Activation of the mouse hair keratin 1a promoter by Lef1 and β-catenin. Primary mouse keratinocytes were transfected and processed as described in the legend to Fig. 2, except HK1Flash, containing the mouse hair keratin 1a promoter/enhancer, was used instead of TOPFlash. β-catenin had little effect on the hair keratin promoter unless cotransfected with Lef1. Both ΔNLef1 and ΔNTcf3 failed to activate the promoter when cotransfected with ΔNβcat.

Tcf3 requires the Grg-binding domain but not the CtBP-binding domains for repressor activity in keratinocytes. (A) Diagram of Tcf3 mutants and Tcf3–Lef1 fusions used in frame B (see below). For fusion proteins, Tcf3 regions are white, and Lef1 regions are gray. Numbers above each fusion correspond to the amino acids of Tcf3 (black numbers) and Lef1 (gray numbers) at the junctions of the fusions. (B) Primary human keratinocytes were transfected and processed as described in Fig. 2. Note that Tcf3-mediated repression of basal TOPFlash activity required the Grg-interaction domain, but not the carboxy-terminal segment. Note also that the ΔNβcat-mediated activation of TOPFlash required the carboxy-terminal segment, but not the Grg-interacting domain. The requirement of the carboxy-terminal segment for ΔNβcat-mediated activation was influenced by residues 183–341 of the Tcf3 protein, as judged by the fact that Fusion 3 activated, whereas Fusion 1 did not. (B‘) The amino-terminal 71 amino acids of Tcf3 were not required for repression, but were required for ΔNβcat-mediated activation. Transfections in B′ were conducted in the presence of ΔNβcat.

Model suggesting roles of Lef1 and Tcf3 in controlling cell fate in the skin stem cell lineage. Each large circle represents a different cell type arising from the stem cell. The color of sectors in the center of the inner circles represents nuclear localization of Lef1, Tcf3, or β-catenin as indicated by the legend. The outer ring represents the cytoplasm and the status of TOPGAL activity in each cell type (blue: ON). See text for detailed description of events depicted. Note that in the model, Tcf3 is associated with maintaining stem cell and ORS cell fate, and Lef1 and β-catenin promote hair cell fate. Blocking a Wnt signal response (Lef1–β-catenin activation) through ΔNLef1 expression allows induction of sebocyte cell fate during the hair cycle. Note also that it remains unclear whether Tcf3–β-catenin and/or Lef1–β-catenin complexes are responsible for the hair cycle-dependent induction of TOPGAL activity in the bulge cells.

Transgenic expression of Tcf3 perturbs epidermal barrier formation and late-stage epidermal differentiation. (A–D) Barrier function assays. An endogenous β-galactosidase activity in embryonic skin allows for detection of permeability to small molecules (i.e., Xgal). (A,B) Wild-type embryos acquire barrier function between E16.5–18.5 (Hardman et al. 1998). The dorsal–ventral progression is characteristic of nontransgenic littermates. (C,D) Barrier formation in K14–Tcf3 was incomplete, even at E18.5. Note the Zorro-like mask of blue staining and the opened eyelids. (E–H) Hematoxylin and eosin stained sections of newborn back skins of mice with genotypes indicated in the upper right corners. Basal (Bl) and spinous (Sp) layers of the epidermis appear histologically normal in each sample. The basophilic keratohyalin granules (brackets in E,G,H) are distinctly absent from the K14–Tcf3 epidermis (asterisks in F).

Expression of K14–Tcf3, K14–Tcf3*, and K14–Lef1 in transgenic mice. (A) Western blot analysis of total skin protein from transgenic mice. Equal levels of protein extracts were resolved by 7% SDS-PAGE gel, and transgene products were detected by anti-myc monoclonal antibody. (B–E) Double immunofluorescent staining of newborn mouse back skin sections. Mouse anti-myc (green) was used to detect transgene expression and rabbit anti-K5 (red) used to highlight the basal layer of the epidermis and the ORS/bulge. Note that transgenic protein was stable even in suprabasal layers (arrowheads). The broken lines demarcate dermal–epidermal boundaries. (F–H) Localization of β-catenin in newborn back skin sections. (F) β-catenin (green) was restricted to the cell borders where it colocalized with E-cadherin (red) in wild-type epidermis. (G) Double immunofluorescence using anti-β-catenin (green) and anti-Tcf3 (red) antibodies shows colocalization of Tcf3 and β-catenin in epidermal nuclei (arrowheads) of K14–Tcf3 transgenic mice. (G, inset) Nuclear localization of β-catenin (green; arrowheads) was distinct from cell borders identified by E-cadherin staining (red). (H) Double immunofluorescent staining using anti-β-catenin (green) and anti-Lef1 (red) antibodies shows that transgenic expression of Lef1 did not appear to cause nuclear localization of β-catenin in K14–Lef1 mice.

