAs by combining nanopore sequencing of yeast mutants with mass spectrometry. Using this technique, we were the first to validate the presence of a tRNA modification circuit, where Ψ55 promotes the formation of m1A58, in all yeast tRNA isoacceptors. Furthermore, we showed that m1A58 is a dynamic modification that can change under stress. This method is a first step in being able to comprehensively detect and eventually quantify modifications in tRNAs. Being able to accurately detect these modifications will help us understand their complex roles.
Embargo
Understanding Evolution with Simulations: Three Tales about Trees
(University of Oregon, 2024-08-07) Rodrigues, Murillo; Kern, Andrew
Evolutionary processes impact patterns of genetic variation, so there is an opportunity to reverse this relationship and use genetic data to learn about past evolutionary events. Traditional evolutionary inference from genetic data is plagued by a few issues: (i) different processes can impact a particular feature in similar ways, making it difficult to disentangle them; (ii) there is a growing need for modeling interactions between processes; and (iii) many models do not make full use of genomic data and instead assume that loci are unlinked. Simulation-based evolutionary inference can help alleviate many of these issues. It is now possible to simulate complex evolutionary scenarios, and these can be used to approximate analytically intractable likelihoods, for example by using supervised machine learning. The major downsides to using simulations is the computational cost, but recent advancements both in hardware and software have lessened this cost. In this dissertation, I pushed the boundaries on how simulation-based inference can be applied in evolutionary genetics. To mitigate the costs associated with simulations, I made a few contributions to the tskit ecosystem of evolutionary simulation tools. First, I developed a way to partially parallelize the simulation of multiple populations to make inference using multi-population genomic datasets more feasible. Second, I helped create standards for reproducible simulations with natural selection within the Stdpopsim consortium. I implemented the ability to simulate using previously published distribution of fitness effects (DFEs) and to simulate selective sweeps. I demonstrate the utility of this tool by tackling the long-standing question of whether the power to detect sweeps varies along realistic chromosomes. Next, I used simulations to better understand the behavior of a complex multi-population model. Species can be thought as semi-independent realizations of the same (or very similar) evolutionary process. Thus, by looking at multiple species at once it may be possible to better disentangle the processes that shape variation along genomes. Using simulations, I show that positive selection is necessary to explain the genetic data obtained from multiple great ape species. Further, I lay down a framework for leveraging multi-species information to better understand the effects of different processes on a group's evolutionary history. Lastly, I present a new method that uses whole-genome genealogies for evolutionary inference. This data structure efficiently and sufficiently encodes evolutionary processes. I develop a machine-learning framework, tsNN, that takes whole-genome genealogies as inputs and is flexible enough to perform tasks at different scales (e.g., inferring mutation times, demographic parameters, etc.). I demonstrate that tsNN can learn to predict mutation times accurately, outperforming current likelihood-based methods. tsNN, represents an important step in genealogy-based evolutionary inference, but there still much work to be done in applying deep learning to gain new insights into past evolutionary events.
