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doi: 10.1242/10.1242/dev.00447

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Erbb2 regulates neuromuscular synapse formation and is essential for muscle spindle development

Marco Leu¹, Elena Bellmunt², Martin Schwander¹, Isabel Fariñas², Hans Rudolf Brenner³ and Ulrich Müller^1,*,

¹ Friedrich Miescher Institute for Biomedical Research, Maulbeerstr. 66, 4058 Basel, Switzerland
² Departamento de Biologia Celular, Universidad de Valencia, 46100 Burjassot, Spain
³ Department of Physiology, University of Basel, 4051 Switzerland
^* Present address: The Scripps Research Institute, 10550 North Torrey Pines Road, ICND 222, La Jolla, CA 92037, USA

View larger version (52K): [in a new window]   Fig. 1. Erbb2flox and HSA-CRE mice. (A) The Erbb2flox locus and the gene targeting strategy. A loxP-neo/tk-loxP cassette was inserted into the intron 3' of the first coding exon of the Erbb2 gene, and additional loxP-site was inserted into the intron 5' to the exon (loxP-sites are shown as red triangles; the start site of transcription by an arrow). ES cells were transfected with a DNA fragment extending from the EcoRI site to the BamHI site. Targeted ES clones were identified by Southern blot, using a probe located immediately 3' of the targeting vector. One positive clone was retransfected with a Cre-expressing plasmid, and clones that had lost the neo-tk cassette, but retained two loxP sites flanking the first coding exon of the Erbb2 gene were identified by Southern blot using the same probe as described above. One positive clone was used to generate germ line transmitting chimeric mice. (B) Southern blot analysis of targeted ES cells containing two loxP sites; wt, no targeting; clones 173, 243, properly targeted ES cell clones. (C) The HSA-Cre construct used to generate the transgenic mouse lines. HAS, human skeletal -actin; NLS, nuclear localization signal; pA, polyadenylation signal. (D) Whole-mount lacZ staining of E12.5 (left panel) and E14.5 (right panel) embryos obtained from intercrosses between a Rosa26-lacZflox tester mouse with a HSA-Cre mouse. Arrows in the left panel indicate intercostal muscles, arrowheads highlight muscle groups in the forelimb; the arrow in the right panel marks recombined cells in the skin, the arrowheads mark muscle groups in forelimb. (E) Cross-section through forelimb of a E12.5 mouse embryo stained for lacZ. All muscle groups within the forelimb were lacZ positive (arrows), as well as some cells in the skin (arrowhead). (F) Extra- and intrafusal muscle fibers show strong nuclear lacZ staining (arrows), whereas perineural cells forming the spindle capsule are lacZ negative (arrowhead). Scale bars: 200 µm in D (left panel) and E; 500 µm in D (right panel); 50 µm in F.

View larger version (84K): [in a new window]   Fig. 2. Inactivation of the Erbb2 gene. (A) At the NMJ of soleus muscle in wild-type mice Erbb2 (red) formed co-clusters with AChR (green) as identified by -bungarotoxin staining (a-Btx). (B) No Erbb2 protein was detected at the NMJ of Erbb2-deficient mice. Diffuse staining of Erbb2 was seen in terminal Schwann cells. (C-E) Myoblasts from wild-type and mutant mice were isolated from P2 mice and induced to form myotubes in culture. Agrin was added to induce AChR clusters. In wild-type mice, Erbb2 (red) co-localized with AChR clusters (green) (C). In the mutants, agrin induced AChR clusters, but Erbb2 was absent from the clusters (D). Utrophin (red) still co-clustered with AChR (green) in the mutant muscle fibers (E). Scale bars: 15 µm in A,B; 10 µm in C-E.

View larger version (55K): [in a new window]   Fig. 3. Formation and function of NMJs. (A) -bungarotoxin (red) stained NMJ of diaphragm muscle of 4-weekold mice. Mature NMJ in wild-type (left) and mutant (right) mice showed the typical pretzel-like structure. (B) ß-dystroglycan (green) and (C) utrophin (green) were expressed in the postsynaptic membrane of NMJs of soleus muscle and co-localized with -bungarotoxin (red) in both wild-type (left) and mutant (right) mice. (D) Miniature endplate currents were decreased in amplitude by 15% in mutant mice when compared with wild-type mice, while decay time constants (E) were similar (means±s.e.; data from 37 wild-type and 39 mutant endplates, from five wild-type and six mutant diaphragms), as shown in these representative recordings. (F) Quantitative immunofluorescence of -bungarotoxin-labeled AChRs at NMJs of diaphragm muscle reveals decrease in AChR density by about 20% in mutant mice (means±s.e.; data from 27 wild-type and 27 mutant endplates, from five wild-type and five mutant diaphragms), consistent with the reduction in mepc. amplitude observed in electrophysiological recordings. Scale bars: 3 µm in A-C.