The appearance of differentiation features characteristic of ORS/bulge in transgenic epidermis expressing Tcf3, but not Lef1 nor Tcf3*. Each panel (A–P) shows double immunofluorescent staining of newborn back skin sections from the mice with the genotype indicated in upper right corner. Mouse anti-myc (green) staining was used to detect transgene expression, and rabbit polyclonal antibodies (red) were used to detect filaggrin, loricrin, K17, or K6 as indicated. (A–D) Compared with the normal punctate pattern of anti-filaggrin staining (red in A,C,D), filaggrin staining was less intense and more diffuse in K14–Tcf3 epidermis (B). (E–H) Compared with the normal pattern of anti-loricrin staining (red in E,G,H) which is localized to cell borders and distinct from keratohyalin granules, the loricrin staining of K14–Tcf3 epidermis was more intense and nearly uniform in suprabasal layers (F). (I–L) Expression of K17 (red) was restricted to the ORS of nontransgenic (I), K14–Tcf3* (K), and K14–Lef1 (L) newborn back skin. In contrast, K17 was expanded to all layers of the epidermis in K14–Tcf3 mice (J). (M–P) K6 (red) was restricted to the inner layer of the ORS in non-transgenic (M), K14–Tcf3* (O), and K14–Lef1 (P) newborn back skin, but it was induced in suprabasal layers of the epidermis in K14–Tcf3 (N) mice.

Expression of Tcf3, Lef1, nuclear β-catenin, and TOPGAL activity in the hair follicle. (A) Structure of the hair follicle. (B) The bulb region of an anagen hair follicle. Double immunofluorescent staining of Lef1 (red in B, B‘) and β-catenin (green in B‘) showed that β-catenin is present at cell borders in all cells of the hair follicle, but nuclear β-catenin localization is apparent only in the precortex cells, which contain nuclear Lef1. (B“) X-gal staining of TOPGAL transgenic mice showed that activation of this reporter construct was associated with the colocalization of nuclear β-catenin and Lef1. (C) Immunofluorescent staining of Tcf3 with a mouse monoclonal antibody (green; large panel) showing prominent staining in the bulge with staining extending to the ORS. (C, inset) Double immunofluorescent localization of Tcf3 with a guinea pig polyclonal antibody (red) and β-catenin (green) showed Tcf3 present in bulge and ORS cell nuclei and β-catenin present primarily at cell borders. (D–F) The bulge region of the hair follicle during the hair cycle. (D) X-gal staining of a TOPGAL single transgenic hair follicle showed the activation of the reporter in a small number of bulge cells (arrowheads). (E) In TOPGAL/K14–ΔNβcat double transgenic hair follicles, most bulge cells of the hair follicle displayed TOPGAL activity. (F) Immunofluorescent staining of Lef1 in skin sections from K14–ΔNβcat mice. Note that Lef1 protein was up-regulated in the bulge region and secondary hair germ of K14–ΔNβcat skin. (precx) Precortex; (DP) dermal papilla.

Repressor activity of Tcf3 is required for its potent effects in transgenic mice. (A) Schematic diagrams as described in the legend to Fig. 2. (B,C) Hematoxylin and eosin stained sections of back skin. Note the lack of keratohyalin granules in K14–Tcf3ΔC (asterisks in B) and the normal granular layer in K14–Tcf3ΔGrg (bracket in C). (D–G) Double immunofluorescent detection of transgene protein (green) and either filaggrin (red; D,E) or loricrin (red; F,G). Note that defects in anti-filaggrin and anti-loricrin staining were even more severe in K14–Tcf3ΔC skin than in K14–Tcf3 skin. Note normal staining in K14–Tcf3ΔGrg skin. (H–K) SDS-PAGE and Western blot analysis of equal amounts of total skin protein extracts from 4-day-old nontransgenic, K14–Tcf3ΔC and K14–Tcf3ΔGrg mice. (H) Coomassie-stained gel showing comparable loading of samples. (I) Anti-myc Western blot to illustrate levels of transgene expression. (J) Anti-filaggrin Western blot. Note that mature filaggrin and proteolytically processed pro-filaggrin were decreased in K14–Tcf3ΔC mice but were normal in K14–Tcf3ΔGrg mice. (K) Anti-loricrin Western blot confirming that loricrin levels were diminished in K14–Tcf3ΔC mice but normal in K14–Tcf3ΔGrg mice. (L) Immunohistochemical detection of Grg proteins in nontransgenic epidermis using a panTLE antibody that recognizes all known Grg/TLE proteins. Note that staining was restricted to nuclei and was more intense in suprabasal layers compared with the basal layer. Anti-Grg antibody staining was also seen in the nuclei of cultured keratinocytes. (M) Hematoxylin and eosin stained skin section from K14–ΔNTcf3 mouse. Note the lack of keratohyalin granules.

