ErbB2 is required for muscle spindle and myoblast cell survival

Abstract

Signaling mediated by ErbB2 is thought to play a critical role in numerous developmental processes. However, due to the embryonic lethality associated with the germ line inactivation of erbB2, its role in adult tissues remains largely obscure. Given the expression of ErbB2 at the neuromuscular junction, we have created a muscle-specific knockout to assess its role there. This resulted in viable mice with a progressive defect in proprioception due to loss of muscle spindles. Interestingly, a partial reduction of ErbB2 levels also reduced the number of muscle spindles. Although histological analysis of the muscle revealed an otherwise normal architecture, induction of muscle injury revealed a defect in muscle regeneration. Consistent with these observations, primary myoblasts lacking ErbB2 exhibit extensive apoptosis upon differentiation into myofibers. Taken together, these results illustrate a dual role for ErbB2 in both muscle spindle maintenance and survival of myoblasts.

FIG. 1.

Targeting of the loxP-flanked erbB2 allele. (A) A schematic representation of the targeting construct and the genomic allele is shown. The 2.5-kb 5′ arm of homology (Sph to Nar) and the 8-kb 3′ arm of homology (Kpn to Sal) were employed to direct the homologous recombination to the wild-type allele, illustrated by the dashed lines. Exon 1 of the endogenous allele was replaced by a loxP (triangle)-flanked neuN cDNA followed by a simian virus 40 poly(A) (Neu) and a PGK-neomycin-herpes simplex virus poly(A) (Neo). The targeted allele has replaced exon 1 contained within the Nar/Kpn fragment. The size of the genomic HindII-restricted fragment when detected by a probe 5′ to the site of homologous recombination is also depicted. (B) The recombinant allele containing the loxP-flanked neuN allele in place of exon 1 and the corresponding size of the HindIII restriction fragment detected by the external probe are shown. (C) A representative Southern blot of tail DNA from mice that are wild type (WT) and heterozygous for the knockin (KI) allele illustrates the various alleles. (D) Immunoprecipitation of ErbB2 followed by immunoblotting for ErbB2 revealed that the ErbB2^Flox/Flox muscle expresses a reduced level of protein compared to the wild-type controls (lanes 1 and 2 versus lanes 3 and 4).

FIG. 2.

Mck/Cre-mediated excision of loxP-flanked ErbB2. (A) The schematic of the recombinant allele under the control of the endogenous promoter is shown, as is the effect of Cre-mediated recombination. Using a HindIII digest and the PstI fragment of the neomycin cassette as a probe, the size of the recombinant and null alleles in a Southern analysis are shown. (B) A Southern analysis on pooled hind limb muscle from eight Mck/Cre ErbB2^Flox/Flox mice is shown. Also included are a control for no excision in the ErbB2^Flox/Flox (lane 1) and a control to illustrate when excision is complete for one allele (lane 2). This complete excision control is a knockin/Flox knockout (K-In/Flox K-out), has one recombinant allele and one null allele, and was gen-erated from an embryonic control (Chan and Muller, unpublished observations). Quantification of the extent of excision by PhosphorImager analysis revealed that excision varied from 30 to 40% complete for the muscle samples (lanes 3 to 10). The extent of the observed phenotype is stated above the lanes and does not correlate with an increased level of excision (severe, lanes3 to 6; mild, lanes 7 to 10). (C) To examine the specificity of the excision, DNA was harvested from various organs and was examined through a Southern analysis for excision of the loxP-flanked cDNA. This reveals that excision is limited to skeletal and cardiac muscle. (D) To determine whether excision was complete in the myofibers, several myoblast cell lines were prepared from the Mck/Cre ErbB2^Flox/Flox mice and were allowed to differentiate. The number of days in differentiation medium is shown from day 0 (D0) to day 6 (D6). Upon initiation of differentiation, the Mck promoter is activated, and excision is noted by day 2 of differentiation (lane 4). However, when differentiation is complete at day 6, excision remains incomplete (lane 8).

FIG. 3.

Altered gait and posture in ErbB2^Flox/Flox Mck/Cre mice. (A) The hind limbs of a wild-type mouse were immersed in India ink, and the mouse was permitted to move across a sheet of blotting paper. The mouse was moving from left to right across the sample area in a normal gait where only the footpad was in contact with the surface that it moved across. (B) The track from a mildly affected Mck/Cre ErbB2^Flox/Flox mouse as it moves across the blotting paper reveals several distinct differences. In addition to the footpad coming in contact with the surface the mouse was traversing, a large region of the leg also was in contact due to incomplete extension of the leg during movement (closed arrow). Additionally, due to abnormal weight bearing on the hocks, the toes are turned in during movement, resulting in the pigeon-toed track (open arrow). These alterations in the gait appear to be due to a lack of complete extension of the hind limbs during movement. In addition to the altered gait, the Mck/Cre ErbB2^Flox/Flox mice have an abnormal posture at rest. In comparison to wild-type (C) and ErbB2^Flox/Flox controls, the severely affected ErbB2^Flox/Flox Mck/Cre mice (D) suffer from an extensor posture, extending their limbs and resting upon the abdomen.

FIG. 4.

