Foxi3 Deficiency Compromises Hair Follicle Stem Cell Specification and Activation

Abstract

The hair follicle is an ideal system to study stem cell specification and homeostasis due to its well characterized morphogenesis and stereotypic cycles of stem cell activation upon each hair cycle to produce a new hair shaft. The adult hair follicle stem cell niche consists of two distinct populations, the bulge and the more activation-prone secondary hair germ (HG). Hair follicle stem cells are set aside during early stages of morphogenesis. This process is known to depend on the Sox9 transcription factor, but otherwise the establishment of the hair follicle stem cell niche is poorly understood. Here, we show that that mutation of Foxi3, a Forkhead family transcription factor mutated in several hairless dog breeds, compromises stem cell specification. Further, loss of Foxi3 impedes hair follicle downgrowth and progression of the hair cycle. Genome-wide profiling revealed a number of downstream effectors of Foxi3 including transcription factors with a recognized function in hair follicle stem cells such as Lhx2, Runx1, and Nfatc1, suggesting that the Foxi3 mutant phenotype results from simultaneous downregulation of several stem cell signature genes. We show that Foxi3 displays a highly dynamic expression pattern during hair morphogenesis and cycling, and identify Foxi3 as a novel secondary HG marker. Absence of Foxi3 results in poor hair regeneration upon hair plucking, and a sparse fur phenotype in unperturbed mice that exacerbates with age, caused by impaired secondary HG activation leading to progressive depletion of stem cells. Thus, Foxi3 regulates multiple aspects of hair follicle development and homeostasis. Stem Cells 2016;34:1896-1908.

Keywords: Ectodermal dysplasia; Hair follicle stem cells; Morphogenesis; Shh.

Figure 1

Foxi3 displays a dynamic expression pattern during hair morphogenesis and cycling. Radioactive in situ hybridization (left column) and immunostaining (right column) for Foxi3 at indicated stages of hair morphogenesis and cycle. (A): Foxi3 was detected in epithelial cells of the hair placode. (B, C): In more advanced follicles, Foxi3 was detected in the lower part of the hair peg and later only in cells in the center of the follicle above the matrix likely representing the hair shaft precursors. Note absence of protein expression in the upper part of the outer root sheath (pre-bulge) (arrowhead). (D): Foxi3 was no longer detected in fully formed hair follicles. (E): Foxi3 reappeared at catagen, in the cells of the regressing epithelial strand. (F): At telogen, Foxi3 was restricted exclusively to the cells of the hair germ between bulge and dermal papilla. (G): At the onset of anagen Foxi3 expression expanded into the growing portion of the follicle. Unspecific staining observed in the hair shaft is indicated by a star. Scale bar=50 µm. Abbreviations: E=embryonic day; P=postnatal day.

Figure 2

Foxi3 expression colocalizes with stem cell markers only at the beginning of hair morphogenesis and later in telogen follicle secondary hair germs. Co-localization of Foxi3 (red) with stem cell markers Lhx2, Nfatc1, and Sox9 (green) at different stages of hair morphogenesis and at the first telogen was analysed by double immunostaining. (A): Foxi3 was co-expressed with Lhx2 in hair placodes and in the leading front of the growing hair follicle. At advanced stages of development, Foxi3+ cells in the center were negative for Lhx2. At telogen, Foxi3+ cells in the hair germ also expressed Lhx2. (B): Nfatc1 was undetectable at the placode stage, but co-localized with Foxi3 at the hair peg stage (arrowhead). In advanced hair follicles (E18.5), Foxi3+ cells in the center were negative for Nfatc1, which was expressed in the pre-bulge region (arrowhead). At telogen, Nfatc1marked the quiescent SCs of the upper bulge and was not co-expressed with Foxi3 in the hair germ. (C): Some cells of the placode were positive for both Foxi3 and Sox9. At the hair germ stage, Sox9 did not co-localize with Foxi3+ cells at the leading front, but a small number of cells positive for both Foxi3 and Sox9 appeared in the middle of the growing hair peg. In addition, Sox9 was detected in the upper part of the follicle (arrowhead). At telogen, Foxi3+ cells in the hair germ were also positive for Sox9, but the latter was expressed also in the bulge. Scale bar=50 µm. Abbreviations: E=embryonic day; P=postnatal day.

