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M. Dellett, K.A. Stevenson, J.H. P. Brogan, D.A. C. Simpson, T. Cogliati; Targeted Deletion of the Basic Helix–Loop–Helix (bHLH) Transcription Factor Nhlh2 in Mouse Affects Cone Photoreceptors . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2774.
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Nhlh2 is a member of the bHLH family, which is expressed in retinal ganglion cells (RGCs) and amacrine cells in the developing murine retina. The purpose of this study is to gain insight into the role of Nhlh2 in differentiating retinal neurons by characterising the phenotype of the Nhlh2 knockout (Nhlh2–/–) mouse.
Cryostat sections (10–14 µm) were obtained from young wild type (wt) and Nhlh2–/– mouse eyes. Retinal morphology was revealed by Hematoxylin and Eosin (H&E) staining. Immunocytochemistry (ICC) was performed on 2 months old retina cryosections utilizing a panel of antibodies as specific markers for rod photoreceptors (α–Rho 4D2), cone photoreceptors (α–S–opsin, α–M–opsin), amacrine cells (e.g. α–calretinin, α–Islet–1, α–HuC/D, α–Pax6, α–Gad65, α–calbindin), and RGCs (e.g. α–Brn3a, α–Brn3b, αNF160, α–NF200, α–Pax6). Cone counts were performed on postnatal day (P)21 and 3 month old immunostained cryosections as well as on 9 month old mouse eye flatmounts (n=3 wt and Nhlh2–/– in both assays)
Loss of Nhlh2 does not affect overall retinal morphology. All three cell layers are present in Nhlh2–/– retinas and their thickness is comparable to wt. Developmental expression of Nhlh2 in amacrine and RGCs suggested the possibility that targeted deletion might have affected these cell types in the mature retina. However, no obvious abnormalities in the total number or location of amacrine or RGCs were observed. Initial analysis with specific markers for retinal cell subpopulation revealed no differences in staining patterns for Brn3a, calretinin and Gad65 in Nhlh2–/– compared to wt mice. Cone ICC analysis revealed an increase in the proportion of S–opsin immunoreactive cones, but not of M–opsin immunoreactive cones, in the Nhlh2–/– compared to wt retina. This difference is already apparent in the newly mature retina (P21).
Normal retinal morphology in Nhlh2–/– mice, together with altered proportions of S–opsin immunorective cones suggests that Nhlh2 contributes to specification of retinal cell subtypes.
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