Figure 1.
Hair Follicle Morphogenesis and Differential Expression of Lef1-RFP and K14-H2BGFP in All Cells of the Mature Follicle
(A) Schematic depicts follicle morphogenesis, which begins in waves in mouse backskin at approximately embryonic day 15 (~E15) and is complete at approximately postnatal day 4 (~P4).
(B) Transgene constructs and mice. The transgenes used for injection are shown in the stick diagrams. Nucleotide residues encompassing the gene fragments cloned are noted (+1 is the transcription initiation site). For each transgene, three transgenic lines were engineered; all were phenotypically normal. Data shown are from P4 backskin follicles of mice harboring both transgenes. Left and middle images are phase contrast and epifluorescence images, respectively, of hair follicles after dispase and collagenase treatment. Right image shows 3D reconstruction of a confocal Z-stack of a follicle showing that most of the RFP resides in the center of the follicle bulb. DP, dermal papilla; Mc, melanocytes; Md, medulla; Mx, matrix; ORS, outer root sheath.
(C) Section of K14-H2BGFP follicle, which at P4 (shown) is fully mature. Images shown are DAPI and epifluorescence channels, separately, and merged. Note the approximately 3-fold higher levels of GFP in ORS nuclei (arrows) compared to Mx nuclei and its progeny. White lines demarcate the mesenchymal–epithelial boundaries, which are separated by a basement membrane of ECM.
(D) Four-color confocal image of a section of a K14-H2BGFP and Lef1-RFP double transgenic follicle at P4. In addition to H2BGFP (green) and RFP (red) epifluorescence, the follicle was labeled with DAPI (blue) and Abs against tyrosinase (secondary Abs are against Cy5 in the far-red and white was used as a pseudocolor). Note that the anti-tyrosinase antibody labels the RFP-positive Mc and demarcates them from the RFP-positive, anti- tyrosinase negative DP. Note also that Mc are located on the epithelial side of the basement membrane, denoted by the white lines.
Figure 2.
Isolation and Purification of Mx, ORS, DP, DF, and Mc Populations
(A) Schematic of isolation procedure. After removing subcutaneous fat by dissection, and epidermis/upper follicle segment by enzymatic digestion, single-cell suspensions were prepared from pure dermis and subjected to three FACS schemes to purify five populations of cells: Mx, GFPlowRFP−; ORS, GFPhighRFP−; DP, RFPhighGFP−CD34−CD45−CD117−; DF, RFP−GFP−CD34−CD45−CD117−; Mc, RFPhighGFP−CD117+.
(B) Immunofluorescence analyses of FACS isolated cell populations. Frozen skin sections (hair bulb) and relevant cytospin populations were stained with Abs as color-coded and indicated. At the right of each set is quantification of percentage of cells that expressed the marker. Note: ~10% of DP and DF cells lysed on cytospin. and hence did not stain with any markers. β4, β4 integrin; Tyr, tyrosinase; Vim, vimentin; white line, basement membrane.
(C) RT-PCR: cDNA fragments were resolved by agarose gel electrophoresis, and the gene detected is denoted at left. All fragments were of the expected size. Expression of Msx2, vimentin, and β4 in multiple populations was later confirmed.
(D) Cell cycle differences in cell populations. Profiles of the five purified populations were performed by FACS. Anti-BrdU immunofluorescence is from a P4 backskin follicle from a mouse injected intraperitoneally with 50 μg/g 5-bromo-2′-deoxyuridine (BrdU) (Sigma-Aldrich) and analyzed 4 h later. Note greatest incorporation in Mx and ORS.
Figure 3.
Gene Expression Patterns of the Five Hair Follicle/Skin Populations
(A) Overall correlation of gene expression profiles between different cell types. A high correlation coefficient was observed between the DP-DF and Mx-ORS populations, consistent with their mesenchymal versus epithelial characters. Intriguingly, the least similar populations were the Mx and DP, which are strongly engaged in reciprocal exchange of signaling necessary for the maintenance of both compartments. Inset: Dendrogram outlining the correlation of gene expression between the populations.