Figure 3

Normal patterning but impaired downgrowth of hair follicles in Foxi3 KO embryos. (A): Primary hair follicle formation was not affected in Foxi3 KO, whole mount in situ hybridization using a probe specific for placode marker Dkk4. (B): Impaired invagination of hair buds in Foxi3 KO was detected in hematoxylin and eosin stained histological sections of E15.5 back skins. (C): The downgrowth defect of Foxi3 KO hair follicles was evident also at E18.5. (D): Dot plots of hair follicle length at late embryogenesis measured from Foxi3 KO (N=183 follicles from 5 embryos), and control littermates (N=178 follicles from 4 embryos); p=4.27 E-5. (E): Quantification of the number of hair follicles per optic field revealed decreased number of hair follicles in Foxi3 KO (n=62 OFs from 4 embryos; 575 hair follicles were scored) compared to control littermates (n=59 OFs from 4 embryos; 669 hair follicles were scored) at late embryogenesis; p=0.00093. (F): Quantification of percentage of BrdU+ cells per hair follicle at E15.5 showed decreased proliferation in Foxi3 KO (n=4, 48 hair follicles, 1271 cells scored) compared to control littermates (n=4, 48 hair follicles, 1610 cells scored), p=0.0026; no differences were found at E19.5 between Foxi3 KO (n=3 embryos, 82 HFs, 3217 cells scored) and control littermates (n=3 embryos, 70 hair follicles, 3079 cells scored). (G, H): BrdU staining in Foxi3 KO and littermate control embryos at E15.5 (G) and at E19.5 (H). (I): Impaired HF downgrowth in skin grafts of E18.5 Foxi3 KO (n= 5) compared to control explants (n=6) transplanted onto back skin of immunocompromised Nude mice, 10 days post grafting. (J): Macroscopic pictures at 25 days post grafting. (K): Expression of Lef1 was not affected in Foxi3 KO hair follicles at E18.5. Scale bar=50 µm. Data are shown as mean±SD. P-values were estimated using unpaired two-tailed Student t-test. Abbreviations: E=embryonic day; OF=optic field; HF=hair follicle; ns=non-significant (p>0.05).

Figure 4

Several SC signature genes were downregulated in Foxi3 KO hair follicles during morphogenesis. (A): qRT-PCR analysis of indicated genes (selected based on differential expression in the microarray) in8ä ä E15.5 epithelia isolated from Foxi3 KO and wild-type littermates. (n=6 per genotype; *p<0.05, related-samples Wilcoxon signed ranked test, IBM SPSS Statistics). (B, C): Expression of Lhx2 (B) or Nfatc1(C) was not detected in Foxi3 KO hair follicles at E15.5. (D, E): At later stages, Foxi3 KO hair follicles showed relatively normal staining for Lhx2 (D) and Sox9 (E). (F–H): In contrast, Nfatc1 and K15 were downregulated in Foxi3 KO embryos at E18.5-E19.5. However, K15 staining in the basal epidermis was indistinguishable in control and Foxi3 KO embryos. (F): Quantification of percent of Nfatc1+ and K15+ hair follicles per optic field in Foxi3 KO and control littermates at late morphogenesis. Nfatc1: Foxi3 KO (n=182 OFs from 3 embryos; 760 hair follicles were scored) and control littermates (n=164 OFs from 3 embryos; 881 hair follicles were scored); p=2.21 E-82. K15: Foxi3 KO (n=47 OFs from 3 embryos; 410 hair follicles were scored) and control littermates (n=42 OFs from 2 embryos; 502 hair follicles were scored), p=2.67 E-19. Data are shown as mean±SD. P-values were estimated using unpaired two-tailed Student t-test. (G, H): Representative images of Nfatc1 and K15 staining at late embryogenesis in control and Foxi3 KO hair follicles. Scale bar=50 µm. Abbreviations: E=embryonic day; OF=optic field; ns=non-significant (p>0.05).

