Molecular characterisation of primary wool follicle initiation in Merino sheep.

Abstract

Primary wool follicles are initiated in the skin of sheep foetuses at approximately day 50 of gestation as the result of complex reciprocal molecular interactions between the mesenchyme and overlying epithelium. The lifetime wool production potential and fibre diameter of the Merino sheep is dependent on the total number of follicles initiated in utero. Understanding the molecular events that surround primary wool follicle initiation may provide approaches to enhance or manipulate this process in order to maximise the profitability of wool production enterprises. In order to study the morphological and molecular changes occurring during early wool follicle development, a foetal skin series spanning primary follicle initiation was generated. Foetal skin was sampled from the shoulder, midside and rump of four foetuses at 8 time points between day 43 and day 68 of gestation. Histological characterisation of the shoulder skin samples revealed that primary epidermal placodes emerged at around day 53, dermal condensates were visible from day 57 and downgrowth of the follicle began at day 68. An equation relating age of the foetus (day of gestation post AI) and crown-rump length, specific to Merino foetuses, was developed for use in future studies of this nature. Molecular markers of fibroblast migration, epidermal and dermal stem cells and cell proliferation were selected to test the hypothesis that dermal condensates are initiated at discrete sites beneath the epidermis as a result of a combination of migration and arrangement of multipotent pre-papilla cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of RAC1 and RHOa (migration markers), β1-integrin and alkaline phosphatase (stem cell markers), proliferative nuclear cell antigen and cyclinB1 (proliferation markers), patched-1, selected tumor necrosis factor (TNF) signalling molecules and eleven reference genes was conducted using midside and rump skin samples from each of four foetuses from the 8 time points. geNorm analysis of the reference and target genes revealed that the migration markers RAC1 and RHOa along with GAPDH were the most stably expressed genes in this sample series. Significant changes in mRNA expression were detected for β1-integrin, alkaline phosphatase, patched-1 and the TNF members EDA, EDAR, TROY and TRAF6. Many of these significant differences in expression coincided with key morphological events. Significant differences in expression were also detected between the midside and rump samples for numerous transcripts. Laser capture microdissection (LCM) was implemented for analysis of the target transcripts within particular structures of foetal sheep skin. Frozen tissue sectioning, staining, LCM, RNA extraction and cDNA synthesis were optimised for qRT-PCR analysis of endogenous controls and selected TNF transcripts. Several RNA extraction methods and reverse transcription approaches were trialled to ensure optimum extraction and reverse transcription efficiency for this tissue type. Exogenous mRNA transcripts were also incorporated prior to RNA extraction and reverse transcription to track reaction efficiency between samples. A comparison of different slide types revealed that laser pressure catapulting from membrane slides was an absolute requirement for foetal skin tissue studies. Follicle regions (including the epidermal placode and dermal condensate) and the adjacent non-follicle regions were laser captured from foetal skin, and the mRNA expression levels of patched-1 and selected TNF members was compared. Preliminary qRT-PCR analysis using this technique revealed that EDAR, TROY and PTCH1 mRNA levels were higher in the follicle regions than the non-follicle regions. The TNF signalling pathway appears to play an important role in primary wool follicle initiation and patterning at different sites on the body. Spatial differences in expression of some of these regulators may be involved in initiating different types of follicles. The molecular events surrounding primary wool follicle initiation also show a high degree of conservation between sheep, humans, and mice. Considering the high degree of DNA sequence conservation as well as the histological, signalling and cycling similarities between sheep and humans, sheep may represent a better model for the study of human hair follicle initiation and disease than the currently used mice and rat models.