<i>Fat1</i> controls the shape of subsets of scapular muscle by modulating myoblast polarity during planar migration.

<p>(<b>A–C</b>) Reporter gene expression in the forelimb and flank of mouse embryos between E11.5 and E13.5. (<b>A</b>) <i>Gdnf-lacZ</i> staining labels myoblasts of the <i>latissimus dorsee</i> (LD) and <i>cutaneous maximus</i> (CM). CM myoblasts migrate away from the brachial plexus to form a subcutaneous muscle sheath, composed of radially-oriented chains of myoblasts. (<b>B</b>) At E13.5, <i>MLC3f-lacZ</i> staining reveals the characteristic fan-shaped form of the CM (dotted white purple line) as compared to other limb muscles. (<b>C</b>) <i>Fat1</i> expression detected using the <i>lacZ</i> gene trap allele KST249 (<i>Fat1^LacZ</i>) is selectively localized within the CM and in surrounding tissue (pink arrow). (<b>D</b>) CM myoblasts express <i>Fat1</i> and migrate towards an increasing gradient of <i>Fat1</i> expression. Alternate vibratome cross-sections of a wild type E12.5 embryo were hybridized with <i>Fat1</i> (left column) and <i>MyoD</i> (purple, right column) RNA probes. Photographs of adjacent sections were superimposed (photoshop) after conversion of <i>Fat1</i> staining color in pink (right column; <i>Fat1</i> in pink, <i>MyoD</i> in purple). <i>MyoD</i> expression is used as a marker of the muscle lineage. Superimposition was meant to compare the relative levels of <i>Fat1</i> expression within and around the <i>cutaneous maximus</i> (CM) muscle (indicated with purple arrows), at three consecutive antero-posterior positions, respectively within the CM (top row), at the posterior end (middle row), and posterior to the caudal extremity of the CM at that stage. CM myoblasts, migrating from anterior to posterior, express lower levels of <i>Fat1</i> RNA than the surrounding subcutaneous cell layer (pink arrows). Intensity of <i>Fat1</i> staining in this subcutaneous layer increases gradually in caudal sections. (<b>E–H</b>) Orientation of CM myoblast migration in whole-mounts of E12.5 <i>Fat1^LacZ/LacZ</i> and control embryos detected using <i>MyoD in situ</i> hybridization. In all panels anterior is to the left, dorsal is to the top. (<b>E</b>) The CM muscle (purple dotted line) in <i>Fat1^LacZ/LacZ</i> embryos displays reduced size and altered shape as compared to wild type. Higher magnification images (right hand panels) show that within the CM muscle, radial organization of myoblast chains was perturbed by <i>Fat1</i>-deficiency, resulting in a fuzzy migration front and irregular distribution of myoblasts (red arrows). In addition, ectopic clusters of myoblasts (orange arrows) are detected in the shoulder area (dotted orange line). (<b>F</b>) Quantification of the abnormal orientation of <i>Fat1</i> mutant myoblasts. The angle between the longest diameter of each myoblast nucleus and the axis of the closest myoblast chain was measured on flat-mounted CM muscles. The bar graph presents mean (± s.e.m.) percentages of <i>myoD</i>⁺ nuclei displaying a given angle (by angle ranges of 10°) for wild type (gray) and <i>Fat1^LacZ/LacZ</i> (black) embryos. (<b>G, H</b>) High magnification images of <i>MyoD</i>-expressing myoblasts in equivalent positions – within the chains (<b>G</b>) or at the leading edge (migration front, <b>H</b>) – in the CM of mutants and controls. Scale bars: (<b>A–C</b>), 0.8 mm; (<b>D</b>) 300 µm; (<b>E</b>), left: 0.5 mm; (<b>E</b>), right: 100 µm; (<b>G, H</b>) 10 µm.