Figure 5

Foxi3 cKO mice show sparse fur and asynchronous hair cycle. (A): Foxi3 cKO mice (marked with *) displayed a sparse coat by P7. The phenotype was stable throughout life and was further exacerbated in older mice (1 year old). (B): Scanning electron microscopy image of an awl hair shaft of control and Foxi3 cKO littermates at 2.5 months of age showed no changes in external hair structure. (C–F): Hematoxylin and eosin stained histological sections of control and Foxi3 cKO back skin at indicated time points. (C): At P10, a large fraction of Foxi3 cKO follicles displayed morphological abnormalities (arrowheads). (D): Many Foxi3 cKO hair follicles were delayed in proceeding through catagen. (E): At first telogen, a big amount of Foxi3 cKO follicles displayed abnormal morphology, including absence of clearly discernible bulge and HG (arrowheads), enlarged sebaceous glands, widened hair channels, and cyst formation. (F): At P25, control follicles had proceeded to first anagen, whereas many Foxi3 cKO follicles had not yet completed catagen and failed to initiate a new growth phase. Scale bar=20 µm (B), 50 µm (C–F). Abbreviations: P=postnatal day.

Figure 6

Stem cells are progressively depleted in Foxi3 cKO hair follicles. (A, B): Reduced number of cells positive for stem cell markers Sox9 (A) and Lhx2 (B) in Foxi3 cKO hair follicles at first telogen, P20. (C): Quantification of percent of hair follicles which contained at least some Lhx2+ cells at the first telogen and at 1 year of age showed a progressive phenotype. At first telogen: control littermates (n=47 OFs from 3 mice; 296 follicles were scored) and Foxi3 cKO (n=40 OFs from 3 mice; 257 hair follicles were scored). At 1 year of age: control (n=61OFs from 3 mice; 142 hair follicles were scored) and Foxi3 cKO (n=61 OFs from 3 mice; 147 hair follicles); p (Control^{1st telogen} vs. Foxi3 cKO^{1st telogen})=5.0 E-10; p (control^{1y old} vs. Foxi3 cKO^{1y old})=8.7 E-14; p (Foxi3 cKO^{1st telogen} vs. Foxi3 cKO^{1y old})=0.0031. (D): Quantification of Lhx2+ cells per SC niche (bulge and HG) in control (n=16 follicles from 3 mice, 636 cells were scored) and per SC niche residuals in Foxi3 cKO (n=17 follicles from 2 mice; 275 cells were scored) at 1^st telogen; p=1.2 E-6. (E): Lef1 (red) and P-cadherin (green) staining at anagen onset showed decreased Lef1 activation Foxi3 cKO compared to control mice (F): Representative images of Lhx2 staining in 1 year old mice. (G): Quantification of percent of hair follicles with malformations, as cysts and enlarged sebaceous glands, per optic field, at first telogen and at 1 year of age. The number of samples analyzed is the same as in (C). p (Control^{1st telogen} vs. Foxi3 cKO^{1st telogen})=2.08 E-19; p (control^{1y old} vs. Foxi3 cKO^{1y old})=9.0 E-25; p(Foxi3 cKO^{1st telogen} vs. Foxi3 cKO^{1y old})=0.013. Scale bar=50 µm. Data are shown as mean±SD. P-values were estimated using unpaired two-tailed Student t-test. The dotted line shows epithelial-mesenchymal border. Abbreviations: P=postnatal day; OF=optic field; ns=non-significant (p>0.05).

Figure 7

Foxi3 deficiency compromises stem cell activation in a plucking-induced hair regeneration model. (A): Upon hair plucking, Foxi3 cKO (marked with *) regrew hairs more slowly than controls. (B): Analysis of BrdU incorporation at day 2 post plucking (dpp2) revealed decreased cell proliferation in Foxi3 cKO hair follicles. (C): Quantification of percent of BrdU+ cells in the lower third of the hair follicle at dpp2. Control (n=23 hair follicles, 1021 cells scored) and Foxi3 cKO (n=28 hair follicles, 1149 cells scored); p=1.32 E-9. Data are shown as mean±SD. P-value was estimated using unpaired two-tailed Student t-test. (D): At dpp15, Foxi3 cKO mice showed only few follicles that had progressed to anagen while in littermate controls hair follicles had reached late anagen. (E): Shh expression in control and Foxi3 cKO at the early stages of 1^st anagen analyzed by radioactive in situ hybridization. In contrast to control littermates, Shh was not detectable in Foxi3 cKO hair follicles. (F): A schematic summary describing the consequences of Foxi3 deficiency in embryonic hair follicle morphogenesis and in telogen hair follicles. The dermal component of the hair follicle was omitted for clarity. Scale bar=50 µm. Abbreviations: Dpp=day post plucking; P=postnatal day.