A Forward Genetic Study on Neural Tube Development

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

T-type Calcium Channel Regulation of Neural Tube Closure and EphrinA/EPHA Expression

SummaryA major class of human birth defects arise from aberrations during neural tube closure (NTC). We report on a NTC signaling pathway requiring T-type calcium channels (TTCCs) that is conserved between primitive chordates (Ciona) and Xenopus. With loss of TTCCs, there is a failure to seal the anterior neural folds. Accompanying loss of TTCCs is an upregulation of EphrinA effectors. Ephrin signaling is known to be important in NTC, and ephrins can affect both cell adhesion and repulsion. In Ciona, ephrinA-d expression is downregulated at the end of neurulation, whereas, with loss of TTCC, ephrinA-d remains elevated. Accordingly, overexpression of ephrinA-d phenocopied TTCC loss of function, while overexpression of a dominant-negative Ephrin receptor was able to rescue NTC in a Ciona TTCC mutant. We hypothesize that signaling through TTCCs is necessary for proper anterior NTC through downregulation of ephrins, and possibly elimination of a repulsive signal

Eri1 regulates microRNA homeostasis and mouse lymphocyte development and antiviral function.

Natural killer (NK) cells play a critical role in early host defense to infected and transformed cells. Here, we show that mice deficient in Eri1, a conserved 3'-to-5' exoribonuclease that represses RNA interference, have a cell-intrinsic defect in NK-cell development and maturation. Eri1(-/-) NK cells displayed delayed acquisition of Ly49 receptors in the bone marrow (BM) and a selective reduction in Ly49D and Ly49H activating receptors in the periphery. Eri1 was required for immune-mediated control of mouse CMV (MCMV) infection. Ly49H(+) NK cells deficient in Eri1 failed to expand efficiently during MCMV infection, and virus-specific responses were also diminished among Eri1(-/-) T cells. We identified miRNAs as the major endogenous small RNA target of Eri1 in mouse lymphocytes. Both NK and T cells deficient in Eri1 displayed a global, sequence-independent increase in miRNA abundance. Ectopic Eri1 expression rescued defective miRNA expression in mature Eri1(-/-) T cells. Thus, mouse Eri1 regulates miRNA homeostasis in lymphocytes and is required for normal NK-cell development and antiviral immunity

Interleukin-4 production by follicular helper T cells requires the conserved Il4 enhancer hypersensitivity site V

FollicularhelperT cells (Tfh cells) are the major producers of interleukin-4 (IL-4) in secondary lymphoid organs where humoral immune responses develop. Il4 regulation in Tfh cells appears distinct from the classical Thelper 2 (Th2) cell pathway, but the underlying molecular mechanisms remain largely unknown. We found that hypersensitivitysiteV (HS V; also known as CNS2), a 3? enhancer in the Il4 locus, is essential for IL-4production by Tfh cells. Mice lacking HS V display marked defects in type 2 humoral immune responses, as evidenced by abrogated IgE and sharply reduced IgG1 production in vivo. In contrast, effector Th2 cells that are involved in tissue responses were far less dependent on HS V. HS V facilitated removal of repressive chromatin marks during Th2 and Tfh cell differentiation and increased accessibility of the Il4 promoter. Thus, Tfh and Th2 cells utilize distinct but overlapping molecular mechanisms to regulate Il4, a finding with important implications for understanding the molecular basis of allergic disease