Fig 2: Lrig1 and Lrig2-ßgeo are co-expressed in the non-sensory region of the cochlea.Transverse sections through Lrig2+/- tissue at E12.5 (A), E16.5 (B), and P15 (C) were double labeled with antibodies to Lrig1, ß-galactosidase, and NF. (A) At E12.5, staining was evident in the non-sensory region of the cochlear epithelium (asterisk) and the mesenchyme surrounding the spiral ganglion. (B) At E16.5, Lrig1 was detected in the medial wall of the cochlea, which will form the inner sulcus and Reissner's membrane (asterisk). (C) At P15, Lrig1 was found in the base of Reissner's membrane (asterisk), with localization to the cell surface (inset). In contrast, at E12.5 and 16.5, Lrig2-ßgeo was found broadly in the cochlear epithelium and surrounding mesenchyme (A'–B'). At P15, expression was enriched in spiral ganglion neurons and in the organ of Corti (C'). cd = cochlear duct, m = mesenchyme, oC = organ of Corti, rm = Reissner's membrane, sg = spiral ganglion. Scale bar = 40 µm.

Fig 3: JunB expression during skin maturation and upon stress. a Representative microphotographs with double immunostaining for JunB (green) and FABP5 (red), indicative of sebaceous glands, in skin derived from 1, 3, and 5 days old mice. Inset showing magnified view of a sebaceous gland. Scale bars, 50 µm. b Immunostaining of JunB (green) and FABP5 (red) in 60 days old mice. Inset showing magnified view. c Representative microphotographs with double immunostaining of JunB (green) and FABP5 (red) in dorsal skin of 60 days old mice following hair plucking or d after topical TPA application, a potent inducer of proliferation. e Immunostaining of JunB (green) and LRIG1 or CD34 (red) in dorsal skin of 60 days old mice following hair plucking. f Immunostaining of JunB (green) and FABP5 (red) in adult human skin. E, epidermis; D, dermis; HF, hair follicle; SG, sebaceous gland; SD, sebaceous duct; HS, hair shaft. Asterisk indicates hair shaft autofluorescence. Dashed line indicates the epidermal-dermal junction. Scale bars, 50 µm

Fig 4: Repairing Intestinal Epithelium Has a Cellular and Molecular Profile Distinct from the Normal Epithelium(A) Macroscopic images of the distal part of the colon in an untreated mouse (left) and a mouse 2 weeks post-administration of DSS (right). The demarcated area indicates regions undergoing active re-epithelialization. Scale bar, 2 mm.(B) H&E staining of homeostatic tissue and tissue in the repair phase at 2 weeks following DSS administration. Scale bar, 100 µm.(C) The mucosal/submucosal thickness at homeostasis and repair phase (2 weeks). Shown are mean distances ± SEM (n = 4 animals; p = 0.007 based on two-sided Student’s t test).(D) Sca1 (green) expression in colonic epithelium in homeostatic and repair phase. Sections are counterstained with DAPI (blue). The demarcated line indicates the epithelial structure. Scale bar, 100 µm.(E) Flow cytometric analysis of cells from the colonic epithelium during homeostasis and in repair phase (2 weeks post-administration of DSS). Diagrams show representative plots for Sca1-PECy5 in live CD45-veCD31-veEpcam+ve cell population.(F) Quantification of the percentages of Sca1high cells in homeostasis and repair phase. Diagram shows average ± SEM (n = 3; p = 4 × 10-5 based on two-sided Student’s t test).(G) qRT-PCR analyses in Epcam+Sca1high cells sorted via FACS from the repairing epithelium and in homeostatic epithelial cells (Epcam+). Bars represent average levels ± SEM (n = 3; Ly6a, p = 1.3 × 10-4; Reg3b, p = 9.7 × 10-3; Reg3g, p = 8.5 × 10-3 based on two-sided Student’s t test).(H) Heatmap analysis of differentially expressed probe sets (fold change > 2.0; FDR < 0.05), comparing the expression profile of epithelial cells isolated from homeostatic tissue and Sca1high cells from the repairing epithelium.(I) Detection of Lrig1 (green) in homeostasis, early and late repair phase counterstained for E-cadherin (red) with DAPI (blue). Scale bar, 50 µm.(J) GSEA of repairing and homeostatic epithelium using Lgr5 intestinal stem cell gene signature.(K) Organoid formation for Epcam+ cells isolated from normal homeostasis, as well as Epcam+ Sca1low and Sca1high cells during tissue repair showing representative images of formed organoids. Bars represent average number of organoids formed ± SEM (n = 3 animals).See also Figure S1 and Table S1.

Fig 5: Cochlear structure and patterning is normal in Lrig1-/-;Lrig2-/- double mutants but cochlear innervation is disrupted.(A–C) Cochlear tissue from adult animals was immunostained and then imaged as a flat-mount by confocal microscopy. In controls (A), neurofilament (NF)-positive neurites from both afferent and efferent neurons align in distinct radial bundles. Efferent projections also travel non-radially along the length of the cochlea in the inner spiral bundle (ISB) (bracket in A). The regular spacing normally found between these axonal bundles (A, arrowheads) is disrupted in Lrig1-/-;Lrig2-/- double mutants (C, arrowheads). In addition, the ISB is reduced (brackets). Innervation was grossly normal in Lrig1-/-;Lrig2+/- animals. (D–F) Efferent axons and their terminals were visualized by staining for Choline acetyltransferase (ChAT) (D–F and D'–F') and synaptophysin (Syp) (D?–F?). High power images of the boxed regions illustrate the obvious reduction in efferent innervation in double mutants (F', F?) compared to controls (D', D?). Lrig1-/-;Lrig2+/- animals showed an intermediate effect (E', E?). ISB = inner spiral bundle, oC = organ of Corti, sg = spiral ganglion. See Figure S4 for images of additional genotypes. (A–F) Scale bar = 50 µm. (D'–F?) Scale bar = 10 µm.