To explore the importance of erbB2
in muscle development, we employed Mck/Cre transgenic mice (6
) to delete a loxP-flanked erbB2
cDNA in skeletal muscle. The initial phenotypic analyses of these strains revealed that these mice exhibited a background-dependent deficit in proprioception due to the loss of muscle spindle cells. It should be noted that the 80% penetrance of the proprioception defect was not related to the extent of excision, since affected and unaffected mice exhibited comparable levels of excision. Despite the dramatic phenotype and prior reports of the utility of the Mck/Cre transgenic mice (6
), only 40% of pooled skeletal muscle tissue exhibited excision of the LOXP1-flanked allele. Consistent with these observations, examination of myoblast lines from these mice revealed comparable levels of excision that correlated with residual levels of ErbB2 protein. Since the ErbB2Flox/Flox
mice already express reduced levels of ErbB2, the observed muscle-specific excision results in a further 40% reduction in ErbB2 levels and results in the dramatic phenotype. The progressive nature of the proprioception phenotype in the ErbB2Flox/Flox
Mck/Cre mice and the complete lack of spindles in the EDL suggest an essential role for ErbB2 in spindle maintenance. Interestingly, the number of muscle spindles observed in the ErbB2Flox/Flox
mice was significantly reduced compared to that in wild-type mice. Taken together, these observations suggest a crucial threshold of ErbB-2 is required to maintain the muscle spindles. This argument is further supported by the detection of ErbB2 expression in the intrafusal muscle fibers at the equatorial region of the muscle spindle. Additionally, a global reduction from 10% of wild-type ErbB2 levels to 5% was achieved by interbreeding the ErbB2Flox/Flox
mice with the ErbB2 knockout mice. Interestingly, this resulted in embryonic lethality in the heterozygotes, which again supports the argument for a critical threshold of ErbB2 for proper development (R. Chan and W. J. Muller, unpublished data).
The reduction of spindle numbers, without a discernible alteration in gait or posture, in the ErbB2Flox/Flox
mice has raised the important question of how many spindles are required for normal proprioception. However, the loss of proprioception observed in Mck/Cre ErbB2Flox/Flox
mice bears a striking similarity to that in other knockout mutants. For example, germ line elimination of EGR3 results in a proprioception defect associated with a loss of spindles (47
). Given the similarity of two phenotypes, it is conceivable that EGR3 and ErbB2 are on an identical signaling pathway that is required for normal spindle maintenance. Interestingly, previous work has shown that EGR3 is a downstream target of ErbB2-mediated signaling (44
). In addition, it has recently been reported that ErbB2 and another EGR family member known as EGR2 are required for Schwann cell development (17
). These observations suggest that a conserved ErbB2/EGR signaling axis may be required for development or maintenance of multiple cell lineages.
Previous studies have implicated a role for ErbB2 in the induction of acetylcholine receptor subunits at the NMJ. Although clear defects in cell survival were noted in the ErbB2-deficient myoblast cell lines, we failed to note any effect on induction of acetylcholine receptor subunits in ErbB2-null myoblasts or myofibers (data not shown). These observations argue that the presence of functional ErbB2 is dispensable for induction of acetylcholine receptor subunits. Further, given the presence of other members of the EGFR family at the NMJ, including ErbB3 and ErbB4, these data argue that the other EGFR family members may compensate for loss of ErbB2 in the induction of acetylcholine receptor subunits. Future studies with muscle cell-specific eliminations of other EGFR family members should allow this hypothesis to be tested. However, the previous studies that described ErbB2 expression at the NMJ did not document expression of ErbB2 in the muscle spindle. Given the lack of spindles in the mice with a targeted deletion, we examined ErbB2 expression in the muscle spindle itself. In addition to expression in the primary afferent adjacent to the spindle, we noted expression of ErbB2 in the capsule and in the intrafusal muscle fibers in the equatorial region of the spindle.
Apart from the dramatic loss of muscle spindle cells, histological sections of the muscle of Mck/Cre ErbB2Flox/Flox mice did not reveal any apparent abnormalities. However, upon induction of muscle injury the mice exhibited incomplete regeneration, with numerous areas containing encapsulated cellular debris. Consistent with these in vivo observations, induction of differentiation in ErbB2-null myoblast cell lines stimulated a large increase in the number of apoptotic cells. These results suggest that although differentiation can occur in the absence of a functional ErbB2, it provides an important cell survival signal that is critical during myoblast differentiation. However, terminal differentiation is still possible for the myoblasts that survive the early stages of differentiation.
In addition to EGFR family, other receptor tyrosine kinases have been implicated in muscle spindle development. For example, signaling through specific neurotrophins and their receptors plays an integral role in muscle spindle formation. Consistent with this view, germ line elimination of NT-3 and its receptor, TrkC, both resulted in a loss of muscle spindle cells (15
). Our results indicate that the reciprocal signaling loop proposed to occur between intrafusal fibers and their afferents to maintain the spindle (30
) may well funnel through an ErbB2/ErbB3 heterodimer. Indeed, our observations suggest that other distinct growth factor signaling pathways may cooperate with ErbB2 in promoting development and maintenance of this cell lineage. Further exploration of the role of cross talk between these distinct growth factor signaling pathways should provide important insight into molecular basis for muscle spindle development and maintenance.