Research ArticleDevelopmentStem cells Open Access | 10.1172/JCI180160

Elnaz Ghotbi,1 Edem Tchegnon,1,2 Zhiguo Chen,1 Stephen Li,1,3 Tracey Shipman,1 Yong Wang,1 Jenny Raman,1 Yumeng Zhang,1 Renee M. McKay,1 Chung-Ping Liao,1,4,5 and Lu Q. Le1,2,6,7,8

1Department of Dermatology,

2Genetics, Development and Disease Graduate Program, and

3Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

4Graduate Institute of Medical Sciences and

5International PhD Program in Cell Therapy and Regenerative Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.

6Hamon Center for Regenerative Science and Medicine and

7Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

8Department of Dermatology, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Address correspondence to: Chung-Ping Liao, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Email: chungpingliao@tmu.edu.tw. Or to: Lu Q. Le, Department of Dermatology, University of Virginia School of Medicine, 1221 Lee Street, Room 3512, Charlottesville, Virginia 22908, USA. Email: bkn6qd@uvahealth.org.

Authorship note: EG and ET contributed equally to this work.

Find articles by Ghotbi, E. in: JCI | PubMed | Google Scholar

1Department of Dermatology,

2Genetics, Development and Disease Graduate Program, and

3Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

4Graduate Institute of Medical Sciences and

5International PhD Program in Cell Therapy and Regenerative Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.

6Hamon Center for Regenerative Science and Medicine and

7Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

8Department of Dermatology, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Address correspondence to: Chung-Ping Liao, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Email: chungpingliao@tmu.edu.tw. Or to: Lu Q. Le, Department of Dermatology, University of Virginia School of Medicine, 1221 Lee Street, Room 3512, Charlottesville, Virginia 22908, USA. Email: bkn6qd@uvahealth.org.

Authorship note: EG and ET contributed equally to this work.

Find articles by Tchegnon, E. in: JCI | PubMed | Google Scholar

1Department of Dermatology,

2Genetics, Development and Disease Graduate Program, and

3Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

4Graduate Institute of Medical Sciences and

5International PhD Program in Cell Therapy and Regenerative Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.

6Hamon Center for Regenerative Science and Medicine and

7Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

8Department of Dermatology, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Address correspondence to: Chung-Ping Liao, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Email: chungpingliao@tmu.edu.tw. Or to: Lu Q. Le, Department of Dermatology, University of Virginia School of Medicine, 1221 Lee Street, Room 3512, Charlottesville, Virginia 22908, USA. Email: bkn6qd@uvahealth.org.

Authorship note: EG and ET contributed equally to this work.

Find articles by Chen, Z. in: JCI | PubMed | Google Scholar |

1Department of Dermatology,

2Genetics, Development and Disease Graduate Program, and

3Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

4Graduate Institute of Medical Sciences and

5International PhD Program in Cell Therapy and Regenerative Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.

6Hamon Center for Regenerative Science and Medicine and

7Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

8Department of Dermatology, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Address correspondence to: Chung-Ping Liao, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Email: chungpingliao@tmu.edu.tw. Or to: Lu Q. Le, Department of Dermatology, University of Virginia School of Medicine, 1221 Lee Street, Room 3512, Charlottesville, Virginia 22908, USA. Email: bkn6qd@uvahealth.org.

Authorship note: EG and ET contributed equally to this work.

Find articles by Li, S. in: JCI | PubMed | Google Scholar

1Department of Dermatology,

2Genetics, Development and Disease Graduate Program, and

3Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

4Graduate Institute of Medical Sciences and

5International PhD Program in Cell Therapy and Regenerative Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.

6Hamon Center for Regenerative Science and Medicine and

7Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

8Department of Dermatology, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Address correspondence to: Chung-Ping Liao, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Email: chungpingliao@tmu.edu.tw. Or to: Lu Q. Le, Department of Dermatology, University of Virginia School of Medicine, 1221 Lee Street, Room 3512, Charlottesville, Virginia 22908, USA. Email: bkn6qd@uvahealth.org.

Authorship note: EG and ET contributed equally to this work.

