1996;7:1209C1224. test this hypothesis in vivo, we generated mice in which the gene was disrupted specifically in muscle stem cells (satellite cells) and we found that this resulted in impaired myotube formation during muscle regeneration after injury. To examine the role of FAK in the fusion of myogenic cells, we examined the expression of FAK and the effects of FAK deletion around the differentiation of myoblasts in vitro. Differentiation of mouse primary myoblasts was accompanied by a rapid and transient increase of phosphorylated Baloxavir marboxil FAK. To investigate the requirement of FAK in myoblast fusion, we used two loss-of-function approaches (a dominant-negative inhibitor of FAK and FAK small interfering RNA [siRNA]). Inhibition of FAK Baloxavir marboxil resulted in markedly impaired fusion but did not inhibit other biochemical measures of myogenic differentiation, suggesting a specific role of FAK in the morphological changes of cell fusion as part of the differentiation program. To examine the mechanisms by which FAK may be regulating fusion, we used microarray analysis to identify the genes that failed to be normally regulated in cells that were fusion defective due to FAK inhibition. Several genes that have been implicated in myoblast Baloxavir marboxil fusion were aberrantly regulated during differentiation when FAK was inhibited. Intriguingly, the normal increases in the transcript of caveolin 3 as well as an integrin subunit, the 1D isoform, were suppressed by FAK inhibition. We confirmed this also at the protein level GNG7 and show that direct inhibition of 1D subunit expression by siRNA inhibited myotube formation with a prominent effect on secondary fusion. These data suggest that FAK regulation of profusion genes, Baloxavir marboxil including caveolin 3 and the 1D integrin subunit, is essential for morphological muscle differentiation. INTRODUCTION Skeletal muscle terminal differentiation is usually a temporally ordered process characterized by the expression of the myogenic regulatory factor myogenin, cell cycle withdrawal, the expression of muscle-specific proteins, and myoblast fusion (Andres and Walsh, 1996 ). Even though the molecular mechanisms regulating myoblast fusion remain largely unknown, many proteins have been shown to be essential for the fusion process to occur normally (Horsley and Pavlath, 2004 ; Chen embryo, the cellular and subcellular events described during fusion include cell recognition, adhesion, alignment, the recruitment of electron-dense vesicles and plaques, and membrane merging (Doberstein have suggested the importance of pathways involving cytoskeleton remodeling during fusion (Chen gene disruption could be induced in satellite cells by tamoxifen treatment. To obtain these knockouts, mice carrying floxed alleles of FAK (disruption, 2-mo-old mice received 150 l of tamoxifen (20 mg/ml) by intraperitoneal injections for five consecutive days, and injury was performed at least 3 d after the last tamoxifen injection. Mice were genotyped by polymerase chain reaction (PCR) analysis of tail DNA by using the following primers for FAK: P2 (5-GAATGCTACAGGAACCAAATAAC-3) and P3 (5-GAGAATCCAGCTTTGGCTGTTG-3) (Beggs allele was detected with primer P1 (5-GACCTTCAACTTCTCATTTCTCC-3) and P2 (5-GAATGCTACAGGAACCAAATAAC-3) PCR primers (Beggs assessments. Differences were considered to be statistically significant at the p 0.05 level. RESULTS Deletion of FAK In Vivo in Satellite Cells Affects Muscle Regeneration The requirement of 1 1 integrins for myoblast fusion has been exhibited both in vivo and in vitro (Schwander in satellite cells was obtained by crossing mice (carrying alleles of FAK in which the second exon encoding the kinase domain name was flanked by loxP sites with mice in which an inducible Cre (CreER) was knocked into the exon encoding the 3-UTR of the Pax7 gene (Brack in skeletal muscles only after Baloxavir marboxil tamoxifen treatment (Physique 1A). Hindlimb muscles were then injured by BaCl2 injection. Regeneration was analyzed 3 d after injury when new myofibers started to form. Early regenerating myofibers expressing eMyHC were.