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. 2020 Sep 15;10:15119. doi: 10.1038/s41598-020-71865-3

Figure 4.

Figure 4

Sema3A inhibits axonal outgrowth in dissociated cell cultures and gut explants. (A) Schematic illustrating the coculture of enteric neurons with transfected COS-7 cells in matrigel. (B) Enteric neurons aggregated in matrigel were co-cultured with eGFP- or Sema3A-transfected COS-7 cells and were immunolabeled for TuJ1. In cocultures with eGFP-transfected COS-7 cells, numerous axonal fibers extend out of the matrigel dot (arrows). Scale bar 100 µm. (C) Quantification of the number of axons extending outside the matrigel dot. Data represent means ± SEM (n = 8 from 4 independent experiments); ***p = 0.0002, Mann–Whitney test. (D) Schematic illustrating the coculture of gut explants with transfected COS-7 cells in matrigel. (E) Gut explants co-cultured with eGFP- or Sema3A-transfected COS-7 cells were immunolabeled for Hu and TuJ1. The dotted red line corresponds to the enteric neurons migration wavefront from the explant. The dotted blue line corresponds to the border of axonal projections. Scale bar 250 µm. (F) Quantification of the thickness of the neuronal cell body layer. (G) Quantification of the thickness of the axonal layer. Data represent means ± SEM (n = 7 from 3 independent experiments); **p = 0.0012, Mann–Whitney test. (H) Gut explants co-cultured with eGFP- or Sema3A-transfected COS-7 cells were immunolabeled for SOX10 to assess the migration of precursor and glial cells out of the explants. Data represent means ± SEM (n = 7).