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Ion channels and pumps play critical roles during sea urchin development including mediating the blocks to polyspermy, regulating left-right and dorsal-ventral axis specification, directing ventral PMC migration, and controlling biomineralization of the larval skeleton. We performed a screen of pharmacological ion channel inhibitors, and we chose two inhibitors to investigate further. First, we found that tricaine, a potent inhibitor of voltage-gated sodium channels (VGSCs), induces aberrant skeletal patterning in Lytechinus variegatus larvae. The larval skeleton is secreted by the primary mesenchyme cells (PMCs), which migrate within the blastocoel into a stereotypical pattern. We show that VGSC activity is required for normal PMC migration and skeletal patterning. Timed inhibitor studies identified VGSC activity as specifically required from early gastrula to the onset of late gastrula for normal skeletal patterning. Tricaine inhibits the voltage-gated sodium channel LvScn5a which is strongly expressed in the developing nervous system in pluteus larvae. We found that exogenous expression of an anesthetic-insensitive version of LvScn5a is sufficient to rescue hallmark tricaine-mediated skeletal patterning defects, demonstrating the specificity of the inhibitor. LvScn5a exhibits a ventrolateral ectodermal expression domain in gastrulating embryos that is spatiotemporally congruent with triradiate formation in the ventrolateral PMC clusters at the onset of skeletogenesis. This ectodermal territory normally expresses the patterning cue Wnt5, and we find that the expression of Wnt5 is dramatically spatially expanded by tricaine treatment. We also observe ectopic PMC clusters in tricaine-treated embryos. We found that knockdown of Wnt5 expression is sufficient to rescue tricaine-mediated skeletal patterning defects. These results are consistent with a model in which LvScn5a activity in the ventrolateral ectoderm functions to spatially restrict the expression of the ectodermal patterning cue Wnt5 that in turn induces PMC cluster formation. Together, these findings show that spatially restricted sodium channel activity regulates ectodermal cue expression that, in turn, regulates PMC differentiation and skeletal morphogenesis. Second, we show that V-type H⁺ ATPase (VHA) activity is required for specification of the dorsal-ventral (DV) axis. DV specification is controlled by the TGF-β signal Nodal that specifies the ventral territory and indirectly activates dorsal specification via induction of BMP 2/4 expression. Nodal expression occurs downstream of p38 MAPK, which is transiently, asymmetrically inactive on the presumptive dorsal side of the blastula embryo. VHA activity is required for that transient inactivation of p38 MAPK, and it is required for the subsequent spatial restriction of Nodal expression. We show that VHA inhibition is sufficient to induce global Nodal expression during the blastula stage, resulting in ventralization of the embryo. We show that this phenotype can be rescued by experimentally imposing asymmetric Nodal expression at the 4-cell stage. We discover a VHA-dependent voltage gradient across the DV axis and find that VHA activity is required for hypoxia inducible factor (HIF) activation. We show that neither hyperpolarization nor HIF activation is sufficient to perturb DV specification, which implicates a third unknown pathway connecting VHA activity and p38 MAPK symmetry breaking. These results are consistent with a model in which dorsal VHA activity is required to inhibit Nodal expression and signaling, potentially via dorsal p38 MAPK inhibition. Together, these studies demonstrate that ion channels are required for both DV specification and for normal skeletal patterning.

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<p>I have put my half title as the main thesis title here. I would like to use that as the title displayed online.</p> / <p>The Drosophila heart arises from two bilateral rows of cardioblasts (CB) that migratedorsally towards the midline and contact their contralateral partners to form the dorsal vessel.Generally, migrating cells rely on the extensions at the leading edge domain. Like other migratingcells, we show that the leading edge of the CBs extends finger-like processes which might play arole in sensing guidance cues during guided migration. Expressing an mCherry-Moesin transgenein the CBs enabled us to characterise the dynamic nature and genetic requirements of thesefilopodial processes. While studying the role of filopodial activity during heart assembly weobserved that CBs extended cellular protrusions towards the internalizing amnioserosa cells.Filopodial activity is low during migration, and rises when the CBs are near the amnioserosacells. However, filopodial contacts are stabilized by interaction with contralateral CBs, not theamnioserosa cells. CB cell bodies can contact their contra lateral partners only after theamnioserosa is fully internalized. We propose that filopodia are generated in response to thepresence of sensory guidance molecules excreted by the amnioserosa cells.Robo/Slit signalling has been previously shown to play a role in CB migration, adhesionand lumen formation. Additionally, studies have shown that Robo/Slit signalling plays a role infilopodial extension in the Drosophila nervous system development. We observed that in embryosin which Robo signaling in the CBs was reduced or absent, the CBs were less active at the LE. Inaddition, the migration speed of CBs in mutant embryos was notably decreased. Based on theseresults, we hypothesize that Robo/Slit signaling plays a role in filopodial extensions.</p> / Master of Science (MSc)

Primary sensory axons fail to regenerate into the spinal cord following dorsal root injury leading to permanent sensory deficits. Re-entry is prevented at the dorsal root entry zone (DREZ), the CNS-PNS interface. Current approaches for promoting DR regeneration across the DREZ have had some success, but sustained, long-distance regeneration, particularly of large-diameter myelinated axons, still remains a formidable challenge. Our lab has previously shown that induced expression of constitutively active B-RAF (kaBRAF) enhanced the regenerative competence of injured DRG neurons in adult mice. In this study, I investigated whether robust intraspinal regeneration can be achieved by selective expression of kaBRAF alone or in combination with deletion of the myelin-associated inhibitors or neuron-intrinsic growth suppressors (PTEN or SOCS3). To this end, I used LSL-kaBRAF: brn3a-CreERT2 transgenic mice in which kaBRAF can be induced selectively in sensory neurons. I have also bred LSL-kaBRAF: brn3a-CreERT2 mice with triple knock-out mice lacking Nogo, Mag and OMgp or mouse lines carrying floxed alleles of PTEN or SOCS3. Single, double, and triple conditional mice were subjected to cervical DR crush and AAV2-eGFP vectors were used to selectively label regenerating axons of large-diameter neurons. I compared the extent of regeneration at 3 weeks or 2 months after DR injury using conventional anatomical and behavioral analyses. I found that kaBRAF alone promoted axon regeneration across the DREZ but did not produce significant functional recovery by two months. Supplementary deletion of Nogo, MAG, and OMgp did not improve kaBRAF-induced regeneration. Deletion of PTEN or SOCS3 individually or in combination failed to promote axon regeneration across the DREZ. In marked contrast, simultaneous deletion of PTEN, but not SOCS3, dramatically enhanced kaBRAF-mediated regeneration enabling many more axons to penetrate the DREZ and grow deep into the spinal cord. This study shows that dual activation of BRAF-MEK-ERK and PI3K-Akt signaling is an effective strategy to stimulate robust intraspinal DR regeneration and may lead to recovery of sensory function after DR injury. / Biomedical Sciences