Tail Epidermis Wholemount Data for Git2 (MAKH)
Skin screen collaboration (Mice were fed on Mouse Breeder Diet (5021, Labdiet) from weaning): Tails were collected in duplicate from mutant and wild type mice of the same genetic background (≥ 14 weeks). Epidermal whole mounts were prepared by separating the epidermis from the underlying dermis as an intact sheet following EDTA digestion. The epidermis was fixed in 4% paraformaldehyde and permeabilised, then stained using a rapid labelling protocol with antibodies to keratin 14 and 15 (K14, K15) that were directly conjugated with fluorophores (Alexa 555 (red) for K14 and 488 (green) for K15). Whole mounts were counter-stained with DAPI to reveal cell nuclei (blue). Preparations’ were imaged using a Leica SP5 Confocal microscope and Maximum Intensity Projections produced from ~150um Z-series at 4um spacing. DAPI, Alexa 488 and Alexa555 channels were merged. Images are x10 magnification unless otherwise indicated.
Evaluation of Phenotype
A tick-list was used to record the presence (red cross) or absence (green tick) of a phenotype in the epidermis.
K14 is expressed in basal layer epidermal cells in the interfollicular epidermis (IFE), sebaceous gland (SG) and hair follicles (HF). DAPI staining provides an indication of cell density and K15 is most highly expressed in the HF bulge, which is a stem cell reservoir.
In tail epidermis melanocytes reside in the HF and also in the IFE immediately adjacent to the HF (parakeratotic scale). In some mutant mice pigmentation in the IFE is altered and this is recorded in bright-field images.
In the tick-list, abnormal K14 expression would indicate loss of expression. Abnormal K15 expression indicates either abnormal bulge morphology or altered K15 expression (for example, reduced in the bulge or up-regulated outside the bulge). In tail epidermis the HF are normally arranged in groups of three (triplets) and in staggered rows; abnormalities in HF patterning include changes in number of HF per group and distance between rows. The hair growth cycle in tail epidermis is less highly synchronised than in the back and the mice analysed are not closely age-matched. Therefore, detailed evaluation of the hair cycle has not been performed; nevertheless, obvious disturbance, such as all HF being in catagen, is recorded. The SG are K14-positive sack-like structures that lie above the bulge; changes in SG size, number or morphology are recorded as abnormal. Changes in the IFE include hyperproliferation (detected as increased basal layer density and enveloping of the junction with the HF), hypocellularity and altered pigmentation.
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The screen is being performed by Emma Heath in the laboratory of Fiona Watt, assisted by Elizabeth MacRae and Kifayathullah Liakath Ali. Our website is: http://www.bio.cam.ac.uk/~wattlab/. We gratefully acknowledge the financial support of Cancer Research UK, the Medical Research Council, the Wellcome Trust and the European Union. The references below are the original description of the whole mount tail epidermal preparation (Braun et al., 2003) and a review on different epidermal stem cell populations (Watt and Jensen, 2009).
Braun, K.M., Niemann,C., Jensen, U.B., Sundberg, J.P., Silva-Vargas, V. and Watt, F.M. (2003) Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in wholemounts of mouse epidermis. Development 130:5241-5255.
Watt, F.M. and Jensen, K.B. (2009). Epidermal stem cell diversity and quiescence. EMBO Molecular Medicine 1:260-267.