(A) Dorsal view schematic of contralateral migration phases. Dotted lines and arrows indicate the midline and direction of migration, lines represent leading processes. (B) Transverse view projection of an Isl1:GFP embryo immunostained against GFP. TN, trochlear nerve. Scale bars = 20 μm. (C–D) Dorsal view maximum intensity projections of ventral half of wild-type Isl1:GFP and cdh2ΔEC embryos immunostained against GFP and RFP at 50 hpf. Dotted lines represent the midline. (E–F) Single sagittal confocal sections of nIII GFP fluorescence at the midline in wild-type and cdh2ΔEC fish. Scale bars = 20 μm. (G) Area of GFP fluorescence measured in sagittal sections of nIII. Error bars represent median and range, n = 7 (wild-type) from two clutches and n = 14 from two clutches (cdh2ΔEC), *p=0.0125. There was no difference in the number of nIII cells (medians for wild types (n = 4)=95 and cdh2ΔEC (n = 9)=103, p=0.414). Both tests are two-tailed Mann-Whitney U. (H) Labelling of ocular motor subnuclei by retro-orbital dye fill. MHB, midbrain-hindbrain boundary. (I–J’) Ventral view maximum intensity projections of DiI-filled ventral nIII neurons in Isl1:GFP and cdh2ΔEC larvae at 5 dpf. Scale bars = 20 μm. White arrows indicate neurons which have crossed to the contralateral side, asterisks represent mCherry+ signal. (K–L’’) Dorsal view maximum intensity projections of sparsely labelled neurons located in ventral nIII in Isl1:GFP and cdh2ΔEC larvae at 5 dpf. White and blue arrows indicate GFP-positive and GFP-negative dye-filled neurons. The left eye is the side of dye fill. Alx647 = AlexaFluor 647 dye. Scale bars = 20 μm. Note that in K-K’’, small magenta puncta represent non-specific dye spread, while in K-K’’ these likely also include punctate mCherry signal from cdh2ΔEC expression, due to spectral overlap. (M) Total observed number of pooled dye-filled cells belonging to each category in wild types and cdh2ΔEC at 5 dpf. There is a significant relationship between genotype and distribution of dye-filled neurons, (p<0.0001; chi squared test). There was no significant effect of genotype on the total number of dye-filled neurons across groups per larva, medians for wild types = 31, cdh2ΔEC = 40.5, p=0.674, 2-tailed Mann-Whitney U. Wild-type larvae are from four clutches and cdh2ΔEC larvae are from five clutches. (N–Q) Percentage proportions of dye-filled neurons per larva for each genotype separated by category. All tests are two-tailed Mann-Whitney U. (N) Percentage proportions of contralateral GFP-positive SR cells. n = 15 larvae (wild-type) and n = 16 (cdh2ΔEC) larvae, **p=0.0015. (O) Percentage proportions of ipsilateral GFP+ cells. n = 15 larvae (wild-type) and n = 16 (cdh2ΔEC) larvae, *p=0.0109. (P) Percentage proportions of ipsilateral GFP negative IO cells. n = 15 larvae (wild-type) and n = 16 (cdh2ΔEC) larvae, p=0.5131. (Q) Percentage proportions of contralateral nIV cells. n = 19 larvae (wild-type) and n = 20 (cdh2ΔEC) larvae, p=0.9391. (N–P) Proportions are relative to the total number of nIII cells filled per larva, for (Q) proportions are relative to combined total of dye-filled nIII and nIV neurons. Larvae in which less than 10 nIII cells were filled in total were excluded from analysis in (N–P), whereas larvae in which less than 10 combined nIII and nIV cells were filled in total were excluded from analysis in (Q).