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. 2013 Dec;140(24):4870-80.
doi: 10.1242/dev.101725. Epub 2013 Nov 6.

Keratin 79 Identifies a Novel Population of Migratory Epithelial Cells That Initiates Hair Canal Morphogenesis and Regeneration

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Free PMC article

Keratin 79 Identifies a Novel Population of Migratory Epithelial Cells That Initiates Hair Canal Morphogenesis and Regeneration

Natalia A Veniaminova et al. Development. .
Free PMC article

Abstract

The formation of epithelial tubes underlies the development of diverse organs. In the skin, hair follicles resemble tube-like structures with lumens that are generated through poorly understood cellular rearrangements. Here, we show that creation of the hair follicle lumen is mediated by early outward movement of keratinocytes from within the cores of developing hair buds. These migratory keratinocytes express keratin 79 (K79) and stream out of the hair germ and into the epidermis prior to lumen formation in the embryo. Remarkably, this process is recapitulated during hair regeneration in the adult mouse, when K79(+) cells migrate out of the reactivated secondary hair germ prior to formation of a new hair canal. During homeostasis, K79(+) cells line the hair follicle infundibulum, a domain we show to be multilayered, biochemically distinct and maintained by Lrig1(+) stem cell-derived progeny. Upward movement of these cells sustains the infundibulum, while perturbation of this domain during acne progression is often accompanied by loss of K79. Our findings uncover previously unappreciated long-distance cell movements throughout the life cycle of the hair follicle, and suggest a novel mechanism by which the follicle generates its hollow core through outward cell migration.

Keywords: Epithelial stem cells; Hair follicle; Infundibulum; Krt79; Lumen; Tube morphogenesis.

