Nerve growth factor is expressed by postmitotic avian retinal horizontal cells and supports their survival during development in an autocrine mode of action

Abstract

Cell death in the developing retina is regulated, but so far little is known about what factors regulate the cell death. Several neurotrophic factors and receptors, including the neurotrophins and Trk receptors, are expressed during the critical time. We have studied the developing avian retina with respect to the role of nerve growth factor (NGF) in these processes. Our starting point for the work was that NGF and its receptor TrkA are expressed in a partially overlapping pattern in the inner nuclear layer of the developing retina. Our results show that TrkA and NGF-expressing cells are postmitotic. The first NGF-expressing cells were found on the vitreal side of the central region of E5.5-E6 retina. This pattern changed and NGF-expressing cells identified as horizontal cells were later confined to the external inner nuclear layer. We show that these horizontal cells co-express TrkA and NGF, unlike a subpopulation of amacrine cells that only expresses TrkA. In contrast to the horizontal cells, which survive, the majority of the TrkA-expressing amacrine cells die during a period of cell death in the inner nuclear layer. Intraocular injections of NGF protein rescued the dying amacrine cells and injection of antisense oligonucleotides for NGF that block its synthesis, caused death among the TrkA-expressing horizontal cells, which normally would survive. Our results suggest that NGF supports the survival of TrkA expressing avian horizontal cells in an autocrine mode of action in the retina of E10-E12 chicks. The cells co-express TrkA and NGF and the role for NGF is to maintain the TrkA-expressing horizontal cells. The TrkA-expressing amacrine cells are not supported by NGF and subsequently die. In addition to the effect on survival, our results suggest that NGF plays a role in horizontal cell plasticity.

Fig. 1

Analysis of early NGF and TrkA expression in embryonic retina. mRNA expression was analysed using in situ hybridisation analysis. Micrographs show cross sections of central region retina and are oriented with vitreal side facing downwards. (A) Analysis of E3 retina. No labelling was found. (B) Analysis of NGF mRNA expression in E5.5–E6 central retina. (C) Overview of a section of an E7 retina with labelling for NGF mRNA. Ventricular/external side (ext), vitreal side (vitr) and area of retina with dispersed NGF cells (disp). Note that the pigment epithelium is not included. b, f and i indicate regions of the retina that are representative for B, F and I, respectively. (D) E7 control (ctr) retina hybridised with sense probe for NGF mRNA. (E) Combination of NGF in situ hybridisation and BrdU immunohistochemistry analyses of E6 retina. (F) Analysis of NGF and Brn3a mRNA expression in the E6.5 retina using double in situ hybridisation. (G) High magnification of cells shown in F (NGF, red and Brn3a, blue). (H) E6.5 control retina hybridised with sense probes for NGF and Brn3a mRNA. (I) Combination of NGF in situ hybridisation and BrdU immunohistochemistry analyses of E7 retina. (J) Analysis of NGF and Brn3a mRNA expression in E7 retina. (K) Analysis of Brn3a mRNA expression in E7 retina. (L) Analysis of TrkA (fluorescein) and BrdU immunoreactivity (Texas Red) in the E6.5 retina. (M) In situ hybridisation and (N) immunohistochemical analyses of TrkA expression in E7 retina. Scale bar in A, 75 μm for A; in B, 80 μm for B, D–F, I–N; in C, 200 μm for C; in G, 25 μm for G; in H, 50 μm for H.

Fig. 2

NGF and TrkA mRNA expression in the E12 and E17 developing chicken retina. In situ hybridisation analysis of NGF and TrkA mRNA expression in E12 (A–D) and E17 retina (E–H). Micrographs show cross sections of central region retina and sections are oriented with vitreal side facing down. (A, E) Analysis of NGF mRNA expression. (B, F) Analysis of TrkA mRNA expression. (C, D, G, H) Double-in situ hybridisation analysis of NGF (red) and TrkA (blue) mRNA expression. (C, G) High-power magnification of the regions shown in D, H, respectively. Arrows indicate double-labelled cells. gcl, ganglion cell layer; inl, inner nuclear layer. Scale bar in A, 50 μm for A, B, D–F, H; in C and G, 25 μm.

Fig. 3

Number of TUNEL, TrkA and BrdU immunoreactive cells in the inner nuclear layer of E5-E18 retina. Cell density plotted against the age of the developing retina. The number of cells was counted in cross sections of central retina and density was calculated as number of cells per mm² of transverse section of inner nuclear layer. The size, including thickness of the retina, increased from E4 to E6 as a result of extensive cell proliferation, decreased cell death and increased cell size. This resulted in a drop of cell density between E5 and E6. TrkA-expressing amacrine cells (AC) and horizontal cells (HC) were counted from E9 as earliest age when those cells are clearly distinguishable. The increase in TrkA-labelled horizontal cell density seen after E10 is a result of decreased thickness of the inner nuclear layer and not a result of more cells. Error bars are s.d. (n=4).

Fig. 4

TrkA immunohistochemistry in combination with TUNEL in normal and injected retinas. TrkA immunohistochemistry was combined with TUNEL on sections of retina. (A–K) TrkA immunoreactivity is shown in red (Texas Red) and TUNEL in green (fluorescein isothiocyanate). Analysis of normal E9 (A), E12 (B) and E17 (C) retinas. (D–F) Analysis of E12 retinas after intraocular injection of AS1 antisense NGF PON (D), control PON (E) and NGF (F). (G–L) Analysis of E17 retina after intraocular injection of AS1 antisense NGF PON (G, H), control PON (I, J) and NGF (K, L). TrkA immunoreactivity is visualised using epifluorescence (G, I, K), as well as using the diaminobenzidine reaction (H, J, L). Arrowheads in B, D, E indicate TrkA amacrine; small arrows in D indicate TrkA horizontal cells that are labelled with TUNEL. Asterisks in G, H indicate the disrupted horizontal dendritic structures. gcl, ganglion cell layer; inl, inner nuclear layer; prl, photoreceptor layer. Scale bar in A, 50 μm for A–C; in D, 50 μm for D–F; in G 25 μm for G–L.

Fig. 5

Effect on NGF protein levels and density of TrkA amacrine and horizontal cells in E12 retinas after intraocular injections of antisense NGF PON or NGF protein. (A) The level of NGF protein was analysed using an enzyme immunoassay in normal (untreated, n=4), AS1 antisense NGF PON- (AS1, n=5) and nonsense PON- (ctr, n=5) treated retinas. Density of amacrine cells (B) and horizontal cells (C) in E12 retina after treatment with AS1 (n=4), nonsense (ctr, n=4) and NGF protein (NGF, n=4). Stippled bars indicate the total number of TrkA immunoreactive cells and white bars indicate TrkA-and TUNEL-positive cells. Graphs show mean densities±s.d. *P=0.02, ***P<0.0005 (t-test). White bars for ctr and NGF in C are zero.