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. 2021 Sep 10;7(37):eabi8787. doi: 10.1126/sciadv.abi8787

Fig. 3. Optogenetic modeling of neuromuscular circuits in DMD with CRISPR-corrected isogenic controls.

Fig. 3.

(A) Timeline of mouse ESC-MN and ESC-AC differentiation. RA, retinoic acid; SAG, smoothened agonist. (B) Timeline of the neuromuscular circuit assembly and medium changes in the microdevices. (C) Schematic of the microdevice. MPCs were plated in the central compartment. The two outer compartments held MN/AC spheroids. Motor axons projected through microchannels to innervate myofibers. (D) Representative images of YFP-positive motor axons in central compartments of microdevices containing MPCs at 0 hours and converted grayscale images. Scale bars, 250 μm. (E) Quantification of YFP-positive area in central compartments. n = 16. Values are means ± SEM; unpaired t test. (F) Particle image velocimetry (PIV) analysis in central compartments containing DMD- and CORR-R3381X neuromuscular circuits following optogenetic stimulation on day 5. Representative images show before, during, and after optogenetic stimulation. Green arrows represent velocity vectors. Scale bars, 250 μm. (G) Quantification of mean velocity in the central compartments containing DMD- and CORR-R3381X myofibers in response to optogenetic stimulation. Blue shading indicates the time during optogenetic stimulation. n = 24. Values are means ± SEM; two-way ANOVA and Sidak’s multiple comparisons test, ****P < 0.0001. (H) Representative 3D reconstruction images of immunocytochemistry of TUBB3 (blue), SV2 (red), and acetylcholine receptor (AChR; turquoise) in central compartments containing innervated DMD- and CORR-R3381X myofibers. Scale bars, 50 μm. (I) Quantification of TUBB3, SV2, AChR, and NMJs in central compartments containing DMD- and CORR-R3381X neuromuscular circuits. n = 5 for DMD-R3381X and n = 6 for CORR-R3381X. Values are means ± SD; unpaired t test, *P < 0.05, **P < 0.01, and ***P < 0.001.