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Abnormal Hair Development and Apparent Follicular Transformation to Mammary Gland in the Absence of Hedgehog Signaling

Summary We show that removing the Shh signal tranducer Smoothened from skin epithelium secondarily results in excess Shh levels in the mesenchyme. Moreover, the phenotypes we observe reflect decreased epithelial Shh signaling, yet increased mesenchymal Shh signaling. For example, the latter contributes to exuberant hair follicle (HF) induction, while the former depletes the resulting follicular stem cell niches. This disruption of the niche apparently also allows the remaining stem cells to initiate hair formation at inappropriate times. Thus, the temporal structure of the hair cycle may depend on the physical structure of the niche. Finally, we find that the ablation of epithelial Shh signaling results in unexpected transformations: the follicular outer root sheath takes on an epidermal character, and certain HFs disappear altogether, having adopted a strikingly mammary gland-like fate. Overall, our study uncovers a multifaceted function for Shh in sculpting and maintaining the integrity and identity of the developing HF.


The Heterotopic Glands (hg) Differentiate into Mammary Glands In Vitro and Express Mammary Gland Markers
After in vitro culture in the presence of 17β-estradiol and progesterone, the hg and mid-ventral skin explants generated well developed glandular ductal structures Both the hg associated with the mutant follicles and the hg that emerged from the IFE expressed genes that are normally expressed during mammary gland development, including Barx2, Sox9, Tbx3 (Krasner et al., 2000;Pask et al., 2002;Davenport et al., 2003) and Wnt7b (this study), and the expression levels were similar to those of control orthotopic mammary glands. Finally, similar to orthotopic mammary glands, the hg were devoid of Shh, Ptc1 and Ihh (Figures S6J-M and data not shown).

Absence of Shh and Absence of Major Skin Changes Following Partial Removal of Epithelial Smo Activity
Four to 6 weeks post-grafting, dorsal and ventral grafts from control fetuses produced normal furs with the ventral ones being less dense than the dorsal, consistent with  Figure S7M-N), some abnormal follicles developed in ventral skin grafts ( Figure S7O). Furthermore, in the latter, one graft displayed a hg arising directly from the epidermis ( Figure S7P).  Table S1).
Hybridization signals appear as black or bright grains in bright-field and dark-field images, respectively. In some panels, silver grains were pseudo-colored red using PhotoShop.

In Vitro Culture of Skin Biopsies
All culture media and supplements were purchased from Invitrogen, hormones from Sigma. Small pieces of dorsal and mid-ventral skin fragments (excluding mammary glands/ducts) were dissected out from 3-5 days post-partum ( in a humidified atmostphere of 5% CO 2 in air at 37°C and in the presence of α-MEM medium, 2 mM Glutamax, 10% fetal bovine serum and antibiotics. After 24 hours, the medium was changed into a serum-free basal medium, a mix (1:1) of Dulbecco's MEM medium and M-199 supplemented with 2 mM Glutamax, antibiotics, 2 µl/ml bovine pituitary extract, 40 µg/ml bovine transferrin, 5 µg/ml insulin, 150 µg/ml ascorbic acid, 0.2% BSA, 12 ng/ml EGF. After 1 or 2 weeks, the explants were cultured for an additional 2 or 3 weeks in the above basal medium supplemented with 1 ng/ml 17-β-estradiol and 1 µg/ml progesterone. After estrogen/progesterone treatments, some explants were cultured for an additional 1 or 2 weeks in a medium containing the lactogenic hormones prolactin (5 µg/ml) and dexamethasone (1 µM) and devoid of estrogen/progesterone. Cultured explants were processed for paraffin embedding, histology and in situ hybridization.

Skin Grafting
Full-thickness midventral and dorsal skin biopsies taken from 18.5 days post-coitum (dpc) embryos were grafted onto similar size (≈ 8 x 6-8 mm) back skin recipient sites in anesthesized male or female nu/nu immunodeficient mice. The grafts were protected by surgical tape and bandage which were removed one week post-surgery.