(B,C) The Venn diagram demonstrates the degree of similarity based on absolute present calls. Probe-sets were considered present only if they were called present in both replicates. Note that for each cell type, ~200–400 probe-sets showed selective hybridization under these criteria, while ~2/3 of all present probe-sets were represented in at least four or all five of the fractions and ~4,000 genes (~6,000 probe-sets) were expressed and unchanged in all five populations (“Molecular backbone”). When all five fractions were cross-compared, ~150–300 genes (“Molecular signatures”) scored as being upregulated by ≥ 2× selectively in only cell type. The lists show array predictions for differential expression of mRNAs encoding some established, defining markers for each cell type, when compared against an adjacent or related cell population. The total number of mRNAs that show at least a 2× difference between the two cell populations is provided at the top, and the fold difference in specific mRNA levels is given next to each marker. DF, dermal fraction; DP, dermal papilla; Mc, melanocytes; Mx, matrix; ORS, outer root sheath.
Figure 4.
GO Analyses and Functional Grouping of the Molecular Backbone and Signatures
(A) GO analyses of ~4,000 genes present and unchanged in all five fractions irrespective of lineage or cell type. Shown is the percentage of genes in a given GO category, compared to all genes of the signature of a given cell type. Note that the genes were enriched mostly in categories involved in basic cell functions representing the molecular backbone. Asterisks denote a significant increase over a whole genome prediction. NC, not changed.
(B) GO analyses of the molecular signatures. The signature was defined as the genes whose expression was upregulated by ≥ 2× in only one of the five hair/backskin populations. Each signature was categorized into groups of genes depending upon their putative cellular functions. Shown is the percentage of genes in a given GO category, compared to all genes of the signature of a given cell type. Asterisks denote a significant increase over a whole genome prediction.
(C) The molecular signatures. The gene abbreviations and/or accession numbers are according to the NCBI listings. # denotes genes implicated in skin/hair disorders. (P) denotes genes with appreciable signal but higher levels in one of the other four populations. For multiple genes in a signature, the abbreviation is listed once, followed by –x, where x is the specific gene number.
Figure 5.
Implementation of Array Analyses to Examine Characteristics and Dynamics of the Follicle DP Niche
(A) Semi-quantitative RT-PCR on mRNAs isolated from each population. Shown are representative data from molecular signature genes (see Figure 4) whose expression patterns in the DP niche environment had been previously uncharacterized. In this case, categories were consolidated into three groups: Signal transduction, transcription/nuclear, and cytoskeleton/ECM/cell adhesion. For each primer set, at least three different cycles were employed, and the resulting cDNA fragments were resolved by agarose gel electrophoresis along with DNA size markers to confirm that bands were of the expected sizes. For each gene, the data presented were from the cycle that provided the most meaningful comparisons. Note: bands seen in > 1 fraction accurately reflect mRNA expression at the differences in levels shown.
(B) Immunohistochemistry and in situ hybridizations. Skin sections were taken from 2-mo-old K14-GFPactin mice [49] whose follicles were at the transition from the resting to growing (telogen to anagen) stage of the hair cycle (8 wk) or from P4 WT mice (full anagen follicles) (all others). Sections 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).
Figure 6.
Detailed Expression Analysis of Hair/Skin Disease Genes Found in the Molecular Signatures of the Epithelial and Mesenchymal Populations of the Hair Bulb
Real-time PCR confirmation of 24 different signature genes of the Mx, ORS, or DP, which have previously been implicated in genetic disorders of the hair. Many of these genes have not been well-studied at the level of expression and function. In each case, the highest level of mRNA expressed corresponded to the cell population in which the signature gene appeared. Moreover, in cases where more than one cell population showed appreciable mRNA levels, this was also reflected in our microarray comparisons.
Figure 7.
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.