All tissue is from P22 animals, and all images are z-stack projections of confocal images. A–B”’) Skin from Atoh1^LacZ/+ (A-A”’) and Atoh1^CKO (B-B”’) animals. Brackets denote a single touch dome. Subpanels in A-A”’ show Merkel cells (arrows) from the wildtype touch dome denoted by the bracket, while none are seen in the Atoh1^CKO animal (subpanels B-B”’). D – dermis, GH – guard hair. C–D”’) Hind foot pad from Atoh1^LacZ/+ (C-C”’) and Atoh1^CKO (D-D”’) animals. Brackets denote epidermal rete ridges, asterisks denote areas shown in insets, and arrows in (C-C”’) mark individual Merkel cells. Cells positive for Keratin 8 and β-galactosidase are absent from Atoh1^CKO body skin and foot pad. E–F”’) Whisker pad from Atoh1^LacZ/+ (E-E”’) and Atoh1^CKO (F-F”’) animals. There is no difference in the immunostaining patterns between the two genotypes. Dotted boxes outline areas shown in insets. HS – whisker hair shaft. All Keratin 8-positive cells were also β-galactosidase-positive and vice versa in all tissues of both genotypes. Scale bar: 25µm in all panels, 12.5 µm in subpanels and insets.

Wholemount X-gal staining of E16.5 embryos revealed β-galactosidase expression driven from the ROSA26^R26R (A-A”) or Atoh1^LacZ (B–C’) loci. A) Hoxb1^Cre/+; ROSA26^R26R embryo. Hoxb1^Cre expression is absent from the majority of the head. Boxes denote regions shown in (A’) and (A”). A’) and A”) Body skin and whisker pad, respectively, from a Hoxb1^Cre/+; ROSA26^R26R embryo counterstained with nuclear fast red. Staining is present throughout the epidermis and dermis of the body skin, while virtually no staining is seen in the whisker pad. Bracket in (A’) denotes a developing touchdome. WF – whisker follicle. B) Atoh1^LacZ/+ embryo showing β-galactosidase expression in the whisker pad (bracket) and touch domes of the skin. Box denotes region shown in (B’). B’) Hoxb1^+/+; Atoh1^LacZ/flox embryo body skin showing staining in individual touch domes. C) Atoh1^CKO embryo showing β-galactosidase expression driven from the Atoh1 locus in the whisker pad (bracket), but absent from touch domes of the skin (box). C’) Atoh1^CKO embryo body skin. Touch domes are present but lack the wildtype staining pattern. Scale bar: 5 mm (A, B, C); 400 µm (B’, C’); 100µm (A’, A”).

A, B) Touch dome sections from P22 Atoh1^LacZ/+ (A) and Atoh1^CKO (B) animals immunostained for NF200 (red) and VGLUT2 (green). Arrows mark nerve branches innervating the touch dome, white arrowheads mark nerve terminal branches contacting individual Merkel cells, and green arrowheads mark VGLUT2-positive, NF200-negative nerve terminal branches. Counterstain (blue) is TOTO3. Note the lack of cellular staining but increased nerve branching pattern in (B). GH – guard hair, TD – touch dome. C, D) In vivo FM 1–43 dye labeling of adult Atoh1^LacZ/+ (C) and Atoh1^CKO (D) touch domes. Dotted lines delineate touch dome boundaries. Arrows (C) show sensory nerve branches; arrowheads mark Merkel cells. Note the excessive sensory nerve branching but absence of Merkel cells in (D). Scale bar: 12.5µm. E) Semi-intact recordings from touch-sensitive afferents innervating hairy skin of wildtype mice. Top: Displacement trace showing a 5-s touch to the skin surface. Bottom: slowly-adapting type I (SAI) response to the 5-s mechanical stimulus shown above. F–I) Representative plots of instantaneous firing frequency vs. time for 5-s mechanical stimuli of wildtype afferent fibers. F) Aβ SAI fiber. G) Aβ slowly-adapting type II (SAII) fiber. H) Aδ down hair fiber (D-Hair). I) Aβ rapidly-adapting (RA) fiber. J, K) Atoh1^CKO fiber populations did not differ from wildtype littermates in mechanical sensitivity (by von Frey threshold, J) or conduction velocity (K) (Atoh1^CKO n=38, wildtype n=97; p>0.10, Mann-Whitney U-test). L) A survey of all mechanosensitive afferents revealed that fiber type proportions (Aβ, Aδ and C) were not significantly different in Atoh1^CKO mice compared to wildtype littermate control animals (p>0.10, Fisher exact test). M) Directed survey of Aβ-subtypes in Atoh1^CKO and control mice. No SAI responses were found among Aβ fibers in Atoh1^CKO mice (p<0.02, Fisher exact test). Number of fibers in (L) and (M) are shown in parentheses.