剖析Semaphorin 3D-Neuropilin 1A引導疆核軸突目標辨認的分子機制

雙邊的疆核(HA)及其聯接到interpeduncular核(IPN)的結構是在脊椎動物腦中最保守的神經迴路，其扮演了許多重要的角色如調控認知,厭惡反應及回饋反應。之前的研究指出在斑馬魚胚胎發育時，分泌性的指引線索Semaphorin 3D(Sema3D)會吸引位於左邊表現Neuropilin 1A(Nrp1A)的神經元軸突延伸到中腦IPN的背面區塊。然而如何去闡釋由Sema3D所引起疆核神經元軸突吸引的下游分子機制仍不清楚。利用收尋法，我已經確定collapsin response mediator proteins (CRMPs) 1, 4, 5a,及Ras-related in brain 6b like (Rab6bl)在斑馬魚受精後72及96小時胚胎中的疆核表現。尤其，Rab6bl和Nrp1A的轉錄產物會共位於斑馬魚胚胎左邊疆核在受精後HA-IPN 迴路形成的72到96小時。利用顯微注射過量表現野生型(WT)及負顯性型(DN)的Rab6bl在1到2個細胞時期並沒有造成可以被偵測到的分子缺陷。這顯示Rab6bl在早期胚胎發育中可能沒有顯著的功能。這個結果跟我在受精後48小時前所觀察到沒有明顯的表現量一致。為了特別去破壞Rab6bl受精後72到96小時在左邊疆核的功能，我採用了電穿孔的技術並且在目前的裝備(Axoporator)設立及實驗條件下，利用100伏特(V)，200赫茲(Hz)及2毫秒寬度的方形波重複七次電刺激可以達到最高的成功率。

關鍵字

HA-IPN迴路； Neuropilin1A-Semaphorin3D 訊息傳遞； CRMP ； Rab6bl ；電穿孔

並列摘要

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.

並列關鍵字

habenula-interpedunclur nucleus circuit ； Neuropilin1A-Semaphorin3D signaling ； CRMPs ； Rab6bl ； electroporation

參考文獻

Opdam, F.J., Echard, A., Croes, H.J., van den Hurk, J.A., van de Vorstenbosch, R.A., Ginsel, L.A., Goud, B., and Fransen, J.A. 2000. The small GTPase Rab6B, a novel Rab6 subfamily member, is cell-type specifically expressed and localised to the Golgi apparatus. J Cell Sci 113 ( Pt 15): 2725-2735.

Arimura, N., Inagaki, N., Chihara, K., Menager, C., Nakamura, N., Amano, M., Iwamatsu, A., Goshima, Y., and Kaibuchi, K. 2000. Phosphorylation of collapsin response mediator protein-2 by Rho-kinase. Evidence for two separate signaling pathways for growth cone collapse. J Biol Chem 275(31): 23973-23980.

Arimura, N., Menager, C., Kawano, Y., Yoshimura, T., Kawabata, S., Hattori, A., Fukata, Y., Amano, M., Goshima, Y., Inagaki, M. et al. 2005. Phosphorylation by Rho kinase regulates CRMP-2 activity in growth cones. Mol Cell Biol 25(22): 9973-9984.

Bagnard, D., Thomasset, N., Lohrum, M., Puschel, A.W., and Bolz, J. 2000. Spatial distributions of guidance molecules regulate chemorepulsion and chemoattraction of growth cones. J Neurosci 20(3): 1030-1035.

Barnekow, A., Thyrock, A., and Kessler, D. 2009. Chapter 5: rab proteins and their interaction partners. Int Rev Cell Mol Biol 274: 235-274.

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剖析Semaphorin 3D-Neuropilin 1A引導疆核軸突目標辨認的分子機制

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