Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy
Figure 2
The transmembrane domain of FAT1 is required to polarize muscle migration.
(A) Schemes representing the main protein product expected from a wild type, a Fat1LacZ, and a Fat1ΔTM locus. Positions of the epitopes for three antibodies are also shown, with a color code matching that used in the western blots below. (B) Western blot analysis of the FAT1 protein products observed in total lysates from E12.5 Fat1LacZ/LacZ, wild type, and Fat1ΔTM/ΔTM embryos using indicated antibodies, which targeted epitopes are positioned in (A). (C) Whole mount LacZ staining of E12.5 Fat1LacZ/LacZ mutant embryo. (D) Skeletal muscle groups were visualized in E12.5, E13.5, and E18.5 control and Fat1ΔTM/ΔTM embryos carrying the MLC3f-2E transgene, by X-gal staining. Whole mount analysis of skeletal muscles confirms the presence of a reduced CM (red dotted lines) at E12.5, leading to a misshaped CM one day later (E13.5), and the systematic presence of ectopic muscles in the shoulder area (yellow arrow), most frequently inserting between the deltoid and triceps muscles. Flat mounted preparations of the CM dissected from an E18.5 Fat1ΔTM/ΔTM embryo, showing the reduced density as well as randomly oriented multinucleated myofibres (right panels). (E) Whole mount in situ hybridization on E10.5 embryos with an RNA probe matching the Floxed exons (exons 24–25, the probe is indicated in yellow in Figure S4A). The profile of Fat1 RNA expression in a wild type embryo matches previously reported expression domain, including staining in the limb, somites, branchial arches, telencephalon, midbrain, eye, tail bud, and neural tube roof plate. Fat1ΔTM/ΔTM embryos are entirely devoid of staining, apart from the otic vesicle, a known site of substrate trapping (yielding background staining). In contrast, varying amounts of residual RNA were consistently observed in Fat1LacZ/LacZ embryos, in the telencephalon, midbrain, limbs, tailbud, and somites. Two examples are shown with different RNA levels detected.