利用基因轉殖異位超量表現方法分析Sonic Hedgehog在斑馬魚眼睛視網膜發育過程中所扮演之功能

Abstract

Sonic Hedgehog訊息傳遞在斑馬魚眼睛的發育過程中扮演相當重要的角色，從anterior neural plate分裂形成兩個optic primordia (morphogenesis)直到神經視網膜上各層細胞分化形成(neurogenesis)以及後續眼球因應生長所需之細胞增生(CMZ，位於ciliary marginal zone)都需要此蛋白之作用。因此為了更進一步了解此蛋白在眼睛發育過程中所扮演之角色，我構築Cr1.3-Shh-IRES-cDNA結構體，利用顯微注射方法將此結構體注入斑馬魚受精卵中，以斑馬魚水晶體專一啟動子βB1-crystallin來啟動Shh蛋白在晶體專一表現，並藉由擴散方式來影響神經視網膜各層細胞之發育；同時藉由綠色螢光蛋白之表現觀察得知此轉殖斑馬魚帶有此結構體。 我篩選得到兩個轉殖恆定品系1號及19號，觀察發現在魚苗瞳孔部位有黑色細胞點塊之形成，在統計7 dpf (days post fertilization)轉殖恆定品系19號之結果發現，在瞳孔黑色細胞點塊出現頻率約有9成，推測其出現有三種原因︰CMZ轉分化(progenitor cells transdifferentiation)、色素視網膜細胞增生(RPE proliferation)及Shh生化引誘作用(chemoattraction)。以黑色素細胞專一基因標的物MITF (microphthalamia-associated transcription factor) 啟動子接上紅色螢光蛋白(DsRed)之結構體進行過渡性轉殖實驗，根據結果推測此黑色細胞點塊為黑色素細胞。而後經由定時追蹤觀察以及in vitro移植實驗，證明Shh為ㄧ生化吸引劑(chemoattractant)，在水晶體中異位過量表現而導致體表色素視網膜上移動中的黑色素細胞(migrating melanocyte)受到其生化引誘功能而遷移至瞳孔部位。 另一方面，透過觀察發現轉殖恆定品系斑馬魚之瞳孔似乎有縮小之現象，經過實際測量其寬度發現確實有縮小之現象。而在組織學上冷凍切片之觀察發現異位過量表現之Shh蛋白斑馬魚視網膜發育似乎也造成各主要細胞層次(GCL、INL及ONL)的增厚，同時Shh蛋白似乎也促使CMZ之幹細胞增生，而導致轉殖恆定品系斑馬魚瞳孔縮小，然而這些在CMZ中增生之神經前驅細胞(neural progenitor cells)接收其他的訊號刺激便分化成視網膜中特定之神經細胞，因此造成在發育上與野生型斑馬魚出現不同的結果。但更詳細之相關機制必須以in situ hybridization及immunohistochemistry方法才能得知視網膜中各類神經細胞之RNA及蛋白質層面之變化，對於Shh在眼睛發育過程中之功能作更完整之了解，以及釐清其分子間之相互調控的機制。

Sonic hedgehog signaling transduction plays crucial roles in the development of zebrafish eye. From the split of anterior neural plate into two bilateral optic primodia (morphogenesis) to the differentiation of seven major neurons in the neural retina (neurogenesis), and then the neural progenitor cells proliferation in the CMZ (ciliary marginal zone) corresponding to eye growth region at later embryonic stage, all depends on the shh signaling pathway. To dissect the functional roles of shh in the eye development of zebrafish in detail, I have constructed the Cr1.3-Shh-IRES- GFP chimera gene and microinjected into the zebrafish eggs. The lens-specific βB1-crystallin promoter drives the expression of shh protein in the lens ectopically. We assumed that the Shh protein may affect the development of retina by diffusion; and simultaneously the transgenic fish containing this construct can be screened by the expression of GFP. Two zebrafish transgenic stable lines No.1&No.19 were obtained from 28 F0 transgenic fish. Both lines express Shh protein ectopically in the lens, we found that some melanocyte-like cells clustering in the pupil region. This observation inferred three hypothetical possibilities that may lead to such a phenotype: the transdifferentiation of neural progenitor cells (from CMZ), cells of RPE proliferation or the chemoattraction role of shh. We suggested that the melanocyte-like cell aggregates were indeed melanocytes as identified by transient transgenic assay using the promoter of melanocyte-specific gene marker, MITF (microphthalamia -associated transcription factor). Furthermore, I proved that the Shh protein has the role of chemoattraction to guide the melanocyte migration by in vitro transplantation. On the other hand, I found that the pupil of transgenic stable line fish seemed to be smaller than wild type fish, and the three major layers of neural retina (GCL, INL and ONL) seems to be more proliferated. We suggested that shh protein promoted the proliferation of neural progenitor cells in CMZ, and resulted in the smaller pupil. Then, these cells received some other signals from developing eye and specified into seven types of neurons in the neural retina. In the future, in situ hybridization and the immunohistochemistry may pave the ways to elucidate the more detailed fuction of Shh during the eye development of zebrafish and to unravel the molecular mechanism underlying this process.