Abstract
The bilateral habenula nuclei (HA) and their connections to the interpeduncular nucleus (IPN) structure one of the most highly conserved circuits in vertebrate brains that plays many important roles such as regulating cognition, aversive response and reward processing. Prior study showed that during zebrafish embryonic development, secreted guidance cues Semaphorin 3D (Sema3D) attract subset of Neuropilin 1A (Nrp1a) positive neuronal axons from the left HA to their targets in the dorsal IPN domain in the midbrain. However, the downstream molecular mechanism of how HA neurons interpret this Sema3D-mediated axonal attraction event still remains elusive. Utilizing candidate gene search approaches, I have identified that collapsin response mediator proteins (CRMPs) 1, 4, 5a, and Ras-related in brain 6b like (Rab6bl) are expressed in HA in zebrafish embryos at 72 and 96 hours post fertilization (hpf). Particularly, the Rab6bl and the Nrp1a transcrips are co-localized within the left HA during the formation of HA-IPN connections between 72-96 hpf. Over-expressions of wildtype (WT) and dominant-negative forms of Rab6bl (DN-Rab6bl) by injecting in vitro transcribed mRNAs into 1-2 cell-stage embryos caused no detectable molecular defect suggesting that Rab6bl has no obvious function during early embryonic development. This result is consistent with my observation that there is no detectable expression of rab6bl before 48 hpf. In order to specifically perturb the function of Rab6bl in left HA during 72-96 hpf, I have adopted the electroporation technique, and under my current equipment set-up and experimental condition, the use of 100V, 200Hz and 2.0mS squire wave width for 7 times exhibited the highest successful rate with the Axonporator 800 apparatus.
Published Version
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