Fig 4. Fat1 is expressed in CM progenitors and the surrounding subcutaneous mesenchyme.

(A) Fat1 expression is visualized in an E12.5 Fat1^LacZ/+ embryo by X-gal staining. Left panel: whole embryo picture; right panel: higher magnification of the forelimb and flank region in which the CM spreads. In the right panel, the approximate CM shape is highlighted by red dotted lines, and the level of sections shown in (C) is indicated by vertical lines. (B) Gdnf expression is visualized in an E12.5 Gdnf^LacZ/+ embryo (S1 Table) by Salmon-Gal staining. Left panel: whole embryo left side view. Right panel: higher magnification of the upper forelimb and flank region, showing that the CM exhibits a high level of Gdnf^LacZ+ expression (highlighted with red dotted lines). The level of sections shown in (D) is represented by vertical bars. (C) Cross sections of an E12.5 Fat1^LacZ/+ embryo at anterior and posterior CM levels were immunostained with antibodies against Pax7 (red), Myh1 (green), and β-galactosidase (white). The right panels show neighboring sections of the same Fat1^LacZ/+ embryo in which β-galactosidase activity was revealed by Salmon-Gal staining. (D) Comparison between expression of Gdnf^LacZ (visualized with an anti-β-galactosidase antibody [red]) and that of Fat1 (green, Ab FAT1-1869 Sigma) on two cross sections of an E12.5 Gdnf^LacZ/+ mouse embryo at middle and posterior CM levels, as indicated in (B). Fat1 protein is detected both within and around the Gdnf^LacZ/+ CM progenitors. Scale bars (A, B): 1 mm (left), 500 μm (right); (C, D): 200 μm (low magnification), 50 μm (high magnification). β-Gal, β-galactosidase; CM, cutaneous maximus.