Figure 8. Soluble MsContactin fusion proteins inhibit EP cell migration in an APPL-dependent manner. A–E, Examples of the developing ENS in cultured embryos immunostained with anti-pan Fas II (redrawn from camera lucida images). FG/MG, Foregut–midgut boundary; EN, esophageal nerve on the foregut. Arrowheads indicate the leading EP cell processes on each midgut band pathway; only the four dorsal pathways are shown. Scale bar, 40 μm. A, Preparation fixed and immunostained at experimental onset (∼50 hpf); at this stage, EP cells had begun to align with the muscle bands (b) while avoiding the interband regions (ib). B, Embryo treated with control MOs for 24 h before fixation and immunostaining; the migratory EP cells and their axons remained largely confined to the band pathways. C, Embryo treated with MsCont–Fc fusion proteins; neuronal migration and axon outgrowth were markedly reduced, compared with controls. D, Embryo treated with APPL-specific MOs; a substantial number of EP cells migrated and elaborated processes onto the interband regions of the midgut (arrows), similar to preparations treated with MsContactin-specific MOs (Fig. 6). E, Embryo treated with a combination of APPL-specific MOs plus MsCont–Fc fusion proteins. The overall extent of EP cell migration and axon elongation was similar to control preparations, accompanied by a moderate increase in ectopic migration and outgrowth onto the interband regions (arrows). F, Quantification of the overall extent of migration (black bars) and axon outgrowth (gray bars) on the band pathways; distances were normalized to untreated and vehicle-treated controls in each experiment. The distance traveled by the EP cells along the bands was not affected by APPL-specific MOs (compared with controls) but was significantly reduced in preparations treated with MsCont–Fc fusion proteins. In embryos treated with a combination of APPL-specific MOs plus MsCont–Fc, migration and outgrowth distances were largely restored to control levels. For the extent of neuronal migration, F(3,63) = 72.8, p < 0.001; for the extent of axon outgrowth, F(3,62) = 33.0, p < 0.001. N ≥ 11 for each condition; histograms indicate means ± SD. G, H, The number of neurons (G, hatched bars) and the extent of processes (H, stippled bars) that grew into the interband regions were significantly increased by treatment with APPL-specific MOs, compared with control MOs. Treating embryos with control MOs plus MsCont–Fc did not alter the low levels of interband growth seen in untreated embryos, whereas treatment with APPL-specific MOs plus MsCont–Fc induced a significant increase in the number of ectopic neurons (G) and a more moderate increase in ectopic outgrowth (H); histograms indicate means ± SD. For the number of interband neurons, F(3,14) = 17.0, p < 0.001; for the extent of interband outgrowth, F(3,14) = 7.2, p = 0.004. With Bonferroni's corrections, p values are as follows: 1: t(34) = 11.7, p < 0.001*; 2: t(34) = 8.1, p < 0.001*; 3: t(30) = 8.0, p < 0.001*; 4: t(29) = 4.7, p < 0.001*; 5: t(26) = 3.6, p = 0.004*; 6: t(25) = 2.3, p = 0.087; 7: t(7) = 4.9, p = 0.005*; 8: t(7) = 5.0, p = 0.005*; 9: t(6) = 4.4, p = 0.014*; 10: t(7) = 4.0, p = 0.015*; 11: t(6) = 3.2, p = 0.055; 12: t(14) = 2.6, p = 0.063. *, Student's t test results that are significant at the α = 0.05 level.