Muscle spindle loss due to ErbB2 reduction. (A) Longitudinal sections of the EDL muscle were examined for the presence of muscle spindles through urea-silver nitrate staining. This method results in readily visible muscle spindles in the thick sections. (B) Muscle spindles are easily detected in wild-type muscle when the nonselective silver stain is preferentially taken up by the neurons. The primary sensory annulospiral endings surrounding the equatorial region ofintrafusal muscle fibers are shown (A and B). (C) While neurons were readily observed in EDL from the ErbB2^Flox/Flox Mck/Cre mice, no muscle spindles were found in serial sections through the entire muscle. Only motor innervation was observed in the serial sections. Interestingly, it was also difficult to detect muscle spindles in the EDL muscle from ErbB2^Flox/Flox mice. Accordingly, we counted the number of spindles in EDL muscles from ErbB2^WT/WT, ErbB2^Flox/Flox and ErbB2^Flox/Flox Mck/Cre mice. (D) The result of the spindle count is shown. Strikingly, the number of spindles observed in ErbB2^Flox/Flox mice was significantly reduced compared to those observed in wild-type mice, although no phenotypic effects were visible in these mice. The further reduction in ErbB2 levels in ErbB2^Flox/Flox Mck/Cre mice correlated with a complete loss of spindles and the associated proprioception defects. Error bars, standard deviations.

FIG. 5.

Histological comparison of ErbB2^WT/WT and Mck/Cre ErbB2^Flox/Flox muscle. (A) A cross section of TA muscle reveals the presence of numerous muscle spindles in the wild-type controls after hematoxylin and eosin staining (arrows). However, these muscle spindles were not observed in serial sections of Mck/Cre ErbB2^Flox/Flox mice (B). In all other aspects, there are no readily discernible differences between histology of wild-type and conditional null muscle (compare panels A and B). (C) Since the targeted deletion of ErbB2 resulted in a loss of muscle spindles, we examined ErbB2 expression in the muscle spindle through immunohistochemistry. ErbB2 expression was readily observed in wild-type intrafusal muscle fibers, in the capsule surrounding the equatorial region, and in the neuron adjacent to the muscle spindle. (D) A serial section is shown after hematoxylin and eosin staining. (E) In contrast to the expression in the intrafusal muscle fibers in the equatorial region of the spindle, ErbB2 is not detected in the intrafusal muscle fibers towards the distal terminus of the muscle spindle. (F) The corresponding hematoxylin-and-eosin-stained serial section is also shown.

FIG. 6.

Impaired regeneration after an induced muscle injury. Two weeks after a muscle crush injury in the TA muscle, serial sections were examined. Serial longitudinal sections of ErbB2^WT/WT and ErbB2^Flox/Flox muscle revealed that regeneration was essentially complete, with continuous fibers of normal caliber. (A) The only evidence of the crush injury are the areas with numerous centrally located nuclei shown in the ErbB2^Flox/Flox muscle. In contrast, regeneration at the same time point is not complete in the ErbB2^Flox/Flox Mck/Cre mice. (B) Numerous regions with encapsulated cellular debris are observed at the site of the injury and there are fewer continuous fibers.

FIG. 7.

Increased apoptosis in ErbB2-null myoblasts during differentiation. (A) Myoblast cells were prepared from adult FVB wild-type controls, ErbB2^Flox/Flox, and Mck/Cre ErbB2^Flox/Flox mice. Given the residual ErbB2 expression in Mck/Cre ErbB2^Flox/Flox cell lines (not shown), we infected ErbB2^Flox/Flox and Mck/Cre ErbB2^Flox/Flox (Aff2) cell lines with Ad-Cre. A mock infection and Ad-Bgal were included as controls. At day 5 of differentiation, ErbB2 was detected in the wild-type and ErbB2^Flox/Flox infection controls (lanes 1 to 3) but was not seen in the ErbB2^Flox/Flox Ad-Cre or Aff2 Ad-Cre cells (lanes 4 and 5). ErbB3 was found to be present at equal levels in all cells, and Grb2 was included as an internal loading control. (B) During differentiation, it was noted that a large number of cells were lifting off the plates in cell lines lacking ErbB2. Quantification of the percentage of cells in suspension at each differentiation time point revealed that in cells containing ErbB2, usually less than 5% of the cells were in suspension (•, FVB; □, mock; ▴, Ad-Bgal). In contrast, by day 3 of differentiation, on average 12 and 25% of cells were in suspension in the ErbB2^Flox/Flox Ad-Cre (♦) and Aff2 Ad-Cre (▪) cell lines, respectively. Apoptosis was confirmed through annexin-propidium iodide staining for FVB (C and D) and Aff2 Ad-Cre (E and F) (by light microscopy [C and E], annexin fluorescence [D and F], and propidium staining [not shown]). Although many cells lacking ErbB2 underwent apoptosis, the remaining myoblasts were still capable of terminal differentiation. Mock-infected (G), Ad-Bgal-infected (H), and Ad-Cre-infected (I) ErbB2^Flox/Flox cells at day 5 of differentiation are shown after immunostaining for myosin heavy chain (MF20). No differences were noted in the ability of the cells to reach terminal differentiation.