Characterising Crim1 in Vertebrate Development

This thesis investigates the role of Crim1, a transmembrane protein that is expressed in a number of areas in the vertebrate embryo including the developing kidney, eye, testis and spinal cord, which we believe may be a regulator of vertebrate tissue development. To dissect the function of Crim1 in normal mammalian development, two vertebrate models were used, zebrafish and mice. The results show that in zebrafish, crim1 is expressed early in development from the 16-cell stage through to 30 hours post fertilisation (Chapter 3). At 24 hours post fertilisation crim1 is expressed in the intermediate cell mass (icm), the site of haemangioblast development. Haemangioblasts are precursor cells that contribute to the formation of the blood and endothelial cell lineages. Injection of crim1 antisense oligonucleotides into zebrafish embryos (crim1 morphants) lead to an expansion of the icm and defects in the trunk, tail, somites and vasculature. The injection of crim1 antisense oligonucleotides into transgenic fli:GFP zebrafish revealed defects in the intersegmental, dorsal longitudinal anastomotic and parachordal vessels. Although crim1 is expressed during haemagiogensis the primary defect in the crim1 morphant zebrafish appears to be vascular. Further experiments used a ‘knock-in’ mouse, Crim1KST264, in which a loss of functional Crim1 leads to defects in limb (syndactyly), skeleton, eye, vascular, kidney and placental development. Analysis of the kidney phenotype in the embryonic Crim1KST264 homozygotes showed that a loss of Crim1 affects ERK1/2 and phosphorylated-Smad1/5/8 protein expression, although has no direct effect on BMP or TGFβ protein expression (Chapter 4). Analysis of the adult Crim1 outbred kidneys revealed they have albuminuria and leaky vasculature. The complex phenotype presented by the Crim1KST264 homozygote kidneys suggests Crim1 may be regulating multiple growth factor pathways. As Crim1 was shown to be expressed in the placenta, we characterised the role of Crim1 in placental development using the Crim1KST264 mouse (Chapter 5). Crim1KST264 homozygote placentas and embryos are smaller than their wild-type littermates. Our investigations revealed that Crim1 is expressed in trophoblast giant cells and in spongiotrophoblasts. A loss of Crim1 causes a developmental defect in that the junctional zone (region of the placenta containing spongiotrophoblasts and glycogen cells) is expanded, although this phenotype does not appear to be due to a defect in proliferation or apoptosis. Further analysis of E15.5 Crim1KST264 homozygote placentas revealed there was a reduction in the number of labyrinth trophoblast gaint cells. Thus, by using zebrafish and mouse as two model organisms of vertebrate development, this thesis has showed that Crim1 is clearly important for normal embryonic development. To dissect the complex phenotype presented by the Crim1KST264 mouse, further studies of Crim1 and its interaction with other growth factor pathways is needed to elucidate how and to what extent they interact with Crim1 to determine its biological effect on vertebrate tissue