Open Access
Fish Out of Water: Understanding the Impacts of Regional Species Pool Variation on Local Community Assembly in a Host-Microbiome Model System
(University of Oregon, 2024-08-07) Evens, Kayla; Cresko, William
The host-microbiome is essential to many aspects of host health and function, and acts as a useful model system in which to investigate broader questions of community assembly. Since the composition of host-microbiomes is, in part, determined by the input of microbes from the surrounding environment, it is integral to understand how variation in the environmental microbiome may influence host-microbiome assembly. For my dissertation research, I used a species pool conceptual framework to investigate drivers of variation in the microbiomes of aquaculture research facilities and the fish they house. Zebrafish (Danio rerio) are used extensively as model organisms in scientific research, especially in studies investigating host-microbiome dynamics. For in-vitro experimentation utilizing model organisms, we rely on the reproducibility of results to make broadly applicable conclusions about the host-microbiome. However, evidence suggests that inter-facility variation may influence zebrafish gut microbiome composition via acquisition of microbes from the environment, potentially leading to phenotypic differences among fish housed at different aquaculture facilities. To investigate the relationship between aquaculture facility water and fish gut microbiomes, I first characterized spatial and temporal variation across multiple aquaculture facilities on the University of Oregon campus. Facility water microbiota not only varied over time, but patterns of spatial variation in each facility were consistent despite differences in host species. I then used this information to guide an expanded, intensive sampling of water and fish across four zebrafish facilities in Oregon and Norway. I observed significant variation in microbiome composition both within and between facility water systems and zebrafish gut samples. Further, there was evidence that variation in the water microbiome was a source of variation in zebrafish gut microbiomes. Finally, as differences in facility management and technical specifications can make directly linking microbiome variation in the water to variation in fish difficult, I attempted to isolate the effects by experimentally manipulating the water microbiome in a laboratory study using germ-free larval fish. My results indicate that microbial inputs from live feed overwhelmed any potential influence of water microbiome variation in early-life microbiome assembly. Overall, this dissertation provides a comprehensive look at the drivers of environmental microbiome variation and how these may mediate aspects of host-associated microbiota. My results have implications for fish microbiome research and suggest that research conducted with zebrafish sourced from a single facility may be heavily influenced by facility-specific effects.
Open Access
Ion Signaling for Organ Size and Scaling During Zebrafish Fin Regeneration
(University of Oregon, 2024-08-07) Le Bleu, Heather; Stankunas, Kryn
Organs “know” how to develop to a specific size and shape conferring optimal function. In humans, very few organs and appendages show a natural ability to repair. Exemplifying this fundamental mystery, adult zebrafish fins regenerate to their original size and shape regardless of injury extent. Therefore, zebrafish fin regeneration provides a tractable system to investigate “organ scaling” mechanisms. Bioelectricity, or ion flow across cell membranes, is long-associated with both organ size control and regeneration. However, the links between ion signaling and their effectors to specific cell behaviors determining organ size are limited. Perturbed ion signaling, notably by elevated voltage-gated K+ channel activity and inhibited Ca2+-dependent calcineurin signaling, leads to dramatic overgrowth of regenerating zebrafish fins. A unique distal population of mesenchymal cells within the fin’s regenerative blastema sustains fin outgrowth. The classic zebrafish mutant longfint2 develops and regenerates dramatically elongated fins and underlying ray skeleton. We show ectopic expression of the kcnh2a K+ channel in fin ray fibroblast-lineage cells enhances fin outgrowth in late regeneration rather than at early blastema establishment. Epistasis experiments suggest that Kcnh2a likely blocks Ca2+-dependent calcineurin signaling to end fin outgrowth. Mechanisms of putative Ca2+ signaling during fin size acquisition has not been explored. Using a new Ca2+ responsive GCaMP6s transgenic reporter line, we show fibroblast-lineage cells are the nexus of dynamic voltage-gated Ca2+ channel activity and Ca2+ signaling events during zebrafish fin regeneration. Single cell transcriptomics identifies upstream voltage-gated Ca2+ channels cacna1c (CaV1.2, L-type), cacna1ba (CaV2.2, N-type), and cacna1g (CaV3.1, T-type) as candidate mediators of fibroblast-lineage Ca2+ signaling in vivo. Dual chemical inhibition reveal that L/N-type voltage-gated Ca2+ channels are actively required for fin outgrowth during regeneration. Genetic analysis demonstrates cacna1g mutants regenerate extraordinarily long fins, indicating Cacna1g has a key in fin cessation and scaling. Accordingly, live imaging of regenerating animals suggests Cacna1g channel activity in distalmost mesenchymal cells is essential for Ca2+ flux. We conclude that a cadre of ion channels act within fibroblast-lineage cells to fine-tune Ca2+ signaling events and restore fin size and shape.This dissertation includes previously published and unpublished co-authored material.
