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

DEV018119 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Distribution of AChRs in the absence of the γ-subunit. (A) Whole-mount diaphragm muscles from E15.5 wild-type and γ^-/- embryos were incubated with Texas Red-conjugated α-bungarotoxin (α-bgt). Images were collected with a low-power objective (10×) so the entire length of muscle fibers could be viewed under the same field. The α-bgt labeled AChRs were clustered in the central region of the wild-type muscle, but remained diffuse in the γ^-/- muscle. Despite the diffused pattern, the central region (marked by the broken lines) of the γ^-/- muscle was still more intensely labeled, compared with its flanking region. (B) The difference in fluorescence intensity was presented in a line-scan. The entire muscle fiber was manually traced from one end to the other (∼800 µm) using NIH ImageJ software. The gray levels of each pixel were measured and plotted against its position along the muscle fiber (wild-type, black line; γ^-/-, grey line). The central region of the γ^-/- muscle appeared more intensely labeled than its flanking region.

• Supplemental Figure S2 -

  Fig. S2. Innervation pattern of hind limb muscles in wild-type and γ<^b>^-<^/b>^/<^b>^- mice. (A,B) Cross-section of E18.5 hind limb, stained with Hematoxylin and Eosin (H&E); note the apparent atrophy of the γ^-/- muscles (B). (C,D) Low-magnification view of hind limb sections immunostained with synaptotagmin 2 (Syt2) antibody. Note a dramatic increase of innervation in all muscles in the γ^-/- embryos (D), when compared with the wild-type muscles (C). (E-H) Higher magnification views of EDL and soleus are shown in the bottom row: nerve terminals in the wild-type muscles localized to small regions of the muscle (arrowheads in E,F), whereas nerve terminals in the γ^-/- muscles were broadly distributed (arrows in G,H). TA, tibias anterior muscle; EDL, extensor digitorum longus muscle; MG, medial gastrocnemius muscle; LG, lateral gastrocnemius muscle; Sol, soleus; F, fibula. Scale bars: 200 µm in A-D; 100µm in E-H.

• Supplemental Figure S3 -

  Fig. S3. Differentiation of the presynaptic nerve terminals at E16.5. Whole-mount diaphragm muscles labeled with synaptotagmin 2 (Syt2) antibody (A,D) and Texas-Red conjugated α-bungarotoxin (B,E). AChR clusters in the γ^-/- muscle (arrowheads in E) were markedly reduced, compared with those in the wild-type muscles (E). However, individual nerve terminals were intensely labeled by Syt2 antibody in both wild-type (arrowheads in A) and in γ^-/- muscles (arrowheads in D), although the nerve terminals in γ^-/- muscles were distributed across a much broader region. The majority of the nerve terminals in the wild-type muscle were closely apposed by AChR clusters (arrowheads in C), whereas the majority of the nerve terminals in the γ^-/- muscles were not apposed by AChR clusters (arrows in F). Note, however, that aneural AChR clusters were still present in the wild-type muscles (arrows in B,C). Scale bar: 30 µm

• Supplemental Figure S4 -

  Fig. S4. Low magnification views of whole-mount diaphragm muscles immunolabeled by synaptotagmin 2 antibody. In wild-type muscle, nerve terminals (arrowhead in A) were localized along the central region of the muscle; occasionally, some nerve sprouts (arrows in A) extended beyond the central region. In the γ^-/- muscle, nerve terminals (arrowheads in B) were distributed across a broad region of the muscle, especially at the dorsal quadrant of the diaphragm. Excessive nerve sprouts were seen across the entire region (arrows in B). Scale bar: 500 µm.