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
Date Issued
2006
Date
2006
Author(s)
Huang, Song-Wei
DOI
zh-TW
Abstract
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.
Subjects
斑馬魚
眼睛發育
過量表現
抑制方法
TGF8208
β3
Shh
Over-expression
Down-regulation
Zebrafish
Type
other
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