Open Access
Reducing Overstory in Pacific Northwest Forests Enhances Forage for Bumble Bees Without Increasing Microparasite Prevalence
(University of Oregon, 2024-08-07) Fan Brown, Jesse; Ponisio, Lauren
Bumble bees (Bombus spp.) are the most effective pollinator group in temperate and boreal regions, but habitat loss and disease are contributing to steep declines of several forest-associated species. Forest bumble bees benefit when forest management decreases overstory cover and enhances understory forage plant species, but effects can be short-lived. Forest restoration techniques that prioritize canopy openness may prolong forage availability through later successional stages. Management that increases floral resources may aggregate bumble bee populations and increase disease risk. We examined effects of forest management strategies on understory plant and bumble bee communities and evaluated whether plant and bee community characteristics were correlated with bumble bee disease dynamics. We surveyed the abundance and diversity of flowering plants and bumble bees within stands of varied canopy cover in the Coast Range (n = 98 stands) in Oregon, USA and screened bumble bees for six microparasite taxa (n = 191 bees). We found that canopy openness was positively correlated with flowering plant abundance and diversity and flowering plant communities were positively correlated with bumble bee richness and diversity. Our parasite prevalence rates were comparable to those of other North American bumble bee populations and were not correlated with characteristics of flowering plant or bee communities. Our data suggest that thinning in dense forests can enhance bumble bee habitat without increasing disease prevalence, informing efforts to conserve, restore, and expand forest habitat for imperiled bumble bee species.
Open Access
THE RNA-BINDING PROTEIN, IMP, GENERATES NEURAL DIVERSITY IN THE DROSOPHILA TYPE 2 NEUROBLAST LINEAGE
(University of Oregon, 2024-08-07) Munroe, Jordan; Doe, Chris
Neural diversity generated during development is required to produce a fully functioning nervous system. Thousands of neurons precisely target their axons to the relevant post-synaptic partners to create neural circuits that generate proper sensory and motor behavior at the organismal level. A lack of neural diversity can cause improper circuit formation. In Drosophila, temporal patterning within neural stem cells aids in the correct regulation and generation of neurons. Drosophila neural stem cells, or neuroblasts (NBs), within the central brain express opposing temporal gradients of the RNA-binding proteins, Imp and Syp. Here I show that a subset of central brain NBs, known as type 2 NBs (T2NBs) all express Imp in a high-to-low expression pattern early in Drosophila neurogenesis, while Syp expression is dependent upon the T2NB lineage. Unique to T2NBs, compared to other Drosophila neuroblasts, is the generation of intermediate neural progenitors (INPs) which are necessary for expansion of neural number and diversity in the Drosophila central complex (CX), an adult brain structure required for celestial navigation. Upon their generation, I have shown that newborn INPs express equivalent Imp and Syp levels as T2NBs and form high-to-low expression gradients throughout the INP lineage. However, Imp levels increase in old INPs where I show it is required for the proper generation of E-PG and PF-R neurons in the CX. Loss of Imp in old INPs causes morphological defects while Imp overexpression causes abnormal neurite morphology. Finally, I highlight Imp’s minor role in post-mitotic morphogenesis of PF-R and P-FN neurons in the adult CX. Loss or overexpression of post-mitotic Imp causes only minor changes in PF-R and P-FN neuropil volume. This dissertation includes previously published co-authored material. Supplemental Video 3.1 Imaris reconstruction of T2NBs in larval brain
Open Access
Evolution of Developmental Pattern and Larval Form in the Ophiuroids of Oregon and Beyond
(University of Oregon, 2024-03-25) Nakata, Nicole; Emlet, Richard
Despite the ubiquity and known impacts of transitions in developmental pattern in marine invertebrates, many taxa have been insufficiently analyzed. In Chapter II, I report the effects of larval feeding in an undescribed facultative planktotroph, Amphiodia sp. opaque. By culturing larvae with and without food and observing development, I found that larval feeding led to faster development times, greater percent metamorphosis, and larger juveniles able to evade starvation longer than individuals that did not receive food. We compared metrics of early life stages using a series of generalized linear models, and model fitting was determined using Akaike’s Information Criterion (AIC). Scores for each model and test are included as supplementary tables. In