Figures

Fig. 1.
Fig. 1.
The INF is multilayered and biochemically distinct. (A) Schematic summarizing hair follicle development and regeneration. The cellular mechanism for generating the hair canal lumen during morphogenesis and anagen is unclear, but is thought to involve outward movement of differentiated inner root sheath (IRS) cells (green). Note the morphological similarity between the hair germ (HG) during development and the secondary hair germ (SHG) during anagen. After generating a mature hair shaft, the follicle regresses during catagen (dashed arrow) and enters the telogen resting phase. The infundibulum (INF) (blue) remains largely unchanged throughout the adult hair cycle. Sebaceous glands, yellow; companion layer (CL), red; dermal papilla, gray. (B) Immunohistochemical staining (IHC) for various skin differentiation markers, as indicated (red). The INF basal layer, highlighted by staining for K5 (green, arrows), is continuous with the basal layer of the epidermis. (C) IHC for various markers, as indicated (red), that are enriched in the suprabasal layers of the INF (sINF) of telogen hair follicles. SG, sebaceous glands. (D) IHC using an antibody against Ag-7195 (red), as shown in standard histological sections (left) and by whole-mount (right). The asterisk indicates the region of the hair canal enlarged in the inset. (E) TEM of telogen hair follicle INF, revealing the presence of multiple layers. The asterisk indicates the region of the INF enlarged in the right panel; the hair shaft is marked by double asterisks. Scale bars: 50 μm, except 10 μm in right panel of E.
Fig. 2.
Fig. 2.
Identification of K79 in the hair follicle. (A) Immunogold TEM showing that Ag-7195 localizes to keratin intermediate filaments in the skin. (B) Flow cytometry plot showing separation of hair follicle suprabasal YFP+ integrin α6- cells (red box) and basal YFP+ integrin α6+ cells (blue box) from telogen back skin of Shh;YFP mice. (C) Quantitative real-time PCR analysis of various poorly characterized keratins in YFP+ basal and suprabasal hair follicle cells isolated by flow cytometry in B. Values are expression fold change in suprabasal integrin α6- cells relative to basal integrin α6+ cells. Green bars, expression values of well-characterized keratins (K14, K10) or integrin α6 (A6) as used to verify the proper sorting of cell populations. Blue bars, keratins displaying reduced expression in suprabasal cells. Red bars, keratins displaying increased expression in suprabasal cells. (D) IHC on sweat glands from paw skin reveals that Ag-7195 (red, left panel) is enriched in suprabasal cells lining the sweat duct. (Right) Enlarged single-channel views of the region marked by the asterisk, with DAPI omitted to enhance clarity. Although K5 (green) is typically a marker of basal cells in the skin, suprabasal sweat duct cells can also express this keratin. (E) GEO2R profile graph of K79 expression in different sweat gland compartments using data collected by Lu et al. (Lu et al., 2012). Myo, myoepithelium; s. basal, suprabasal; lum, luminal epithelium. Two replicates for each compartment were analyzed in their study and are shown here. (F) Western blot (WB) showing that both the Ag-7195 and K79 antibodies recognize overexpressed K79 in 293FT kidney epithelial cells. Blots were also probed for β-actin as a loading control. (G) IHC showing colocalization of Ag-7195 (green) and K79 (red) in the sINF (yellow, merge). Inset, low-magnification view of the same hair follicle. Error bars indicate s.e.m. Scale bars: 50 μm, except 0.25 μm in A.
Fig. 3.
Fig. 3.
The INF is sustained by Lrig1+ stem cells but not by bulge stem cells. (A) IHC revealing colocalization of K79 (green) and Lrig1 (red) near the hair follicle isthmus. Double asterisk marks K79 and Lrig1 double-positive cells near the isthmus; single asterisk marks K79 single-positive sINF cells extending distally beyond the isthmus. SG, sebaceous gland. DAPI was omitted to improve clarity. (Right) Single-channel views of the same follicle. (B) β-galactosidase staining of skin from Lrig1;lacZ mice at the indicated time points after tamoxifen induction. (C) Quantitation of lineage tracing results using Lrig1;lacZ mice. The average percentage labeling within each compartment is shown following a chase period of 2-58 days after tamoxifen induction, as indicated on the x-axis. The INF is subdivided into upper and lower half domains. IFE, interfollicular epidermis. (D) IHC showing that the progeny of Lrig1+ stem cells (YFP, green) contributes stably to the basal (K14+, red, left panel) and suprabasal (K79+, red, right panel) layers of the INF in Lrig1;YFP mice, 50 days after tamoxifen induction. (E) Quantitation of bulge stem cell contribution to the INF in intact skin and in wound-proximal follicles, using K15;YFP mice, 3 or 50 days post injury. (Left) Wound-proximal follicles are defined as the three closest follicles to the site of wounding. Inset, example of IHC staining, depicting bulge-derived YFP+ cells in the INF (green, arrow), with β4-integrin as a basal layer marker (red). Asterisk indicates the wound edge. (Right) Bulge-derived contributions, by follicle, to the INF at the indicated times after wounding. The hair follicle closest to the wound site is designated as ‘1’. Error bars indicate s.e.m. *P<0.05, ***P<0.01; paired Student’s t-test (n=6 independent skin samples per time point). Scale bars: 50 μm.
Fig. 4.
Fig. 4.
