A Bayesian hierarchical model that integrated information about state and observation processes was used to estimate the number of adult Delta Smelt entrained into the southern Sacramento−San Joaquin Delta during water export operations by the California State Water Project and the Central Valley Project. The model hierarchy accounted for dynamic processes of transport, survival, sampling efficiency, and observation. Water export, mark−recapture, and fish facility count data informed each process. Model diagnostics and simulation testing indicated a good fit of the model, and that parameters were jointly estimable in the Bayesian hierarchical model framework. The model was limited, however, by sparse data to estimate survival and State Water Project sampling efficiency. Total December to March entrainment of adult Delta Smelt ranged from an estimated 142,488 fish in 2000 to 53 fish in 2014, and the efficiency of louvers used to divert entrained fish to fish facilities appeared to decline at high and low primary intake channel velocities. Though applied to Delta Smelt, the hierarchical modeling framework was sufficiently flexible to estimate the entrainment of other pelagic species.

Neural tube closure (NTC) is a fundamental process in the development of all chordates. During NTC, the developing central nervous system is transformed from a flat sheet of neural precursor cells into a hollow neural tube, which is the basic form that persists into the mature central nervous system. When the neural tube fails to properly close, neural tube defects (NTDs) result. NTDs are the most prevalent category of human birth defects, and they occur in about 1 out of every 1000 births. Previously, we discovered a NTC mutant (named bugeye) in the basal chordate Ciona savignyi that results from a defect in the gene encoding a T-Type Calcium Channel (Cav3). We found that Cav3s are also required for proper NTC in the amphibian Xenopus suggesting a conserved function among chordates. Loss of Cav3 leads to defects in anterior NTC characterized by the brain protruding from the larval head. Here, I present a study expanding these findings to a new Cav3 mutant in the related species C. robusta. RNAseq analysis of bugeye mutants from both species reveals misregulation of several transcripts including ones that are involved in cell-cell recognition and adhesion. Two in particular, Selectin and Fibronectin leucine-rich repeat transmembrane protein are aberrantly upregulated in the mutant. They are expressed in the closing neural tube and knockdown of either in wildtype larvae phenocopies the bugeye mutant. We speculate that these molecules play a transitory role in tissue separation and adhesion during NTC, and the failure to downregulate them properly leads to an open neural tube. One hypothesis surrounding Cav3’s role in NTC, is that it acts as a mechanical developmental checkpoint, detecting the completion of NTC and signaling through calcium to move on to the next developmental process. To further explore this, I used various pharmacological inhibitors as well as manual disruption of the progression of NTC to recapitulate the open brain phenotype. The RNAseq investigation revealed a suite of transcripts that are abnormally upregulated in the bugeye mutant, but their expression during normal development declines sharply following neurulation. Other transcripts displaying similar expression profiles may play a role in NTC. Using publicly available RNA-sequencing data I identified several additional candidate genes. Further work is necessary to determine what role these genes play in NTC, but this demonstrates a valid approach for identifying genes with roles in specific developmental processes in Ciona.

In my dissertation studies, I have focused on uncovering the neural circuitry driving sensorimotor behaviors in a remarkable animal - Ciona. Understanding the signal transmission properties neurons use to elicit behavior is essential for determining functionality. An advantage to using the Ciona larval tadpole is its relatively simple nervous system. Another, and powerful, advantage is the availability of the completely described connectome for the Ciona larval-stage central nervous system (CNS). Furthermore, Ciona are tunicates, a subphylum of chordates, and are the closest living relatives of vertebrates, making their connectome the sole representative in the chordate phylum. The Ciona tadpole larva shows several vertebrate-like features, including a CNS that shows strong conservation with vertebrate CNSs. Despite having only ~180 neurons, Ciona larvae have a surprisingly complex set of behaviors. Among these behaviors are negative phototaxis and a response to rapid light dimming called the dim response (also known as the looming shadow behavior). Previous work in the Smith lab investigated these two behaviors and showed that they are mediated by distinct groups of photoreceptors. However, the details of the circuits, such as neurotransmitters used, were still unknown. To explore these circuits, I used a range of approaches, including gene expression analysis to discern the distribution of neurotransmitters and their receptors in individual neurons, and behavioral studies using pharmacological agents and behavioral mutants. Together, these approaches allowed me to fill in many of the details of the circuits predicted by the connectome, and to construct models that link circuits to behavior. In my first published study, I found that the two photoreceptor groups have distinct, but overlapping, circuits. The first circuit is excitatory and responds to the direction of light, driving phototaxis. The second circuit is disinhibitory and responds to rapid changes in light, driving the dim response. In my second published study, I found that both circuits detect fold-change differences. In fold-change detection (FCD), behavioral (i.e., swimming) responses scales with relative change in sensory input, and not to the overall magnitude of the stimulus. Furthermore, the two visuomotor behaviors have different input/output relationships, indicating different FCD strategies. Pharmacological manipulation of specific relay neurons in the posterior Brain Vesicle (pBV) led to an extinction of FCD without eliminating the visuomotor behavior, suggesting the FCD circuits lie at the neuronal level outside of the visual organ, as opposed to a mechanism of the photoreceptors. The role of pBV in sensory processing, along with it receiving converging inputs from other sensory systems, has lent further support of the pBV being a vertebrate midbrain homolog. By examining neurotransmitter receptor expression with in situ hybridization, I found broad expression of the glutamate receptor, NMDA-R, in the Ciona CNS, except in the photoreceptors. When NMDA-R is pharmacologically inhibited, the larvae lose their ability to respond to sensory input, suggesting an important role of the receptor in sensory processing. Further work is necessary to determine the specific components involved in visual processing and FCD, as well as the role of NMDA-R across the sensory systems. The work described here established a model to study sensory neural circuits for behavior in a new chordate model system.