Find articles by Shipman, T. in: JCI | PubMed | Google Scholar

1Department of Dermatology,

2Genetics, Development and Disease Graduate Program, and

3Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

4Graduate Institute of Medical Sciences and

5International PhD Program in Cell Therapy and Regenerative Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.

6Hamon Center for Regenerative Science and Medicine and

7Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

8Department of Dermatology, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Address correspondence to: Chung-Ping Liao, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Email: chungpingliao@tmu.edu.tw. Or to: Lu Q. Le, Department of Dermatology, University of Virginia School of Medicine, 1221 Lee Street, Room 3512, Charlottesville, Virginia 22908, USA. Email: bkn6qd@uvahealth.org.

Authorship note: EG and ET contributed equally to this work.

Find articles by Wang, Y. in: JCI | PubMed | Google Scholar |

1Department of Dermatology,

2Genetics, Development and Disease Graduate Program, and

3Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

4Graduate Institute of Medical Sciences and

5International PhD Program in Cell Therapy and Regenerative Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.

6Hamon Center for Regenerative Science and Medicine and

7Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

8Department of Dermatology, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Address correspondence to: Chung-Ping Liao, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Email: chungpingliao@tmu.edu.tw. Or to: Lu Q. Le, Department of Dermatology, University of Virginia School of Medicine, 1221 Lee Street, Room 3512, Charlottesville, Virginia 22908, USA. Email: bkn6qd@uvahealth.org.

Authorship note: EG and ET contributed equally to this work.

Find articles by Raman, J. in: JCI | PubMed | Google Scholar

1Department of Dermatology,

2Genetics, Development and Disease Graduate Program, and

3Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

4Graduate Institute of Medical Sciences and

5International PhD Program in Cell Therapy and Regenerative Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.

6Hamon Center for Regenerative Science and Medicine and

7Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

8Department of Dermatology, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Address correspondence to: Chung-Ping Liao, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Email: chungpingliao@tmu.edu.tw. Or to: Lu Q. Le, Department of Dermatology, University of Virginia School of Medicine, 1221 Lee Street, Room 3512, Charlottesville, Virginia 22908, USA. Email: bkn6qd@uvahealth.org.

Authorship note: EG and ET contributed equally to this work.

Find articles by Zhang, Y. in: JCI | PubMed | Google Scholar |

1Department of Dermatology,

2Genetics, Development and Disease Graduate Program, and

3Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

4Graduate Institute of Medical Sciences and

5International PhD Program in Cell Therapy and Regenerative Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.

6Hamon Center for Regenerative Science and Medicine and

7Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

8Department of Dermatology, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Address correspondence to: Chung-Ping Liao, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Email: chungpingliao@tmu.edu.tw. Or to: Lu Q. Le, Department of Dermatology, University of Virginia School of Medicine, 1221 Lee Street, Room 3512, Charlottesville, Virginia 22908, USA. Email: bkn6qd@uvahealth.org.

Authorship note: EG and ET contributed equally to this work.

Find articles by McKay, R. in: JCI | PubMed | Google Scholar |

1Department of Dermatology,

2Genetics, Development and Disease Graduate Program, and

3Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

4Graduate Institute of Medical Sciences and

5International PhD Program in Cell Therapy and Regenerative Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.

6Hamon Center for Regenerative Science and Medicine and

7Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

8Department of Dermatology, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Address correspondence to: Chung-Ping Liao, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Email: chungpingliao@tmu.edu.tw. Or to: Lu Q. Le, Department of Dermatology, University of Virginia School of Medicine, 1221 Lee Street, Room 3512, Charlottesville, Virginia 22908, USA. Email: bkn6qd@uvahealth.org.

Authorship note: EG and ET contributed equally to this work.

Find articles by Liao, C. in: JCI | PubMed | Google Scholar |

1Department of Dermatology,

2Genetics, Development and Disease Graduate Program, and

3Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

4Graduate Institute of Medical Sciences and

5International PhD Program in Cell Therapy and Regenerative Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.

