Supplementary Figure 5
From: Anisotropic stress orients remodelling of mammalian limb bud ectoderm
(a) Confocal Z sections (top panels) and XY sections (bottom panels) of control Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud ectoderm visualising βcatenin (red) and DAPI (blue). (b) Histogram representing percentage Venus-positive nuclei from Fgfr2f/f and Fgfr2f/f ;Crect mutant limb bud ectodermal cells expressing Tcf/Lef::H2B-Venus (p = 0.0014 (Student’s t-test); WT n = 3, mutant n = 4 20-25 som. embryos). (c) Confocal XY section of Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud ectoderm visualising phospho-ERK (green) and DAPI (blue). (d) Cartilage (Alcian Blue) and bone (Alizarian Red) staining of E18.5 Fgfr2f/f;Crect mutant forelimb (top) and hindlimb (bottom) skeletons. (e) Histogram representing percentage of pHH3-positive cells in Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud ectodermal cells (p = 0.8731). (f) Histogram representing number of Caspase3-positive cells in Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud ectodermal cells (p = 0.6567). (g) Histogram representing percentage of pHH3-positive cells in Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud mesodermal cells (p = 0.9973). (h) Histogram representing number of Caspase3-positive cells in Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud mesodermal cells (p = 0.6032). (For e-h, n = 3 19-21 som. embryos per condition, Student’s t-test) (i) Confocal XY section of Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud ectoderm visualising E-cadherin (red) and DAPI (blue). (j)Fgf10 expression in Fgfr2f/f; Crect mutant limb buds at E9.5 (left) and E10.5 (right). (k) Length-width ratio of WT and Fgfr2f/f;Crect mutant limb bud ectodermal cells (n = e 19-21 som. embryos per condition; p = 0.0189 (Student’s t-test)). (l) Angle of cell orientation in WT and Fgfr2f/f;Crect mutant limb bud ectoderm. Histogram shows distribution of cells grouped into 6 bins of 15° (n = 3 19-21 som. embryos per condition; p = 0.0412 (75-90° bin) (Student’s t-test)). (m) Confocal XY projection of E10.0 (32 somite stage) Fgfr2f/f;Crect mutant forelimb bud expressing Tcf/Lef::H2B-Venus. (n) Confocal images of Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud ectoderm visualising CD44 (green) and DAPI (blue). (o) Confocal XY sections Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud ectoderm visualising phospho-βcatenin (red) and DAPI (blue). Scale bar indicates 20 μm (a,c,o), 10 μm (i,n), 50 μm (m), 100 μm (j), 1 mm (d). Error bars indicate SEM. Asterisk indicate p < 0.05.
