Fig 2: Rspo3GOF mice show changes in hair follicles formation, hair type occurrence and length. (A) Comparison of zigzag and awl hair shaft structure of Rspo3GOF and Control. (B) Comparison of length of different hair types from Control and Rspo3GOF mice. (C, D) Analysis of different hair types occurrence (C) and length (D) in Control and Rspo3GOF mice, conducted at P18. The values are mean ± standard deviation of a three replicates. Student’s t-test was used to test the significance: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Error bars ±SEM. Data are representative of at least three independent experiments. (E) Hematoxyline & Eosine (HE) staining of horizontally cut back skin from Control and Rspo3GOF mice at P0. Scale bar, 1,000 µm. (F) Analysis of hair follicle amount at P0 confirmed decreased hair follicle amount upon Rspo3 overexpression. Error bars ±SEM. Data are representative of at least three independent experiments. (G) HE back skin staining performed during the hair morphogenesis (E17.5; P0) show decreased hair amount and length in Rspo3GOF mice. Scale bar, 1,000 µm. (H, I) Immunofluorescence staining of Rspo3GOF and Control back skin at E17.5 targeting HFSCs markers Sox9 (H) and Lhx2 (I). DAPI counterstaining was used to label cell nuclei. Scale bars, 20 µm. (J, K) Immunofluorescence staining of Rspo3GOF and Control back skin at P0 targeting β-catenin (J) and pSmad1/5 (K). DAPI counterstaining was used to label cell nuclei. Scale bars, 20 µm.

Fig 3: Keratinocytes with Rspo3 overexpression exhibit decreased ability to proliferate and migrate. (A, B) Rspo3 overexpression in keratinocytes was confirmed by RT-qPCR analysis. Error bars ±SEM. Data are representative of at least three independent experiments (A) and Western blot (B). (C) RT-qPCR analysis depicting increased expression of Dkk3, Wnt5a, Wnt7a, Wnt7b, and decreased expression of Bmp4 in Rspo3GOF keratinocytes compared to Control. Results were normalized to β-actin. Error bars ±SEM. Data are representative of at least three independent experiments. (D) Representative pictures from Colony Formation Assay (CFA), performed on control and Rspo3GOF keratinocytes and taken after 9 days of culture. (E) CFA analysis revealed decreased ability of Rspo3GOF keratinocytes to formation of colonies. (F) CFA analysis showed increased size of colonies formed by Rspo3GOF keratinocytes compared to Control. Error bars ±SEM. Data are representative of at least three independent experiments. (G) Representative pictures of Control and Rspo3GOF keratinocytes from the Wound Healing assay, taken during different time points from the start (0, 4, 6, 24, 48 h).

Fig 4: Long-term Rspo3 overexpression sustained prolonged immaturity of bulge HFSCs in aged mice. (A) FACS sorting results depicting population of keratinocytes isolated from the whole epidermis of one-year old (1 year) Control and Rspo3GOF mice. The CD34+, α6-integrin positive cells represented 1% of the whole keratinocytes population in Control and 0.1% in Rspo3GOF mice. (B–D) Immunofluorescence staining of 1 yo Control and Rspo3GOF mice back skin, targeting HFSCs markers: CD34 (B), Sox9 (C), K15 (D), Lhx2 (E). DAPI counterstaining was used to label cell nuclei. Scale bars, 20 µm. (F) Alkaline phosphatase staining (purple) identified a presence of proper dermal papillae (DP) localization in 1 yo Rspo3GOF mice. Scale bar, 500 µm. Abbreviation: Bu, bulge; HG, hair germ.