Tcf3 and Tcf4 are essential for long-term homeostasis of skin epithelia

Abstract

Single-layered embryonic skin either stratifies to form epidermis or responds to Wnt signaling (stabilized beta-catenin) to form hair follicles. Postnatally, stem cells continue to differentially use Wnt signaling in long-term tissue homeostasis. We have discovered that embryonic progenitor cells and postnatal hair follicle stem cells coexpress Tcf3 and Tcf4, which can act as transcriptional activators or repressors. Using loss-of-function studies and transcriptional analyses, we uncovered consequences to the absence of Tcf3 and Tcf4 in skin that only partially overlap with those caused by beta-catenin deficiency. We established roles for Tcf3 and Tcf4 in long-term maintenance and wound repair of both epidermis and hair follicles, suggesting that Tcf proteins have both Wnt-dependent and Wnt-independent roles in lineage determination.

Figure 1

Tcf4 shares an expression pattern similar to that of Tcf3 in skin, where it becomes largely restricted to the slow-cycling hair follicle (bulge) stem cells and their early ORS progeny, (a) Tcf3 cKO mouse and wild-type (WT) littermate have indistinguishable phenotypes. (b) Immunolocalization of Tcf4 reveals temporal expression analogous to that of Tcf3 (ref. 4). Inset shows higher magnification of occasional Tcf4⁺ cell (red arrow) in postnatal epidermis. The most prominent postnatal labeling of Tcf4 is in hair follicle bulge stem cells and their early descendents in the ORS, (white arrow). Hp, hair placode; Hg, hair germ; Bu, bulge; Mx, matrix; Epi, epidermis; Der, dermis. (c) Real-time PCR analyses of mRNAs from flow cytometry–purified populations of cells from P4 skin. When normalized against the epidermis, both Tcf4 and Tcf3 are enriched in ORS cells that include the bulge and early bulge progeny. When normalized against suprabasal epidermal cells, Tcf3 and Tcf4 mRNAs are clearly detected in basal cells. Error bars indicate s.d. (d) Immunofluorescence of PO skin confirms that Tcf4 expression is ablated in Tcf4 knockout (KO) skin and that the antibody to Tcf4 is specific. Scale bars represent 20 μm.

Figure 2

Sustained epidermal expression suggests repressor and activator functions for Tcf4. (a) Left, the B isoform of Tcf4 (referred to here as Tcf4) represses basal TOPFlash Wnt reporter expression, as previously reported for Tcf3 (ref. 2). Right, Tcf4ΔG, which lacks the Groucho binding domain, does not repress TOPFlash, whereas Tcf4ΔN, which lacks the β-catenin-interacting domain, does. Error bars indicate s.d. RLU, relative light units. (b)Top, Tcf4 transgenes used to generate mice. Bottom, Krt14-Tcf4 transgenic (Tcf4Tg) mice die at birth and show repression of the epidermal differentiation phenotype, as previously described for Krt14-Tcf3 transgenic mice. Lor, loricrin; Inv, involucrin. (c,d) This phenotype is independent of the β-catenin-interacting domain but dependent on the Groucho repressor binding domain, (c) Expression of Lor and Inv is reduced in Tcf4ΔN, but not Tcf4ΔG, mice, (d) Krt6 expression is induced in Tcf4ΔN and Tcf4ΔTg, but not Tcf4ΔG, mice. Scale bars represent 20 μm.

Figure 3

Characterization of skin of newborn mice lacking Tcf3 and Tcf4. (a) Tcf3/4-null PO pups differ from wild-type and singly null mice in having thinner skin. (b) H&E staining shows that hair follicle downgrowth in skin from a severely affected PO Tcf3/4-null pup is defective, and the epidermis is abnormally thin compared to control wild-type skin (shown) or singly targeted Tcf3 cKO or Tcf4-null skins (see also Supplementary Fig. 3). (c) Histological analysis of toluidine blue–stained semithin sections reveals that Tcf3/4-null epidermis shows differentiating spinous, granular and stratum corneum layers, but cells in the basal layer are flattened, and differentiation appears morphologically less well developed, (d–h) Immunofluorescence analysis for the indicated markers, (d) Tcf3 and Tcf4 are absent from Tcf3/4-null skin relative to control skin. (e,f) PO Tcf3/4-null hair follicles still express Sox9, an early bulge stem cell marker, and Lhx2, which at this stage marks the leading edge of the downgrowing follicle. (g) Tcf3/4-null skin is also still proliferating at P, as shown by Ki67 immunostaining. (h) However, follicle regions show some TUNEL-positive cells, suggestive of a failure to survive (arrows, middle panel), (i) Quantification of data in g and h. Overall, the percentage of apoptotic cells was still very low (~5%). Error bars indicate s.d. HF, hair follicle. Scale bars represent 10 μm in c and 20 μm in b,d–h.

