Fig. 6

Mmp14a is required for timely differentiation of retinal neurons.

A Toluidine blue stained transverse semi-thin sections show a delay in retinal differentiation and lamination in Mmp14a morphant eyes (Mmp14a^KD). At 48 hpf, control embryos show an initial laminated retina, which develops into a fully laminated and differentiated retina at 72 hpf, with a clearly distinguishable RGCL, IPL, INL, OPL, ONL, and PR. Mmp14a morphant retinas, however, completely lack lamination at 48 hpf, while at 72 hpf the degree of lamination is still severely reduced, as compared to control embryos. At 96 hpf, however, morphant retinas appear fully laminated and differentiated and are indistinguishable from control retinas. B In situ hybridization for otx2 shows increased expression in the central retina of transverse eye sections of morphant embryos as compared to controls. Nuclear Fast Red was used as counterstain. C–H Immunohistochemical stainings for markers of neuronal differentiation on transverse sections of Mmp14a morphant and control embryos reveal a reduced expression of Zn5 (red), which stains RGCs and their axons, in Mmp14a morphants at 48 hpf (C) and 72 hpf (D, white arrowhead shows RGC dendrites), of Pax6 (green), which labels RGCs and amacrine cells, at 48 hpf (E) and 72 hpf (F), of the Müller glia marker Zrf1 (red) (G), and of the photoreceptor marker Zpr1 (red) (H), both at 72 hpf, all indicative for a disrupted retinal differentiation. DAPI (blue) was used as counterstain in C, E, F. The images in panel D, G and H were taken using confocal microscopy. hpf, hours post fertilization; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; PR: photoreceptor layer; RGCL, retinal ganglion cell layer. Scale bars: 50 μm.