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Proliferationsuntersuchungen zur Entwicklung des Lymphgefäßsystems bei Wnt5a-transgenen Mäusen

Analysis of proliferation on the development of the lymphatic system of Wnt5a-gene-modified mice

by Anna Haarmann
Doctoral thesis
Date of Examination:2018-05-07
Date of issue:2018-04-27
Advisor:Prof. Dr. Jörg Wilting
Referee:Prof. Dr. Jörg Wilting
Referee:Prof. Dr. Claudia Binder
crossref-logoPersistent Address: http://dx.doi.org/10.53846/goediss-6844

 

 

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Abstract

English

Wnt5a is a secreted glycoprotein and represents one of the 19 members of the human WNT-gene-family, which regulate embryonic development. In contrast to most other members of the Wnt-family, Wnt5a also is a regulator of embryonic angiogenesis, but is expressed at higher levels in lymphatic endothelial cells (LECs) as compared to blood vascular endothelium. Wnt5a also has important roles for the development of other organs, and homozygous Wnt5a-knock-out (ko) mice die immediately after birth. Here, I studied the impact of Wnt5a on the development of the lymphatic vascular system of the dermis and analyzed mouse embryos of different genotypes: wild-type mice (+/+), heterozygous Wnt5a-ko-mice (+/-) and homozygous Wnt5a-ko-mice (-/-). I studied mice at embryonic days (ED) 12.5, 14.5, 16.5 and 18.5. Thereby, my focus was the proliferation of the LECs, because previous studies on ED18.5 mice have not been able to clearly discriminate if Wnt5a regulates the morphogenesis of the initial lymphatic vessels in the dermis or the proliferation of LECs. With immuno-fluorescence techniques I analyzed large numbers of serial cryo-sections of the embryos. Thereby, antibodies against the transcription factor Prox1 were used to identify the nucleus of LECs, anti-Ki67 was a marker for proliferating cells, and DAPI was used as a pan-nuclear marker. Ki67-positive and -negative LECs were counted with a microscope at 40-fold magnification, and the results were expressed as percent values. Statistical analyses were performed with One-way Anova and the Post-hoc-test Tukey. There were no statistically significant differences in the number of proliferating LECs between the three genotypes at any developmental stage. The most active phase of lymphangiogenesis in murine embryos is at ED14.5 with 55% of proliferating LECs. Already at ED 16.5 the lymphatic vessels of the homozygous Wnt5a-ko-embryos were clearly deformed. This leads to the conclusion that Wnt5a controls the morphogenesis of dermal lymphatic vessels during embryonic development, but not proliferation. Wnt5a might regulate migration of LECs, their polarity, or elongation during vascular network formation.
Keywords: Wnt5a
 

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