xvii, 125 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / MicroRNAs are small regulatory RNAs that control various developmental and physiological processes in animals and plants. To study the involvement of microRNAs in skeletal development, I manipulated the expression of miR-140, which is strongly expressed in the developing skeleton, and miR-196, which is located among the body patterning Hox cluster genes. I found that miR-140 regulates zebrafish palate formation by interfering with neural crest cell migration through the inhibition of the expression of the platelet derived growth factor receptor alpha ( pdgfra ) gene. I also found that miR196 regulates zebrafish pectoral fin initiation by regulating the expression of the retinoic acid receptor alpha b ( rarab ) gene and that miR-196 is involved in the patterning of zebrafish pharyngeal arches and vertebrae. These results illuminate previously unknown regulatory mechanisms of skeletal development. I also reviewed current knowledge concerning microRNAs in skeletal development and evolution and discussed potential relationships between microRNAs and skeletal disease. This dissertation includes previously published and unpublished coauthored material. / Committee in charge: Judith Eisen, Chairperson, Biology; John Postlethwait, Advisor, Biology; Charles Kimmel, Member, Biology; William Cresko, Member, Biology; J. Andrew Berglund, Outside Member, Chemistry

The subventricular zone (SVZ) of the mammalian brain serves as one of only two sources of adult born neurons. Adult born neural progenitors - known as neuroblasts - acquire the ability to migrate and travel large distances to their destination in the olfactory bulb. Disruption of neuroblast migration is associated with learning and memory deficiencies and, following injury, neuroblasts are re-routed to promote neurodegeneration. While a lot of research has attended to augmenting the production and survival of neuroblasts, the body of evidence for pharmacological targets or compounds that promote migration is comparatively sparse. This thesis set out to identify novel strategies to modulate neuroblast migration for brain repair by studying proteins known to modulate migration and identifying new ones through compound screening. Firstly, an explant migration assay from the mouse SVZ was used to investigate the potential to use growth factors to stimulate neuroblast migration. This confirmed that that insulin-like growth factor 1 (IGF1) and IGF2 regulate neuroblast migration, as previously reported by other research groups. The role of IGF2 is investigated further using a mouse model in which the binding of IGF2 to IGF2R is disrupted, resulting in increased proliferation in the embryonic cortical SVZ, brain overgrowth and perinatal lethality. In the second half of this thesis I try to tackle one of the major bottlenecks limiting the search in for pharmaceutical interventions targeting neuroblast recruitment: the lack of high-fidelity in vitro migration assays. Drawing concepts from existing in vitro migration assays and cerebral organoid models, I developed a novel neuroblast spheroid migration assays that permits the investigation of large numbers of interventions, concurrently, in 3 dimensions. Using the spheroid assay I successfully screened 1012 small molecule kinase inhibitors for their effects on neuroblast migration. Several compounds were identified that significantly increased or decreased neuroblast migration. Two genes: MUSK and PIK3CB were selected from the screen as putative biological targets and genetic knockdown of these genes validated that interruption of their activity increased neuroblast migration. In the future these compounds could be studied further to explore their potential for augmenting the recruitments of new neurons to sites of injury so support neuroregeneration, or for decreasing invasion of brain malignancies.

Calcium is a ubiquitous intracellular signal responsible for controlling numerous cellular responses including development and proliferation. Calcium (Ca²⁺) is stored in both neutral and acidic stores and its release through gated channels has been implicated in regulating development in Dictyostelium discoideum. This thesis aims to understand the roles of the calcium channel proteins, in particular the two-pore channel proteins (TPCs), found on acidic stores in Ca²⁺ signalling during Dictyostelium development. Bioinformatic analysis indicates conservation of a gene encoding an orthologue of TPC2 in Dictyostelia and, similar to plant TPCs, a Ca²⁺ sensing domain is predicted along with a novel potential calmodulin binding site. To investigate the role of intracellular Ca²⁺ channels, a series of strains was generated, disrupted in one or more of genes encoding the channels TPC2 and mucolipin (TRP-ML), predicted to be located on acidic stores, and IplA, located on neutral stores. All disrupted strains, including one lacking all three channels, are able to complete development. However, strains lacking TPC2 show a pronounced delay in early development, correlating with reduced expression of some early developmental genes. Vesicles derived from tpc2-null cells show normal Ca²⁺ release compared to parental cells but an increased rate of Ca²⁺ uptake. During early development, the pH of acidic vesicles is increased in the absence of TPC2. However development of tpc2-null cells showed increased sensitivity to weak bases in producing fewer aggregates but resistance to sodium chloride and weak bases in later development suggesting a complex role for TPC during development. In vivo cytosolic Ca²⁺ responses were analysed in strains expressing an ultra-sensitive Ca²⁺ indicator YC-Nano 15. Growing tpc2^- and iplA^- cells have lower basal cytosolic Ca²⁺ than parental Ax2 cells. Intercellular Ca²⁺ waves were observed in aggregates from Ax2, mcln^- and tpc2^- cells but were greatly reduced in iplA- aggregates, as was the increase in cytosolic Ca²⁺ in response to extracellular cAMP. In tpc2- aggregates, wave frequencies were reduced and the response to cAMP addition abolished after treatment with caffeine, a known adenylyl cyclase inhibitor in Dictyostelium. This work demonstrates that TPC2 plays a role in the early stages of Dictyostelium development. TPC2 is important for pH regulation in acidic vesicles and cytosolic Ca²⁺ levels, either or both of which could influence development either directly or via changes in early developmental gene expression.

