Dependence of mesenchyme-only patterning on restricted TGFβ availability.

<p>(A) Western blot detection of phospho-SMAD2, total SMAD2 and γ-tubulin in E13.5 skin cultures treated with recombinant transforming growth factor (TGF) β2 (100 ng/ml), the TGFβ receptor inhibitor LY2109761 (25 μM,) or both agents for 8 h. (B) Effects of TGFβ2 supplementation and LY2109761 on normal and mesenchyme-only patterning. Condensates are slow to appear in LY2109761, and expression of the placode marker <i>Dkk4</i> expands through the epidermis. Mesenchyme-only patterning in FGF^HiBMP^Lo conditions is abolished upon either suppression or augmentation of TGFβ signalling. Scale bars: 250 μm. (C) Whole-mount in situ hybridisation (top panel) and corresponding transverse section (bottom panel) detecting spatial arrangement of <i>Tgfb2</i> expression in E14.5 mouse embryos. Expression is most intense at sites of dermal condensate formation. Scale bars: top panel = 1 mm, bottom panel = 50 μm. (D) At E13.5, phospho-SMAD2 immunofluorescence detects signal throughout the dermal mesenchyme (De.) and epidermis (Ep.), with this signal becoming intensified in the nascent dermal condensate at E14.5 (arrowhead). Epidermis is demarcated by dotted lines. Scale bar: 25 μm. (E) Dermal mesenchymal cell attraction (arrows) to sources of TGFβ2. Images of bovine serum albumin (BSA) control and TGFβ2 loaded beads placed on E12.5 TCF/Lef::H2B-green fluorescent protein (GFP) skin for 48 h. Scale bars: 250 μm.