Bifunctional ectodermal stem cells around the nail display dual fate homeostasis and adaptive wounding response toward nail regeneration

Yvonne Leunga,b,1, Eve Kandybaa,b,1, Yi-Bu Chenc, Seth Ruffinsa, Cheng-Ming Chuongb,d, and Krzysztof Kobielaka,b,2

aEli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research, bDepartment of Pathology, and cNorris Medical Library, University of Southern California, Los Angeles, CA 90033; and dInternational Research Center of Wound Repair and Regeneration, Institute of Clinical Medicine, Cheng Kung University, Tainan City 70101, Taiwan

Edited by Mina J. Bissell, E. O. Lawrence Berkeley National Laboratory, Berkeley, CA, and approved August 28, 2014 (received for review October 7, 2013) Regulation of adult stem cells (SCs) is fundamental for organ fingertip (Fig. 1A). Found at the “root” of the NP is the actively maintenance and tissue regeneration. On the body surface, different proliferating nail matrix (Mx) with a keratogenous zone (KZ) ectodermal organs exhibit distinctive modes of regeneration and the directly above where Mx cells differentiate into the overlying NP dynamics of their SC homeostasis remain to be unraveled. A slow (Fig. 1A). Pulse–chase studies have confirmed that Mx cells move cycling characteristic has been used to identify SCs in hair follicles and superficially into the NP and also distally toward the nail bed (4). sweat glands; however, whether a quiescent population exists in The proximal end of the NP is covered by the proximal fold (PF), continuously growing nails remains unknown. Using an in vivo label which is a continuation of the epidermis that folds inward (Fig. retaining cells (LRCs) system, we detected an unreported population 1A). Interestingly, epidermal differentiation is “switched off” just of quiescent cells within the basal layer of the nail proximal fold, after the PF invaginates inward at the eponychium, where cells organized in a ring-like configuration around the nail root. These nail continuing beyond this point (including adjacent proximal fold LRCs express the hair stem cell marker, 15 (K15), and lineage A tracing show that these K15-derived cells can contribute to both the cells, Mx, KZ, and nail bed) differentiate to form a NP (Fig. 1 ). nail structure and peri-nail epidermis, and more toward the latter. Previously, the slow cycling, label-retaining cells (LRCs) charac- CELL BIOLOGY Thus, this stem cell population is bifunctional. Upon nail plucking teristic has been successfully used to identify SCs in various organs injury, the homeostasis is tilted with these SCs dominantly delivering including the hair follicle, cornea, and sweat gland; however, exis- progeny to the nail matrix and differentiated nail plate, demonstrat- tence of a quiescent SC population in continuously growing nails ing their plasticity to adapt to wounding stimuli. Moreover, in vivo has not been revealed so far (2, 3, 5–7). Here, we aim to identify the engraftment experiments established that transplanted nail LRCs can location of LRCs in mouse nails, characterize their molecular fea- actively participate in functional nail regeneration. Transcriptional tures, and evaluate their stem cell potential in vivo. This study is profiling of isolated nail LRCs revealed bone morphogenetic important in the light of recent discoveries that the tips of mam- signaling favors nail differentiation over epidermal fate. Taken malian digits may regenerate after amputation, depending on its together, we have found a previously unidentified ring-configured interaction with the nail appendage (8). Therefore, defining the population of bifunctional SCs, located at the interface between the quiescent reservoir of nail SCs and their regenerative potential nail appendage organ and adjacent epidermis, which physiologically might help fill in this newly opened research area with novel un- display coordinated homeostatic dynamics but are capable of redi- derstanding of nail organ biology and provide novel forms of verting stem cell flow in response to injury.