Doctor of Philosophy / Genetics Interdepartmental Program / Susan J. Brown / In Drosophila, all segments form in the blastoderm where morphogen gradients spanning the entire anterior-posterior axis of the embryo provide positional information. However, in the beetle Tribolium castaneum and most other insects, a number of anterior segments form in the blastoderm, and the remaining segments form sequentially from a posterior growth zone during germband elongation. In this work, I show that segmentation at both blastoderm and germband stages of Tribolium is based on a segmentation clock. Specifically, I show that the Tribolium primary pair-rule gene, Tc-even-skipped (Tc-eve), is expressed in waves propagating from the posterior pole and progressively slowing until they freeze into stripes; such dynamics are a hallmark of clock-based segmentation. Phase shifts between Tc-eve transcripts and protein confirm that these waves are due to expression dynamics. Such waves, like their counterparts in vertebrates, are assumed to arise due to the modulation of a molecular clock by a posterior-to-anterior frequency gradient. I provide evidence that the posterior gradient of Tc-caudal (Tc-cad) expression regulates the oscillation frequency of pair-rule gene expression in Tribolium. I show this by correlating the gradient of Tc-cad expression to the spatiotemporal dynamics of Tc-even-skipped expression in WT as well as in different knockdowns of Tc-cad regulators. Specifically, the spatial extent, frequency, and width of Tc-eve waves correlate with the spatial extent, expression level, and slope of Tc-cad gradient, respectively, as predicted by computer modeling. These results pose intriguing evolutionary questions, since Drosophila and Tribolium segment their blastoderms using the same genes but different mechanisms, and highlight the role of frequency gradients in pattern formation.

abstract: A genetically engineered line of human induced pluripotent stem cells was used to study the effects of gene expression on cell fate. These cells were designed to activate expression of the gene GATA6 when exposed to the small molecule doxycycline. This gene was chosen because it plays an important role in the developmental biology stages of liver formation. Because of the way the cells were engineered, a given population would have a heterogeneous expression of GATA6 because each cell could have a different copy number of the exogenous gene. This variation allows for the differentiation of multiple cell types, and is used to grow liver organoids. The early liver organoid samples were studied via immunofluorescent staining, imaging, and quantitative image analysis. It was originally hypothesized that absolute gene expression was not the most important factor in determining cell fate, but relative gene expression was. This meant that the spatial location of the cells and their local environment were critical in determining cell fate. In other words, the level of GATA6 of a cell is important, but so is the level of GATA6 in the surrounding cells, or neighborhood, of that cell. This hypothesis was analyzed with the creation of various Neighborhood Impact Factor (NIF) methods. Multiple time points of growth were analyzed to study the temporal effect, in addition to the gene expression and NIF influence on a cell’s fate. Direct gene expression level showed correlation with certain cell fate markers. In addition to GATA6 expression levels, NIF results from early and late time point experiments show statistical significance with relatively small neighborhood radii. The NIF analysis was useful for examining the effect of neighboring cells and determining the size of the neighborhood – how far cells influence one another. While these systems are complex, the NIF analysis provides a way to look at gene expression and its influence in spatial context. / Dissertation/Thesis / Powerpoint presentation used in the defense. / Masters Thesis Bioengineering 2017

