Journal: PLoS Biology
Article Title: Molecular Dissection of Mesenchymal-Epithelial Interactions in the Hair FollicleMolecular Signatures of the Developing Hair Follicle
Figure Lengend Snippet: Neuronal and Neural Crest Genes Expressed by the DP and Hair Follicles (A) Semi-quantitative RT-PCRs were conducted as in the legend to Figure 5 , except in this case, we used oligonucleotides against neuronal and/or neural crest expressed genes. In all cases, these genes either appeared on the DP molecular signature or scored as expressed by the DP as well as one or more of the other four skin populations. (see Figure 4 ) Shown are representative RT-PCR data, which show an excellent correlation with the DP-preferred expression pattern of the majority of these neural genes. (B) Immunohistochemistry and in situ hybridizations of neuronal/neural crest genes in skin. Sections of P4 backskins were subjected to either immunofluorescence using color-coded Abs as indicated or in situ hybridization using the indicated biotinylated cRNA probes (sense controls were negative). Merged images of serial sections were used to compare Ab (red) and in situ (pseudogreen) patterns. Gfra1 , glial derived neurotrophic factor receptor1; Mdk , midkine; Prss12 , serine protease neurotrypsin; Tyr, tyrosinase. (C) Detection of neuronal/neural crest genes in highly enriched hair follicle preparations. Highly enriched follicle preparations were isolated by serial low-speed centrifugation following dispase and collagenase digestion of P4 backskins. After isolation and preparation of their mRNAs, semi-quantitative RT-PCR was conducted using oligonucleotides to those neuronal and neural crest markers that were found in the DP signature. As controls, oligonucleotides were used against Akp2 , Alx4 , Bmp6 , and Fgf7 , which are all markers that we mapped to DP by in situ hybridizations and/or immunofluorescence (see Figure 5 ). Note that the neuronal/neural crest genes appearing on the DP signature showed comparable signals to the documented DP genes.
Article Snippet: DP were obtained after first depleting Mc (CD117+ ), lymphocytes (CD45+ ), and endothelial cells (CD34+ ) with biotinylated Abs (BD Pharmingen, San Diego, California, United States)/magnetic anti-biotin microbeads (Miltenyi Biotec, Bergish Gladbach, Germany), and then selecting for RFPhigh GFP− cells in the FACS.
Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemistry, In Situ, Immunofluorescence, In Situ Hybridization, Derivative Assay, Isolation, Centrifugation, Quantitative RT-PCR