DEV02564 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Identification of the lateral commissural tract in hindbrain labelled in Fig. 1 as the cC-VC tract. (A) Retrograde labelling from a rostrolateral position in a stage 25 hindbrain labelled a group of neurons residing contralaterally across r6/r7 and possibly rostral r8 (white arrowhead). White arrow indicates the position of r4/r5 boundary. (B) A transverse vibratome section of a retrogradely labelled sample as in A at the level of r7 shows the neurons are located in the mantle zone at the lateral hindbrain neuroepithelium (white arrowhead). These neurons give rise to a tightly fasciculated commissure traversing between the matrix and mantle zones. (C) Two neuronal groups were retrogradely labelled from the right caudal cerebellar plate of a stage 28 hindbrain: the rostromedial inferior olive nucleus (IO), and a second group (white arrowhead) positioned rostrolaterally to IO. (D) Confocal imaging showing double labelling in a stage 27 hindbrain with the central projection of gVIII anterogradely-labelled by DiD, and the lateral commissural tract neurons retrogradely labelled by DiI. (E,F) Progressively higher magnification confocal images of a similarly labelled hindbrain as in D. (F) A higher magnification image of the boxed area in E. The descending gVIII afferents send collaterals both laterally and medially, extending into the aggregates of retrogradely labelled neurons, and make close contacts with the neuronal bodies and their dendritic fields (indicated by white arrows in F). The anatomical localisation of these neurons and their commissure, together with the fact that they project to the cerebellum and receive innervation from gVIII central project led us to conclude that these neurons are caudally located contralateral cerebellar-projecting second order vestibular neurons (cC-VC). White lines in A-C indicate the midline. Scale bar: 200 μm in A-D; 100 μm in E; 50 μm in F.

• Supplemental Figure 2 -

  Fig. S2. A functional blocking antibody of chick ephrin-A2 (B3) could reduce the inhibitory activity of caudal hindbrain. Grafting was performed as depicted in Fig. 2B. Inhibition from the caudal graft could be partially alleviated when 20-30 μg/ml blocking antibody (B3) was added to the culture medium (A), whereas almost all cC-VC axons were inhibited when equal amount of Fc fraction of human Ig (hFc) was added (B). (C) Quantification of the data in A,B (P<0.005, Mann-Whitney U test). Scale bar: 200 μm.