Figure 1. L/R asymmetries of the phrenic nerve patterns are established from the onset of diaphragm innervation.

(A) Neurofilament (NF) staining showing the branching patterns of the left and right phrenic nerves in whole-mount E15.5 mouse diaphragm. Left and right primary branches are pseudocolored (middle panel) in green and red, respectively. (See Figure 1—figure supplement 1A, for complete branch traces). L/R asymmetry is especially apparent after superimposing the left and right primary branches (right panel). Arrows point to the nerve entry points. Images are top views of the whole diaphragm, oriented as indicated in the top left hand corner of the left panel (V, Ventral; D, Dorsal; L, Left; R, Right). (B) NF and Bungarotoxin staining showing the asymmetry of acetylcholine receptor clusters and nerve domains on the left (left panel, green frame) and right (right panel, red frame) diaphragm muscles of an E15.5 embryo (see Figure 1—figure supplement 2 for quantification). (C) NF staining showing the patterns of left and right phrenic nerves at E13.5 and E14.5. Green- and red-framed panels show enlarged images of the left and right phrenic nerves, respectively. (D) Schematics showing the method used to quantify the defasciculation distance (shown in blue), from the nerve entry point to the dotted line and histogram of the defasciculation distance at E13.5, E14.5 and E15.5 (E13.5 — left 32.76 ± 11.01, right 94.82 ± 21.94, N = 9, p=0.0106; E14.5 — left 42.56 ± 4.16, right 135.71 ± 10.20, N = 8, p=0.00015; E15.5 — left 77.16 ± 7.32, right 188.51 ± 7.01, N = 18, p=4 E-10, Mann-Whitney). (E) Schematics showing the method used to quantify the secondary branch number by counting the number of NF-positive fascicles that crossed the dotted line positioned at 80% of the defasciculation distance and histogram of the secondary branch number at E13.5, E14.5 and E15.5 (E13.5 — left 5.55 ± 0.96, right 8.88 ± 0.65, N = 9, p=0.0288; E14.5 — left 7.5 ± 0.38, right 10.88 ± 0.69, N = 8, p=0.00117; E15.5 — left 5.94 ± 0.31, right 10.7 ± 0.3, N = 18, p=2.35 E-7, Mann-Whitney). Histograms show the mean ± SEM for each stage. Scale bars: 200 μm (A,C); 100 μm (B). Numerical values used to generate the graphs are accessible in Figure 1—source data 1.

DOI: http://dx.doi.org/10.7554/eLife.18481.003

Figure 1—figure supplement 1. Phrenic nerve patterns and quantification in mice and L/R nerve asymmetry in a human diaphragm.

(A) NF staining showing the branching patterns of the left and right phrenic nerves in a whole-mount E15.5 mouse diaphragm. In the right panel, the primary, secondary and tertiary branches of the left and right phrenic nerves are traced in green and red, respectively. The left and right crural phrenic nerves are traced in blue. (B) Example of quantification on an NF-labelled wholemount diaphragm. (C) L versus R differences of nerve pattern in human diaphragms, the left (green) and right (red) branches are innervating the lateral muscle (grey regions). Reproduced from the original figure of Hidayet et al. (1974). The L/R asymmetry is especially apparent after superimposing the left and right nerve pattern (right panel).

DOI: http://dx.doi.org/10.7554/eLife.18481.005

Figure 1—figure supplement 2. L/R differences of acetylcholine clusters during synaptogenesis.

(A) Bungarotoxin staining on the left and right sides of an E15.5 mouse diaphragm and plot profile showing the asymmetry of the clusters of acetylcholine receptor indicative of the endplate thickness (left and right in green and red, respectively). (B) Histogram showing the quantification of the endplate thickness (left: 254.9 ± 22.2, right: 529.3 ± 53.0, N = 11, p=0.00097, Wilcoxon signed rank). Scale bars: 200 μm. Numerical values used to generate the graphs are accessible in Figure 1—figure supplement 2—source data 1.

DOI: http://dx.doi.org/10.7554/eLife.18481.006

Figure 1—figure supplement 3. Stereotypy and variability of L/R asymmetry of the phrenic nerve patterns.

(A) NF staining showing the patterns of left and right phrenic nerves at E12.5 and E18.5. Green- and red-framed panels show enlarged images of the left and right phrenic nerves, respectively. Note that at E12.5, the dorsal and ventral branches have already split with different angles on the left and right sides (left: 166° ± 4°; right: 132° ± 4°; N = 8). (B) Ladder graph showing the stereotypy of the left and right defasciculation distances for eight E14.5 embryos (E1 to E8) (ratio shown in brackets). (C) NF staining of whole-mount diaphragms from E14.5 mouse embryos showing the phrenic nerve pattern variability at that stage. Left (green) and right (red) primary branch traces are shown in the lower panels. Scale bars: 200 μm, 100 μm for enlargement panels. The numerical values used to generate the graphs are accessible in Figure 1—figure supplement 3—source data 2.

DOI: http://dx.doi.org/10.7554/eLife.18481.008