斑馬魚腦部gnrh3的結構、發育與功能探討

Abstract

性釋素(gonadotropin-releasing hormone, GnRH)為下視丘分泌之一種神經性荷爾蒙，主要的功能在於刺激腦下腺促性腺激素之合成及分泌，在生殖上扮演重要角色。GnRH也表現在下視丘外的腦部及性腺等組織，但在這些組織所扮演的角色了解很少。下視丘GnRH神經元在胚胎發育時會從嗅板遷移至前腦，其起源及遷移機制尚待研究，所以本論文以斑馬魚為實驗對象，了解GnRH細胞的起源及在遷移過程中的機制。首先利用3’-RACE及5’-RACE的方式初步確定斑馬魚腦部有兩種形式的性釋素，即gnrh2 (cGnRH-II)及gnrh3 (sGnRH)。接著產生帶有gnrh3 promoter的基因轉殖斑馬魚，利用gnrh3 antisense oligonucletide morpholino -knockdown、mRNA misexpression等技術，可知gnrh3的表現對於本身的發育是必要的。gnrh3沒有表現在cyc、oep及smu等突變魚，進一步分析可知PKA等訊息分子可能參與GnRH細胞的起源及分化。未來可利用此帶有螢光蛋白之基因轉殖斑馬魚來研究表現gnrh3神經細胞在胚胎發育起源及遷移的機制。

Expression of gnrh3 neurons started in the olfactory pit at 24-26 hours post-fertilization (hpf) and migrated into forebrain region during early larvae stage. We generated transgenic zebrafish expressing green fluorescent protein (GFP) and LacZ driven by the gnrh3 promoter. In gnrh3-transgenic fish, the expression of GFP and LacZ were similar to gnrh in the olfactory placode, olfactory bulb and telencephalon. To understand the function of GnRH at gnrh3 neuron development, we have done the morpholino knockdown and over-expression analysis. Reduced GFP and gnrh3 cell numbers, and delayed migration were caused by gnrh3 morpholino knockdown. The gnrh2 can rescue the morphants partially. Over-expression of gnrh3 can induce the earlier development of gnrh3 neurons. The gnrh3 expression was not in cyc, oep and smu mutant. PKI misexpression can rescue the gnrh3 defect in oep mutant. PKA and PKI misexpression caused the GFP cell numbers decrease and GFP ectopic expression, respectively. The normal gnrh3 expression and PKA pathway were involved in the development of gnrh3-expressing neurons during embryos. Transgenic zebrafish with specific gnrh3-GFP-LacZ expression in the brain offers a genetic tool to study the origin and migration of gnrh3 neuron lineage.