Fig. 3. Isthmic tissue is a source of a diffusible attractant for trochlear motor axons. (A) A schematic representation of how the collagen gel co-culture was assembled, showing the dissected MHB tissue explant containing trochlear neurons (orange) and the dorsal isthmus (blue) explant. Mb, midbrain; r1-r4, rhombomeres 1-4. (B) Scoring system for detecting deflection of axons towards a source (isthmus tissue, blue sphere). The number of axons were counted growing in two sectors (1, 2) of a T-bar placed parallel and central to the initial trajectory of the extending nerve. (C-F) Rat E11.5 explants into collagen gels that were cultured for 48 hours and stained with F84.1 antibody. (C) Trochlear axons grow from an isolated MHB explant defasciculate upon entering the gel but do not deviate greatly from their original trajectories. (D,E) MHB explants cultured at a distance from a piece of isthmus tissue: axons turn towards the isthmus tissue (i) and grow within it. (F) MHB explant cultured at a distance from a piece of posterior rhombomere 1 (rl) tissue: axons are not affected by the r1 tissue.

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