Figure 4

Medial longitudinal fasciculus axons shift toward the midline in a Slit dose-dependent manner. (A-C) Close-up views of the midline in rhombomere 4 (r4) in embryonic day 10.5 mouse embryos stained with βIII-tubulin antibody. The position (yellow line) of the most ventral medial longitudinal fasciculus (MLF) axons was quantified. (A) Embryos with four Slit doses as littermate controls for the normal MLF distance from midline in r4. (B) Slit mutants with only one remaining dose of Slit2 caused the most ventral MLF axons to shift toward the midline. (C) One dose of Slit3 caused a severe phenotype in which a subset of MLF axons had angling trajectories that crossed the midline. In these cases, subsets of axons which did not cross but maintained longitudinal trajectories were used to measure the MLF distance from midline. (D) Average normalized distance from the midline to the most ventral MLF fibers in r4 decreased gradually with loss of Slit doses. The number of embryos (n) is a sum of different combinations of Slit2 and 3 wild-type alleles, with 6 to 11 analyzed for each genotype (indicated on each bar). Error bars show SEM. Differences between genotypes were statistically significant by analysis of variance (P < 0.005). The trend in MLF position was significant by an ordered heterogeneity test (P < 0.0001) (see Methods). (E-H) Slit triple mutants. Bilateral diI tracing of MLF axons in littermates with decreasing Slit doses. Embryos with only one dose of either Slit2 or Slit 3 causes a subset of axons to cross the midline. Slit1/2/3 triple mutant embryos (n = 6) had a complete collapse of MLF axons into and projecting longitudinally within the floor plate. Scale bar in (C) also applies to (A-C): 100 μm; scale bar in (H) also applies to (E-H): 100 μm.