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First published online 23 April 2008
doi: 10.1242/dev.018119

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Essential roles of the acetylcholine receptor -subunit in neuromuscular synaptic patterning

¹ Department of Neuroscience, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75235-9111, USA.
² Centre National de Genotypage, 91057 Evry Cedex, France.
³ Department of Neurobiology, The University of Chicago, Chicago, IL, USA.
⁴ Department of Biology, University of Utah, Salt Lake City, UT, USA.
⁵ Department of Pharmacology and Physiology, UMDNJ-New Jersey Medical School, Newark, NJ, USA.

View larger version (101K): [in this window] [in a new window]   Fig. 1. Absence of AChR clusters during initial stages (E13-E15.5) of neuromuscular synaptogenesis in -/- embryos. Clustering of AChRs was detected by Texas Red-conjugated -bungarotoxin. In whole-mount diaphragm muscles (A-C,F-H), AChR clusters were distributed along a central region of the muscle in the wild type (arrowheads in A-C), but were absent from the -/- muscles (F-H); instead, diffused fluorescence was observed on the surface of the muscles. Broken lines in H indicate the central region of the muscle. Similarly, AChR clusters were detected in EDL or soleus muscles in the wild-type (D,E), but not in the -/- muscles (I,J). Scale bars: 25 µm in A-C,F-H; 20 µm in D,E,I,J.

View larger version (66K): [in this window] [in a new window]   Fig. 2. Delayed occurrence of AChR clustering in the absence of the -subunit. (A-F) Whole-mount diaphragm muscles (E16.5-E18.5) labeled with Texas Red-conjugated -bungarotoxin. AChR clusters were detected in the -/- muscles (arrowheads in D-F), although they were less numerous and appeared dimmer than those observed in the wild-type muscle (arrowheads in A-C). (G) Comparison of the fluorescence intensity (mean gray value), area, perimeter and Feret's diameter (the length of the greatest axis) of individual AChR clusters in the -/- muscles with those of the wild-type muscles. *P<0.05 (Student's t-test), Scale bar: 50 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 3. Expression of the AChR -subunit in embryonic muscles. (A) RT-PCR analyses in wild-type and -/- embryos (E13.5, E15.5, E16.5 and E18.5). In both wild-type and -/- muscles, the expression of the -subunit and GAPDH was readily detectable from E13.5 to E18.5, but the expression of -subunit was detectable only after E16.5 (*), and its level of expression was sharply increased at E18.5 (**). (B) Quantitative real-time PCR analysis using specific TaqMan probes for -subunit gene. The expression levels of -subunit gene in both wild-type and -/- muscles (E18.5) were similar (wild-type, 3.1±2.9%, n=3 embryos; -/-, 3.4±2.7%, n=3 embryos), both at 3% of level compared with the adult muscle (100%).

View larger version (22K): [in this window] [in a new window]   Fig. 4. Distinct pharmacological properties of EPPs recorded from wild-type and -/- muscles. (A) Sample EPP traces recorded from wild-type or -/- muscles in normal Ringer's, or in Ringer's with A-conotoxin OIVB (10 µM). (B) Quantification of normalized EPP amplitudes. EPPs in wild-type muscles, but not -/- muscles, were sensitive to A-conotoxin OIVB. (C) Sample EPP traces recorded from wild-type or -/- muscles in normal Ringer's, or in Ringer's with waglerin 1 (1 µM). (D) Quantification of normalized EPP amplitudes. Bath application of waglerin 1 (1 µM) in Ringer's completely blocked the EPPs in the -/- muscles, but was much less effective in the wild-type muscles. *P<0.05, ***P<0.001 (Student's t-test).

