Cxcr4 is transiently expressed in both epithelial and mesenchymal compartments of nascent hair follicles but is not required for follicle formation

Abstract

Hair follicle (HF) morphogenesis relies on the coordinated exchange of signals between mesenchymal and epithelial compartments of embryonic skin. Chemokine receptor Cxcr4 expression was recently identified in dermal condensates (DCs) of nascent HFs, but its role in promoting HF morphogenesis remains unknown. Our analyses confirmed Cxcr4 expression in condensate cells, and additionally revealed transient Cxcr4 expression in incipient epithelial hair placodes. Placodal Cxcr4 appeared prior to detection in DCs, representing a switch of expression between epithelial and mesenchymal compartments. To explore the functional role of this receptor in both compartments for early HF formation, we conditionally ablated Cxcr4 with condensate-targeting Tbx18(cre) knock-in and epidermis-targeting Krt14-cre transgenic mice. Conditional knockouts for both crosses were viable throughout embryogenesis and into adulthood. Morphological and biochemical marker analyses revealed comparable numbers of HFs forming in knockout embryos compared to wild-type littermate controls in both cases, suggesting that neither dermal nor epithelial Cxcr4 expression is required for early HF morphogenesis. We conclude that Cxcr4 expression and chemokine signaling through this receptor in embryonic mouse skin is dispensable for HF formation.

Keywords: dermal papilla cells; hair follicle morphogenesis; hair follicle stem cells; mesenchymal-epithelial interactions; stem cell niche.

Figure 1. Cxcr4 expression in embryonic skin

(a–c) Immunofluorescence staining for CXCR4 shows differential expression in epithelial placodes (arrowheads) and mesenchymal dermal condensates (arrows) of forming HFs at E14.5, E16.5, and E18.5. LAMININ labels basement membranes; Dapi highlights nuclei. (d) qRT-PCR of FACS sorted cells at E14.5 shows high Cxcr4 expression in dermal condensates (“DC”), compared to embryonic dermis (“D”) and epidermis (“E”). Cxcr4 ligand chemokine Cxcl12 is expressed in mesenchymal cells. (e) Immunofluorescence of E14.0 skin confirms CXCR4 expression in EDAR⁺ placodes of earliest forming HFs. (f) EDAR (placode marker) and SOX2 (condensate marker) expression highlight the stage- and compartment-specific localization of CXCR4 expression. In more advanced stage 2 follicles (i) CXCR4 co-localizes with SOX2. In early stage 0 follicles (ii) CXCR4 co-localizes with EDAR. (g) Quantification of CXCR4 localization in early forming HFs by stage at E15.0 (mean and SD of 3 embryos). CXCR4 expression switches from epithelial to mesenchymal cells. Dotted line denotes basement membrane. Scale bar = 50µm.

Figure 2. Targeted ablation of Cxcr4 in placodes or condensates does not affect HF formation

(a) Schematic of Tbx18^cre and Cxcr4^fl/fl lines to target dermal condensates at E14.5 for Cxcr4 ablation. (b) Immunofluorescence staining confirms CXCR4 ablation at the protein level in condensates (arrows) at E14.5 and E16.5 stage 1–2 follicles, while epithelial CXCR4 expression remains intact (arrowheads). (c) Quantification of first-wave HF numbers per field of view (FOV). Comparable HF numbers form in P10 cKO skin compared to controls. (d–f) Krt14-cre targets epidermal cells including the placode for Cxcr4 ablation. (e) Immunofluorescence staining confirms absence of CXCR4 in placodes (arrowheads) as early as E14.0 in cKO skin. Note expression in condensates is still present (arrows). (f) Comparable numbers of first-wave follicles form in P12 cKO skin compared to controls. (g–h) Hematoxylin/eosin staining of skin sections and macroscopic view of external hair shafts. Hair follicles and shafts form normally in both Tbx18^cre (g) and Krt14-cre (h) Cxcr4^fl/flcKO pups. c,f: Data are mean and SD of 2 WT and 2 cKO sex-matched littermates (–10 FOV). Dotted lines denote basement membranes. Scale bar = 50µm.