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|Title:||ADP-Ribosylation Factor-like 6 Interacting Protein在斑馬魚眼睛發育功能的新發現|
Novel Function of ADP-Ribosylation Factor-like 6 Interacting Protein in the Ocular Development of Zebrafish Embryos
|Publication Year :||2008|
|Abstract:||ADP-ribosylation factor-like 6 interacting protein (Arl6ip) 是母體效應基因，在原腸胚期前的斑馬魚胚胎中有全身性表現，發育到咽期後則會局部表現在特定組織；在Arl6ip缺失的斑馬魚中可以觀察到斑馬魚腦部、眼睛、心臟、肌肉有明顯的發育不良，特別是眼睛，出現小眼睛及視網膜細胞分化不良的情形。過去的研究認為Arl6ip與細胞的分化有密切關係，但它在發育上的功能卻完全未知；於是我們利用斑馬魚在發育生物學上的優勢來觀察眼部發育相關基因的表現情形，進而探討Arl6ip在眼睛發育時期調控的分子機制。當Arl6ip缺失時，早期決定眼部區域的基因表現受到影響，包括shh在頭部過量表現，以及rx1與pax6表現量降低，並且pax2高量表現於視柄，這都導致眼部區域的縮小。而在視網膜形成時期，觀察與視網膜發育相關的基因之表現情形，發現Arl6ip缺失後，斑馬魚胚胎視網膜細胞不能正常表現shh、six3a及six6等基因，代表視網膜細胞分化的過程受到影響；而HuC、neurolin及HNK-1等蛋白在視網膜的表現也都受到抑制，另外感光受體專一表現的基因crx也出現缺失，僅有少量訊號出現在視網膜腹側，顯示視網膜結構的確沒有出現；再者，利用deltaC標定視網膜前驅細胞，也可發現Arl6ip缺失後，眼部區域的細胞的確大量處於命運未被決定的形態。最後觀察眼部區域細胞增生的情形，我們也發現在Arl6ip缺失的斑馬魚胚胎中，眼部區域有大量BrdU的訊號，並且細胞大量表現cyclin D1，而不表現p57kip2，說明這些細胞無法離開細胞週期且無法進行分化，而處於早期形態。總結，當Arl6ip缺失時，斑馬魚視網膜的前驅細胞的分化會受到影響，使眼睛的發育停留在早期，並且這群眼睛的前驅細胞又因為細胞週期無法被停止而持續進行分裂；因此，我們認為Arl6ip影響視網膜前驅細胞離開細胞週期，並且擔任了眼部區域及視網膜細胞分化與成熟過程的重要功能。|
ADP-ribosylation factor-like 6 interacting protein (Arl6ip) was a maternally inherited gene in zebrafish, and it was expressed throughout the whole embryo before the gastrula period and restrictedly expressed in certain tissues after the pharyngula period. As losing Arl6ip, many phenotypic defects occurred in many tissues of the zebrafish embryo, such as brain, eyes, heart, and trunk. In particular, the occurrence of microphthalmos of eye and incomplete differentiation of retina were found. According to previous researches, Arl6ip was thought closely related to cell differentiation, but its biological function in development was unknow. Here we confer the molecular mechanism that Arl6ip involved in by observing the expression of related genes releated to zeberfish eyes development. First, we found that the expressions of early patterning genes were affected while losing Arl6ip, including overexpression of shh mRNA in the head, down-regulated of rx1 and pax6 mRNA in the eye field, and elevated expression of pax2 mRNA in optic stalk. These change in gene expression all result in the reduction of eye field. Then, we focued on the expressions of specific genes as retinal development. As losing Arl6ip, zebrafish embryos could not express shh, six3a, and six6 normally; that affected the differentiating processes of retina progenitor. Therefore, we could observe the specific proteins such as HuC, Neurolin, and HNK-1 were repressed in retina, and the photoreceptor specific gene crx was crowded at the ventral site of eyes. Furthermore, we labeled the cells in the eye field with deltaC and confirmed the cell fate of retinal progenitor kept indetermination while losing Arl6ip. Finally, we examined the level of cell proliferation in the eye field. While losing Arl6ip, we could observe the BrdU signals were higher in the eye, and the retinal progenitor kept expressing cyclin D1 but not p57kip2, suggesting the eye progenitor cell stayed as an early progenitor and could not exit cell cycle to process differentiation. Thus, inhibition of Arl6ip leads to arrest eye development at the early stage. These mal-functional eye progenitors keep proliferating and continually express early marker gene. Taken together, we conclude that arl6ip not only affects the signals controlling eye development, but also plays an important role in cell differentiation.
|Appears in Collections:||分子與細胞生物學研究所|
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