Chapter III, I present a summary of developmental diversity in the ophiuroids of the northeast Pacific Ocean. We used DNA barcoding to identify eighteen species from the plankton of the southern Oregon coast. We found four species with reduced plutei, one with vitellaria, three with pelagic direct development, and ten with planktotrophic ophioplutei (including one followed by a vitellaria). This diversity of larval forms suggests multiple transitions from feeding to nonfeeding larvae in the Amphiuridae, which I tested for in Chapter IV using comparative phylogenetic analyses. To do so, I constructed a four-gene phylogenetic hypothesis for species with known development pattern in the family Amphiuridae. Single-gene trees were made to check for congruence between loci and the multi-gene dataset and are included as supplementary figures. This analysis inferred a brooding ancestor, an instance of re-acquisition of feeding, and multiple transitions back to nonfeeding larvae. The analysis inferred multiple transitions from brooding to nonfeeding planktonic development via pelagic direct development. Altogether, this work introduces new examples of the developmental patterns in Ophiuroidea, including an example of facultative planktotrophy, a rare developmental pattern that may represent an evolutionary intermediate between feeding and nonfeeding larvae. We show the effectiveness of DNA barcoding for identifying the early life stages of benthic marine invertebrates. Finally, I found that reconstructed ancestral states of developmental pattern are influenced by tree topology and completeness of the dataset. This dissertation includes previously published and unpublished co-authored material.
Open Access
SYNERGIZING GENETIC ENGINEERING AND EVOLUTIONARY BIOLOGY: ANIMAL-BASED LINEAGE TRACKING TO STUDY ADAPTIVE MUTATIONS
(University of Oregon, 2024-03-25) Stevenson, Zachary; Phillips, Patrick
The fitness effects of new mutations are one of the central drivers of evolutionary change. Mutation is the ultimate source of novel genetic diversity, yet only a small fraction of new mutations provides an adaptive advantage. Additionally, many new adaptive mutations provide only a slight advantage and are challenging to identify and quantify their selection coefficient. Furthermore, a given mutation may be advantageous in one environment and disadvantageous in another environment. Natural selection acts upon the phenotype produced by the new mutation and if adaptive, the mutation can increase in frequency. Evolution acts though the lineage–the fundamental unit of evolution–because it is the lineage which changes overtime in response to selection. Over the past 150 years a robust and comprehensive set of theory has been developed around evolutionary biology and adaptive mutations. However, comprehensive estimation of the fitness effects of new mutations has remained challenging, but recent developments in genetic engineering open new opportunities to significantly advance the field. Within this dissertation, I present several novel approaches for editing the genome using a large genomic library, which allows the implementation of barcoding approach for evolutionary lineage tracking in an animal system for the first time. I apply this methodology to measure the fitness of a known Ivermectin resistant strain of Caenorhabditis elegans in what is the largest animal experimental evolution to date and highlight important new directions in evolution and genomics that this new approach allows.
Open Access
Investigating Protein Evolution Through Sequence Space Using a Biophysical Lens
(University of Oregon, 2024-03-25) Shavlik, Michael; Harms, Michael
How do the underlying biophysical properties of proteins dictate the “rules” that govern molecular evolution? Understanding the principles and mechanisms that determine which evolutionary trajectories proteins take is crucial to protecting humans against viral protein evolution and developing therapeutic, custom, drugs through protein engineering. Although many approaches have been developed to investigate the process of protein evolution, a deep understanding of the relationship between sequence space and protein biophysics can alleviate key deficiencies in our knowledge. What is the underlying distribution of functional proteins in sequence? Do specific biophysical properties dictate the interconnectedness of these functional proteins? How does the protein energy landscape change across evolutionary time and how can that inform our understanding of evolution? This dissertation will explore two methods of answering these questions: 1) High-throughput mutagenesis and phenotype characterization to explore sequence space using fluorescent proteins and 2) Ancestral Sequence Reconstruction linked to a biophysical lens using protein energy landscapes.
