Gene Expression in mESC Deficient in FGFR Signalling and the Identification of the Novel Gene Ened

Sammanfattning: Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of blastocyst stage embryos, and upon differentiation into embryoid bodies (EBs) they recapitulate the genetic, cellular and morphological events occurring in early embryogenesis. Among several signalling pathways and inductive factors, the fibroblast growth factors (FGFs) and their receptors (FGFRs) have been shown to be important during early embryogenesis. Previously it has been shown that ESCs expressing a mutant form of FGFR failed to form the two characteristic cell layers of EBs: the endoderm and the ectoderm. In addition, due to the impaired FGF/FGFR signalling, the mutant EBs failed to synthesize laminin-111 and collagen type IV, the main building blocks of the basement membrane (BM) protein network. The aim of this thesis was to elucidate the molecular and biological changes occurring due to impaired FGF/FGFR signalling in differentiating EBs. A large scale microarray study was performed that provided insights into the global gene expression changes occurring in both wild-type (normal) and FGF/FGFR signalling deficient EBs, which resulted in an extensive catalogues of significantly expressed genes in both cell lines. Results showed that during wild-type EB differentiation a rapid down-regulation of pluripotency related genes and up-regulation of genes related to morphogenesis and development occurred. Analysis of FGF/FGFR deficient EBs showed a significant decrease of genes encoding endodermal and BM related proteins. In addition, an increase of mesodermal and pluripotency related gene transcripts was observed. Such transcripts are normally activated later during EB development. The assumption was that the most significantly decreased genes could be involved in FGF/FGFR signalling. To further test this theory, we chose to elucidate the expression pattern of the hitherto uncharacterized gene 1110032E23Rik whose expression was significantly decreased in FGF/FGFR mutant EBs. By in situ hybridization analysis, the expression of 1110032E23Rik was examined in both mouse and Xenopus laevis (frog) embryonic development. Results showed that the expression of 1110032E23Rik in mouse embryos was restricted to epithelia such as the lining of the gastrointestinal tract, urogenital tract, heart, skin and lens, and in addition to the peripheral nervous tissues. Expression analysis of the mouse 1110032E23Rik orthologue in frog embryos showed that mice and frogs share several expression domains such as the eye, heart, kidney and epidermis. The expression was observed at locations where active FGF/FGFR signalling takes place and BMs are present. Based on the expression pattern observed in mice, we named this uncharacterized gene Ened (Expressed in Nerve and Epithelium during Development).