View larger version (114K): [in this window] [in a new window]   Fig. 5. Increased nerve branching and broadening of the innervation band in -/- muscles. Whole-mount diaphragm muscles were immunostained with antibodies against neurofilament (A,B), syntaxin (C,D), synaptophysin (E,F) or synaptotagmin 2 (Syt2) (G,H). (A,B) The hemi-diaphragm; (C-H) the dorsal quadrant diaphragm. In E13 wild-type muscle, collateral axons emanated from the nerve trunk in an orderly fashion and were mainly confined to the central region of the muscle (arrowheads in A); in E13 -/- muscle, noticeable increases of axons were observed and distributed across a broad region of the muscle (B). Similarly, at E15.5, intramuscular nerves were confined to the central region of wild-type muscle (C,E,G), but projected aberrantly to a broad region of the -/- muscles (D,F,H). Numerous puncta labeled by either synaptophysin or Syt2 antibodies at the nerve terminals are observed in both the wild-type and -/- muscles (arrowheads in E-H, also see inset in G,H). Scale bar: 200 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 6. Progressive accumulation of synaptic vesicle protein at the nerve terminals. Whole-mount diaphragm muscles (E18.5) were double-labeled with synaptotagmin 2 (Syt2) antibody (A,D) and Texas-Red-conjugated -bgt (B,E). The merged images are shown in C and F. In both wild-type (A) and -/- muscles (D), individual nerve terminals (arrowheads in A,D) were heavily labeled by Syt2 antibody. AChR clusters are present in the end-plates of the -/- muscles (arrowheads in E), but they are less intensely labeled by -bgt, compared with those observed in the wild-type muscles (arrowheads in B). Merged images show that nerve terminals are juxtaposed with AChR clusters in both -/- (arrowheads in F) and wild-type muscles (arrowheads in C); however, there were marked increases of nerve sprouting in the -/- muscles (arrows in D,F), compared with those in the wild-type muscle (arrows in C). Scale bar: 50 µm in A-F.

View larger version (204K): [in this window] [in a new window]   Fig. 7. Aberrant distribution of AChE clusters in -/- muscles. Whole-mount diaphragm muscles from the wild-type (A,B) and -/- embryos (C,D) were processed for AChE staining. While AChE clusters (arrowheads) were distributed along the central region of the wild-type muscles at E15.5 (A) and E18.5 (B), the AChE clusters were aberrantly distributed across a broad region in the -/- muscles at E15.5 (C) and E18.5 (D). Scale bar: 200 µm.

View larger version (81K): [in this window] [in a new window]   Fig. 8. Localization of MuSK in the absence of the -subunit. Whole-mount E15.5 diaphragm muscles from the ventral costal region (A-D) and sternal region (E-H) of wild-type (A-B,E-F) and -/- (C-D,G-H) embryos were doubly labeled with MuSK antibody and -bungarotoxin. In wild-type muscles, MuSK staining (arrowheads in B and F) was detected at the central region of the muscle, corresponding to AChR clusters labeled by -bungarotoxin (arrowheads in A,E). In the -/- muscles, MuSK staining was also seen at the central region of the muscle (arrows in D,H), despite the lack of AChR clustering (C,G). Scale bar: 10 µm.

View larger version (117K): [in this window] [in a new window]   Fig. 9. Absence of rapsyn at synaptic sites in E15.5, but not E18.5, -/- muscles. Diaphragm muscle sections at E15.5 (A-F) and E18.5 (G-L) from wild-type (A-C,G-I) and -/- embryos (D-F,J-L) were triple-labeled with anti-Syt2 (A,D,G,J), -bgt (B,E,H,K) and anti-rapsyn (C,F,I,L). At E15.5, rapsyn was highly localized to synaptic sites in wild-type muscles (arrowhead, C), but absent from synaptic sites in -/- muscles (F). There were also no AChR clusters (E) at synaptic sites (arrowhead in D) in the E15.5 -/- muscle. At E18.5, however, rapsyn (arrowheads) was localized to synaptic sites in both wild-type (I) and -/- muscles (L). Scale bar: 10 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 10. Increased motoneuron survival in -/- embryos. Cross-sections of cervical spinal cords (C3-C8) from wild-type (A) and -/- (B) embryos (E18.5) were stained with Cresyl Violet. Motoneurons were identified in the ventral horn (within the region marked by broken lines) and their numbers were counted for each genotype. The total numbers of motoneurons from these segments (C3-C8) were presented in the bar graph (C). There were significantly more (65% increase) motoneurons in the -/- embryos (7473±283, n=3) than in the wild-type embryos (4539±247, n=3) (**P<0.001, Student's t-test). Scale bar: 100 µm.

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