abstract: The Erk/MAPK pathway plays a major role in cell growth, differentiation, and survival. Genetic mutations that cause dysregulation in this pathway can result in the development of Rasopathies, a group of several different syndromes including Noonan Syndrome, Costello Syndrome, and Neurofibromatosis Type-1. Since these mutations are germline and affect all cell types it is hard to differentiate the role that Erk/MAPK plays in each cell type. Previous research has shown that individual cell types utilize the Erk/MAPK pathway in different ways. For example, the morphological development of lower motor neuron axonal projections is Erk/MAPK-independent during embryogenesis, while nociceptive neuron projections require Erk/MAPK to innervate epidermal targets. Here, we tested whether Erk/MAPK played a role in the postnatal development of lower motor neurons during crucial periods of activity-dependent circuit modifications. We have generated Cre-dependent conditional Erk/MAPK mutant mice that exhibit either loss or gain of Erk/MAPK signaling specifically in ChAT:Cre expressing lower motor neurons. Importantly, we found that Erk/MAPK is necessary for the development of neuromuscular junction morphology by the second postnatal week. In contrast, we were unable to detect a significant difference in lower motor neuron development in Erk/MAPK gain-of-function mice. The data suggests that Erk/MAPK plays an important role in postnatal lower motor neuron development by regulating the morphological maturation of the neuromuscular junction. / Dissertation/Thesis / Masters Thesis Biology 2017

abstract: Sparsity has become an important modeling tool in areas such as genetics, signal and audio processing, medical image processing, etc. Via the penalization of l-1 norm based regularization, the structured sparse learning algorithms can produce highly accurate models while imposing various predefined structures on the data, such as feature groups or graphs. In this thesis, I first propose to solve a sparse learning model with a general group structure, where the predefined groups may overlap with each other. Then, I present three real world applications which can benefit from the group structured sparse learning technique. In the first application, I study the Alzheimer's Disease diagnosis problem using multi-modality neuroimaging data. In this dataset, not every subject has all data sources available, exhibiting an unique and challenging block-wise missing pattern. In the second application, I study the automatic annotation and retrieval of fruit-fly gene expression pattern images. Combined with the spatial information, sparse learning techniques can be used to construct effective representation of the expression images. In the third application, I present a new computational approach to annotate developmental stage for Drosophila embryos in the gene expression images. In addition, it provides a stage score that enables one to more finely annotate each embryo so that they are divided into early and late periods of development within standard stage demarcations. Stage scores help us to illuminate global gene activities and changes much better, and more refined stage annotations improve our ability to better interpret results when expression pattern matches are discovered between genes. / Dissertation/Thesis / Ph.D. Computer Science 2013

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第17557号 / 工博第3716号 / 新制||工||1566(附属図書館) / 30323 / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 安達 泰治, 教授 楠見 明弘, 准教授 井上 康博, 教授 琵琶 志朗 / 学位規則第4条第1項該当

The vertebrate vasculature is formed by populations of endothelial and mural cells that arrange into functionally and molecularly distinct arterial, venous and capillary beds. Although a number of signalling pathways and transcriptional regulators have been implicated in these processes of vascular differentiation, a clear picture of how arterial-specific gene regulation is achieved is yet to emerge. In this study I have investigated the transcriptional regulation of arterial identity from two different directions: characterisation of enhancers to identify the transcription factors that bind and direct arterial specification; and direct study of the function of one particular transcription factor expressed specifically in the arterial vasculature. I have identified a novel gene enhancer that directs arterial-specific expression of Flk1 (Vegfr2) in transgenic mouse and zebrafish models. Dissection of inputs from individual transcription factor binding sites within this enhancer shows a requirement for Gata factors for enhancer function in endothelial cells, whereas arterial-specification is directed by Rbpj-mediated repression of enhancer activity in veins. This work demonstrates that Flk1 expression in arterial endothelial cells is downstream of the Notch/Rbpj pathway, and also describes a novel transcriptional mechanism of arterial differentiation. In parallel, I have uncovered a novel role for the transcription factor Tbx2 in the regulation of arterial mural cell identity. Histological analysis demonstrates the previously unreported expression of Tbx2 exclusively in mural cells of peripheral arteries and microvessels, and genetic deletion experiments in mice suggest a role for Tbx2 in mural cell recruitment, survival, proliferation, and differentiation upstream of Notch3 and Pdgfrβ. Together, these results contribute valuable insights into our understanding of the establishment of vascular identity by identifying novel transcriptional regulators of arterial fate in both endothelial and mural cells.

The mammalian neocortex contains diverse neuronal and glial cell types. Among them lies an important subclass, the subcerebral projection neurons (SCPN) that project to distant targets like the spinal cord. Aiming at identifying molecular controls over the postnatal development of SCPN, I focus my investigations on the role of Ppargc1á because its function remains relatively unknown in the brain while it is important for metabolism and survival in other tissue systems.