斑馬魚第四型酪胺酸磷酸水解酶對早期胚胎細胞移動能力之影響

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Title:	斑馬魚第四型酪胺酸磷酸水解酶對早期胚胎細胞移動能力之影響
Other Titles:	Functional analysis and its effects on cell migration of zebrafish ptp4a3 during early embryogenesis
Authors:	鄭凱文;Cheng, Kai-Wen
Contributors:	淡江大學化學學系碩士班陳曜鴻;Chen, Yau-Hung
Keywords:	斑馬魚;第三型酪胺酸磷酸水解酶;細胞移動;Zebrafish;ptp4a3;Cell;Migration
Date:	2011
Issue Date:	2011-12-28 18:11:29 (UTC+8)
Abstract:	第三型酪胺酸磷酸水解酶（ptp4a3）在各類的研究中指出與細胞的生長發育、增生以及癌細胞的轉移有關。然而其在胚胎早期發育上的功能卻仍不清楚，在本篇論文中我們利用斑馬魚做為模式物種來研究ptp4a3在早期胚胎發育上的生物功能。藉由原位雜交法我們可以發現斑馬魚的ptp4a3在單細胞時期就有所表現，而在胚胎的原腸期其訊號表現於胚環及胚盾上，在胚胎發育的後期其訊號則遍佈全身；包括腦部、體節、咽弓、嗅基板、視網膜以及blood island等。由這些觀察結果我們可以得知ptp4a3在斑馬魚早期胚胎上的重要性。此外，我們利用顯微注射施打了反股的morpholino藉以觀察ptp4a3缺失所造成的畸形。包括了胚胎外觀上由圓形變的狹長、卵黃因受力而腫脹向外凸出以及尾芽部分因細胞堆積形成的囊泡狀。這些缺陷的比例隨著morpholino的劑量而增減且能藉由注射ptp4a3的mRNA來進行補救回覆。為了進一步剖析ptp4a3表型的分子機制，我們使用了細胞移動的探針（gsc, hgg1, ntl）進行研究，且由結果發現了gsc及hgg1的訊號在胚胎腦部原基處難以聚集表現，而ntl訊號則是難以由胚盾處向上延伸。此外phalloidin的染色顯示了在下列兩種胚胎主要細胞的明顯缺陷，分別為胚胎細胞邊緣處的eYSL由纖維性肌動蛋白（F-actin）所構成的點狀寬帶以及EVL上細胞周圍的環狀F-actin訊號。由這些觀察結果可知ptp4a3會影響細胞移動且在斑馬魚早期胚胎發育的原腸期中扮演了非常重要的角色。 Protein tyrosine phosphatase 4a3 plays a role in the regulation of cell growth, proliferation and metastasis, but its function during early embryogenesis is still unclear. In this thesis, we used zebrafish model to study the biological function of ptp4a3 during early development. Whole mount in situ hybridization revealed that zebrafish ptp4a3 transcript was first observed at 1-cell-stage and extended its expression to germ ring and embryonic shield during gastrula period. Later, ptp4a3 signals were detected at head, somite, blood island, pharyngeal arch, olfactory placode, retina as well as at head muscle precursors. These observations highlight the importance of ptp4a3 during early development. Furthermore, we injected antisense morpholino (MO) to knockdown ptp4a3 expression for studying ptp4a3 loss-of-function phenotypes. Ptp4a3-morphant displayed several classic gastrulation defects, such as longitudinal shapes, swelling yolk and bubble-like tail bud vesicles. These defects were does-dependent and can be rescued by injection of ptp4a3 mRNA. To further dissect the molecular mechanism underlying ptp4a3-MO-induced gastrulation defects, three cell migration markers (gsc, hgg1 and ntl) were investigated. Results showed that gsc and hgg1failed to express at the head primordium, and ntl was difficult to extend from the embryonic shield. In addition, phalloidin stain showed that a significant reduction of the F-actin punctate band of the eYSL and the peripheral F-actin ring within the EVL in the ptp4a3-morphant. On the basis of these observations, we conclude that ptp4a3 affects cell migration and plays an important role during gastrulation period of zebrafish embryogenesis.
Appears in Collections:	[Graduate Institute & Department of Chemistry] Thesis

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