Tcf3 and Tcf4 are essential for long-term homeostasis of skin epithelia - PubMed (original) (raw)
. 2009 Oct;41(10):1068-75.
doi: 10.1038/ng.431. Epub 2009 Aug 30.
Affiliations
- PMID: 19718027
- PMCID: PMC2792754
- DOI: 10.1038/ng.431
Tcf3 and Tcf4 are essential for long-term homeostasis of skin epithelia
Hoang Nguyen et al. Nat Genet. 2009 Oct.
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.
Figures
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 (_Tcf4_Tg) 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 _Tcf4_ΔN, but not _Tcf4_ΔG, mice, (d) Krt6 expression is induced in _Tcf4_ΔN and _Tcf4_ΔTg, but not _Tcf4_ΔG, 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 Tcf3fl/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.
Comment in
- Tcf proteins are deeply rooted in skin.
Owens DM. Owens DM. Nat Genet. 2009 Oct;41(10):1050-1. doi: 10.1038/ng1009-1050. Nat Genet. 2009. PMID: 19786951 No abstract available.
Similar articles
- Tcf proteins are deeply rooted in skin.
Owens DM. Owens DM. Nat Genet. 2009 Oct;41(10):1050-1. doi: 10.1038/ng1009-1050. Nat Genet. 2009. PMID: 19786951 No abstract available. - In vivo transcriptional governance of hair follicle stem cells by canonical Wnt regulators.
Lien WH, Polak L, Lin M, Lay K, Zheng D, Fuchs E. Lien WH, et al. Nat Cell Biol. 2014 Feb;16(2):179-90. doi: 10.1038/ncb2903. Epub 2014 Jan 26. Nat Cell Biol. 2014. PMID: 24463605 Free PMC article. - A dynamic exchange of TCF3 and TCF4 transcription factors controls MYC expression in colorectal cancer cells.
Shah M, Rennoll SA, Raup-Konsavage WM, Yochum GS. Shah M, et al. Cell Cycle. 2015;14(3):323-32. doi: 10.4161/15384101.2014.980643. Cell Cycle. 2015. PMID: 25659031 Free PMC article. - Transcriptional and signalling regulation of skin epithelial stem cells in homeostasis, wounds and cancer.
Guan Y, Yang YJ, Nagarajan P, Ge Y. Guan Y, et al. Exp Dermatol. 2021 Apr;30(4):529-545. doi: 10.1111/exd.14247. Epub 2020 Dec 11. Exp Dermatol. 2021. PMID: 33249665 Free PMC article. Review. - Epithelial stem cells: an epigenetic and Wnt-centric perspective.
Gu B, Watanabe K, Dai X. Gu B, et al. J Cell Biochem. 2010 Aug 15;110(6):1279-87. doi: 10.1002/jcb.22650. J Cell Biochem. 2010. PMID: 20564229 Free PMC article. Review.
Cited by
- Suppression of TCF4 promotes a ZC3H12A-mediated self-sustaining inflammatory feedback cycle involving IL-17RA/IL-17RE epidermal signaling.
Jiang Y, Gruszka D, Zeng C, Swindell WR, Gaskill C, Sorensen C, Brown W, Gangwar RS, Tsoi LC, Webster J, Sigurðardóttir SL, Sarkar MK, Uppala R, Kidder A, Xing X, Plazyo O, Xing E, Billi AC, Maverakis E, Kahlenberg JM, Gudjonsson JE, Ward NL. Jiang Y, et al. JCI Insight. 2024 Mar 12;9(8):e172764. doi: 10.1172/jci.insight.172764. JCI Insight. 2024. PMID: 38470486 Free PMC article. - SOX9 switch links regeneration to fibrosis at the single-cell level in mammalian kidneys.
Aggarwal S, Wang Z, Rincon Fernandez Pacheco D, Rinaldi A, Rajewski A, Callemeyn J, Van Loon E, Lamarthée B, Covarrubias AE, Hou J, Yamashita M, Akiyama H, Karumanchi SA, Svendsen CN, Noble PW, Jordan SC, Breunig JJ, Naesens M, Cippà PE, Kumar S. Aggarwal S, et al. Science. 2024 Feb 23;383(6685):eadd6371. doi: 10.1126/science.add6371. Epub 2024 Feb 23. Science. 2024. PMID: 38386758 Free PMC article. - Reproducible strategy for excisional skin-wound-healing studies in mice.
Yampolsky M, Bachelet I, Fuchs Y. Yampolsky M, et al. Nat Protoc. 2024 Jan;19(1):184-206. doi: 10.1038/s41596-023-00899-4. Epub 2023 Nov 29. Nat Protoc. 2024. PMID: 38030941 Review. - Transcriptome analysis reveals genes associated with wool fineness in merinos.
Ma S, Long L, Huang X, Tian K, Tian Y, Wu C, Zhao Z. Ma S, et al. PeerJ. 2023 May 23;11:e15327. doi: 10.7717/peerj.15327. eCollection 2023. PeerJ. 2023. PMID: 37250719 Free PMC article. - Downregulation of Lhx2 Markedly Impairs Wound Healing in Mouse Fetus.
Takaya K, Sunohara A, Aramaki-Hattori N, Sakai S, Okabe K, Kishi K. Takaya K, et al. Biomedicines. 2022 Aug 31;10(9):2132. doi: 10.3390/biomedicines10092132. Biomedicines. 2022. PMID: 36140233 Free PMC article.
References
- Merrill BJ, et al. Tcf3: a transcriptional regulator of axis induction in the early embryo. Development. 2004;131:263–274. - PubMed
- Nguyen H, Rendl M, Fuchs E. Tcf3 governs stem cell features and represses cell fate determination in skin. Cell. 2006;127:171–183. - PubMed
- DasGupta R, Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development. 1999;126:4557–4568. - PubMed
Publication types
MeSH terms
Substances
Grants and funding
- R01 AR031737/AR/NIAMS NIH HHS/United States
- R01 AR031737-27/AR/NIAMS NIH HHS/United States
- HHMI/Howard Hughes Medical Institute/United States
- R01-AR31737/AR/NIAMS NIH HHS/United States
LinkOut - more resources
Full Text Sources
Molecular Biology Databases