Fig. 1. Development of a lateral commissural tract in chick hindbrains. (A) Schematic showing an open-book hindbrain preparation, with DiI anterograde labelling. DiI crystals were inserted into the lateral margin of the caudal hindbrain, 700-1200 µm caudal to the gVIII root. (B-D) DiI anterograde labelling on fixed hindbrains at stage 24 (n=3), stage 26 (n=3) and stage 27 (n=4), respectively, showing the progression of a laterally located commissural tract. (D) The labelled commissural tract grew to the base of the cerebellar plate. High magnification of the blue-boxed area in D shows that some axons from the tract turn to invade the cerebellar plate (white arrow in the inset in D). (E-H) DiI anterograde labelling of organotypically cultured hindbrains at stage 21 (n=3), stage 23 (n=8), stage 25 (n=10) and stage 27 (n=3), cultured for 2 days in vitro (div). The lateral commissural tract labelled by DiI anterograde labelling in the organotypic cultures closely resemble that in the fixed samples both in its appearance and developmental course. At stage 27+2 div (H), axons could be seen to leave the longitudinal tract and invade the developing cerebellar plate. White arrowheads in C,D,G,H indicate retrogradely labelled neurons contralateral to the DiI injection sites. (I) An organotypically cultured stage 26 hindbrain, with the lateral commissural tract labelled by DiI (white arrow) and the central projection of the right gVIII labelled by DiD (white arrowhead). The lateral commissural tract was located lateral to the central projections of gVIII (n=6). (J,K) Immunohistochemistry using an antibody against a neurofilament-associated protein (3A10) on a stage 24 flat-mounted hindbrain. (K) A higher magnification view of the boxed area in J. Blue arrowheads in J,K indicate the position of the lateralmost longitudinal tract. This lateralmost tract appears to converge from a turning point 900-1000 µm caudal to gVIII (indicated by a red arrow in J). Three longitudinal tracts located in the lateral extreme of the hindbrain can be seen in K: the central projection of gV (purple arrow) and of gVIII (root of gVIII indicated by red asterisk), and the lateral commissural tract (blue arrowhead). White vertical line in B indicates the midline. The asterisks in B-I indicate the injection sites of DiI. Scale bar: 400 µm in B-J; 100 µm in the inset in D and in K.