溶血磷脂酸第一型受體在嗅覺發育之調控角色探討

Abstract

嗅覺系統發育是一準確且複雜的過程。在由嗅覺感覺神經(olfactory sensory neuron)投射至嗅球(olfactory bulb)的過程中，軸突準確的定位及生長為嗅覺系統正確建立所必需。斑馬魚卵在受精4天內可形成完整嗅覺系統，在此期間嗅覺感覺神經會被特化並遷移，而其上的嗅覺受體(odorant receptors) 會表現在特定的嗅覺感覺神精元，並佔據嗅覺上皮細胞層(olfactory epithelium)上特定的區域，同時，軸突開始投射並集中於前驅神經細胞(precursor neurons)，最終，表現不同的嗅覺受體的感覺神經元叢集將各自投射至嗅球內的特定區域並形成嗅小體叢集(glomeruli)，使得嗅球內產生具物種間保守性的神經叢集圖譜，此嗅覺系統的建立過程雖已有許多在生理、解剖、組織學上的證據，但對於在分子層面上嗅覺系統的建立是如何被精準控制目前尚未明白。 在前人小鼠的研究中已發現剔除溶血磷脂酸第一型受體(Lysophosphatidic acid receptor 1, Lpar1)會造成新生小鼠因吸奶不足而死亡(Contos, et al, 2000)。該文作者推測其可能因嗅覺缺損所造成，然該研究團隊或他人並未針對Lpar1在嗅覺發育機制之角色做進一步之探討，因此本篇研究乃利用斑馬魚為生物模式對該議題進行相關研究。 結果發現Lpar1在不同發育時期循序表現於前發育基板區域(pre-placodal region)及嗅覺發育基板(olfactory placode)，顯示其與嗅覺發育的相關性；利用反義嗎啉基寡核苷酸(Morpholino, MO)使斑馬魚胚 Lpar1蛋白質生成量降低，並藉由DiI的順行性軸突追蹤發現，Lpar1的早期表現量下降會造成嗅覺感覺神經軸突往前腦嗅球的投射集叢消失，嗅小體功能區LG1, LG2 LG3 LG4, DZ, MG2 及CZ縮小，並降低幼魚對丙胺酸(L-analine)之食慾反應(appetitive response)，但其嗅上皮細胞層卻無缺失，亦不影響嗅覺感覺神經元數量及個體發育進程。因此在本論文中我證明了Lpar1為斑馬魚嗅覺系統發育所必需之基因。

Zebrafish olfactory system development is a precise and complex process, initiating from the olfactory sensory neurons (OSNs) fate determination and assembling, followed by OSNs differentiation and specific odorant receptors expression, and ended by OSNs axon projecting through pioneer neuron (PN) tangle together into olfactory bulb (OB) and forming the glomeruli specific map. Although many lines of evidence have revealed signal pathways in each step of olfactory system establishment, the complete mechanism of how OSNs project to OB and construct topographic map is largely unknown. We found the expression pattern of Lysophosphatidic acid receptor 1(Lpar1) matches the pre-placode region and olfactory placode which suggests the possible regulatory role of Lpar1 in the olfactory system development. Using morpholino (MO) to eliminate Lpar1 expression also caused appetitive olfactory behavior reduction which showed high relativity between Lpar1 signal and olfactory system development. We further traced OSNs and found that the loss of olfactory behavior may result from the loss of OSNs projection bundle and disorder of glomeruli map. Moreover, the lpar1 MO-induced projection defect is not due to the change in OSN cells fate determination and the loss of placodal cells migration in the early stage of olfactory development. By using Tg(claudinB-EGFP), the size of different parts of glomeruli (i.e. LG2, DZ, MG2 and CZ) were significantly reduced in lpar1 MO-treated larvae. We further used Ki16425 and HA130 to pharmacologically inhibit the Lpar1 downstream signal to disturb the production of LPA in 12~24 hpf. Similar results were obtained for this short term inhibition as that in lpar1 MO-treated larvae. It suggests that Lpar1 signal may play a critical role in establishing OSNs projection bundle. In conclusion, I demonstrate that Lpar1 plays an essential role in olfactory development.