In keratinocytes, Tcf3 and Lef1 have different effects on target gene activation and on nuclear localization of β-catenin. (A) Schematic diagrams of constructs illustrating key protein domains of Tcf3, Tcf3*, and Lef1. The α-helical DNA-interacting segment is denoted in blue and the mutated amino acids in Tcf3* are indicated in red. The two CtBP-binding motifs in the carboxyl terminus of Tcf3 and Tcf3* (PLSLT and PLSLV) are inidicated. (Int.) Intron; (myc) myc-epitope tag; (β) β-catenin-binding domain; (Grg) Grg-binding domain; (CtBP) CtBP-binding region. (B, top) EMSA using recombinant 6×HisTcf3 HMG and 6×HisTcf3* HMG proteins. Recombinant Tcf3 HMG retarded the mobility of the ³²P-labeled TCRα oligo in a concentration dependent manner, whereas Tcf3* HMG did not bind the DNA. (B, bottom) Western blot using anti-pentaHis monoclonal antibody to detect the Tcf3 HMG and Tcf3* HMG at the concentrations used for the EMSA. (C) Primary human keratinocytes were transfected with TOPFlash reporter, the indicated Lef1/Tcf3, and either empty vector (open bars) or K14–ΔNβcat (solid bars, inset). Values represent the average of duplicate transfections +/− standard deviation, and this graph is a representative from three separate experiments. Tcf3 repressed TOPFlash activation, whereas Lef1 activated it. Tcf3* had no major effect on TOPflash activity. (D–G) Keratinocytes grown on coverslips were transfected with either K14–Tcf3 (D,E) or K14–Lef1 (F,G), given high Ca⁺⁺ to induce junction formation, and processed for immunofluorescence to detect endogenous β-catenin (green) and transfected Lef1 or Tcf3 (anti-myc; red). Note endogenous β-catenin was restricted to the junctions in nontransfected and Lef1 transfected cells; note also that in Tcf transfected cells, β-catenin also displayed nuclear localization.

K14–ΔNLef1 expression promotes sebocyte cell-fate during the hair cycle. (A–E) Secondary hair germ growth in wild-type littermate. (A) Hematoxylin and Oil Red-O stained section of tail shows a primary hair follicle with sebaceous glands (SG; red stain) and secondary hair germ extending below the club hair. (B) Hematoxylin and eosin stained section shows the formation of the developing new follicle, replete with matrix surrounding the dermal papillae (DP), and a discernable outer root sheath (ORS). (C) Immunofluorescent staining using a β-catenin antibody showing nuclear accumulation of β-catenin (arrowheads) in cells that formed a crescent around the dermal papilla (DP); note that the demarcation between matrix and precortex is not yet complete, but the ORS is already distinct. (D) Similar stage to panel B, revealing more prominent nuclear β-catenin in precortex (precx) and more diffuse staining in matrix (mx). (E) Double immunofluorescent detection of K5 (red) and hair keratin (AE13; green) indicates precortex (precx) formation in the region containing nuclear β-catenin; Hair keratins are extremely stable and can also be seen in the hair shaft, containing dead hair cells (see club hair). (F–I) Abnormal cell fate determination in K14–ΔNLef1 skin. (F) Hematoxylin and eosin stained section from the toe of a K14–ΔNLef1 mouse shows the formation of vacuous structures encased by the epithelial cells of the secondary hair germs. (F, top inset) Hematoxylin and Oil Red-O staining of back skin sections show that the vacuous structures appeared to be filled with sebum, as judged by the fact that they accumulate Oil Red-O stain. (F, bottom inset) Immunofluorescent staining for transgene product (anti-myc) shows that it is present in the crescent of secondary hair germ (HG) cells that abut the dermal papilla (arrowheads). (G) Hematoxylin and Oil Red-O staining of tail follicles shows the presence of oil-producing cells (arrow) at the base of the newly developed follicle, which is replete with an outer root sheath (ORS), but lacks hair structures. Note also the oil-producing cells of the sebaceous glands (SG), similar to those seen in A. (H) Immunofluorescent staining for β-catenin shows that nuclear β-catenin is still found in the crescent of cells surrounding the dermal papilla (DP). At this stage, the secondary hair germ is similar to that seen in the wild type (cf. with C). (I) Anti-K5 and AE13 double staining of developing secondary follicle. Note that the cells surrounding the dermal papilla formed, but they did not give rise to hair keratin-expressing cells, as judged by the absence of AE13 staining. In the place of the hair shaft are differentiated cells (*) that appear to be the ones that stain with Oil Red-O.