Links between signal transduction, transcription and adhesion in epithelial bud development

Abstract

The morphogenesis of organs as diverse as lungs, teeth and hair follicles is initiated by a downgrowth from a layer of epithelial stem cells. During follicular morphogenesis, stem cells form this bud structure by changing their polarity and cell-cell contacts. Here we show that this process is achieved through simultaneous receipt of two external signals: a Wnt protein to stabilize beta-catenin, and a bone morphogenetic protein (BMP) inhibitor to produce Lef1. Beta-catenin then binds to, and activates, Lef1 transcription complexes that appear to act uncharacteristically by downregulating the gene encoding E-cadherin, an important component of polarity and intercellular adhesion. When either signal is missing, functional Lef1 complexes are not made, and E-cadherin downregulation and follicle morphogenesis are impaired. In Drosophila, E-cadherin can influence the plane of cell division and cytoskeletal dynamics. Consistent with this notion, we show that forced elevation of E-cadherin levels block invagination and follicle production. Our findings reveal an intricate molecular programme that links two extracellular signalling pathways to the formation of a nuclear transcription factor that acts on target genes to remodel cellular junctions and permit follicle formation.

Figure 1

Wnt and noggin induce a transcriptionally competent Lef1 complex that is absent in Nog ^−/− mice. a, Cell–cell signalling (denoted by arrows) in developing follicles: noggin is expressed by mesenchyme and Wnts by ectoderm. b, SDS–PAGE immunoblots showing Wnt3a-induced β-catenin upregulation and noggin-induced Lef1 induction in lysates from keratinocytes treated with control (−) or conditioned (+) media. c, Immunofluorescence of keratinocytes, revealing nuclear Wnt3a-induced β-catenin and noggin-induced Lef1. d, RT–PCR and immunoblot analyses revealing BMP2 and BMP4, but not noggin, in keratinocytes. e, Effect of Wnt3a and/or noggin on TOPFLASH (test) and FOPFLASH (control) in transiently transfected keratinocytes. E, low calcium media. f, Dependency of nuclear β-catenin, Lef1 and TOPGAL expression on noggin in vivo. E16.5 skins from the knockout/transgenic (Tg) mice as indicated at top. Note that in epithelial buds lacking noggin, neither Lef1 nor TOPGAL are expressed and β-catenin is diffuse; these features are rescued on the K14-Lef1 transgenic background. Dotted lines denote mesenchymal–epithelial boundaries between epidermis (epi) and dermis (der). Bottom row are low-magnification views; lower right is of Nog ^−/− intestinal epithelium, which uses Tcf4, rather than Lef1, to regulate Wnt signalling. For b, c, f, antibodies or TOPGAL activity assays are denoted at left.

Figure 2

During follicle morphogenesis and cycling, skin stem cells change cadherin expression in a fashion dependent upon noggin and Wnt signalling. Wild-type skins from TOPGAL reporter mice at E16.5 (a–d, h, i) or adult (e–g) (left) or mutant E16.5 skins from the genetic backgrounds indicated (j–r) (right) were processed for double immunofluorescence, TOPGAL (β-galactosidase) activity or in situ hybridizations, using the markers indicated (colour coding denotes secondary antibodies). Wherever noggin and Wnt signalling were active, E-cadherin was downregulated, and nuclear Lef1 was present, indicating β-catenin activated Lef1 complexes. This was true even for Shh-null skin (o, p), arrested at the bud stage, and for the Lef1-positive epithelial invaginations (arrowheads) of K14-ΔNβcat transgenic (Tg) skin, expressing stable β-catenin (q, r). Without noggin, few buds formed and E-cadherin remained high (j–m), but this was rescued by K14-Lef1 (n). lam, laminin 5 antibody, demarcating the epithelial–mesenchymal boundary (elsewhere delineated by dotted lines); K5 antibody, marking the basal layer of epidermis and outer root sheath of the hair follicle. epi, embryonic epidermis; der, dermis; DP, dermal papilla.

Figure 3

Lef1 and β-catenin transcriptionally downregulate E-cadherin in vitro. a, b, d, Promoter activity assays on extracts from keratinocytes treated without (a) or with (b, d) control (E) or conditioned media as indicated. Cells were co-transfected with E-cadherin-β-galactosidase (WT (wild-type) or mt (mutant) Lef1-site; a, b) or HK1-luciferase (d) ± K14-expression vectors encoding Lef (Lef1), βcat (ΔNβcat), Sna (Snail), or control (empty vector). c, Chromatin IP analyses of Lef1-expressing keratinocytes ± Wnt-conditioned medium. Fragmented, crosslinked DNA from chromatin immunoprecipitated with Lef1 or control antibody was subjected to PCR with primers encompassing the known Lef1 site or control sequence in the E-cadherin promoter. DNA, genomic control.

Figure 4

Levels of adherens junction proteins influence follicle morphogenesis. a–d, Immunofluorescence and haematoxylin-eosin staining of frozen back-skin sections from a representative newborn mouse mosaic for the K14-Ecadherin-HA transgene. Transgene-negative (Tg −) and positive (Tg +) regions were identified by anti-HA (HA), and anti-laminin 5 (lam) delineated the basement membrane. e–h, Haematoxylin-eosin staining of back-skin sections from wild-type and α-catenin K14-Cre conditional-null (αcat ^−/−) embryos. Boxed areas are shown at higher magnification to right of each frame. Note that in α-catenin-null skin, follicle buds (arrows) begin to form at E16.5, but cellular disorganization arrests their downgrowth by E18.5.