6Hamon Center for Regenerative Science and Medicine and

7Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

8Department of Dermatology, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Address correspondence to: Chung-Ping Liao, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Email: chungpingliao@tmu.edu.tw. Or to: Lu Q. Le, Department of Dermatology, University of Virginia School of Medicine, 1221 Lee Street, Room 3512, Charlottesville, Virginia 22908, USA. Email: bkn6qd@uvahealth.org.

Authorship note: EG and ET contributed equally to this work.

Find articles by Le, L. in: JCI | PubMed | Google Scholar |

Authorship note: EG and ET contributed equally to this work.

Published October 3, 2024 - More info

Published in Volume 134, Issue 23 on December 2, 2024
J Clin Invest. 2024;134(23):e180160. https://doi.org/10.1172/JCI180160.
© 2024 Ghotbi et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published October 3, 2024 - Version history
Received: February 8, 2024; Accepted: October 1, 2024 View PDF Abstract

Epidermal stem cells control homeostasis and regeneration of skin and hair. In the hair follicle (HF) bulge of mammals, populations of slow-cycling stem cells regenerate the HF during cyclical rounds of anagen (growth), catagen (regression), and telogen (quiescence). Multipotent epidermal cells are also present in the HF above the bulge area, contributing to the formation and maintenance of sebaceous gland and upper and middle portions of the HF. Here, we report that the transcription factor KROX20 is enriched in an epidermal stem cell population located in the upper/middle HF. Expression analyses and lineage tracing using inducible Krox20-CreERT showed that Krox20-lineage cells migrate out of this HF region and contribute to the formation of the bulge in the HF, serving as ancestors of bulge stem cells. In vivo depletion of these cells arrests HF morphogenesis. This study identifies a marker for an epidermal stem cell population that is indispensable for hair homeostasis.

Graphical Abstract Introduction

Adult stem cells play a critical role in normal tissue homeostasis and regeneration (1). Correction of mutant gene expression in epidermal stem cells can reverse the disease state of the genetic blistering disorder epidermolysis bullosa (2), highlighting the importance of stem cell plasticity and its role in the treatment of genetic diseases. The precise identification of stem cell locations or niches is essential to elucidate how these cells contribute to tissue health and function (3, 4). In the skin, the hair follicle (HF) bulge is occupied by slow-cycling cells marked by CD34 and K15 (5–7). Bulge stem cells were originally thought to be the universal stem cells for the HF and interfollicular epithelium (IFE), because they were identified as the only label-retaining cell population in the epidermis (5). However, subsequent studies showed that bulge stem cells only give rise to the HF but not the IFE during normal homeostasis (8), and immediate stem/progenitor cells for IFE are generally believed to reside in the basal layer of epidermis.

The HF structures from the bulge to the IFE are composed of the isthmus, junctional zone, and infundibulum. Including the bulge, these HF compartments are collectively defined as the permanent portion of the HF, as they do not change throughout the hair cycle (9). Previously reported markers for permanent HF niches include LGR6 (isthmus) (10), LRIG1 (junctional zone) (11), and SCA1 (infundibulum) (12). During adult homeostasis, Lrig1-positive cells give rise to the infundibulum and sebaceous glands (13), and Sca1-positive cells contribute to the maintenance of IFE and infundibulum (12). Nevertheless, to the best of our knowledge, there is no documented stem cell population within the permanent regions of the upper and middle HF that contributes to the formation of the bulge during normal adult homeostasis.

In this study, we discovered that the transcription factor Krox20 (EGR2) is enriched in an epidermal stem cell population located in the upper and middle HF. Using genetic labeling for cell lineage tracing, we found that Krox20-lineage cells contribute to the formation of the bulge and hair shaft, and that their depletion in vivo disrupts hair homeostasis.

