Aberrant development of motor axons and neuromuscular synapses in erbB2-deficient mice

Abstract

Receptor tyrosine kinase erbB2, which is activated by neuregulin, is expressed in Schwann and muscle cells in the developing neuromuscular junction (NMJ). In vitro studies have shown that neuregulin promotes the survival and migration of Schwann cells and stimulates acetylcholine receptor gene transcription in cultured muscle cells. These findings suggest an important role for erbB2 in the development of the NMJ. Here we examine erbB2-deficient mice to determine whether erbB2 is required for NMJ development in vivo. Our analysis shows that there are pre- and postsynaptic defects of developing NMJ in erbB2-deficient embryos. The presynaptic defects include defasciculation and degeneration of the motor nerves, and an absence of Schwann cells. The postsynaptic defect features an impairment of junctional folds at the neuromuscular synapse in the mutants. These results demonstrate that erbB2 is essential for in vivo development of the NMJ.

Figure 1

Aberrant innervation of the diaphragm muscle in mutant embryos from E12 to E16.5. All diaphragm images were shown as the top view. The orientation of the diaphragm is indicated in the upper right corner (D, dorsal; V, ventral; R, right; and L, left). In the controls (A), the intramuscular nerve trunk remained as a bundle, reached roughly at the middle of the dorsal diaphragm (arrow); the Inset is the low-power view. In the mutant, the phrenic nerves also reached the dorsal part of the diaphragm (D) but became aberrantly defasciculated (arrowhead). At E12.5, the phrenic nerve in the controls remained as a tight bundle with defined branches emanating perpendicularly from the main nerve trunk (arrows in B); the Inset is the low-power view. In the mutants, the phrenic nerve became more dramatically defasciculated (arrowheads in E) and distributed aberrantly across the entire dorsal surface of the diaphragm; the Inset is the low-power view. At E13.5, the phrenic nerves in the mutant (F) remained grossly defasciculated and aberrant, as compared with that in the control (C). At E14.5, the phrenic nerves in the controls (G) extended more toward dorsal and ventral portions of the diaphragm, innervating a ring of central band on the diaphragm, whereas the phrenic nerves in the mutants (J) were aberrantly distributed throughout the diaphragm and some nerves began to withdraw at this stage. By E15.5, only a small number of nerves remained in the diaphragm in the mutants (K). At E16.5, no phrenic nerve was observed in the entire diaphragm of the mutants [compare (I and L).] Note background staining associated with the esophagus (*) structure in some panels. [Bars = 100 μm (A, B, D, and E) and 200 μm (C, F, and G–L).]

Figure 2

Transient targeting of motor axons and disorganization of nerve terminals in the diaphragm. (A–C) Wild type and (D–F) mutant. At E14, axons terminated at the central endplate band (arrow in B) in the controls (A and B; A, low power; B, higher power). In the mutant embryos, axonal terminals were also located at the central band (arrow in E) of the muscle (D, low-power view; E, high-power view). However, the preterminal nerves (arrowhead in E) and terminals (arrow in E) were disorganized in the mutants, as compared with those in the wild type (B). At E15.5, nerve terminals degenerated in the mutant (F) as compared with the wild-type embryo (C). [Bars = 200 μm (A and B) and 100 μm (C–F).]

Figure 3

Defasciculation of intercostal nerves and abnormal axonal arborization in the intercostal and limb muscles. (A–E) controls and (F–J) mutants. In the control (A), the intercostal nerve trunk formed bundles, with many collateral branches emanating from the main nerve trunk (arrowheads in A); in the mutants (F), the intercostal nerves were defasciculated and nerve branches were disorganized (arrowheads). Note the tip of intercostal nerves terminated aberrantly in the mutants (arrow in F), as compared with the controls (arrow in A). B, C, G, and H show E18.5 intercostal muscles double-labeled with neurofilament antibodies (B and G) and Texas Red-conjugated α-BTX (C and H). In the controls, nerve terminals extended from the nerve bundle (arrow in B) and terminated at a central band labeled by α-BTX (C). In the mutants, the intercostal nerves were defasciculated (arrow in G), and nerve terminals aberrantly projected to the center of muscle fibers (G). As in control embryos, an AChR clustering band was present at the center of muscle fibers in the mutant (H). D, E, I, and J show sections of leg muscle, double-labeled with neurofilament antibodies and α-BTX. In the controls (D and E), neurofilament antibody-labeled nerve terminals were colocalized with α-BTX-labeled AChR clusters (arrowheads in D and E); in the mutants (I and J), only a small number of nerve terminals remained (arrowheads in I and J). [Bars = 100 μm (A and F), 50 μm (B, C, G, and H), and 50 μm (D, E, I, and J).]

Figure 4

Absence of Schwann cells in erbB2-deficient embryos. (A–F) Whole-mount preparations from controls (A, C, and E) and erbB2 mutants (B, D, and F) were labeled with antibodies against S100. In the control preparations, S-100-labeled Schwann cells delineated the entire phrenic nerves as early as E13.5 (A), and were readily detectable at E15.5 (C). S100-positive cells were also found in the intercostal nerve in the controls (arrowheads in E). In contrast, there were no Schwann cells detectable in the mutants in the phrenic nerves (B and D), or in the intercostal nerves (F). Ribs (r) also stained positively for S100. (G and H) Electron micrographs of the intercostal nerves at E18.5. In the control (G), every single axon (ax) was wrapped by Schwann cell processes (SC). Axons were separated apart by extracellular matrix (ec). In the mutant (H), axons were tightly packed together. There were no Schwann cells or extracellular matrix. Note that the density of axons in the mutants is higher, and overall axon diameters are smaller. [Bars = 50 μm (A, B, E, and F), 100 μm (C and D), and 1 μm (G and H).]

Figure 5

Impairment of junctional folds at the synaptic site of NMJ in erbB2-deficient embryos. In the controls (A), nerve terminals (T) were capped by Schwann cell (SC), and junctional folds (arrows) developed in apposition with axon terminals. In the mutants (B and C), Schwann cells were absent in the nerve terminals. No junctional fold was observed in the NMJ from the mutant (B and C). Note that synaptic vesicles were present in both control and mutant nerve terminals. Multiple nerve terminals (T) were present at the synaptic site in both the wild type and mutant. [Bar = 1 μm (A–C).]

Figure 6

AChRα gene transcription in erbB2-deficient embryos. A whole-mount in situ hybridization of E18.5 diaphragm (A and B) and intercostal muscle (C and D) labeled with a digoxygenin-cRNA probe for the AChRα subunit. Labeling was concentrated in a central band of muscle in both the wild type [arrowheads in (A and B)] and mutant [arrowheads in (C and D)].