My dissertation work has focused on mapping and characterizing a spontaneous neural tube closure mutant, bugeye, in the marine, invertebrate chordate organism, Ciona savignyi. The work involved developing a new and rapid method of mutation mapping by next-generation DNA sequencing. This new method of mapping was tested on a previously identified Ciona intestinalis mutant whose genomic data was used to calibrate mapping parameters and the mapping program for Ciona genomes. After successful testing of the mapping program, it was applied to the new bugeye mutant in Ciona. Mapping revealed a unique and narrow signature of linkage in the Ciona bugeye mutant genomes over a predicted T-type calcium channel gene. Requirement for the T-type calcium channel gene in neural tube closure was tested by CRISPR mediated knockout in wild type embryos and definitively established causality of the gene for the bugeye mutant phenotype. The bugeye mutant was investigated for previously known characteristics of neural tube closure mutants such as neural specification, cellular polarity and apical constriction defects. My results did not support any of these possibilities and rather support the hypothesis that bugeye is a failure to maintain a sealed anterior neuropore. To further explore and expand this previously uncharacterized role for T-type calcium channel genes, I tested for conservation of function in the vertebrate model Xenopus. Knockdown of the orthologous and earliest expressed T-type calcium channel gene, CAV3.2, in Xenopus laevis embryos via splice-disrupting morpholinos resulted in an open brain phenotype with characteristics similar to that of the bugeye mutants – a failure to seal the anterior neuropore. The function of T-type calcium channels in neural tube closure was tested in both Xenopus and Ciona: cellular calcium fluctuations, using the genetically encoded calcium indicator, GCaMP3, were investigated in the context of neurulation and T-type calcium channel disruption. I also investigated a downstream target of T-type calcium channels and a previously known neural tube closure gene family, the EphrinA family of ligand-receptor signaling molecules. My results found that the bugeye mutant phenotype, open brain, could be rescued by down regulation of EphrinA signaling in the neural tube. The cumulative results allowed us to propose a model of T-type calcium channel function in neural tube closure via EphrinA signaling and suggest future directions to further explore this newly established role for T-type calcium channels in sealing the anterior neuropore.

Additively manufacturing metal parts through laser powder bed fusion (LPBF) is of growing interest to many welds, ranging from medical to aerospace. Typical LPBF systems use a single laser to weld the metal powder, which leads to sharp temperature gradients as metal rapidly melts and solidifies. However, due to the extreme heating and cooling cycles experienced during LPBF, the resulting parts are often plagued with internal residual stresses which can cause warping and a deviation from the original design specifications. This work explores an in situ method of mitigating residual stress by using a dual laser systemwhere a secondary diode laser projects uniformly over a large area to heat the current top layer. With the ability to determine the timing, duration, intensity, and periodicity of the secondary laser projection, the user is allowed full control of the thermal history of their part throughout the build. This selective heating of the welded surface pattern reduces the temperature gradient between layers, thus reducing the residual stress. The capability to control grain growth in LPBF using the secondary surface-heating laser is also explored in this work. Such a system allows the user to control the solidication time, thus controlling the transition through various phases and ultimately the resulting microstructure. This secondary surface heating source provides an alternative to other heat treatment methods such as post annealing and build plate heating. Directly heating the topmost layer allows for temperature control within the region most affected by the welding.