Results

Krox20 marks an epidermal stem cell population in the HF. We recently reported that the transcription factor Krox20 is a lineage marker for hair shaft structural cells during HF morphogenesis (14). To gain further insight into the developmental origin of Krox20-positive cells and explore their fate within the skin, we first set out to determine the exact expression pattern of Krox20 during late embryonic and postnatal development. Subsequently, we aimed to perform lineage tracing of Krox20-positive cells to track their lineage and observe their behavior. We used a Krox20-GFP knockin mouse line (15) to monitor live Krox20 expression with a GFP reporter. Since this line is a “knockin” and not a transgenic, GFP expression faithfully recapitulates endogenous Krox20 expression, as validated through immunohistochemistry using a KROX20 antibody (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI180160DS1). We observed initial expression of Krox20 in the infundibulum of mouse embryonic whisker HF at E14.5 (Supplemental Figure 1B). Subsequently, Krox20 expression was observed in all skin HFs, persisting throughout postnatal development (Supplemental Figure 1B). Histological analysis of mouse skin at various postnatal ages revealed specific regions exhibiting Krox20 expression. The expression was found to be prominent in the infundibulum and upper HF, extending to the sebaceous glands and the middle portions of the telogen and anagen HFs (Figure 1 and Supplemental Figure 1C). Notably, the expression of Krox20 was enriched in the upper HF and extended across various layers of HF but was restricted to the innermost layer of the outer root sheath in the middle HF (Figure 1, A–E, and Supplemental Figure 1C) (16).

Figure 1

Comparative analysis of Krox20 expression in telogen and anagen HFs in relation to other stem cell niches within the HF. (A) Immunofluorescence analysis depicting Krox20 expression with KROX20 antibody in telogen (left) and anagen (middle) and by GFP immunofluorescence in anagen HFs of Krox20-GFP mice (right). (B–E) Colocalization analysis demonstrating the absence of colocalization between Krox20 expression and bulge stem cell markers, K15 and CD34, in anagen (B and C) and telogen (D and E) HFs. (F and G) Colocalization analysis of GFP expression with upper stem cell niche markers such as SCA1 (F) and LRIG1 (G) in telogen HFs of Krox20-GFP mice. All dashed circles and ovals represent the bulge area. Arrows point to autofluorescence from hair shafts. Asterisks represent the sebaceous glands. n ≥ 3. Scale bars: 100 mm.

In order to characterize the relative location of Krox20-positive cells with respect to previously identified stem cell niches within the HF, we performed immunostaining of the skin from Krox20-GFP mice with bulge stem cell markers (K15 and CD34), an infundibulum marker (SCA1), and a junctional zone marker (LRIG1). We observed no colocalization between the KROX20 protein and the bulge stem cell markers, K15 or CD34, in either anagen or telogen HFs (Figure 1, B–E). This indicates that the cell population marked by KROX20 is distinct from the bulge stem cells. Instead, KROX20 exhibited a degree of overlapping expression with LRIG1- and SCA1-expressing cells (Figure 1, F and G). Upon examination of the expression domain of telogen HF, it became apparent that Krox20 expression was enriched in the upper and middle portions of the telogen HF, including the infundibulum and junctional zone, as indicated by the overlap with SCA1 and LRIG1 markers (Figure 1, F and G). Additionally, based on the expression domain of KROX20 itself, it appeared to extend to the isthmus and sebaceous glands (Figure 1).

Krox20-lineage cells give rise to the HF bulge. To determine the fate of these Krox20-positive cells, we performed lineage tracing. As an inducible Cre driver is the gold standard tool for lineage tracing, we first generated an inducible Krox20-CreERT knockin mouse line. Using CRISPR/Cas9, CreERT was inserted after the last coding sequence of Krox20, following a P2A linker. The P2A linker is a short peptide sequence of about 18–25 amino acids that causes ribosome skipping (17, 18), resulting in the separate translation of the upstream protein and the downstream protein, in this case, KROX20 and CreERT. This design ensures that Krox20 expression and function are preserved in mouse lines containing Krox20-CreERT (Supplemental Figure 2A). We crossed this line with R26-tdTomato reporter mice to generate Krox20-CreERT; R26-tdTomato mice. Notably, in the absence of tamoxifen treatment, minimal tdTomato signal was observed, demonstrating tight regulation with minimal leakiness (Supplemental Figure 2, B–D). Mice were then treated with 1 dose of tamoxifen perinatally, and their skin was subsequently analyzed at various time points, starting at 1 day after induction and continuing throughout adulthood (Figure 2A). We observed that Krox20-lineage cells in the Krox20-CreERT; R26-tdTomato lineage tracing were initially enriched in the upper and middle HF perinatally. However, over time, these cells gradually gave rise to the bulge region and were also detected along the IFE (Figure 2A). Costaining of tdTomato and AE13, a marker of hair shaft, indicated that Krox20-lineage cells contributed to the formation of the hair shaft as early as P12–P14 (Figure 2B). Quantification of HF regions labeled with tdTomato at various time points in anagen and telogen HFs (Figure 2C) of Krox20-CreERT; R26-tdTomato mice when induced at P1 is shown in Figure 2D.

