A Cascade of Wnt, Eda, and Shh Signaling Is Essential for Touch Dome Merkel Cell Development

<div><p>The Sonic hedgehog (Shh) signaling pathway regulates developmental, homeostatic, and repair processes throughout the body. In the skin, touch domes develop in tandem with primary hair follicles and contain sensory Merkel cells. The developmental signaling requirements for touch dome specification are largely unknown. We found dermal Wnt signaling and subsequent epidermal Eda/Edar signaling promoted Merkel cell morphogenesis by inducing Shh expression in early follicles. Lineage-specific gene deletions revealed intraepithelial Shh signaling was necessary for Merkel cell specification. Additionally, a Shh signaling agonist was sufficient to rescue Merkel cell differentiation in Edar-deficient skin. Moreover, Merkel cells formed in Fgf20 mutant skin where primary hair formation was defective but Shh production was preserved. Although developmentally associated with hair follicles, fate mapping demonstrated Merkel cells primarily originated outside the hair follicle lineage. These findings suggest that touch dome development requires Wnt-dependent mesenchymal signals to establish reciprocal signaling within the developing ectoderm, including Eda signaling to primary hair placodes and ultimately Shh signaling from primary follicles to extrafollicular Merkel cell progenitors. Shh signaling often demonstrates pleiotropic effects within a structure over time. In postnatal skin, Shh is known to regulate the self-renewal, but not the differentiation, of touch dome stem cells. Our findings relate the varied effects of Shh in the touch dome to the ligand source, with locally produced Shh acting as a morphogen essential for lineage specification during development and neural Shh regulating postnatal touch dome stem cell maintenance.</p></div

Dermal β-catenin is required for TD MC formation.

<p>(A) Schematic diagram of P0 mouse skin illustrating the relationship of TD MCs to primary hair follicles viewed en face from above the skin surface and on parasagittal section of dorsal trunk skin. (B) En face image of K8 whole mount staining in control (<i>En1</i>^<i>Cre/+</i><i>; β-catenin</i>^{<i>flox/+</i>}) and <i>En1</i>^<i>Cre/+</i><i>; β-catenin</i>^{<i>flox/Δ</i>} dorsal trunk skin at E17.5. *, staining in the periderm. (C) H&E staining in control and <i>En1</i>^<i>Cre/+</i><i>; β-catenin</i>^{<i>flox/Δ</i>} mouse dorsal trunk skin at E17.5. Scale bars, 50 μm.</p

Intraepithelial Shh signaling is required for TD MC development.

<p>(A) K8 and K17 whole mount staining in control and <i>K14-Cre; Shh</i>^{<i>flox/flox</i>} skin at P0. (B) K8 and K17 section staining in control and <i>K14-Cre; Shh</i>^{<i>flox/flox</i>} skin at P0. Arrowhead, K17+ TD keratinocytes. Arrow, K17+ hair follicle. Pentagon, abortive K17+ hair germ. (C) H&E staining in sections of control and <i>K14-Cre; Shh</i>^{<i>flox/flox</i>} skin at P0. (D) K8 and K17 whole mount staining in control and <i>K14-Cre; Shh</i>^{<i>flox/flox</i>} skin at E15.5. (E) Confocal maximum projection oblique view of Sox2 whole mount staining in control and <i>K14-Cre; Shh</i>^{<i>flox/flox</i>} skin at E15.5. Red circle, epidermal surface over primary hair germ. Green outline, dermal papilla. (F) K8 whole mount staining in control and <i>K5-tTA; TRE-Cre; Smo</i>^{<i>flox/flox</i>} skin at E18.5. Scale bars, 50 μm.</p

Edar is necessary for TD MC formation.

<p>(A) K8 whole mount staining in control (<i>Edar</i>^{<i>dl-J/+</i>}) and <i>Edar</i>^{<i>dl-J/dl-J</i>} dorsal trunk skin at P0. (B) H&E staining in sections of control and <i>Edar</i>^{<i>dl-J/dl-J</i>} dorsal trunk skin at P0. (C) X-gal whole mount staining in <i>Atoh1</i>^{<i>LacZ/+</i>} and <i>Edar</i>^{<i>dl-J/dl-J</i>}; <i>Atoh1</i>^{<i>LacZ/+</i>} dorsal trunk skin at 2 months old. (D) RT-PCR relative expression levels (mean ± SD) in control and <i>Edar</i>^{<i>dl-J/dl-J</i>} dorsal trunk skin at E15.5. **, <i>p</i><0.01. Scale bars: A & B, 50 μm; C, 0.5 mm.</p

Shh is essential for TD MC development.

<p>(A) K8 whole mount staining in control (<i>Shh</i>^{<i>GFPcre/+</i>}) and <i>Shh</i>^{<i>GFPcre/GFPcre</i>} skin at E18.5. (B) K8 and K17 staining in sections of control and <i>Shh</i>^{<i>GFPcre/GFPcre</i>} skin at E18.5. *, periderm and unspecific epidermal staining. Arrowhead, K8+ Merkel cell (C) H&E staining in sections of control and <i>Shh</i>^{<i>GFPcre/GFPcre</i>} skin at E18.5.(D) RT-PCR relative expression levels (mean ± SD) in skin from E13.5 <i>Edar</i>^{<i>dl-J/+</i>} and <i>Edar</i>^{<i>dl-J/dl-J</i>} embryos cultured for 2 days in the absence or presence of the Smo agonist Hh-Ag1.5. #, undetectable. **, <i>p</i><0.01, compare to untreated. Scale bars, 50 μm.</p

Proposed model of the developmental signaling requirements for TD MC production.

<p>(left) Model of the signaling cascade that regulates the development of TD MCs and their adjacent guard hair follicle, along with experimental interventions that disrupted or rescued the developmental processes. (right) Graphical representation of the sequential signaling mechanisms that drive TD MC specification in embryonic mouse skin. TD MC specification requires mesenchymal Wnt signaling which allows Eda signaling to Edar in the developing primary hair placodes and subsequent Shh signaling from the primary hair placodes/germs to adjacent ectoderm containing the TD anlages. Square cells, surface ectoderm. Trapezoidal cells, hair placode/germ cells. Oval cells, periderm.</p