Functional analysis of wnk1 on angiogenesis in zebrafish
| Autor: | Lai, Ju-Geng, 賴如耕 |
|---|---|
| Rok vydání: | 2010 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 98 Wnk1 (With No lysine (K) 1) is a serine/threonine protein kinase. The increase of Wnk1 expression caused pseudohypoaldosteronism type 2 (PHA2), an autosomal-dominant disease featured by hypertension and hyperkalemia. By using gene disruption and rescue in mice, Dr. Huang’s lab at UT Southwestern Medical Center, Dallas showed that Wnk1 function is required for proper development of the cardiovascular system, especially the remodeling of vessels. To understand the detailed mechanism for the function of Wnk1 in angiogenesis, we took advantage of a fli1-EGFP transgenic fish line, which expresses green florescent protein in the vessel, as a model system to study the effect of knocking down wnk1. Using morpholino antisense oligonucleotide against wnk1, we observed that wnk1 morphant has defects in the growth of intersegmental vessels. Furthermore, wnk1 mRNA can rescue the defect of vascular formation caused by flk1 (vegfr2) (vascular endothelial growth factor receptor-2) or flt4 (vegfr3) (vascular endothelial growth factor receptor-3) knockdown. Moreover, the rescue effect disappears using the wnk1 mutants’ effect in the kinase domain or Akt/PI3K phosphorlation site. The rescue experiments provide evidence that wnk1 is a downstream target gene for vascular endothelial growth factor receptor -2 and 3, and it is downstream of Akt/PI3K affecting the angiogenesis in zebrafish embryos. Further testing whether knock-down of vascular endothelial growth factor receptor -2 (Flk1/VEGFR2) or vascular endothelial growth factor receptor -3 (Flt4/VEGFR3) will result in decreasing of wnk1 expression level. Further we found the wnk1 RNA level was decreased in flt4 or flk1 morphants was using in situ hybridization and qPCR analysis. However, this does not rule out the possibility of reduced phosphorlation of Wnk1 by Akt/PI3 kinase in flt4 and flk1 morphants, thereby reducing the Wnk1 activity. In this study, I analyzed and constructed the signal pathway how vascular endothelial growth factor receptor -2 (Flk1/VEGFR2) regulated to Wnk1, and explained the manner of Wnk1 activation and the mechanism of Wnk1 affected downstream substrate. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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