Figure 2

Lineage tracing with an inducible Krox20-CreERT confirms the contribution of Krox20-lineage cells to the HF bulge. (A) Krox20-CreERT; R26-tdTomato mice were induced with tamoxifen at P1. Lineage tracing shows that Krox20-lineage cells are initially restricted to the upper and middle HF but, over time, they expand downward, contributing to the formation of the bulge and hair shaft. (B) Colocalization analysis of Krox20-lineage cells with hair shaft marker AE13. (C) Diagram of an anagen HF and a telogen HF. (D) Quantification of HF regions labeled with tdTomato at various time points in Krox20-CreERT; R26-tdTomato mice when induced at P1. n ≥ 3. Scale bars: 100 μm. Tom, tdTomato. Statistical significance was determined for D by 2-way ANOVA; statistics represent mean ± SEM, *P < 0.05, **P < 0.01, ****P < 0.0001. Non-significant values were not plotted on the graphs due to space constraints.

The presence of Krox20-lineage cells along the HF, especially their detection in the location of various epidermal stem cell niches (Figures 1 and 2), suggests that the HF Krox20-positive stem cells serve as the source for previously identified stem cells within the HF. To verify this, we stained for bulge stem cell markers in Krox20-CreERT; R26-tdTomato lineage-traced skin. As expected, the Krox20-lineage cells largely overlapped with K15-positive and CD34-positive bulge cells in both anagen and telogen HFs (Figure 3, A–F), consistent with previous reports of the ability of infundibular cells to regenerate the bulge structure (19). This is critical genetic evidence demonstrating that Krox20-positive cells are the parental/ancestral cells of K15-positive bulge stem cells. Quantification of CD34-positive bulge cells arisen from Krox20-lineage cells at different time points in Krox20-CreERT; R26-tdTomato mice induced at P1 is shown in Supplemental Figure 3.

Figure 3

Krox20 marks an epidermal stem cell niche in the HF excluding the bulge region. Colocalization analysis showing Krox20-lineage cells in relation to bulge stem cell markers K15 (A and B) and CD34 (C–F) within anagen (P36, A and D) and telogen (P60, B; P22, C; P60, E; and P100, F) HFs, induced at P1. Dashed circles represent the bulge area, which is shown at higher magnification in the panels at the end of each row (×400 for A–C, E, and F, and ×200 for D). n ≥ 3. Scale bars: 100 μm. TOM, tdTomato.