Here I present a parallel study of mRNA and microRNA expression during oral siphon (OS) regeneration in Ciona robusta, and the derived network of their interactions. In the process of identifying 248 mRNAs and 15 microRNAs as differentially expressed, I also identified 57 novel microRNAs, several of which are among the most highly differentially expressed. Analysis of functional categories identified enriched transcripts related to stress responses and apoptosis at the wound healing stage, signaling pathways including Wnt and TGFβ during early regrowth, and negative regulation of extracellular proteases in late stage regeneration. Consistent with the expression results, I found that inhibition of TGFβ signaling blocked OS regeneration.

A correlation network was subsequently inferred for all predicted microRNA-mRNA target pairs expressed during regeneration. Network-based clustering associated transcripts into 22 non-overlapping groups, then functional analysis of network clusters showed enrichment of stress response, signaling pathway and extracellular protease categories could be related to specific microRNAs. Predicted targets of the miR-9 cluster suggest a role in regulating differentiation and the proliferative state of neural progenitors through regulation of the cytoskeleton and cell cycle.

Additional experiments were performed to identify precursor cells which contribute to OS regeneration. A population of cells expressing the pluripotency marker Piwi ( was observed to accumulate at the distal edge of regenerating OS blastemas, suggesting they may act as multi- or pluri-potent stem cells which contribute to siphon regeneration by differentiating into the functional tissues. Another population of Piwi expressing cells (PECs) has been identified in the transverse vessels of the branchial sac and these cells appear to increase in number following OS amputation. Expansion of the branchial sac (BS) PEC population following amputation could be inhibited by several pharmacological inhibitors of major conserved signaling pathways that I identified in the above-mentioned RNAseq study. This observation supports the hypothesis that signaling occurring within the regenerating OS is capable of inducing expansion of the PEC population in the BS, likely through the action of circulating PECs. However, the function of PECs and contribution to siphon regeneration remains unresolved.

My dissertation focuses on identifying the role of T-type voltage- gated Ca2+ channels (VGCC) in neural development. This research uses invertebrate and vertebrate models - specifically, the ascidian Ciona and the frog Xenopus, to span chordate development. Our lab previously identified a Ciona savignyi (C. savignyi) mutant, bugeye, that phenocopies exencephaly in humans by displaying an open brain. In C. savignyi bugeye, we see a dramatic decrease of Cav3 expression using qPCR. The Cav3 gene encodes for the single T-type VGCC in the Ciona genome. T-type VGCCs are part of a larger family of VGCCs, and are distinguished as being “low- voltage” because they are activated by small depolarizations in membrane potential to increase Ca2+ permeability. T-type VGCCs had not been implicated in embryonic development prior to these findings. Moreover, our lab found that EphrinA-d, a cell repulsion protein, was overexpressed in bugeye embryos, and thus was likely regulated by Ca2+. We found that overexpression of EphrinA-d phenocopied the open brain phenotype in wild-type embryos.

More recently the Smith lab discovered a new exencephaly mutant in a related species, Ciona robusta (C. robusta). To identify the causative gene, I performed linkage analysis and a complementation test which pointed toward Cav3, the causative gene in C. savignyi bugeye. RT-PCR analysis of Cav3 indicated no change in expression levels compared to wild- type siblings. However, sequence analysis of the mutant C. savignyi Cav3 gene revealed multiple amino acid changes, many in areas of functional importance. The Smith lab followed up on these observations and performed RNAseq on both C. savignyi bugeye and C. robusta bugeye embryos to identify novel genes involved in neural tube closure. I extended research on Cav3 by examining a vertebrate model, the African clawed frog Xenopus laevis, which provided more tools to study morphogenesis. In Xenopus, I found that morpholino oligonucleotide knockdown of Cav3.3 caused a wide range of developmental defects, including delays in gastrulation and neurulation, craniofacial defects, a dorsal flexure phenotype, and an apparent mis-migration of melanocytes.

To identify a cellular pathway, I investigated Calpain2, a Ca2+ -dependent protease involved in the Wnt/Ca2+ pathway during gastrulation and neurulation. Knockdown of Calpain2 shows similar phenotypes as knockdown of Cav3.3, and I observed abolishment of tagged Calpain2L fluorescence, mislocalization of Calpain2L in nearby cells, and increased surface area for cells affected by the knockdown. Furthermore, we found loss of apical actin localization along the hinge points of the folding neural tube. This apical constriction is essential for normal neural tube closure. Our results indicate that Cav3.3 is necessary for neural induction and/or differentiation and mechanical aspects of gastrulation and neurulation. Future research will aim to identify the mechanisms through which Cav3.3 affects these events.

Introduction

Materials and methods

Results

Conclusions