View larger version (95K): [in a new window]   Fig. 4. Proprioceptive defects and absence of spindles. (A) Wild-type animals but not the mutants showed an abnormal hind-limb extension reflex (panels 1-3). They also had difficulties in coordinating limb movement when placed on a slippery platform (panel 4). (B) Hematoxylin/Eosin stained cross-sections of P9 soleus muscle. Two muscle spindles of a wild-type mouse are indicated by arrows. In the mutant mice no spindles could be identified. (C) Erbb2 (green) was expressed in intrafusal muscle fibers of muscle spindles. It co-localized with -bungarotoxin labeled AChR clusters in postsynaptic membranes of g-motoneuron contact sites (yellow, arrows). Scale bars: 50 µm in B; 15 µm in C.

View larger version (107K): [in a new window]   Fig. 5. Analysis of muscle spindle development. (A,B) EM pictures of a primary myotube being contacted by Ia afferent sensory fibers at E16 in a wild-type (A) and a mutant (B) embryo. Single myotubes (circled black) were contacted by nerve terminals (circled green) as indicated by the arrow. Schwann cells (circled yellow) accompanied the ingrowing afferent fiber. (C,D) At E17, many muscle spindles in wild-type embryos consisted of two intrafusual fibers (black) contacted by multiple nerve endings (green). Schwann cells (yellow) accompanying the spindle nerve bundle formed a rudimentary unilamelar capsule surrounding the developing spindle. In some cases two nuclei were observed at the equatorial plane of the myotube that was developing into a nuclear bag2 fiber. In muscle in mutant mice (D), myotubes contacted by sensory nerve terminals were still frequently observed close to nerve bundles but muscle spindle differentiation appeared halted. Arrow in D indicates contact between myotube and nerve terminal. Rudimentary spindles were still formed by a single myotube contacted by unexpanded afferent nerve terminals and were not surrounded by Schwann cell processes. Innervated myotubes did not progress towards a nuclear bag phenotype and other myotubes were not recruited as intrafusal fibers. Nevertheless, synaptic contacts by sensory nerve terminals, as revealed by the lack of a basal lamina in the sypnaptic cleft still persisted. (E,F) By E18, spindles in wild-type mice were surrounded by a multi-lamellar spindle capsule, and spindle fibers were innervated by a Ia afferent (boxed contact zone enlarged in E'). Spindles in the mutant mice (F) did not differentiate further. Only one spindle fiber was present, and no capsule had formed. However, contacts between the muscle fiber and Ia-afferent was maintained (boxed area enlarged in F'). (G,H) Plastic sections through P0 muscle of wild-type and mutant mice showed that although muscle spindles in wild-type mice (G) typically consisted of three or four encapsulated multinucleated intrafusal fibers innervated by multiple nerve terminals, spindles in the mutants (H) only consisted of one muscle fiber still surrounded by nerve terminals. Some of the afferents appeared abnormally enlarged and swollen. Scale bars: 2 µm in A-F; 5 µm in G,H.

View larger version (68K): [in a new window]   Fig. 6. Analysis of Ia and Ib afferent projections by parvalbumin staining. (A) Immunohistochemical analysis using an antibody against parvalbumin as a marker for Ia- and Ib afferents. At E18.5, spindles were innervated by sensory afferents in both wild-type and mutant mice (arrows). (B) Two enlarged innervated spindles in mutant mice (right) at E18.5 showing the Ia afferent spiraling around the intrafusal muscle fiber (arrows). Notice the presence of only one intrafusual fiber in mutant mice. Left panel, longitudinal; right panel, cross-section. (C) Golgi tendon organs formed and were properly innervated in wild-type and mutant animals at E18.5 (arrows). (D) At postnatal day 4, spindles in wild-type and mutant mice were innervated, but the nerve terminals in the mutants were weakly stained, appeared irregular and were less frequently found. Scale bars: 100 µm in A; 30 µm in B; 200 µm in C; 40 µm in D.

View larger version (138K): [in a new window]   Fig. 7. Central projections of Ia and Ib afferent neurons. (A) Parvalbumin positive cell bodies of sensory neurons were present throughout the dorsal root ganglia at lumbar levels in both mutant and wild-type mice at P3. (B) Central projections of Ia and Ib neurons were still present at P3 in mutant mice (CC, Clark columns; LMC, lateral motor columns. Scale bars: 50 µm in A; 150 µm in B.