Krox20-positive cells are indispensable for hair development and regeneration. To define the functional role of epidermal Krox20-positive cells during development, we deleted Krox20-expressing cells in the skin by crossing mice harboring the Krox20-lox-Stop-lox-DTA (Krox20-DTA) knockin allele (15) with a K14-Cre line to generate Krox20-DTA; K14-Cre mice. In this model, Krox20-positive cells of the K14 lineage express diphtheria toxin A (DTA) and are ablated. K14 is expressed in the basal layer of the IFE and in the outer root sheath of the upper and middle regions of the HF (14, 20), while K14 lineages contribute to the formation of the entire IFE and HF except for the dermal papillae (14). Krox20-DTA; K14-Cre mice were born phenotypically similar to control mice at birth (Figure 4A). The HF and skin of Krox20-DTA; K14-Cre pups at P1 were histologically normal (Figure 4B) and expressed the markers K14 and K15 in a similar pattern in comparison with the control mice (Figure 4C). These data suggest that epithelial Krox20-positive cells are not essential for normal embryonic skin and initial HF development. However, within 1 week of postnatal development, Krox20-DTA; K14-Cre pups began showing an obvious phenotype. Krox20-DTA; K14-Cre mice were visibly smaller by P3, and their skin pigmentation was significantly lighter by P6 (Figure 4D). As opposed to human skin, where pigmentation is derived from IFE melanocytes, skin pigmentation of postnatal mice is derived from melanin in the anagen HF, suggesting a loss or arrest of anagen HF development in the Krox20-DTA; K14-Cre mice. Consistent with this, histological analysis of the skin of P6 Krox20-DTA; K14-Cre mice showed significantly shorter HFs in comparison with their littermate controls (Figure 4E). Additionally, their HFs grew in discordant directions, while normal mouse anagen HFs grew in a similar direction (Figure 4E). The observation of miniaturized HFs was anticipated, as Krox20-positive cells are distributed within the upper and middle anagen HFs, and their deletion results in shorter HFs.

Figure 4

Krox20-positive cells are indispensable for hair development. (A–C) Krox20-DTA; K14-Cre pups at P1 show normal gross appearance (A), skin histology (B), and K15-positive stem cell distribution (C). (D) Krox20-DTA; K14-Cre pups show no difference compared with littermate controls at P1 but are lighter in skin color and weight by P6. (E) H&E staining at P6 shows that the HFs of Krox20-DTA; K14-Cre mice are shorter and random in orientation. (F) Immunostaining shows the absence of KROX20 protein in the skin of Krox20-DTA; K14-Cre mice. (G–L) Immunostaining reveals that HFs in P6 Krox20-DTA; K14-Cre pups have aberrant expression of Ki67 (G), K15 (H), versican (I), DCT (J), AE13 (K), and P-cadherin (P-CAD) (L). n = 5. Scale bars: 100 μm.

To validate the ablation of Krox20-positive cells by DTA, immunostaining was performed using the KROX20 antibody. The results of the immunostaining clearly demonstrated the complete absence of KROX20 expression, confirming that epithelial Krox20-positive cells were effectively eliminated in the examined samples (Figure 4F). Complete absence of Krox20-expressing cells, despite the partial overlap between K14 and Krox20, suggests that the ablated population of Krox20-positive cells in K14 lineages may give rise to the Krox20-expressing cells that do not express K14. This observation is consistent with our previous report, in which we proposed a pattern indicating that embryonic K14-positive cells serve as the origin for a specific subset of Krox20-expressing cells (14).

Normal anagen HFs are associated with highly proliferative cells in the matrix, also known as transit-amplifying cells, that are derived from bulge stem cells (21). To look for the presence of highly proliferative cells, we performed immunostaining for Ki67. Mice lacking epidermal Krox20-positive cells showed no matrix transit-amplifying cells (Figure 4G), further supporting the role of Krox20-positive cells as HF stem cells upstream of transit-amplifying cells. To explore the effect of Krox20-positive cell depletion on the cells and structures of the HF, we performed immunostaining for K15, a bulge stem cell marker (6). We found that loss of Krox20-positive cells resulted in significantly reduced K15-positive cells (Figure 4H), suggesting that Krox20-positive cells are the ancestral cells of K15-positive cells within the HF.

The dermal papilla is a mesenchymal structure deep in the HF that reciprocally signals with HF stem cells (22) to initiate and maintain normal HF growth state (23). To determine whether loss of epidermal Krox20-positive cells affects the dermal papillae, we stained for the marker versican. We found atrophied dermal papillae in Krox20-positive cell–deficient HFs (Figure 4I). Given that Krox20-positive cells were depleted only in the epidermal K14-positive lineages, our results provide further support for the existence of cellular crosstalk between the HF epidermis and mesenchyme. Melanocytes are another cell type associated with HF development. Melanocyte stem cells reside in the bulge (24), and their differentiation is synchronized with the hair cycle. During normal anagen, differentiated melanocytes (DCT-positive cells) were predominantly located in the HF bulb (Figure 4J). Depletion of Krox20-positive cells resulted in loss of melanocytes from the bulb, suggesting that Krox20-positive cells either give rise to melanocyte stem cells or are essential for their differentiation (Figure 4J).

