The results of these analyses revealed that six independent ES cell lines contained evidence of a successful recombination event. 1B), genomic DNA from individual cell lines was subjected to Southern blot analyses with a probe external to the targeting event. To confirm that a successful targeting event had occurred (Fig. The targeting vector was electroporated into R1 embryonic stem (ES) cells, and independent clonal cell lines were isolated. Using a similar strategy, we replaced the first coding exon of erbB2 with the loxP-flanked rat c- erbB2 cDNA ( neu) followed immediately downstream by a PGK-neomycin ( Neo) selection cassette (Fig. However, it should be noted that these mice express only 10% of the expected amount of ErbB2 protein and are thus hypomorphic for ErbB2 expression ( 11). Replacement of the first coding exon of mouse erbB2 with the wild-type rat erbB2 cDNA completely rescued the embryonic lethality associated with the germ line inactivation of erbB2.

To assess the effect of a lack of erbB2 in muscle development and maintenance, we employed a strategy in which the first coding exon of the endogenous erbB2 gene was replaced by a loxP-flanked wild-type cDNA. These sections were counterstained with hematoxylin. Expression of ErbB2 in intrafusal muscle fibers was detected using an antibody against ErbB2 (C18 Santa Cruz Biotechnology, Inc.), and a biotinylated goat anti-rabbit secondary antibody was used (Vector Laboratories) in conjunction with the Vectastain Elite ABC kit and DAB substrate (Vector Laboratories). After being rinsed three times in PBS, the reaction was developed with DAB (0.6 mg/ml Sigma) and images were captured using the Zeiss Axiovision system. Following incubation with the primary antibody, cells were washed with PBS and were incubated with the goat anti-mouse immunoglobulin G-horseradish peroxidate conjugate secondary antibody (Bio-Rad) for 1 h.

At day 5 of differentiation, cells were washed with PBS, fixed in ice-cold 90% methanol for 2 min, rinsed three times in PBS with 5% skim milk, and incubated with MF20 for 1 h at a 1:10 dilution. However, the precise roles of the individual EGFR family members, including ErbB2, in muscle physiology have not been previously elucidated.Ĭomplete differentiation of myofibers was detected with MF20, an antibody against myosin heavy chain MF20 (Developmental Studies Hybridoma Bank, Iowa City, Iowa). Indeed, ErbB2 is thought to play a central role in the EGFR family signaling pathway, since it is the preferred heterodimeric partner for the other EGFR members ( 22).

ErbB2/ErbB3-mediated activation of phosphatidylinositol 3′-kinase ( 25, 43) has been implicated in stimulation of a number of important cell survival elements, including Akt serine kinase ( 8, 16). In addition to stimulating acetylcholine receptor subunit expression, neuregulin-mediated ErbB2/ErbB3 heterodimers can stimulate the phosphatidylinositol 3′-kinase cell survival pathway ( 45). Heterodimerization and tyrosine phosphorylation of ErbB2/ErbB3 has been shown to regulate the expression of various subunits of the acetylcholine receptor ( 40, 45, 50). Neuregulins serve as ligands for ErbB3 and ErbB4 and are released from the motor neuron end plate, where they are thought to activate these receptor tyrosine kinases ( 34). Indeed, EGFR family members have been implicated as important functional components of the NMJ ( 24). In addition to its role in Schwann cell development, ErbB2 is expressed in skeletal muscle and is concentrated at the neuromuscular junction (NMJ) along with ErbB3 and ErbB4 EGFR family members ( 1, 34, 52).

Muscle spindle