Abstract
Developmental genetics has been a field of interest for over a hundred years. We have set out to perform an ENU mutagenesis screen in the mouse at E10.5. In the first chapter the pathways and developmental processes described in this thesis are introduced. In the second chapter we describe the
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ENU-mutagenesis screen for developmental abnormalities at E10.5 that was the basis of this thesis. We identified a series of novel mutants and alleles that display diverse defects. Cardiac and nuchal edema, neural tube defects, situs-inversus of the heart, shorter anterior-posterior axis and the absence of limbs and lungs are examples of the abnormalities we identified. Due to the nature of the mutagen ENU that induces point mutations we identified both mutants with severe protein truncations and mutants with interesting amino acid substitutions that may tell us more about the functional domains in the proteins. In chapter three, two mutants identified in the ENU-screen are described. The Sec24b and Scribble mutants both have a remarkable similar phenotype; they show craniorachischisis, abnormal arrangement of outflow tract vessels and disturbed development of the cochlea. All are characteristics of defects in the planar cell polarity pathway. Sec24b is involved in the formation of COPII coated vesicles that are responsible for ER to golgi protein trafficking. We show that Sec24b in specifically crucial in the transport of the PCP protein Vangl2. Btaf1, a protein involved in RNAII-polymerase transcription and a negative regulator of TBP, is essential for embryonic development. In the 4th chapter of this thesis we describe a point mutation disrupting Btaf1 function. Btaf1V1330M mutants are characterized by delayed development starting around E8.25 and severe edema in the pericardial sac at E9.5. By western blot we show a reduction in Btaf1 protein levels at E8.5 and 9.5 likely due to a steric clash of Btaf1V1330M with the tryptophan at position 1340. Microarray analysis showed a higher number of down-regulated genes vs. up-regulated genes. Apob and the heart specific gene Titin are amongst the down regulated genes, homozygous mutants for either of the genes die around E10.5. We suggest that the down-regulation of these genes, amongst others, in Btaf1 mutant embryos may cause the lethality. In the chapter 5 we describe a mutant showing severe cardiac edema at E9.5. We identified a point mutation resulting in the substitution of a highly conserved asparagine by lysine at amino acid position 874. The affected gene encoding for the sodium calcium exchanger Ncx1 has been under a lot of investigation and is important for the excitation-contraction coupling in the heart. Knock-out phenotypes in the mouse exhibit highly similar defects compared to our mutant. We demonstrated intrinsic functional abnormalities of cardiomyocytes by ex vivo analysis. By western blot analysis and immunohistochemistry we demonstrated normal levels and subcellular localization of the mutant protein. This suggests that the abnormalities are not caused by a major disturbance of protein structure, but by reduced protein activity. Here we show the first in vivo model for the importance of amino acid N874 in sodium calcium exchange by Ncx1
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