BMP signaling | nail proximal fold stem cells | nail stem cells | stem cells Significance

Skin appendages including hair follicles, sweat glands, and nails dult stem cells are capable of self-renewal and are respon- offer a source of regenerative support following injury. Here, sible for the maintenance of their respective organs during A we focus on the regenerative potential of the peri-nail region homeostasis and repair. The skin contains a number of associated and reveal a population of K15-positive, label retaining cells appendages such as hair follicles, sebaceous glands, sweat glands, (LRCs) within the nail proximal fold with self-renewal capa- and nails that display different modes of renewal, regeneration, bilities. Physiologically, these cells display bifunctional stem cell and stem cell (SC) homeostatic dynamics during normal wear and qualities and contribute to both the nail structure and peri-nail tear (1). For example, the epidermis undergoes continual re- epidermis long term; however, upon injury, the homeostatic newal, hair follicles undergo episodic regeneration, and sweat balance is tilted toward nail regeneration. Molecularly, reduced glands display little turnover as demonstrated by the presence of bone morphogenetic protein signaling tilts nail keratinocytes quiescent label-retaining stem cells in vivo (2, 3). Moreover, nails toward an epidermal fate. Collectively, we demonstrate the exhibit continuous growth under physiological conditions and plasticity of these stem cells: bifunctional under normal ho- can fully regenerate upon removal. During development, these meostasis, but adaptive in response to wounding. Such princi- different skin appendages are derived from a common ectodermal ples may exist in the interface between other ectodermal organs origin and share similar characteristics but ultimately adopt alter- and skin. native, individual homeostatic mechanisms to fulfill the different functions of each distinct miniorgan. Therefore, dissecting the Author contributions: Y.L. and K.K. designed research; Y.L., E.K., and K.K. performed underlying similarities and differences that occur during distinct research; Y.-B.C. and S.R. contributed new reagents/analytic tools; Y.L., E.K., Y.-B.C., skin appendage renewal may prove highly beneficial in unrav- C.-M.C., and K.K. analyzed data; and Y.L., E.K., and K.K. wrote the paper. eling the fundamental principles that govern ectodermal organ re- The authors declare no conflict of interest. generation in vivo. This article is a PNAS Direct Submission. The nail contains a hard keratinized structure called the nail 1Y.L. and E.K. contributed equally to this work. plate (NP), which acts as a protective cover to prevent trauma to 2To whom correspondence must be addressed. Email: [email protected]. the digit tips. Beneath the distal end of the NP lies the nail bed This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. with both regions sealed together at the hyponychium of the 1073/pnas.1318848111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1318848111 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 near conical structure, dorsal-ventral sections revealed two clus- ters of nail LRCs: one in the upper proximal fold (U-PF) above the NP (upper LRCs) (Fig. 1F) and one in the lower proximal fold (L-PF) localized below the NP (lower LRCs) (Figs. 1H and 2C), indicating the LRCs population encircles the proximal end of the NP. Topological localization was emphasized by using hematoxylin and eosin staining (H&E) on the same sections (Fig. S1 C–F). Furthermore, in vivo confocal microscopy (with serial Z- stack 3D reconstruction) revealed multiple rows of LRCs or- ganized in a concave fashion around the outer surface of the proximal NP after a 3-wk chase (Fig. S2A and Movie S1).

Characterization of Nail Proximal Fold LRCs. To specifically localize the LRCs within the nail epithelium, we stained for the basal marker, K5, in 4-wk chased nails and confirmed nail LRCs lo- calized to the β4-integrin–positive basal layer (Fig. 2A) and at- tached to the (Fig. 2B). Although the epidermis contains a stratum granulosum that expressed loricrin, this layer fades away at both upper and lower LRCs positions in the nail PF (Fig. 2C and Fig. S2B). Proliferating Mx progenitor cells are found directly adjacent to the nail PF region and differentiate into cells of the KZ to form the external NP. Previous reports have described the presence of LRCs within the nail Mx (11); however, we observed a clear distinction between slow-cycling, nail LRCs in the PF and the actively dividing Mx cells after only 1 wk of chase (Fig. 2D). Indeed, nuclear Ki67 immunolabeling primarily localized to pro- + liferating Mx cells and a minority of weakly H2BGFP cells + immediately adjacent to Ki67 negative, strongly H2BGFP PF LRCs (Fig. 2D, arrows), indicating increased proliferation within the Mx in comparison with the adjacent PF region. We observed + + Fig. 1. Localization of LRCs in the nail proximal fold. (A) Components of the that the area containing weakly H2BGFP and Ki67 cells mouse nail. Top view, horizontal sections of the fingertip before (C) and or “intermediate zone,” arising from H2BGFP label dilution after (B and D) 4 wk of chase with Dox identifying a population of H2BGFP after cell division, decreased in size with increasing periods of marked LRCs surrounding the nail structure. Side view, perpendicular sec- chase. For example, after a 2-wk chase, minimal “intermediate E G F + + tions of the digit tip before ( and ) and after ( ) 4 wk of chase with Dox zone” of H2BGFP with Ki67 Mx expression and strong H + demonstrating the presence of upper LRCs in the upper PF. ( ) Lower LRCs at H2BGFP expression in PF LRCs (Fig. 2E) was observed and, the lower nail PF. GFP; green fluorescent protein; HF, hair follicle; L-LRCs, lower label-retaining cells; U-LRCs, upper label-retaining cells. Yellow box after 4 wk of chase, a distinctive boundary developed between denotes region of interest in B, and red and blue boxes (G) denote repre- the most quiescent PF LRCs and the proliferating Mx region sentative U-PF and L-PF regions for orientation. (Scale bars: 50 μm.) (Fig. 3E and Fig. S2C). Collectively, we have identified a pop- ulation of LRCs localized within the basal layer of the nail PF where the epidermal granular layer ceases (Fig. S2C) and have therapeutics not limited solely for patients with nail/digit defects, not observed any slow cycling H2BGFP cells in the actively but also for amputees in the future. proliferating Mx (Fig. 3E).

