Fig 1: Teasing out a WNT-sensitive molecular signature based upon transcriptome profiling of skin progenitors possessing different WNT signaling levels.(A) LV construct, epifluorescence imaging and FACS strategy for isolating WNT signaling (GFP+) and WNTlo skin progenitors from LV-transduced E14.5 Apc fl/+ and Apcfl/fl; R26fl-stop-fl-tdTOM embryos. (B) Gene set enrichment analysis (GSEA) of gene sets showing marked differential expression in WNT signaling progenitors from Apc-null vs Apc-het embryos. False discovery rate (FDR) q-values of enrichment are shown for each gene set. (C) Waterfall plot depicting genes markedly influenced (Log2 Fold Change = 1.5, p<0.05) by APC status (color-coding according to B). (D) Volcano plot showing differentially regulated transcripts and WNT-reporter status.Figure 2—source data 1.Source data for the graphs shown in Figure 2D.
Fig 2: Wild-type WNT signaling progenitor cells express high levels of WNT inhibitors.(A) Strategy used to isolate and profile slow-cycling basal progenitors from the epidermal fraction of dispase-treated, wild-type E17.5 skin, which contains epidermis and early hair placodes/buds. Note: LEF+ progenitors are simultaneously LHX2GFP+ and mKO2+. (B) Volcano plot and comparative expression profiling reveals that relative to their epidermal counterparts, wild-type basal placode/bud progenitors share strong signature similarities with Apc-null progenitors. Green dots denote previously reported WNT target genes. (C) In situ hybridizations showing that WNT signaling progenitor cells simultaneously express mRNAs for WNT activators and WNT inhibitors. Black dashed lines, epidermal-dermal boundary; white dashed lines demarcate the dermal condensate (DC). Scale bars, 10 µm.Figure 3—source data 1.Source data for the graphs shown in Figure 3B.
Fig 3: Two-dimensional patterning of hair placodes is severely affected upon sustained autonomous WNT activation.(A) Sagittal views and schematic of skin section depicting a basal hair bud progenitor and underlying dermal condensate (DC, encased by blue dotted lines). Labeling is for LacZ expression knocked into the Axin2 locus and nuclear LEF1, two faithful proxies of WNT signaling. Additionally, nuclear SOX9 marks the overlying WNTlo bud cells, and P-cadherin marks the basal epithelial progenitors. Dashed lines denote the basement membrane (BM) rich in extracellular matrix (ECM) and growth factors at the epidermal-dermal border. Scale bars, 10 µm. (B) (top left and bottom) In utero lentiviral delivery strategy to generate sparse epidermal patches lacking APC, and therefore super-activating WNT signaling. Visual and epifluorescence imaging of mosaically transduced (R26dtTomato+) E14.5 Apc heterozygous and null embryos. Scale bar, 2 mm. (top right) Schematic of whole mount imaging. (C) Planar views of the skin surface of E14.5 embryos. Scale bar, 100 µm. (D) Quantifications showing Apc null clusters of broader size and shape than Apc heterozygous (het) placodes, which were analogous to wild-type in this assay (Circularity = 1 perfect circle). (Placodes and clusters density plot n > 10 mm2 skin area; ****p<0.0001; Mann-Whitney test; Area and Circularity plots n = 130 placodes and 216 clusters; ****p<0.0001; Mann-Whitney test; All n = 3 embryos.). (E). Whole mount (planar) images showing atypically strong nuclear ß-Catenin and LEF1 in Apc-null cell clusters. Scale bar, 20 µm.Figure 1—source data 1.Measurements of placodes and clusters distribution, size and shape, shown in Figure 1D.
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