Figure 4

Skin grafting permits evaluation of the long-term consequences of Tcf3 and Tcf4 ablation in skin. (a) After 17 d of engraftment of female skins onto the backs of male nude mice, Tcf3 cKO and Tcf4 KO skins are indistinguishable from the wild type, whereas Tcf3/4-null grafts (area indicated by dashed line) shrink and show no hair. (b) Y-chromosome FISH shows that the engrafted Tcf3/4-null epidermis is still female and is not derived from the male nude epidermis surrounding the graft. (c) H&E staining reveals absence of hair follicles from Tcf3/4-null skin grafts. (d–g) Differentiation of the epidermis still occurs in Tcf3/4-null skin, as shown by immunolabeling for Krt1 (spinous) and loricrin (granular) markers. (f) Although epidermis of Tcf3/4-null grafts appears thicker, no immunolabeling was detected for Krt6, which marks the hair follicle ORS (companion layer) of normal skin and the suprabasal epidermis of hyperproliferative skin. (g) Ki67 immunolabeling reveals a paucity of proliferative keratinocytes within day 17 Tcf3/4-null skin grafts. (h) Quantification of Ki67 and activated caspase 3 staining. Error bars indicate s.d. (i) Tcf3/4-null engrafted skin is defective in reepithelialization after wounding at day 30 after engraftment. Shown are H&E-stained skins 7 d after wounding. Arrows indicate sites where reepithelialization has occurred to repair the wound, in control but not in Tcf3/4-null skin. Scale bars indicate 20 μm in b and 100 μm in c–g and i.

Figure 5

Measuring the stem cell potential within hair follicles and epidermis in the absence of Tcf3 and Tcf4. (a) Hair follicles in Tcf3/4-null dermis seem to lack the potential to regenerate epidermis. After dispase treatment to remove and discard epidermis, entire wild-type or Tcf3/4-null male skin dermis (containing hair follicles) was engrafted onto female nude mice. After 30 d, control (wild-type) grafts showed Y chromosome–positive epidermal cells, whereas epidermis from Tcf3/4-null dermis was derived from nude epidermal reepithelialization. (b) Chamber graft mixing experiments with purified, single-cell suspensions of sex-marked epidermal or hair follicle cells show that, in contrast to wild type, neither Tcf3/4-null epidermal cells nor hair follicle cells contribute properly to reestablishment of skin epidermis. IFE, interfollicular epidermis, either near to or far from hair follicles. (c) Quantification of male IFE cells from split-thickness grafts. (d) Quantification of female epidermal cells in chamber grafts detected in near or far IFE. Error bars (c,d), s.d.

Figure 6

Loss of Tcf3 and Tcf4 results in inability of cultured epidermal keratinocytes to undergo long-term self-renewal. (a) Tcf3/4-null primary mouse keratinocyte cultures show a growth defect. (b) No differences in cell-substratum adhesion are seen when equal numbers of primary mouse keratinocytes are plated onto various matrices and quantified after 1 h. (c) Rhodamine B staining of primary mouse keratinocytes cultured for 10 d reveals a growth defect in the absence of Tcf3 and Tcf4. (d–h) Inducible deletion of Tcf3 from Tcf4^−/− cells affects their growth potential. Cultured primary mouse keratinocytes from epidermis of Tcf3^fl/fl; Tcf4^−/− P0 mice were passaged twice and then infected with retrovirus expressing either green fluorescent protein (GFP) alone or GFP with tamoxifen-inducible Cre (transgene cre-Esr1; referred to here as ‘CreER’). Infected cells were isolated by flow cytometry based on GFP level, and 1 d after plating, cells were treated with tamoxifen or vehicle control to induce Cre recombinase activity. Twelve days after plating, cells were fixed and stained with rhodamine B to visualize and quantify the numbers and sizes of colonies (those with at least four cells, 72 h after plating). Plating (e,f) and colony-forming (f) efficiencies were comparable between wild-type and Tcf3/4-null cells, but the average size of Tcf3/4-null colonies was markedly smaller than that of wild-type colonies (g) and the morphology of the mutant cells revealed signs of premature differentiation (h). All error bars indicate s.d.

Figure 7

Differences between Tcf3/4-null and Ctnnb1-null skin. (a) Conditional ablation of Ctnnb1 by Krt14-Cre results in death shortly after birth, when hair follicle morphogenesis has already been blocked. (b–e) Notably, epidermis is hyper-thickened (b), and proliferation, as judged by Ki67 (c,e), is not compromised, even when skins are grafted and examined 60 d after engraftment. Overlying epidermis is clearly derived from the engrafted skin, as revealed by lack of β-catenin immunolabeling (d). Scale bars represent 20 μm in a,b,d and 100 μm in e. Error bars in c indicate s.d.

Figure 8

Ablation of Tcf3 and Tcf4 in skin leads to an upregulation of gene expression. (a) Basal epidermal keratinocytes were purified by flow cytometry from E17.5 skins of Tcf3/4-null and wild-type mice, and their mRNAs were subjected to microarray analyses as outlined in Online Methods. Comparative analysis scored genes as being increased (‘Inc’) or decreased (‘Dec’) by the fold indicated. Most genes scored as upregulated upon loss of Tcf3 and Tcf4. (b–d) Real-time PCR analyses of mRNAs from flow cytometry–purified basal epidermal keratinocytes of wild-type, Tcf3 cKO, Tcf3^+/−; Tcf4^−/−, Tcf3/4-null and Ctnnb1 cKO E17.5 embryos. Genes chosen for analyses in b are controls to verify the status of Tcf3, Tcf4 and Ctnnb1 relative to comparable levels of the basal marker Krt5. Genes chosen for analyses in c scored as upregulated in Tcf3/4-null compared to wild-type epidermis and are known to directly bind to Tcf3 in embryonic stem cells. Genes upregulated in E17.5 Tcf3/4-null basal epidermis were mostly unchanged (top) in Ctnnb1-null basal cells. A minority were either slightly increased or decreased (bottom panels). (d) A few genes scoring as decreased in the Tcf3/4-null samples also show reductions in expression when β-catenin is lost. Some of these are Wnt target genes, including Dkkl1 and Sostdc1.