- 學位論文
利用基因轉殖表現方法分析Sonic Hedgehog與Netrin訊息傳遞在斑馬魚顱顏部色素細胞移動所扮演之功能
Functional Analyses of Sonic Hedgehog and Netrin Signaling During Zebrafish Craniofacial Pigment Cell Migration by Transgenic Expression Assay
摘要
Shh (Sonic hedgehog)與Netrin訊息對胚胎早期的正常發育相當重要。已知兩者參與中樞和周邊神經系統的建立，近年來陸續發現兩家族成員對神經系統以外的周邊組織細胞有著廣泛性的影響。 於先前研究中，在IRES (Internal Ribosomal Entry Site)系統之下建構結構體，並利用顯微注射方法將結構體注入斑馬魚受精卵中。其中使用斑馬魚βB1- Crystallin 1.3 kb啟動子(Cr1.3)，於水晶體專一表現異位的Shh蛋白，同時藉由綠色螢光蛋白得知此轉殖斑馬魚帶有該結構體，可發現在Shh過量表現於水晶體時，幼魚瞳孔的位置有黑色細胞的聚集(王偉庭，2005)；經過定時追蹤觀察，in vitro移植實驗，初步證明Shh為ㄧ生化吸引劑(chemoattractant)，在水晶體中異位表現而導致體表黑色素細胞(migrating melanocyte)受到其生化引誘功能而遷移至瞳孔部位。 另外文獻指出，黑色素前驅細胞在班馬魚胚胎24 hpf (hours post fertilization)之前已出現在RPE (Retinal Pigmented Epithelium)的邊緣(Lister, 2002)。然而，內生性的Shh在RPE位置表現的時間較晚(40 hpf)，因此推測在眼睛發育早期有不同的因子負責指引黑色素前驅細胞向眼睛的位置移動。根據原位雜合反應觀察斑馬魚Netrin-1a表現的時空分佈 (林文郎, 2005)，以及利用上述之建構方法於班馬魚水晶體中異位過量表現Netrin-1a蛋白，在轉殖恆定品系觀察當中，外觀上出現可能為黑色素細胞的聚集，藉此我們推測黑色素前驅細胞在早期可能受到Netrin-1a的訊號吸引而向眼睛的位置移動。 為了進一步探討Shh及Netrin-1a訊息對顱顏部黑色素細胞移動的影響，我以Tol2 transposase系統與IRES系統並行，利用mitf啟動子在黑色素前驅細胞異位表現Shh及Netrin-1a訊息傳遞分子以及其負向調控分子Gas1b及antisense Dcc，在過渡性基因轉殖實驗中觀察顱顏部黑色素前驅細胞的移動情形，包括時間與空間上的分佈。 我利用Tol2 transposase系統進行基因轉殖實驗，以斑馬魚six1 promoter進行測試，發現Tol2 transposase系統確實可以很有效率將plasmid DNA嵌入基因體中。然而，利用mitfa promoter及IRES系統進行基因轉殖實驗表現上述訊息傳遞分子，發現IRES無法在黑色素細胞內有效運作。此外，亦無觀察到黑色素細胞異常分佈的現象。由於報導基因綠色螢光蛋白(GFP)無法在黑色素細胞中以IRES系統表現，因此無從判斷訊息傳遞分子是否能夠有效影響黑色素細胞之移動或分化。 由於IRES系統無法於黑色素細胞內正常運作，因而可考慮以chimeric promoters (βB1- Crystallin promoter及mitfa promoter)進行後續基因轉殖實驗，可挑選出水晶體具有螢光之基因轉殖斑馬魚(F1)，從而進行表現型之觀察及研究。 此外，也可利用病毒2A peptide的方式來表現分割二個不同的蛋白質(bicistronic mRNA的轉譯)，以改善上述IRES系統所遭遇的問題。
並列摘要
The Sonic Hedgehog signaling pathway, as well as the Netrin signaling pathway, both play significant roles in early stages of embryonic development. It has been shown that they help building the central and peripheral nervous system. Moreover, as being discovered recently, tissues other than the nervous system can be influenced by their broad effects. According to our previous studies, constructs of plasmid DNA with the IRES (internal ribosomal entry site) system can be employed in trasngenic zebrafish assay. We discovered that melanocytes could be allured to the lens, by the ectopic biscistronic overexpression of Sonic Hedgehog and GFP (green flrorescent protein) reporter gene in the lens. Consecutively, the transplantation experiment suggests that Sonic Hedgehog could function as a chemoattractant. Moreover, studies have demonstrated that melanocyte precursor cells are located at the margin of RPE (Retinal Pigmented Epithelium) before 24 hours post fertilization (24hpf). However, the expression of endogenous Sonic Hedgehog in RPE begins at 40hpf, which indicates factors other than Sonic Hedgehog may lead melanocyte precursor cells instead. We have shown the distribution of Netrin-1a in RPE primordium by in situ hybridization. The lens of zebrafish in transgenic lines with ectopic overexpression of Netrin1a can also attract melanocytes. According to the above data, we assumed that the melanocyte precursor cells could be attracted by Netrin1a, therefore migrating to the eyes. Sonic Hedgehog is negatively regulated by Gas1b, while antisense Dcc inhibits the Netrin1a signaling pathway. To further unveil the impacts of Sonic Hedgehog and Netrin1a on craniofacial melanocytes migration, I created constructs employing mitf promoter to drive downstream genes, sonic hedgehog, netrin1a, gas1b, and antisense dcc, respectively, with the Tol2 transposase system and the IRES system at the same time. I expect to observe the aberrant spatio-temporal distribution of migrating craniofacial melanocytes in transient transgenic assays. Besides, I found that the plasmid DNA containing GFP reporter gene driven by six1 promoter with the Tol2 transposase system can be effectively integrated into the genome of zebrafish. However, constructs that express the aforementioned genes with IRES and mitfa promoter failed to function properly in melanocytes. In addition, there is no apparent difference shown either in melanocytes migration or differentiation. I failed to elucidate the importance of the signaling molecules on melanocytes because the GFP reporting system is unable to work in melanocytes with IRES. Owing to the negative results shown in melanocytes with IRES, we will proceed to the transgenic experiments with the cooperation of chimeric promoters (βB1-Crystallin promoter and mitfa promoter) eventually. By this means, we might observe the phenotypes in transgenic zebrafish. Alternatively, we may replace the IRES system by utilizing the viral 2A peptide to produce two independent proteins.
參考文獻
延伸閱讀
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