<i>Fat1</i> knockout alters expansion of the subcutaneous muscle, CM.

<p>Whole-mount β-galactosidase staining was performed using X-gal as substrate on embryos carrying the <i>MLC3F-2E</i> transgene (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004734#pbio.2004734.s002" target="_blank">S1 Table</a>) <b>(A, B)</b> or using Salmon-Gal as substrate on embryos carrying the <i>Gdnf</i>^{<i>LacZ/+</i>} allele (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004734#pbio.2004734.s002" target="_blank">S1 Table</a>) <b>(D, E)</b>. In each case, two successive stages are shown, E12.5 <b>(A,D)</b> and E12.75 <b>(B, E)</b>, respectively with <i>Fat1</i>^<i>+/+</i> (left) and <i>Fat1</i>^<i>-/-</i> (right) embryos, with the lower panels showing a higher magnification of the flank in which the CM muscle spreads. On upper panels in <b>(A, B)</b>, the yellow dotted line highlights the BW muscles, the area of which is being measured. The white square highlights the area shown in lower panels. Lower panels: the red dotted line highlights the area covered by differentiating <i>MLC3F-2E</i>⁺ muscle fibers constituting the CM muscle in <b>(A, B)</b>, also matching an area of higher <i>Gdnf</i>^<i>LacZ</i> intensity in <b>(D, E)</b>; the white dotted lines highlight the area corresponding to the full shape of the <i>Gdnf</i>^<i>LacZ+</i> area in <b>(D, E)</b>, in which a low density of blue (<i>MLC3F-2E</i>⁺) nuclei can also be observed in <b>(A, B)</b>. <b>(C)</b> Quantifications of the relative expansion of the <i>MLC3F-2E</i>⁺ CM differentiated area. Left plot: for each embryo side, the area of differentiated CM is plotted relative to the BW area. Arrows represent the stages shown in <b>(A)</b> and <b>(B)</b>, respectively. Right plot: for each embryo, the CM area/BW area was normalized to the median ratio of control embryos. Blue dots: <i>Fat1</i>^<i>+/+</i><i>; MLC3F-2E</i> (<i>n</i> = 21); red dots: <i>Fat1</i>^<i>-/-</i><i>; MLC3F-2E</i> (<i>n</i> = 14). Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004734#pbio.2004734.s001" target="_blank">S1 Data</a>. <b>(F)</b> Quantifications of the relative expansion of the <i>Gdnf</i>^<i>LacZ+</i> area. Left plot: for each embryo side, the <i>Gdnf</i>^<i>LacZ+</i> area is plotted relative to the length of the trunk (measured between two fixed points). Arrows represent the stages shown in <b>(D)</b> and <b>(E)</b>, respectively. Right plot: for each embryo, the CM area/trunk length was normalized to the median ratio of control embryos. Blue dots: <i>Fat1</i>^<i>+/+</i><i>; Gdnf</i>^{<i>LacZ/+</i>} (<i>n</i> = 21); red dots: <i>Fat1</i>^<i>-/-</i><i>; Gdnf</i>^{<i>LacZ/+</i>} (<i>n</i> = 11). Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004734#pbio.2004734.s001" target="_blank">S1 Data</a>. Scale bars: 500 μm. BW, body wall; CM, cutaneous maximus; <i>Gdnf</i>, glial cell line-derived neurotrophic factor; <i>MLC3F-2E</i>, <i>Mlc3f-nLacZ-2E</i> line (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004734#pbio.2004734.s002" target="_blank">S1 Table</a>); Salmon-Gal, 6-Chloro-3-indolyl-β-D-galactopyranoside, substrate for β-galactosidase activity; WT, wild-type; X-gal, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, substrate for β-galactosidase activity.