- 學位論文
利用免疫組織染色和原位雜交反應探討TGFβ3和Shh在轉殖斑馬魚眼睛發育過程所扮演的角色
Functional Analyses of TGFβ3 and Shh during Eye Development of Transgenic Zebrafish by Immunohistochemistry and in situ Hybridization
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摘要
Transforming growth factor-β family (TGF-β family)和Sonic Hedgehog在胚胎的早期發育都扮演很重要的角色。以神經管發育為例，TGF-β family在神經管的背部大量表現，Sonic Hedgehog則在神經管的腹面大量表現，這兩種基因產物同時以濃度梯度的方式，向神經管方向擴散並且影響神經管的發育。 在本研究中，我想要探討TGF-β family和Sonic Hedgehog在眼睛的發育中扮演什麼樣的角色。在Shh方面，先前的研究結果(王偉庭, 2005)－βB1晶體蛋白(βB1-crystallin)啟動子在斑馬魚水晶體異位過量表現Shh。觀察所篩選到的轉殖恆定品系19號，發現眼睛視網膜外形呈球狀，不同於野生型的杯狀。而組織切片分析，發現視網膜細胞在CMZ (ciliary marginal zone)區域似乎有增生的現象，然而神經視網膜的分層是否有顯著的影響，則需進行Immunohistochemistry來證明。而TGF-β部分，之前的實驗結果（黃嵩崴,2006）－βB1晶體蛋白(βB1-crystallin)啟動子在斑馬魚水晶體異位過量表現TGF-β，發現在轉殖恆定品系4號的眼睛外觀上，瞳孔有變小的現象，而且將受精後7天的水晶體取下，發現水晶體也比野生型小。此外，除了水晶體大小發生異常之外，也發現在透明的水晶體中央出現混濁且不透明的區域，此為白內障形成的現象。 因此，從先前實驗結果推測Shh可能會對視網膜細胞的增生有所影響，而TGF-β3則影響水晶體的發育，甚至與白內障的形成有關。我在本實驗中便是運用immunohistochemistry、in situ hybridization等方法來了解TGF-β3或Shh在眼睛發育中所扮演的角色。綜合了以上實驗的結果，我發現在Shh異位過量表現的斑馬魚中，其眼睛視網膜的細胞分層並沒有明顯的改變；而在TGF-β3異位過量表現的斑馬魚中，的確有偵測到TGF-β3下游分子的表現。 從目前的實驗結果推論，推測TGF-β3和Shh對於斑馬魚眼睛組織扮演不同的調控角色。TGF-β3的過量表現會影響水晶體的發育，造成白內障的形成。而Shh則是對視網膜細胞的分層並沒有影響，但似乎會造成CMZ區域的增生。而在我所進行的實驗中，對於CMZ是否增生並沒有具體化的結論，但這一部分還必須重複上述實驗才能證明。
並列摘要
Transforming growth factor-β family (TGF-β family) and Sonic Hedgehog play critical roles in early developmental processes of embryo. For example, TGF-β family is expressed in the dorsal neural tube, whereas Sonic Hedgehog is expressed in the ventral neural tube. Both proteins diffuse and pattern the neural tube depending on the function of the concentration gradient. In this study, I want to elucidate the function of TGF-β family and Sonic Hedgehog in the eye development of zebrafish. According to the previous result (Wang, 2005)-using zebrafish lens-specific βB1-Crystallin 1.3 kb promoter fragment (Cr1.3) to drive ectopic overexpression of Shh in the lens, I observed the stable transgenic zebrafish line No.19 for study. I found that the silhouette of retina has changed. Through sectioning, it seems that the CMZ (ciliary marginal zone) has proliferated. And Shh may affect the stratification of neural retina in the line No.19, which depends on the result of Immunohistochemistry to prove it. In TGF-β, according to the previous result (Huang, 2006)-using zebrafish lens- specific βB1-Crystallin 1.3 kb promoter fragment (Cr1.3) to drive ectopic overexpression of TGF-β in the lens, I observed the stable transgenic zebrafish line No.30 for study. I found the pupil of the No.30 stable line seemed to be smaller than wild type fish. Then I excised the lens out of 7 days-post- fertilization (7 dpf) embryo and found the lens was indeed smaller than those of wild type fish. In addition to the abnormal lens size, I also observed that there was a cloudy and opaque region in the eye lens. In the previous results, Shh may affect the proliferation of neural retina cells. And TGF-β3 could influence the lens development, even implicated in the occurrence of cataract. In this study, I use in situ hybridization and immunohistochemistry methods to elucidate the function of TGF-β3 and Shh during the eye development of zebrafish. Conclusively, I found the stratification of neural retina in Shh overexpression zebrafish does not change. And the downstream molecules of TGF-β3 are detected in TGF-β3 overexpression zebrafish. In the preliminary results, TGF-β family and Shh may play different roles in regulating the eye development of zebrafish. TGF-β3 influence the lens development, and implicated in the occurrence of cataract. And Shh do not affect the stratification of neural retina, but result in the expanded CMZ. In my study, we do not have conclusive result. In the future, I have to repeat this method to prove it.
參考文獻
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