利用基因轉殖過量表現或抑制方法探討Transforming Growth Factor‐β3和Sonic Hedgehog在斑馬魚顱顏組織眼睛發育之功能

Functional Analyses of Transforming Growth Factor‐β3 (TGF‐β3) and Sonic Hedgehog (Shh) during Eye Development of Zebrafish by Transgenic Over-expression and Down-regulation Assays

黃嵩崴(Song-Wei Huang)

指導教授 ： 張百恩

國立臺灣大學/醫學院/口腔生物科學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.02740

摘要

Transforming growth factor-β family (TGF-β family)和Sonic Hedgehog在胚胎早期的發育中，扮演很重要的調控角色。以神經管發育為例，TGF-β family在神經管的背部大量表現，而Sonic Hedgehog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。

本研究想要探討TGF-β family和Sonic Hedgehog在眼睛的發育中是否一樣扮演如此重要的角色。之前的研究中指出，TGF-β3會大量表現在斑馬魚的水晶體中，因此我以TGF-β3為研究對象並構築了pCr1.3-TGF-β3-full-length-IRES-hrGFP以及pCr1.3-TGF-β3-antisense- IRES-hrGFP結構體，並利用顯微注射方法將這兩種結構體注入斑馬魚受精卵中。透過斑馬魚βB1-Crystallin 1.3 kb啟動子驅動TGF-β3- full-length cDNA或TGF-β3-antisense cDNA在水晶體專一表現mRNA，使得TGF-β3蛋白過量表現或者抑制內生性TGF-β3蛋白表現，並藉由綠色螢光蛋白之表現觀察得知此轉殖斑馬魚帶有欲表現的結構體。

結果在TGF-β3過量表現方面，篩選到六個轉殖恆定品系分別為4號、5號、9號、10號、11號、21號，其中以4號水晶體綠色螢光蛋白表現最均勻而且強度最強。因此我仔細觀察4號的眼睛外表型，發現瞳孔有變小的現象，並將其受精後7天的水晶體取下，發現水晶體比野生型小，而且二級纖維細胞也尚未形成。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。至於在抑制內生性TGF-β3蛋白表現方面，結果只篩選到一個轉殖恆定品系23號，而且此恆定品系水晶體綠色螢光蛋白表現非常微弱。在眼睛外表型的觀察也與野生型沒有太大的差別，可能是由於表現量不足，使外表型變化不大;另外也可能由於TGF-β family中的其他成員有類似於TGF-β3的作用，因此產生基因功能上的重疊互補性(redundancy)。

而在Sonic Hedgehog方面，利用先前的研究結果(王偉庭, 2005)-藉由βB1晶體蛋白(βB1-crystallin)啟動子在斑馬魚水晶體異位過量表現Shh。藉由觀察其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，不同於野生型呈杯狀。進一步利用組織切片分析，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並沒有顯著的影響。

因此，從目前實驗結果推測TGF-β3以及Shh可能對於斑馬魚眼睛組織扮演不同的調控角色。TGF-β3可能會影響水晶體的發育，甚至與白內障的形成有關。而Shh則對視網膜細胞的增生有顯著的影響。但是更詳細之相關機制必須利用in situ hybridization、immunohisto -chemistry等方法才能對TGF-β3或Shh如何影響眼睛發育有更完整的了解。

關鍵字

斑馬魚 ； 眼睛發育 ； 過量表現 ； 抑制方法

並列摘要

Transforming growth factor-β family (TGF-β family) and Sonic Hedgehog play critical roles in regulating 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.

The aim of this study was to elucidate the function of TGF-β family and Sonic Hedgehog in the eye development of zebrafish. In the previous study, TGF-β3 has been shown to be expressed strongly in the lens of zebrafish. Therefore, I have constructed the pCr1.3-TGF-β3-full-length- IRES-hrGFP and pCr1.3-TGF-β3-antisense-IRES-hrGFP chimeric genes, and these two constructs were micro-injected into the zebrafish eggs. The lens-specific βB1-crystallin promoter drives the expression of TGF-β3- full-length and TGF-β3-antisense cDNA in the lens. In this way, TGF-β3 can be over-expressed or downregulated in the lens, and simultaneously the transgenic fish containing these constructs can be screened by the expression of GFP.

Six stable transgenic zebrafish lines were obtained (No.4, No.5, No.9, No.10, No.11, and No.21) with the pCr1.3-TGF-β3-full-length- IRES- hrGFP construct. The expression of GFP was evenly distributed in the lens of the No.4 stable line, the strongest among the TGF-β3-full- length stable lines. I found the pupil of the No.4 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. Moreover, the secondary lens fibers were not formed normally. In addition to the abnormal lens size, I also observed that there was a cloudy and opaque region in the eye lens.

As regarding antisense approach, only one TGF-β3-antisense stable transgenic zebrafish line was obtained (No.23), and the expression of GFP in the lens of this stable line was very weak. It is probably that the expression of TGF-β3-antisense was not sufficient to affect the phenotype of the lens. An alternative explanation is that the absence of a conspicuous phenotype may reflect the functional redundancy of the TGF-β family in the lens.

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, whereas the stratification of neural retina appears normal in the line No.19.

In the preliminary results, TGF-β family and Shh may play different roles in regulating the eye development of zebrafish. TGF-β3 could influence the lens development, even implicated in the occurrence of cataract. And Shh may affect the proliferation of neural retina cells. In the future, in situ hybridization and other methods may be applied to elucidate the more detailed function of TGF-β3 and Shh during the eye development of zebrafish.

並列關鍵字

TGF‐β3 ； Shh ； Over-expression ； Down-regulation ； Zebrafish

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