Lastly, we explored the role of Krox20-positive cell depletion in hair shaft formation. Loss of Krox20-positive cells resulted in impaired hair shaft abundance and structure, as demonstrated by reduced and aberrant immunostaining for the hair shaft structural markers AE13 and P-cadherin (Figure 4, K and L). Given that HFs of Krox20-DTA; K14-Cre mice are normal at birth, our findings suggest that Krox20-positive cells differentiate to replace the embryonic hair shaft progenitors, as well as maintain normal HF structure after birth. Taken together, our data reveal a requirement for epidermal Krox20-positive cells in HF morphogenesis. They also indicate an essential role for Krox20-lineage cells as supported by the absence of markers for dermal papillae, matrix transit-amplifying cells, differentiated melanocytes, and hair shafts, which are regions devoid of Krox20 expression.

Krox20-positive cells repopulate the bulge and are required for hair regeneration. Krox20-DTA; K14-Cre mice do not survive beyond 1 week of age, most likely because of epithelial dysregulation of vital internal organs, and thus we were unable to determine the adult epidermal phenotype. To examine the effects of Krox20-lineage cell depletion on hair regeneration in adult mice, we used a 4-hydroxytamoxifen–inducible mouse model (Krox20-DTA; Krox20-CreERT) to deplete Krox20-expressing cells in vivo. In this model, Krox20-expressing cells express DTA and are ablated upon induction with 4-hydroxytamoxifen. The expression of Krox20 is preserved in Krox20-DTA; Krox20-CreERT mice because of the design of the Krox20-CreERT line using the P2A linker (Supplemental Figure 2A). We used this model to ablate Krox20-expressing cells in depilated HFs at various stages of the hair cycle: Mice were depilated to cause synchronized induction of new hair generation at P17, P23, P36, P38, P55, and P104. 4-Hydroxytamoxifen dissolved in DMSO was topically administered to the depilated dorsal skin at the same time for a minimum of 3 consecutive days. Interestingly, the effect of depleting Krox20-expressing cells on hair regeneration varied depending on the hair cycle stage at the time of induction. The most prominent phenotype was observed when depilation and 4-hydroxytamoxifen induction were initiated during early telogen I and II, at P17 and P36, respectively (Figure 5): At both hair cycle stages, Krox20-DTA; Krox20-CreERT mice lost their ability to generate new hair, with the hair cycle arrested in telogen, while the HFs of littermate controls progressed to anagen (Figure 5, A and B). Histological analysis of Krox20-positive cell–depleted HFs at telogen II showed severely miniaturized and deformed HFs (Figure 5, C and D) that lacked KROX20 protein expression (Figure 5E), confirming the ablation of Krox20-positive cells. Significantly fewer normal HFs were observed per millimeter of skin cross section from Krox20-DTA; Krox20-CreERT mice compared with littermate controls (Figure 5D). Furthermore, immunostaining revealed complete absence of CD34-positive and K15-positive bulge stem cells (Figure 5, F and G). In addition to suggesting that Krox20-positive cells are the stem/progenitor cells that give rise to HF stem cells, these results also demonstrate that Krox20-lineage cells are essential for maintaining these HF stem cell niches during early adulthood.

Figure 5

Krox20-positive cells are essential for hair regeneration. (A and B) Ablation of Krox20-positive cells via tamoxifen induction in Krox20-DTA; Krox20-CreERT mice during telogen I (P17) (A) and telogen II (P36) (B) hinders hair regeneration following depilation (n = 3). (C) H&E analysis of the skin in telogen II Krox20-positive cell–depleted mice shows hair growth arrest and HF miniaturization. (D) Quantification of number of normal HFs per millimeter of skin cross section of Krox20 cell–depleted and littermate control mice (n = 2). (E–G) Immunofluorescence staining at P56 reveals the absence of KROX20-positive cells (E), and bulge CD34-positive and K15-positive cells (F and G). n = 3. Scale bars: 100 μm. Statistical significance was determined for D by unpaired 2-tailed Student’s t test; statistics represent mean ± SEM, ****P < 0.0001.