+ Results During Normal Nail Homeostasis, K15 Nail Proximal Fold Cells Identification of Nail Proximal Fold LRCs in Vivo: A 3D Ring-Like Demonstrate Bifunctionality Toward the Nail Structure and Peri-Nail Configuration Surrounding the Nail Root. To identify and charac- Epidermis. Because slow-cycling LRCs often possess SCs char- terize mouse nail LRCs, we exploited a transgenic reporter sys- acter, we performed immunofluorescence directed against the tem comprising keratin 5 (K5)-driven tetracycline repressor skin stem cell marker, keratin 15 (K15), expressed by both hair + mice, tTA (K5TetOff) (9), crossed with tetracycline response follicle and sweat gland SCs (2, 3, 12) and detected K15 ex- element-driven histone H2B conjugated green fluorescent pro- pression in the majority of PF LRCs (Fig. 2G). Next, to evaluate tein (TreH2BGFP) mice (K5TetOff/TreH2BGFP) (10). Using the functional contribution of K15-derived cells in nail tissue this approach, H2BGFP is expressed during early embryogenesis over time, we used a K15CrePR-driven lineage tracing approach by K5-specific cells, resulting in efficient labeling of the entire by using either Rosa26Tomato or Rosa26LacZ reporter ex- skin and appendages including nails, hair follicles, and sweat pression (12–14). After topical RU486 (RU) treatment in new- + glands (Fig. 1 C, E, and G and Fig. S1A). To turn off inducible born pups (Fig. S3A), K15-derived Tomato cells were restricted expression of histone H2B–green fluorescent protein (H2BGFP) to the nail PF region at P1 (Fig. 2H and Fig. S3 E and F). and identify nail LRCs, a 4-wk chase with doxycycline (Dox) Subsequent lineage tracing analysis at P5 and P7 revealed in- + treatment, beginning between postnatal day (P) 21 and P28 was creasing numbers of Tomato cells within the contiguous peri- administered, resulting in dilution of H2BGFP within rapidly nail epidermis (Fig. 2J, P7; and Fig. S3 H–L, P5 and P7) forming + dividing cells such that only infrequently dividing cells retained a band of Tomato epidermis surrounding the visible NP (Fig. 2 H2BGFP label (Fig. S1B). After a 4-wk chase, we identified I and I′, P7 and Fig. S3 G and G′, P5). Of note, the corre- + + a population of H2BGFP LRCs orientated in a ring-like structure sponding peri-nail epidermis at P1 lacked visible Tomato ex- surrounding the proximal NP in digits sectioned along the proxi- pression (Fig. S3 D and D′, P1). Comparable results were + mal-distal axis (Fig. 1 B and D, arrows). Specifically, H2BGFP obtained in experiments by using different postnatal RU treat- LRCs were located above the NP at the PF (Fig. 1F, side view). Of ment time points, P18 or P43 (Fig. S3 B and C), with abundant + note, because the mouse nail exists as a three-dimensional (3D) Tomato expression in peri-nail epidermis at P30 and 9 mo

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1318848111 Leung et al. Downloaded by guest on September 25, 2021 alternative K15CrePR/Rosa26LacZ reporter approach also + confirmed LacZ cells in the NP (Fig. 2M). Collectively, these data show that the nail PF region harbors a distinct population of + K15 cells, which possess a bifunctional stem cell characteristic and actively contribute toward the contiguous peri-nail epidermis (Fig. 2 N, 1) and the NP during normal nail homeostasis (Fig. 2N, 2).