Outward migration of K79+ cells during early hair follicle morphogenesis. (A) (Top) IHC showing that K79+ cells (red) form a continuous stream extending from hair germs out into the suprabasal epidermis (asterisks). The basal layer is marked by K5 (green). Beneath are single-channel views of the same hair germs. (B) Lineage tracing in Shh;YFP embryos reveals that K79+ cells (red, asterisks) are also YFP+ (green) and thus hair follicle derived. Note that not all hair follicle cells are YFP+ in Shh;YFP embryos, in agreement with previous observations (Levy et al., 2005). (C) Lineage tracing in Shh;YFP embryos reveals that Plet1+ cell streams (red, asterisk) are also YFP+ (green) and thus hair follicle derived. (D,E) IHC showing that cells at the distal tips of K79+ migratory streams (green) upregulate Mmp9 and cornifin-α (red). Beneath are enlarged single-channel views of the region indicated by the asterisk, with DAPI omitted to enhance clarity. (F) IHC of P3 skin, showing that K79+ cell streams (red) persist in some follicles (asterisk) but are lost in others (arrow). K5 marks the basal layer (green). (G) At P3, persistent K79+ cell streams (asterisk) frequently display weakened K10 expression (green). These streams are eventually lost from the epidermis as K79+ cells become restricted to the sINF, leaving behind epidermal gaps (arrow) above future hair canals. Scale bars: 50 μm.
Fig. 5.
Fig. 5.
Outward migration of K79+ cells during early anagen. (A) IHC showing that suprabasal K79+ cells (red, arrow) appear during anagen I in the SHG, which is highlighted by staining for P-cadherin (green). DAPI has been omitted to enhance clarity. Inset is a low-magnification view of the same follicle. (B) In slightly later stage anagen follicles, suprabasal K79+ cells (green) extend toward the anterior face of the club hair bulge (dotted line). (C) In anagen III follicles, K79+ cells have formed a continuous layer (green) extending from the anagen bulb up to the sINF. The club hair bulge, which is indicated by a dotted line along the anterior face, is magnified in the inset. (D) (Left) IHC of mature anagen follicle showing that K79 (red) localizes to the CL, as marked by K75 (green) (Gu and Coulombe, 2007). Single channels are shown to the right. (E) Gli1;lacZ mice display labeling of the upper bulge and SHG (blue), with a gap of unlabeled bulge cells in between (dotted line) (Brownell et al., 2011). Arrowhead indicates non-specific labeling due to endogenous β-galactosidase activity. (F) (Left) K79+ cells (red) first appear in the SHG during anagen I and express YFP (green) in induced Gli1;YFP skin. (Right) Enlarged single-channel views of the SHG (asterisk). (G) (Left) Lineage tracing in early anagen Gli1;YFP skin reveals that some K79+ cells (red) within the stream are also YFP+ (green, arrow) and therefore likely to be derived from the SHG. (Right) Single-channel reduced magnification views of the same follicle. Asterisk indicates dermal papilla. (H) Serial sections of a later stage anagen hair follicle from depilated Gli1;YFP skin reveals a continuous stream of cells positive for both K79 (red) and YFP (green) extending from the bulb along the anterior face of the club hair bulge. Yellow dotted line indicates regenerated anagen follicle. White dotted line indicates club hair bulge (posterior face). Blue dotted line indicates isthmus/INF. The right-most panel is a magnified view of the region indicated by the asterisk. Scale bars: 50 μm, except 25 μm in A,G.
Fig. 6.
Fig. 6.
Expression of K79 in pathological INF. (A) IHC for cleaved Notch receptor intracellular domain (NICD) (green, arrows) in the sINF. (B) IHC (left) or β-galactosidase staining (middle and right) showing the localization of labeled cells from Hes1;YFP (green) or Hes1;lacZ (blue) mice at the indicated time points after tamoxifen induction. (C) (Top) Hematoxylin and Eosin (H&E) staining of hair follicles containing cystic INF domains (arrows) from Lrig1;dnMAML skin, 20 weeks after tamoxifen induction. (Bottom left) IHC for dnMAML-GFP (green) and K79 (red). Asterisk indicates cystic region in the INF. Arrow indicates progeny of Lrig1+ stem cells expressing dnMAML that have migrated into the epidermis. (Bottom middle) Enlarged view of the region indicated by the asterisk. (Bottom right) Hair follicle from littermate control animal. (D) IHC for K5 (green) and K79 (red) in normal human hair follicles. (E) (Left) Facial acne from two patients. (Middle) H&E staining of acne comedones removed from patients 66 and 74. (Right) IHC for K79 (red) and other differentiation markers, as indicated, in serial sections of the same comedones as stained by H&E. The comedone from patient 66 retained K79, whereas the comedone from patient 74 lost K79. DAPI has been omitted to enhance clarity. Asterisk indicates comedonal cyst. Scale bars: 50 μm.
Fig. 7.
Fig. 7.
Outward migration of K79+ cells throughout the life cycle of the hair follicle. (Top left) During hair follicle morphogenesis, K79+ suprabasal cells (red) are specified within hair germs and stream out into the epidermis. During the hair peg stage, cells at the distal tips of these streams (purple) upregulate Mmp9 and cornifin-α. Concomitantly at this later stage, IRS cells (green) are specified and migrate outwards from the base. K79+ cells are subsequently lost from the epidermis, leaving behind a gap above the future site of the hair canal (asterisk). (Bottom left) During regeneration of the follicle in early anagen, this process is recapitulated when K79+ cells are specified in the SHG and stream out along the anterior face of the club hair bulge. These cells eventually form a continuous layer that links the anagen CL with pre-existing K79 cells in the sINF, effectively joining the new and old hair follicle lumens. (Top right) During homeostasis, Lrig1+ stem cells (blue) produce progeny that move upwards over time to replenish the cells of the INF. (Bottom right) During acne vulgaris, INF differentiation is disrupted, leading to the formation of a comedone in the INF and progressive loss of K79. K79+ cells are in red, except in the upper right panel where Lrig1+ stem cells and their progeny are in blue. Not to scale.

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