In addition to telogen II, we also evaluated how hair regeneration is affected when Krox20-expressing cells are depleted during anagen II (P38). Interestingly, we found that Krox20-positive cell–depleted HFs were initially arrested at anagen II, while HFs in control mice completed the anagen phase and proceeded to telogen (Supplemental Figure 4A, middle). However, signs of hair loss were observed 2 weeks after induction, and these mice showed significant hair loss by P58 (Supplemental Figure 4A). Histological analysis using H&E (Supplemental Figure 4, B and C) and immunofluorescence staining (Supplemental Figure 4D) revealed the presence of both miniaturized and full-length telogen HFs, expressing KROX20, although at reduced levels compared with the control telogen HFs. The presence of KROX20 expression in these HFs was likely due to the incomplete ablation of Krox20-positive cells. Additionally, we immunostained for bulge stem cell markers CD34 and K15. While CD34 expression was lost from the HFs (Supplemental Figure 4E), K15 expression was still detected within the Krox20-positive cell–depleted HFs (Supplemental Figure 4F). However, it is important to note that the expression pattern of K15 in these HFs did not structurally resemble a normal bulge region, which is consistent with the absence of CD34 expression (Supplemental Figure 4, E and F). Moreover, in line with the consequences of Krox20-positive cell depletion during telogen II, a significant reduction was observed in the number of normal HFs per millimeter of skin cross section in the anagen II cycle of Krox20-DTA; Krox20-CreERT mice induced with 4-hydroxytamoxifen, compared with the control mice (Supplemental Figure 4C). Furthermore, ablation at P23, P55, or P104 showed no difference in hair regeneration capacity in the Krox20-DTA; Krox20-CreERT mice compared with the littermate control mice (Supplemental Figures 5 and 6).

To investigate the reasons behind the failure of depilation-induced hair regeneration when Krox20-positive cells were ablated at early telogen I and telogen II, but not at other tested time points, we conducted lineage tracing in Krox20-CreERT; R26-tdTomato mice. Tamoxifen induction was performed at P17, P23, P38, P55, and P98. Interestingly, lineage tracing during both late catagen/early telogen I (P17) and late anagen/early catagen II (P38) revealed labeling of the bulge cells by Krox20-lineage cells within only 4 or 3 days following tamoxifen induction, respectively (Supplemental Figure 4G and Supplemental Figure 5A). Conversely, lineage tracing at mid–telogen I (P23) did not exhibit bulge cell labeling until after telogen II (P55) (Supplemental Figure 5C). Lineage tracing at mid–telogen II (P55) demonstrated that Krox20-lineage cells had not yet reached the bulge even 59 days later (Supplemental Figure 6B). Furthermore, lineage tracing at P98 indicated that Krox20-lineage cells did not reach the bulge until 80 days later (Supplemental Figure 6D). These results suggest that Krox20-positive cells replenish the bulge stem cells during early telogen I and telogen II, which represent highly synchronized hair cycles.

In experiments in which mice older than 1 month were depleted of Krox20-expressing cells, their survival was limited to 3–4 weeks after the last dose of 4-hydroxytamoxifen. However, mice treated during telogen I exhibited a higher mortality rate (note that in Figure 5A, only 1 mouse of the 2 treated survived the 4-hydroxytamoxifen treatment to reach the age of P29) and survived no more than 10–14 days after the final administration of 4-hydroxytamoxifen. Notably, all mice displayed signs of emaciation, kyphosis, shivering, and impaired coordination 2–3 days before death. The observed phenotype was likely due to the crucial role of Krox20 in maintaining the myelinating state of Schwann cells (25). Hence, we were unable to extend our assessment of skin phenotypes in Krox20-DTA; Krox20-CreERT mice beyond the time points documented in this study.

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