Following Injury, Nail Proximal Fold Cells Actively Participate in Nail Regeneration. To test the regenerative potential of the mouse nail skin appendage, we removed the entire visible NP by mechanical plucking (Figs. S4A and S5A) and demonstrated that the nail structure can fully regenerate within 2 wk (Fig. S5B). Physio- + logically, we observed that SCs, which we refer to as K15 “nail CELL BIOLOGY

Fig. 2. Nail proximal fold cells are localized in the basal layer and contribute long term to both peri-nail epidermis and the nail structure. Nail LRCs lo- calized to the K5 (A) and the β4-integrin expressing basal layer attached to basement membrane (B). (C) Nail LRCs are found at the border where lor- icrin expression ends. H2BGFP label becomes progressively diluted out of proliferating matrix cells with a 1-wk chase (D) and 2-wk chase (E). Yellow brackets highlight intermediate (Int.) zone. (F) Model illustrating a gradient of slow- to fast-cycling cells in the nail and position of LRCs in 3D ring-like configuration. Dotted lines mark dermal-epidermal junctions. (G) Nail LRCs express K15. (H) Cryosections of K15-derived Tomato+ cells (red, arrows) in Fig. 3. Nail proximal fold cells participate in nail regeneration in response I I′ the nail proximal fold of P1 nails after RU treatment at P0. ( and ) Whole- A + to plucking injury and upon transplantation. ( ) Whole-mount Tomato ex- mount Tomato expression (red) in the peri-nail epidermis of P7 nails; as- + pression in regenerated nails 2 wk after plucking. Linear streams of Tomato J–L + terisk marks the nail tip. ( ) K15-derived Tomato cells (red, arrows) in the cells (red) in regenerating nails (arrows) extending from the nail base toward nail PF regions at P7, P30, and 9 mo of age, respectively. K15-derived To- B + + + the tip. ( ) Schematic model representing the role of K15 NPFSCs during mato at 9 mo (L) or LacZ lineage tracing (M) demonstrate long-term sur- nail regeneration. (C–C′′) K5 expression (green) in regenerating nails local- vival and contribution of K15-derived cells to the nail structure at 6 mo. (N) + + izing the linear K15-derived, Tomato (red) cell streams emanating from the Schematic model representing the role of K15 NPFSCs during normal nail + basal K5 Mx extending upward (arrows) into the overlying differentiated homeostasis with long-term contribution of K15-derived cells to peri-nail + NP; yellow box denotes region of interest in (C′ and C′′). (D) H2BGFP nail epidermis (1) and the nail structure (2). GFP; green fluorescent protein; Int. LRCs persist in the finger following nail removal. (E) Nail LRCs are quiescent, zone, intermediate zone; L-LRCs, lower label-retaining cells; M, month; P, whereas the nail Mx contains actively proliferating cells marked by BrdU postnatal; U-LRCs, upper label-retaining cells. DAPI counterstaining (blue) incorporation. (F) Upon NP removal, LRCs become activated, indicated by was used to localize cell nuclei in fluorescent images. (Scale bars: A–E, G, H, + Ki67 coexpression. (G) Nail LRCs transplantation strategy. H2BGFP nail cells J–M,50μm; I, 500 μm; I′, 200 μm.) contribute to the nail structure 17 d after transplantation (H), sectioning of 22-d chased transplant (I), demonstrating the presence of remaining LRCs from the transplant. d, day; Epi, epidermis; GFP, green fluorescent protein; (Fig. 2 K and L, respectively). Furthermore, analysis at 9 mo + L-LRCs, lower label-retaining cells; U-LRCs, upper label-retaining cells. DAPI revealed K15-derived Tomato progeny within the NP structure counterstaining (blue) was used to localize cell nuclei in immunofluorescent (Fig. 2L, arrows) and, 6 mo after initial transgene activation, an images. (Scale bars: A and H, 500 μm; C–F and I,50μm.)

