The notch signalling pathway and mesenchymal cell fates during wool follicle initiation in the sheep

Wool follicle development is initiated during foetal life through signals exchanged between the mesenchyme and epithelium. Follicle primordia are initially visible as condensations of dermal prepapilla cells, and are associated with epithelial thickenings. The dermal papilla is believed to secrete signalling molecules that direct the rate of cell division in surrounding bulb cells in the mature wool follicle. This determines the amount of fibre grown. It has been hypothesised that the dermal papilla cell population of the skin is finite and is determined at follicle initiation in the foetus. It is thought that this population defines the total quantity of fibre producing tissue in the mature sheep. The developmental mechanisms that specify this cell population and control their distribution in the growing follicle population is not known. There is however, evidence implicating the Notch signalling pathway in foetal skin development. Some Notch signalling pathway genes have been identified in ovine skin and other tissue, including the incomplete sequences of Notch-1, Jagged-1, Delta—1 and Delta-4. This study reports on the roles of some key Notch genes prior to (d 56) and during (d 70) primary follicle initiation in Merino and Tukidale lines of sheep. Tukidale animals were chosen as a strong-woolled comparison to the fine-woolled Merino, due to their markedly different follicle densities and fibre characteristics. Different experimental procedures were utilised to provide a more thorough understanding of the role of the Notch signalling pathway in wool follicle initiation in sheep. DIG-labelled riboprobes for Delta-1, Delta-3 and Lunatic Fringe were generated from murine cDNA sequences and a PCR-generated ovine cDNA probe for Notch-1 provided information on gene expression using in situ hybridisation studies. Transcripts were detected in sectioned foetal skin prior to and during follicle development. In skin sampled prior to follicle initiation, Delta-l transcripts were detected throughout the epithelium and in some mesenchymal cells. Notch-l was detected in the epithelium, particularly in the basal layer, and in some mesenchymal cells. In skin sampled during follicle initiation, Delta-1 was expressed in the epithlium, basal layer of the epithelium and in some mesenchymal cells. Some Notch-l transcripts were detected in the epithelium, with staining appearing stronger in the basal layer. Notc-l transcripts were also detected in a few cells within the epithelial plug, in some mesenchymal cells and in the dermal condensates in the cells around the periphery of this structure. Counts of Delta-1 and Notch-1 labelled mesenchymal and dermal condensate cells were conducted. An interesting finding of this work was that no significant differences were detected between the numbers of cells labelled for Delta—1 or Notch-1 when the Merino and Tukidale skin samples were compared. Antibodies were used for immunohistochemistry studies in sectioned foetal sheep skin collected from Merino and Tukidale foetuses. Delta—1, Notch-1 and activated Notch protein expression were detected in skin samples. The behaviour of dermal papilla cells was investigated in vitro. An aggregating cell model whereby papilla cells were grown on the extracellular matrix complex MatrigelTM was developed. The cell cultures were shown to aggregate prior to reaching confluency, with the cells being observed over a 31 day period. A floating foetal sheep skin culture system was devised whereby skins were cultured on collagen-coated membranes for 4 days. Cell and skin cultures were examined in the presence and absence of the y-secretase inhibitor DAPT, which was dissolved in DMSO and added to the culture media. Dermal papilla cell aggregation was abolished in all dermal papilla cell cultures in the presence of 50uM DAPT. The formation of new follicle buds within skin harvested on day 70 of foetal development and cultured for 4 days was hindered. An investigation in to the effect of treatment with SOuM DAPT on follicle diameter in day 70 Merino skin cultures suggested that DMSO and DAPT had some effect on follicle diameter, when compared to the untreated controls. The results of this project have provided new insights into the mechanisms by which the wool follicle population is established in a foetus and suggest that some Notch signalling pathway genes are involved in the specification of prepapilla cell fate at follicle initiation