Abstract
Purpose: :
The β2 and y3 chains of the heterotrimeric molecule, laminin, are major constituents of the retinal inner limiting membrane (ILM). The objective of this study is to evaluate the loss of Lamb2 and Lamc3 genes on developing retinal ganglion cells (GCs).
Methods: :
Axonal trajectories and mosaics of GCs in wholemount retinas (WM) were assayed in wild type (WT) and Lamb2, Lamc3 and Lamb2;c3 (DKO) knockout animals using immunohistochemistry (IHC). GC dendrite length, soma size and surface area were studied in retinas from Thy1.1-EYFP mice.
Results: :
To study the effect of laminin chains on GC development, the morphology of GCs was assayed in WT, Lamb2, Lamc3 and DKO mice. Axonal trajectories to the optic nerve were assessed in P5 and P10 WM using a neurotubulin antibody. At both time points, Lamb2 and DKO WM exhibit axon bundles with eccentric curvature; this contrasts with the linear projections in WT and Lamc3 retinas; P0 animals are under study. Mice expressing EYFP controlled by the Thy1.1 promoter were used to study a subset of GCs. P15 WT and DKO WM were assessed for dendrite length, soma size and surface area. There was a shift to smaller average dendrite length distributions in the DKO relative to WT. Additionally, peak GC dendrite length in the DKO was shorter: 110µm (DKO) vs 140µm (WT). Soma size and distribution also differed between the two: WT somas were between 15-30µm and evenly distributed, whereas in DKO, somas were smaller and unevenly spread. In addition, the area of GC dendritic arbors were assayed. WT exhibited a normal Gaussian distribution of 5,000-40,0000 µm2; GCs in DKO had a tighter distribution in the range of 5,000-10,000 µm2. Next we examined a single type of GC; melanopsin IHC was used to study the mosaic of the intrinsically photosensitive GCs (ipGCs). Two types of ipGCs, M1 and M2, were seen with arborizations in the outer and inner IPL, respectively. The distribution of the distances of these cells to their nearest homotypic neighbor were measured in P21 WT and Lamb2 groups. M1 cells in WT exhibited a normal Gaussian distribution of cell distances with a peak spacing of 120-160µm; peak spacing of M1 cells in Lamb2 was 80-120µm. M2 cells spacing in the WT was 40-80µm and 0-40µm in Lamb2 retinas.
Conclusions: :
These results show that absence of laminin β2 and y3 adversely affect the morphology and spacing of GCs.
Keywords: retinal development • retinal degenerations: cell biology • retina