Leung et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 proximal fold stem cells” (NPFSCs), contributed toward NP slower expression profile of the nail LRCs with the surrounding basal, than to adjacent peri-nail epidermis, therefore, we next in- non-LRCs and identified 170 common up-regulated and vestigated NPFSCs responsiveness following nail injury. The NPs 490 common down-regulated genes (Fig. S6G and Dataset S1), were plucked at P7, after initial labeling of nail PF cells at P0 with which were functionally categorized to include signaling, tran- RU (Figs. S3A and S4A), and nail regeneration was monitored scription, and cell adhesion properties (Fig. S6H). Interestingly, over a 2-wk period (Fig. S4B). Significantly, 2 wk after plucking, among the common genes revealed between nail LRCs and non- + multiple linear streams of Tomato cells were observed in regen- LRCs, we observed down-regulation of two bone morphogenetic erating nails (Fig. 3A), which was confirmed by serial sections protein (BMP) signaling inhibitors, Bambi and Decorin, sug- through the entire nail structure (Fig. S4D). K5 immunostaining gesting active BMP signaling may play an important function in + (Fig. 3C) confirmed the presence of K15-derived, Tomato cell the nail appendage (Fig. S6H). progeny within the nail Mx region where multiple linear streams of + Tomato cells, emanating from the Mx, extended upwards and BMP Signaling Guide These Stem Cells Toward Nail Differentiation. differentiated into the overlying, regenerating NP (Fig. 3 C–C′′, To verify canonical BMP signaling activity within mouse nail arrows). In addition, we observed elongation of the nail PF region tissue, we performed phospho-Smad 1/5/8 (pSmad1/5/8) immu- in regenerating nails where the contiguous nail epidermis provided nostaining and detected abundant nuclear pSmad1/5/8 expres- an extended protective covering over the exposed regenerating nail sion in PF LRCs and Mx cells (Fig. S7A). Next, to examine the structure (Fig. S4 C and E). Immunofluorescence confirmed functional significance of BMP signaling during nail formation, coexpression of epidermal markers, K1 (Fig. S4F, arrows) and we specifically deleted BMP receptor 1A (Bmpr1a) in developing + loricrin (Fig. S4G, arrows), with K15-derived, Tomato cells within skin by using a K14Cre-driven approach. Interestingly, in com- the protective PF extension. Taken together, these results parison with control littermates (Fig. 4 A and C), the Mx, KZ, + identify a distinct K15 NPFSCs population capable of nail bed, and NP of Bmpr1a KO nails displayed abnormalities responding to injury to actively deliver progeny into the Mx and (Fig. 4 B and D) with H&E staining indicating decreased cellu- differentiate into NP upon regeneration (Fig. 3B). larity in the Bmpr1a KO Mx region (P8; Fig. 4D vs. Fig. 4C) Next, to verify that the PF LRCs population persisted within and Ki67 immunostaining confirming less proliferation in the the injured nail tissue following mechanical plucking, we sec- Bmpr1a KO Mx than in the control (Fig. S7D vs. Fig. S7C). A KZ + tioned 4-wk chased paws and observed that H2BGFP LRCs zone was not observed above the Mx region in Bmpr1a KO mice remained in the wounded digit tips (Fig. 3D and Fig. S5C, (Fig. 4D vs. Fig. 4C) and hyperplasia of the Bmpr1a KO nail bed + arrowheads). Significantly, although H2BGFP LRCs did not (Fig. 4B vs. Fig. 4A, Insets) with thinner, improperly differenti- + normally overlap with actively proliferating BrdU Mx cells (24 h ated NP was apparent compared with controls (Fig. 4B vs. Fig. after BrdU pulse; Fig. 3E), 24 h after nail removal, nuclear Ki67 4A). Furthermore, the tip of the NP beyond the hyponychium + expression (Fig. 3F) was detected in H2BGFP LRCs, indicating was also absent or broken in Bmpr1a KO nails (Fig. 4B vs. Fig. cell activation and the ability to respond to injury. To validate 4A, arrows). In controls, the granular layer of the epidermis whether nail LRCs actively become fully integrated during nail invaginates into and then ends in the nail PF region (Fig. 4C, + growth, we isolated H2BGFP nail LRC strips from 4-wk chased arrowhead). We observed abnormal extension of the granular layer − mice and transplanted them directly underneath the PF of GFP , throughout the entire Bmpr1a KO nail (Fig. 4 B and D, arrow- immunocompromised NOD SCID mice (in the absence of Dox heads). Furthermore, in control nails, K1 and loricrin immu- to preserve nuclear H2BGFP expression) (Fig. 3G and Fig. S5D). nostaining were observed in the epidermis and nail PF but were + Following engraftment, we detected H2BGFP cells emanating clearly absent in the Mx and overlying NP (Fig. 4 E and G), from the PF region, extending into the growing nail in a direction whereas ectopic K1 and loricrin expression was observed in toward the nail tip (Fig. 