Three-dimensional mapping reveals heterochronic development of the neuromuscular system in postnatal mouse skeletal muscles

Figures

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  Fig. 1. The measurement of motor development in neonatal mice. a Weight (in grams) of postnatal mouse pups. b The average time taken to complete the righting response. When a pup failed to reach the adult pattern within 20 s, its time was recorded as 20 s. c The test for the limb placing response. d The scores for forelimb and hindlimb placing responses after birth. e The walking pattern in the open-field experiment during postnatal development. f The scores for the characteristics of the walking pattern at different time points. g The test for cliff drop avoidance. h The scores for the motor performance test when the pups faced the cliff at different time points. n = 20 independent animals for each behavioral test.

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  Fig. 2. Postnatal development of myofibers in different skeletal muscles. a The selection of muscles from different body parts, including the cleidomastoid (CM) in the cervical part, the biceps brachii (BB) in the forelimb, and the gastrocnemius (GA) in the hindlimb. Scale bar: 5 mm. b Fluorescence imaging of the location of myonuclei (cyan) relative to the membrane (magenta) in the cross-sections of different muscles after birth. The yellow arrowheads indicate myonuclei located at the center of myofibers. Scale bar: 25 μm. c The quantification of the percentage of myofibers with central nuclei in b (n = 4 independent animals for each muscle at each time point). d Fluorescence imaging of proliferative cells (magenta) in the cross-sections. Scale bar: 25 μm. e The quantification of the percentage of KI67 + cells in d (n = 5 independent animals for each muscle at each time point). f Fluorescence imaging of myofiber membranes in the cross-sections; scale bar: 200 μm. The images in the white frames show the detailed morphologies of myofibers; scale bar: 25 μm. g–i The quantification of myofiber numbers in the cleidomastoid (g), biceps brachii (h), and gastrocnemius (i) (n = 5 independent…

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  Fig. 3. 3D visualization of innervating nerves and NMJs in skeletal muscles after clearing. a The entire experimental process of the 3D imaging of intramuscular motor nerves and NMJs in skeletal muscles. b–d 3D reconstructions of intramuscular motor nerves and NMJs in the cleidomastoid (b), biceps brachii (c), and gastrocnemius (d) during postnatal development and adulthood. Scale bar: 200 μm. e–g The quantification of the relative ranges of innervation zones in whole muscles (n = 5 independent animals for each muscle at each time point). All values are presented as the means ± SEM; statistical significance in e–g (n.s. represents not significant) was assessed using one-way ANOVA.

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  Fig. 4. Intramuscular axonal arborization during postnatal development. a A comparison of the motor nerve arborizations in the gastrocnemius during postnatal development. The images in the yellow frames in the second column are enlarged and presented in the third column: they show increased axonal terminals and branches on the selected branch during postnatal development. Scale bar in white frames: 100 μm; scale bar in yellow frames: 30 μm. b qRT-PCR analysis of collagen XXV mRNA expression in embryonic (E13.5 and E17.5), neonatal (P0), and adult hindlimb muscles (n = 3 independent animals for each time point). mRNA levels were normalized to GADPH expression. c qRT-PCR analysis of collagen XXV mRNA expression in postnatal and adult muscles from the cervical part, forelimb, and hindlimb (n = 6 independent animals for each body part at each time point). All values are presented as the means ± SEM; statistical significance in b and c (n.s. represents not significant, *P < 0.05, and ***P < 0.001) was assessed using one-way ANOVA and two-way ANOVA, and followed by Dunnett’s T3 post hoc test and Tukey post hoc test respectively.

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  Fig. 5. 3D analysis of the intramuscular innervation pattern during postnatal development. a–c The spatial conformation of intramuscular motor nerves in a P0 cleidomastoid (a), P6 biceps brachii (b), and P3 gastrocnemius (c). d–f The segmentation of the nerve branches in a–c by 3D tracing using the Imaris software. The primary branches were noted as originating from the nerve branches entering the muscles and extending from proximal to distal, and they were numbered and marked with different colors. g–i The distribution of secondary branches in the 2# primary branch of the cleidomastoid (g), 1# primary branch of the biceps brachii (h), and 2# primary branch of the gastrocnemius (i). j–l The quantification of the primary and secondary branches in the cleidomastoid (j), biceps brachii (k), and gastrocnemius (l) during postnatal development and adulthood (n = 3 independent animals for each muscle at each time point). Scale bar: 100 μm. All values are presented as the means ± SEM; statistical significance in j–l (n.s. represents not significant) was assessed using one-way ANOVA.

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  Fig. 6. 3D analysis of NMJs during postnatal development. a 3D distributions of NMJs in the gastrocnemius at different time points (P0, P3, P6, P9, and adulthood); scale bar: 200 μm. The images in the white frames are enlarged and shown in the second row; scale bar: 100 μm. The images in the third row show the segmentation of NMJs. b–d The quantification of NMJs in the gastrocnemius (b), biceps brachii (c), and cleidomastoid (d) at different time points (n = 5 independent animals for each muscle at each time point). All values are presented as the means ± SEM; statistical significance in (b-d) (n.s. represents not significant and ***P < 0.001) was assessed using one-way ANOVA followed by Tukey post hoc test.