Open Access
INSIGHTS INTO THE REGULATION OF aPKC POLARITY THROUGH PROTEIN-PROTEIN INTERACTIONS
(University of Oregon, 2024-03-25) Vargas, Elizabeth; Prehoda, Kenneth
Cell polarity is a crucial factor in enabling a cell to carry out its specialized functions during animal development and homeostasis. It involves the organized distribution of cellular components into distinct regions, playing a critical role in various processes like asymmetric cell division, cell migration, and epithelial morphogenesis. One critical regulator of animal cell polarity is the protein atypical Protein Kinase C (aPKC), whose catalytic activity is essential for directing the localization of downstream polarity proteins. Consequently, precise regulation of aPKC becomes imperative for the proper control of cell polarity.The regulation of aPKC's polarization and activity involves interactions with several proteins, including Par-6, Cdc42, and Par-3. However, the mechanisms through which these proteins exert their regulatory influence on aPKC have remained a subject of confusion within the cell polarity field. This dissertation investigates the intricate intermolecular interactions responsible for regulating aPKC to establish proper cell polarity. In the first part of this work, we focus on regulation of aPKC by Par-6. Existing models suggest contradictory roles for Par-6 in either activating aPKC or relieving its autoinhibition while keeping it catalytically inactive. To address this ambiguity, we conducted structure/function analyses and in vitro binding assays and found that Par-6 may inhibit aPKC's catalytic activity through a novel interaction involving Par-6's C-terminus. More studies need to be done to address how this new interaction may be regulating aPKC’s kinase domain. We then turn our focus onto how Par-3 interacts with aPKC and Par-6, which together form the Par complex. Previous studies have reported multiple interactions through various biochemical assays. To gain some clarity, we utilized qualitative and quantitative binding assays to understand (1) which domains within Par-3 contribute to its interaction with the Par complex and (2) the overall binding energy contributed by these interactions. Our results indicate that Par-3 PDZ2 and PDZ3 domains bind to the aPKC Kinase domain-PBM region. Lastly, we set out to determine the mechanism behind the transition of the Par complex between its two regulators, Par-3 and Cdc42, to form two distinct complexes. While one biochemical study suggested simultaneous interaction of Par-3 and Cdc42 with the Par complex, in vivo data suggested the formation of separate complexes. Our qualitative binding assays show that Par-3 and Cdc42 negatively cooperate for binding to the Par complex. Questions remain on the detailed mechanism of competition. In summary, this dissertation elucidates the intricacies of aPKC regulation and provides valuable insights into the mechanisms by which Par-6, Cdc42, and Par-3 contribute to the control of aPKC’s localization and activity. This work contains both unpublished and published co-authored materials.
Open Access
An Actin Cross-Linking Effector of the Vibrio Type Six Secretion System Increases Intestinal Motility Through Macrophage Redistribution
(University of Oregon, 2024-03-25) Ngo, Julia; Guillemin, Karen
Host-microbe interactions within the gastrointestinal tract have long been recognized as pivotal for maintaining physiological balance. However, the intricate mechanisms underlying these interactions remain enigmatic. This study delves into the complex world of host-microbe relationships, focusing on the Vibrio type VI secretion system (T6SS), particularly the actin cross-linking domain (ACD) of the valine-glycine repeat G (VgrG-1) protein. We explore the role of the ACD in altering intestinal motility in larval zebrafish. Our findings reveal a fascinating mechanism underlying these interactions. Vibrio, known for its pathogenic potential, instigates cellular death and tissue damage within the vent region of the zebrafish intestine. This destructive action triggers an immune response within macrophages from their typical habitat in the midgut to the affected vent region. This revelation emphasizes the disruptive influence of bacterial pathogenesis on macrophages and, by extension, their role in regulating intestinal motility. Our findings provide valuable insights into the intricacies of intestinal motility regulation in the context of host-microbe interactions. In conclusion, this research broadens our understanding of the mechanism by which gut microbes influence host physiology, specifically in the context of intestinal motility. The presence of bacterial pathogenesis and its influence on macrophages, coupled with insights into the intricate dynamics of host-microbe interactions, underscores the significance of this work. This intricate interplay between microbes and host systems not only reveals microbial influences on host physiology but also highlights the mechanisms employed by the host to maintain homeostasis.