3H and Fig. S5E, 17 d and 10 d after Bmpr1a KO NP (Fig. 4 F and H, arrows) but without detectable engraftment, respectively). To determine whether engrafted AE13 staining (Fig. 4H, unlike controls in Fig. 4G). Thus, in + H2BGFP cells could preserve LRC characteristics, we performed mice lacking BMP signaling activity, proper NP differentiation is a 3-wk Dox chase in the host NOD SCID mouse (Fig. S5F) and compromised with the adoption of an epidermal fate in vivo. + observed H2BGFP donor cells within the transplanted nail PF of recipient mouse digits (Fig. 3I). Collectively, these trans- Discussion + plantation studies demonstrate that engrafted H2BGFP LRCs In this study, we examine the regenerative potential of the peri- functionally participate in nail regrowth and can maintain a qui- nail region and discover a previously unidentified population of escent, slow-cycling SC characteristic (Fig. 3I). K15-positive LRCs within the nail PF with self-renewal capa- bilities. Physiologically, these cells display bifunctional SC qual- Isolation and Transcriptional Profiling of Nail Proximal Fold LRCs. To ities and contribute to both the nail structure and peri-nail examine the transcriptional properties of the PF nail LRCs, we epidermis long term; however, upon injury, the homeostatic + optimized a method to specifically isolate the H2BGFP nail balance is tilted toward nail regeneration. We were able to LRCs population by using surgical microdissection with sub- demonstrate the plasticity of nail SCs: bifunctional under normal sequent enzymatic digestion for live cell FACS analysis and pu- homeostasis, but adaptive in response to wounding. rification (Fig. S6 A–D and SI Materials and Methods). We used + α6-integrin (CD49f) costaining and collected viable H2BGFP LRCs Within the Nail. In prior studies investigating the precise lo- + − + α6 (LRCs) and neighboring H2BGFP α6 basal cells (non- calization of mouse nail LRCs, there is conflicting data from LRCs) for RNA isolation and microarray analyses (Fig. S6E)and, BrdU pulse–chase experiments that localize LRCs to the distal + as a control, also isolated α6 basal cells from footpad epidermis Mx (11); however, we did not detect Mx LRCs by using the (SI Materials and Methods) (2). Total RNA from each cell frac- K5TetOff/TreH2BGFP LRCs system after a 4-wk chase. Instead, + tion (from two independent experiments) was extracted and we observed a ring-like cluster of nonproliferative H2BGFP compared with control basal cells from the footpad to identify the LRCs within the basal nail PF region (Figs. 1 B and D,2F, and nail expression signature (Fig. S6F). Transcriptional pro- Fig. S2A) and demonstrated that the nail Mx is highly pro- + + filing of H2BGFP LRCs revealed consistent up-regulation of liferative (Ki67 ), incorporates BrdU, and rapidly dilutes nuclear 664 genes and down-regulation of 857 genes by at least twofold H2BGFP expression within 1–2 wk of Dox chase (Figs. 2 D and (Datasets S1 and S2). Similarly, 504 genes were consistently E and 3E). This discrepancy may have arisen because of the use up-regulated and 707 genes down-regulated in the non-LRCs of different experimental methods and time points used to label fraction (Datasets S1 and S3). Next, we compared the gene the LRCs population. In our study, the K5TetOff/TreH2BGFP

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1318848111 Leung et al. Downloaded by guest on September 25, 2021 nail PF compared with the Mx after 7 d. Indeed, after 1 mo, increasing numbers of labeled PF cells were detected, suggesting nail PF cells do proliferate, albeit at a slower rate (8), thereby supporting our findings regarding a slower (but still active) characteristic. Thus, the short time point of the experiment and lower labeling efficiency may explain the smaller number of LacZ-positive cells within the PF and their lack of association with streaks of cells forming the NP. Here, we used K15-driven lineage tracing experiments to directly address the contribution of PF cells during nail homeostasis and regeneration in vivo. Significantly, we reveal a distinct physiological + function for K15 PF cells in the delivery of progeny into the contiguous peri-nail epidermis and NP structure thus demonstrat- ing bifunctional SCs characteristics (Fig. 2 H–M). Under physio- logical conditions, K15-derived PF cells display a divergent SC dynamic, contributing faster to peri-nail epidermis (Fig. 2 H–K and Fig. S3 G–L) and slower to NP regrowth (Fig. 2 L and M). Nev- ertheless, we demonstrated K15-derived PF cells participated in the nail structure long term, expanding within the PF region over time (Fig. 2 L and M). Furthermore, we functionally demonstrated that, following injury, K15-derived PF cells could respond, acti- vate, and become recruited to deliver progeny into the nail Mx to participate