Wnt signaling maintains the hair-inducing activity of the dermal papilla - PubMed (original) (raw)

. 2000 May 15;14(10):1181-5.

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Wnt signaling maintains the hair-inducing activity of the dermal papilla

J Kishimoto et al. Genes Dev. 2000.

Abstract

The formation of the hair follicle and its cyclical growth, quiescence, and regeneration depend on reciprocal signaling between its epidermal and dermal components. The dermal organizing center, the dermal papilla (DP), regulates development of the epidermal follicle and is dependent on signals from the epidermis for its development and maintenance. GFP specifically expressed in DP cells of a transgenic mouse was used to purify this population and study the signals required to maintain it. We demonstrate that specific Wnts, but not Sonic hedgehog (Shh), maintain anagen-phase gene expression in vitro and hair inductive activity in a skin reconstitution assay.

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Figures

Figure 1

Figure 1

DP cells lose anagen-specific gene expression in culture. (A) Structure of the hair follicle. The dermal papilla (DP, green) is located at the bottom of the follicle, surrounded by undifferentiated epidermal matrix cells (MC, blue) that give rise to precursor cells (PC, red) and, ultimately, the differentiated hair shaft (HS, brown) and cuticle (CU, yellow). The hair shaft and DP are also surrounded by root sheath cells (RSC, orange) and the connective tissue sheath (CTS, black). (B) Isolated transgenic anagen hair follicles show GFP fluorescence in the DP. (C) Flow cytometric analysis of GFP expression in DP cells immediately after isolation or after 90 hr in culture. The horizontal line demarcates the gate defining GFP positive and negative subpopulations of DP cells.

Figure 2

Figure 2

DP cells respond to Shh. (A) After dissociation of newborn skin, RT–PCR revealed Shh mRNA in the GFP-negative population (HF) that includes the follicular epithelium. Shh mRNA was not detected in the GFP-positive population (DP). β-actin (β-act) serves to compare input RNA levels in this and subsequent panels. (B) ptc and Gli1 are expressed in freshly isolated DP cells, but transcript levels decrease upon passage in culture (P3), as do those from the GFP transgene.

Figure 3

Figure 3

Wnt signaling to DP cells maintains hair inductive activity. (A) Flow cytometric analysis of DP cells cocultured with feeder cells producing Wnt3a or Shh, or infected with a control vector. The horizontal line demarcates the division between GFP-positive (green) and -negative (red) DP cells. The feeder cells found below the lower gate were excluded from this analysis. (B) The ratio of GFP-positive to GFP-negative cells is shown for each treatment. (C) RT–PCR analysis of cocultured DP cells. GFP mRNA levels were maintained by exposure to Wnt3a but not Shh or control feeder layers. ptc and Gli1 expression was induced in DP cells cocultured with Shh-expressing feeder cells. DP cells cultured with control feeders that do not express Shh or feeders expressing Wnt3a express low levels of ptc and undetectable levels of Gli1. (D) Wnt3a-treated cells maintain hair inductive activity. Three weeks after grafting to nude mouse hosts, only occasional hairs were observed in grafts of control or Shh-treated DP cells. DP cells exposed to Wnt3a directed formation of a dense patch of hair in the graft.

Figure 4

Figure 4

Expression of Wnts and their signal transduction cascade in the hair follicle. (A) Wnts 3a, 4, 5a, and 7a were expressed in the GFP-negative population from the initial sort that includes follicular epithelial cells from anagen hair follicles (HF). Note that the Wnt4 reaction was extended an additional six cycles to detect lower transcript levels. Of the Wnts analyzed, only Wnt5a was detected in the GFP positive population (DP). (B) Expression of genes encoding the Wnt signal transduction cascade was analyzed in DP cells immediately after isolation (DP) or after three passages in the absence of other cell types or exogenous factors (P3). frizzled7 (fz7), disheveled2 (dsv2), GSK3, β-catenin (β-cat), and Lef1 were all expressed in freshly isolated DP cells. After three passages, expression of frizzled7, disheveled2, and Lef1 was reduced.

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