Supplemental References
Adolphe, C., Narang, M., Ellis, T., Wicking, C., Kaur, P., and Wainwright, B. J. (2004). An in vivo comparative study of Sonic, Desert and Indian Hedgehog reveals that Hedgehog pathway activity regulates epidermal stem cell homeostasis.   Dermal hypercellularity Abbreviations: dc/DP, dermal condensate(s)/dermal papilla(e); HF, hair follicle; IFE, interfollicular epidermis; ORS, outer root sheath; NdP, not determined but possible phenotypic manifestation; U; unclear due to delayed growth; (*) not applicable to de novo HF formation from the IFE at these early stages; (**), only rare follicles in grafts from ventral but not dorsal skin show the indicated phenotypic changes; (K17), outer root sheath phenotype determined only by histology and loss of Keratin17 expression; 18.5 dpc, analysis up to 18.5 dpc due to lethality at birth; 1dpp, analysis up to 1 dpp due to lethality 24 hours after birth.        Hair follicular fate is induced in the basal layer of the ectoderm by a putative dermal signal (signal 1), the hair placode induces (signal 2) dermal cells to aggregate into dermal condensates (dc, magenta). The dc are thereafter encased by the follicular epithelium as dermal papillae (DP). Hair follicle (HF) growth, morpho-genesis and differentiation depend on messages (signal 3) from the dc/DP and on intraepithelial signals. The hair matrix (dark green) at the base of the mature follicle (anagen 1) consists of highly proliferating cells. Upon differentiation, matrix cells generate the different layers of differentiating cells of the inner root sheath (IRS, light brown) and hair shaft (dark brown). The outer root sheath (ORS, red) is continuous with the basal layer of the interfollicular epidermis (IFE, dark blue).The companion layer (orange) of the ORS separates it from the IRS. The bulge region, home of follicular stem cells, is situated under the sebaceous gland (SG, yellow). The neural-crest-derived melanocytes (black) and their progenitors colonize the follicular epithelium. In the mature HF, melanocyte stem cells populate the bulge/sub-bulge (not represented) while their amplifying progeny and differentiated melanocytes are located in the upper matrix. The entire HF is surrounded by cells of the connective tissue sheath (CTS, violet). During catagen, the HF undergoes a progressive destructive phase culminating in the removal of its lower twothirds (cycling segment). After completion of catagen, the permanent segment of the HF enters the resting telogen phase. Subsequently, a new HF formative phase is initiated following proliferation of follicular stem cells in response to signals from the DP (not represented). Cells of the suprabasal layers of the epidermis, dc, DP and CTS are not , absence of development of a follicular stem cell niche and an impressive expansion of alkaline-phosphatase (APase)-positive dc/DP. The latter two defects are responsible for induction of de novo HF formation (nHF) from pre-existing follicles. The number of IRS and shaft precursors with nuclear β-catenin and P-Smad staining is decreased, whereas β-catenin protein accumulates at cell-cell borders in the HF epithelium and IFE (not represented). A subset of cells in the dc/DP shows peculiar nuclear β-catenin and P-Smad accumulation. As development proceeds, the mutant follicles convert into epidermal cysts filled with cornified debris (light blue). The IFE is hyperplastic, expresses Keratin 17 suprabasally and generates de novo HF (nHF) in response to mesenchymal cells. The rest of the dermis is hypercellular and contains ectopic melanocytes (black) and their progenitors (magenta). Only rare mutant follicles enter the telogen phase (not represented). (C and D) Shh signaling in control (C) and K14-Cre; Smo f/f (D) skins. Cells producing and responding to Shh (green). Cells responding to Shh (orange) and cells producing Shh (cyan; only in the mutants). (C) In control skin, Shh is expressed in a subset of follicular epithelial cells and signals both the HF epithelium and its associated mesenchyme (dc/DP/CTS). (D) Removal of Smo in the follicular epithelium results in abrogation of Shh responsiveness in the epithelium and increased orthotopic and ectopic Shh signaling (due to Shh protein spillover) in the dc/DP/CTS and in the dermis underlying the IFE (dIFE), respectively. (E) In the mutant ventral skin, in addition to the alterations shown in (B), some HF convert partially or totally into heterotopic glands (hg, violet) as a result of loss of epithelial Shh responsiveness and mesenchymal influences. (a) A hg that developed from the IFE at the expense of a HF. (b) A hg that developed from a hair germ. (c) A hg that developed from the hair matrix. (d) A hg that developed from the ORS. The hg express mammary-gland markers including Barx2, Tbx3, Wnt7b and Sox9, keratin 6 (K6) and keratin 8 (K8). (F) Shh signaling in the ventral skin. The hg and are devoid of Shh/Ihh, Ptc1 and Gli1 expression. Additional abbreviations: efc, ectopic follicular cells; K15, Keratin 15; LRC, label-retaining cells.