in linear streams of cells toward NP differentiation during regeneration (Fig. 3 C–C′′). This faster response to injury could represent adaptive activation of the normally quiescent PF nail LRCs population (Fig. 3 E and F). Indeed, when we + tested the regenerative ability of the H2BGFP LRCs, we confirmed that engrafted PF LRCs actively contribute to nail CELL BIOLOGY regrowth (Fig. 3H and Fig. S5E) and retain slow cycling, LRCs qualities after an additional chase period (Fig. 3I). In agree- ment with our study, Sellheyer et al. recently reported that the human nail PF region expresses hair follicle stem cell (hfSC) markers during nail embryogenesis, suggesting that the PF may represent the human nail SC niche instead of the nail Mx (Fig. Fig. 4. BMP signaling is required for proper nail development and differenti- + ation. (A) H&E staining of control (Con) nail at P8; Inset shows magnification of S8) (15), thereby supporting our finding that K15 PF LRCs may the nail bed. (B) Corresponding Bmpr1a KO nails display abnormal NP, absent KZ, represent a distinct mouse NPFSCs population in vivo. nail bed (NB) hyperplasia (Inset), and a broken nail tip. Magnification of the P8 Our data demonstrate that NPFSCs have different proliferation Con (C)andBmpr1a KO (D) nails highlighting the absent KZ and ectopic granular characteristics, with slower cycling cells in the PF and accelerated layer beneath the Bmpr1a KO NP. (E) K1 staining (blue) in the PF of Con nails. (F) proliferation in the adjacent intermediate zone and proximal Mx Ectopic K1 expression (blue) in the Bmpr1a KO NP, arrows. (G) Loricrin (blue) is regions (Fig. 2F). Collectively, our model supports the recent report expressed in the Con nail PF and absent in the NP region marked by AE13 (red) of nail SCs in the proximal Mx by Takeo et al. (8) and now inte- H staining. ( ) Ectopic loricrin (blue) expression in the Bmpr1a KO NP; arrow with grates the adjacent neighboring PF as a defined location of bi- I + an absence of AE13 expression (red). ( ) Summary of the nail defects upon functional K15 ,NPFSCs(Figs.2N and 3B). Bmpr1a depletion. Bmpr1a, bone morphogenetic protein receptor 1; Con, con- trol; GFP, green fluorescent protein; KO, knock out; P, postnatal; U-LRCs, upper label-retaining cells. (Scale bars: 50 μm.) BMP Signaling Activity Tilts Nail Proximal Fold Progenitors Toward Nail Plate Differentiation. Transcriptionally, we observed a down- regulation of two BMP signaling inhibitors, Bambi and Decorin. reporter system uniformly marked all nail cells with Dox-regu- In hair follicles, BMP signaling is critical for maintaining quies- lated H2BGFP expression from embryogenesis (Fig. 1C), and cent SC homeostasis and regulating proper hair differentiation + – we did not observe any overlap between LRCs and the Ki67 nail (16 21) and, more recently, we have demonstrated the require- Mx after 4 wk of chase (Fig. 3E). However, by performing ment for BMP signaling in sweat gland development in vivo (2). shorter chases, we demonstrated that within the nail Mx, there is Msx2 and Foxn1, both downstream targets of BMP signaling, + − a gradient of rapidly proliferating, Ki67 H2BGFP cells, but regulate normal nail differentiation (22) and compound mutations of Msx2 and Foxn1 result in severe nail abnormalities. Here, we also less proliferative cells labeled by both Ki67 and weak + show that Bmpr1a-mediated signaling is essential for proper nail H2BGFP expression arising from less cell division (H2BGFP + differentiation (Fig. 4I and Fig. S7B), and that without it, the nail dilution) after 1 wk of chase (Fig. 2D, bracket). The Ki67 + adopts an epidermal fate. In nails lacking Bmpr1a-mediated H2BGFP intermediate zone Mx cells next to the LRCs signaling, we observed hyperplastic nail beds, broken nail tips, diminishes in size as the Mx H2BGFP label is continually di- and an irregular NP structure (Fig. 4 A and B) (22). Moreover, luted over time during Dox chase (Fig. 2E,bracket). the KZ was absent in Bmpr1a-deficient nails, indicating a more severe phenotype than the Msx2 and Foxn1 double mutant (Fig. Nail Proximal Fold LRCs Express K15 and Exhibit Bifunctional Stem 4D vs. Fig. 4C). In both Msx2 and Foxn1 single and double mutant Cell Fates. Recently, Takeo et al. used a K14CreER/R26LacZ nails, Mx proliferation was comparable to controls (22); how- + lineage tracing system to predominantly mark proliferating Mx ever, we observed reduced nuclear Ki67 staining in Bmpr1a KO cells to demonstrate that Mx cells could persist long term and nail Mx (Fig. S7C vs. Fig. S7D). We also demonstrated that NP form streaks in the NP, suggesting long-lived, SCs characteristics differentiation in Bmpr1a KO is compromised without AE13 (8). However, similar contributions were not observed from PF expression (Fig. 4H) and instead ectopically expresses epidermal cells (8) possibly explained by a lower labeling efficiency of the differentiation markers, K1 and loricrin (Fig. 4 F and H). In

