Role of notch-delta signalling in cell fate determination during cutaneous epithelial differentiation

The notch signalling pathway is essential for the development and growth of all mammalian cells. The canonical pathway works by cell to cell communication and influences gene expression directly. Notch signalling plays an important role in cell differentiation processes in the embryo as well as in adult tissues. In Drosophila, signalling requires a single notch receptor and two ligands (delta and serrate). However, in mammals signalling is more complex and there are four notch receptors (NOTCH1-4), three delta like ligands (DLL1, DLL3, DLL4) and two jagged ligands (JAG1, JAG2). The interaction between a notch receptor at the cell surface and its ligand on a neighbouring cell, leads to sequential proteolytic cleavages which release the notch intracellular domain (NICD) from the cell surface receptor (Estrach et al., 2008). The NICD enters the nucleus and forms a complex with CSL and Mastermind to activate target genes (HES and HEY family members). Notch receptors have modified EGF-like repeats where fucose has been added to a serine or threonine residue and this O-linked fucose can be elongated by the action of an enzyme (fringe), enhancing or blocking ligand binding. There have been extensive studies on notch signalling in embryos but less is known in adult tissues, especially with regard to non-canonical pathways. The aim of this project was to examine the expression of notch receptors, ligands and specific target genes during epidermal differentiation in human skin. This was examined both in human epidermis (in vivo) as well as in culture models using HaCaT, a keratinocyte cell line. Examining notch signalling activity at different stages of keratinocyte differentiation could help to understand the precise role of notch signalling in cell fate determination during normal cutaneous epithelial cell growth and development, and may indicate what role, if any notch signalling might play in the pathology of skin disease. Most of the work on delta ligands has been done in embryonic tissues with little work on adult skin. Thus, our approach focused on expression of DLL1, DLL3 and DLL4 in human epidermis and cultured HaCaT cells. DLL1 expression was confirmed (mRNA and protein level including sequence) but no convincing evidence of DLL3 or DLL4 expression was found. Evidence was also obtained for JAG1 and JAG2 expression in human epidermis and cultured cells, and there was some indication that differential expression might occur during terminal differentiation. RT-PCR results indicated possible changes in notch ligand levels with calcium-induced differentiation in HaCaT cells and these were quantitated by real time PCR (qPCR). Expression levels could not be normalised to a housekeeping gene (β-Actin, human ARP, human TF2H, GRP58, B2H, RPL13 and TBP) as all those tested were not stable enough. Thus, cDNA probes for two notch receptors (N1 and N3), three ligands (DLL1, JAG1 and JAG2), six responsive genes (HES1, HES5, HES7, HEY1, HEY2 and HEYL) and two keratin genes (KRT14, KRT10) were cloned and used to construct standard curves. This allowed quantitative estimation of expression levels in terms of copy number and fold change in both HaCaT cells and human epidermis. K14, Notch1 and DLL1 changed very little in HaCaT cells but K10, Notch3, JAG1, JAG2 and HES1 levels increased with differentiation. It was also concluded that signalling via Notch3 and JAG1 may influence the progress of terminal differentiation in human keratinocytes.