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Role of the notch pathway in regulating cell fate in human epidermis

Student thesis: Doctoral ThesisDoctor of Philosophy

The Notch signalling pathway plays an essential role in skin homeostasis and disease. Previous studies have shown that distinct ligands and receptors are expressed in different layers of human epidermis, suggesting separate roles for each ligand. In vivo, expression of the Notch Delta-Like Ligand 1 (Dll1) is confined to the stem cell compartment in the interfollicular epidermis (IFE). In vitro, cells overexpressing Dll1 can induce neighbouring cells to differentiate into corneocytes. Since expression of this ligand is low, both in vivo and in vitro, it is unknown as to what extent Dll1 is in involved in IFE homeostasis. 
In this thesis, I explore how different keratinocytes subpopulations express Notch pathway components, showing that JAG1 and 2 are highly expressed compared with Dll1. In order to explore the importance of JAG1 and 2, I exposed keratinocytes to these ligands and showed that both can independently induce differentiation. To mimic the IFE stem cell compartment, I exposed keratinocytes overexpressing Dll1 to recombinant JAG1 and 2, observing a reduction in differentiation when compared with control. This data reinforces the hypothesis that DLL1 could be acting on stem cells inducing cis-inhibition and preventing these cells from differentiating. 
To address how keratinocytes can discriminate and respond to distinct ligands I investigated regulators of Notch signalling. Using a sc-RNAseq dataset, I identified fringe proteins as potential modulators of the Notch pathway in human keratinocytes. Fringe proteins are glycosyltransferases that transfer N-acetylglucosamine to O-linked fucose residues on Notch EGF repeats, changing the affinity of these receptors to Notch ligands. Mammals have three different fringe proteins: lunatic (LFNG), manic (MFNG) and radical fringe (MFNG). High expression of LFNG and RFNG was observed in culture. In vitro, overexpression of LFNG showed a decrease in Notch signalling and differentiation, and increased proliferation. Moreover, knock- down using siRNA decreased differentiation, but a reduction in the proliferative potential was observed. 
My findings imply that Dll1 induces cis-inhibition in human keratinocytes. Hence, I suggest that LFNG play a role in modulating Notch signalling in IFE increasing cis-inhibition of Notch and inducing undifferentiated cells to receive signals from Dll1 positive cells.
Original languageEnglish
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Award date1 Mar 2020

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