Leung et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 addition, similar to Foxn1 and Hoxc13 single mutants, we also and the example here. At the molecular level, how the injured noted aberrant extension of the stratum granulosum within the microenvironment affects the diversion of SC fates remain to be nail structure of Bmpr1a KO mice (Fig. 4 B and D) (23, 24). investigated. One pathway we showed here is the level of BMP Collectively, our findings draw similarities for a key role of signaling that can modulate progenitor cells toward a nail or Bmpr1a-mediated signaling for the proper adoption of nail fate epidermal fate. From the Evo-Devo perspective, all ectodermal and differentiation similar to findings reported in hair follicle organs share the same origin in development and in evolution and sweat gland appendage development (2, 17, 20). (28). It is not surprising that different ectodermal organs can share SCs and support each other during regeneration following SCs at the Interface Between Ectodermal Organs. The proposed injury. From a translational point of view, the results described label-retaining, NPFSCs described here present a dynamic hi- here imply the plasticity of ectodermal organ SCs and the need erarchy similar to that described in hfSCs in which some hfSCs to know the control of their specificity, which is likely to be participate in the new hair cycle while other hfSCs of the upper mediated by the dermal microenvironment. Together, our data outer root sheath (ORS) of growing follicles, reestablish the new provide a unique platform from which novel therapeutics could bulge with quiescent hfSCs (25). During this bulge activation, a develop for the treatment of nail and digit defects and, in gradient of slow- to fast-cycling cells are observed within the ORS a broader sense, therapies for patients with severe skin injury extending from the bulge region toward the Mx, respectively and amputation. (Fig. S2D). In a similar manner, the nail contains a gradient of slow- to fast-cycling cells, with slow-cycling LRCs in the PF, Materials and Methods more active cells in the intermediate zone, and finally highly Mice and RU/Doxycycline Treatment. Previously described K5TetOff/TreH2BGFP proliferative cells in the nail Mx (Fig. 2F). Interestingly, we ob- animals were fed 1 mg/g Dox food for 4 wk starting at 3–4 wk of age (P21–P28) served that nail LRCs display faster turnover than hfSCs high- (9, 10). For Nail LRCs lineage tracing, K15CrePR/Rosa26LacZ mouse (12) were lighted by stronger H2BGFP expression in bulge hfSCs compared topically treated with 5 mg of RU [(wt/vol) in 100% ethanol] daily for 16 d with nail LRCs after 4 wk of chase (Fig. 1B). This faster from P43 to P59 as reported (18). turnover is perhaps expected because nails undergo constant Generation of Bmpr1a KO Mice. Bmpr1a floxed mice (29) were mated with growth, whereas hair follicles experience periodic regeneration. K14Cre mice (30) and also crossed onto the K14H2BGFP reporter mouse line From our studies, we postulate that LRCs closer to the Mx di- (10) to ablate Bmpr1a in the skin epithelium as described (17). vide more to fuel the rapidly proliferating transit amplifying Mx cells and establish the intermediate zone, between slow-cycling ACKNOWLEDGMENTS. We thank Dr. Tudorita Tumbar (Cornell University) LRCs and rapidly dividing Mx cells (Fig. 2F), containing slower for help with H2BGFP mice model optimization; Dr. Agnieszka Kobielak cycling Mx cells that overlap with the proximal Mx, recently [University of Southern California (USC)] for manuscript discussion; Dr. Colin Jamora [Institute for Stem Cell Biology and Regenerative Medicine (inStem)] proposed as nail SCs by Takeo et al. (8). for provision of K5, K1, and loricrin antibodies; and the Genomics Core In summary, SCs at the interface between two different ec- Facility, Children’s Hospital Los Angeles, USC Flow Cytometry Core, and USC todermal organs exhibit unique multipotentials. Under physio- Animal Facility. This study was supported by National Institute of Arthritis and logical condition, they can be under one state of homeostasis, Musculoskeletal and Skin Diseases (NIAMS) of the National Institutes of Health Grants R03-AR061028 (to K.K.) and R01-AR061552 (to K.K.). E.K. is a Fellow of whereas in wounding, the change of the SCs environment would the California Institute for Regenerative Medicine Research Training Program II lead to redirection of SCs flow (26) as demonstrated in the in Stem Cell Biology. C.-M.C. is supported by NIAMS Grants AR42177, AR transient emigration of K15 lineage cells to the epidermis (27) 047364, and AR060306.

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