NF-Protocadherin Regulates Retinal Ganglion Cell Axon Behaviour in the Developing Visual System
Fig 4
Perturbation of NFPC binding leads to tectum entry defects.
Open brain embryos were incubated with Con-Fc (A) or NFPC-Fc (B) from stage 35. At stage 40, retinal axons were labelled with DiI. Brains were then dissected and mounted in the contralateral view to enable visualization of the optic tract. Inverse greyscale images show that the axon bundle trajectories of Con-Fc-treated brains appeared normal (A, higher magnification shown in A’), with RGC axons entering the tectum normally (the tectum is delineated with a dashed line in A’). Brains treated with the NFPC-Fc ectodomain construct (B), however, exhibit various phenotypes including axons avoiding the tectum and growing along the anterior tectal boundary (B’). (C) Graph showing the proportion of brains incubated with the NFPC-Fc peptide that display axons avoiding either the anterior or posterior boundary of the tectum. Statistical significance was calculated against the Con-Fc proportions. * p<0.05, ** p<0.01, 1p = 0.0934, Fisher’s exact test (6 independent experiments). Brains electroporated with the Con-MO within the optic tectum (D) do not exhibit defects in axon pathfinding, as RGC axons grew through the electroporated region into the tectum (D’ is an inverse greyscale image showing the trajectory of DiI-filled RGC axons). Panel E shows a representative image of a brain electroporated with the NFPC-MO within the tectum. Perturbation of NFPC binding culminated in phenotypes including looping and projection along the posterior tectal boundary (E’ is an inverse greyscale image showing the trajectory of DiI-filled RGC axons). Panel F reveals the proportion of brains loaded with the NFPC-MO that exhibit abnormal projections into the tectum. Statistical significance was calculated against Con-MO proportions. *** p<0.001, Fisher’s exact test (7 independent experiments). Scale bar in A: 300 μm (A, B, D, E), 75 μm (A’, B’), 50 μm (D’, E’).