The Zebrafish dll4 Mutation Causes Defects in Lymphatic Vessels and Intestine
Date Issued
2007
Date
2007
Author(s)
Young, Hui-Sheng
DOI
zh-TW
Abstract
Abstract
We use the transgenic zebrafish TG(fli1：EGFP)y1 that express GFP in vessel endothelial cells to study the function of dll4 (delta-like 4) gene in vascular development. Previous research suggested that dll4j16e1 is a dominant mutation causing disconnected arteries in the caudal fins of adult fish. The dll4/dll4 homozygotes, however, died between 7 to 14 days post fertilization without evident deficiencies in vessels. Hence, my project is to identify causes to the embryonic lethality of dll4/dll4 homozygotes.
We discovered abnormalities in the lymphatic and intestinal vessels of dll4/dll4 mutants. In dll4/dll4 homozygote embryos, most of the lymphatic vessels appear fragmental or even absence, while less than 10% of them developed lymphatic vessels seemly without lumens. It is known that VEGF-C and Prox1 are indispensable for lymphatic vessel development in the mouse. Therefore, we next investigated the interplay between Prox1, VEGF-C, and dll4. First, we tested whether VEGF-C or Prox1 are also required for lymphatic vessel development in zebrafish by using morpholino knockdown. The results show more than 90% of VEGF-C or prox1 morpholino injected zebrafish embryos develop no lymphatic vessels. Besides, over-expression of VEGF-C in zebrafish embryo by microinjecting the plasmid containing VEGF-C cDNA resulted in excess lymphatic endothelial cells and enlarged lymphatic vessels. These data demonstrated that both VEGF-C and Prox1 play important roles in zebraifsh lymphatic vessel development. We next tested whether overexpression of VEGF-C would rescue Prox1 MO. While almost 99% embryos developed no lymphatic vessels in Prox1-MO alone; 62% of VEGF-C; prox1 MO develop normal lymphatic vessels, suggesting VEGF-C might function downstream to Prox-1. Using the same methods, we further found that dll4 can rescue the lymphatic defects of Prox1-MO and VEGF-C-MO. In contrast, the overexpression of Prox1 or VEGF-C could not rescue the dll4/dll4 lymphatic vessels phenotypes. Accordingly, we proposed that Prox1 might act upstream of VEGF-C followed by dll4 during the development of lymphatic vessels. In consistent with this idea, we found the expression of VEGF-C and Prox1 by QPCR is not significantly altered in dll4/dll4 embryos. Interestingly, the Prox1 expression level is elevated in Prox1 MO suggesting a negative feedback regulation by Prox1 itself.
In dll4/dll4 intestine, the intestinal vessels appear larger. In light of the recent finding that delta-notch signaling controls the intestinal cell fate specification, we went on to examine whether dll4 mutation also causes cell fate defects. Using the early marker for intestinal differentiation cdx-1b gene we showed normal pattern of cdx-1b in dll4 mutants. Interestingly, the expressions of a series of delta/notch genes suggested there were cell fate changes happened in the middle and late intestinal differentiate progression. Finally, the expression of IFABP (instestinal fatty acid binding protein) gene detected by in situ and QPCR is not significantly different between wt and dll4/dll4 embryos suggesting normal function in fatty acid absorption in dll4/dll4.
We conclude that dll4 mediated Delta-Notch signaling also plays a role in lymphatic vessel development which is downstream to Prox1 and VEGF-C. dll4 might involve in the cell fate differentiation of intestinal epithelial cells. It is likely that the defects in lymphatic vessels and intestine cause the embryonic lethality of dll4/dll4 zebrafish embryos.
Subjects
轉基因斑馬魚
淋巴管
腸道血管
細胞分化命運
Transgenic zebrafish
delta-like 4
lymphatic vessel
intestinal vessel
cell fate
Type
other
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