Figure 2. Cellular activities of cluster differentiation (CD)44, vimentin (VIM), and glial fibrillary acidic protein (GFAP) promoters in chicken retina. Lentiviral vectors were injected into the ventricles of chicken embryos (embryonic day 2–E2) in ovo. The retinas of the injected embryos were harvested on E19–20 and the cells expressing the fluorescent reporter proteins were identified using native fluorescent and immunofluorescent microscopy. The viruses injected were as follows: A-I pFIN-CD44-GFP-WPRE; J-Q pFM-VIM-GW; R-V pFM-GFAP-GW. All sections were counterstained with DAPI, and all scale bars shown equal 50 µm. Abbreviations are as follows: ONL represents outer nuclear layer; INL represents inner nuclear layer; IPL represents inner plexiform layer; GCL represents ganglion cell layer. CD44: A: Photograph of whole mount of retina that had been treated with pFIN-CD44-GFP-WPRE. Clusters of GFP-positive photoreceptors (arrows) were detected across the surface of the whole mount. B: This image was produced by re-photographing the boxed region shown in A using a focal plane just below that used to obtain the image shown in A. Horizontal cells (asterisks) were the predominant GFP-positive cell type observed in this focal plane. C-I These images represent sections of retinas showing the cell types (arrows) in which the CD44-GFP transgene was active (C photoreceptors, D horizontal cells, E amacrine cells, F-H Müller cells, I ganglion cells). Section shown in F was counterstained with an antibody against chicken carbonic anhydrase II (CAII), a marker for Müller cells (G). The merged image (H) shows that the GFP-positive cells also expressed carbonic anhydrase II. VIM: J, K Photographs of a whole mount of a retina treated with pFM-VIM-GW and viewed from the photoreceptor side of the whole mount. J Numerous GFP-positive horizontal cells were detected in the transduced retina (arrow). K Enlargement of the region in image J (box) that contains GFP-positive horizontal cells (arrow). L This image was produced by re-photographing the boxed region shown in K using a focal plane just below that shown in K. Müller cell bodies are the predominant cell type observed in this image plane (asterisk). The horizontal cell indicated in J, K, and L by the arrow is the same cell. M,N Images of sections of the retinal whole mount shown in J and K. GFP-positive horizontal (M, arrow), Müller (M, asterisk), and photoreceptor (N, ONL) cells were detected in several sections. O-Q A section containing GFP-positive cells located in the INL (O, arrow) was counterstained with an antibody against chicken carbonic anhydrase II (P). The merged image (Q) shows that the GFP-positive cells also expressed carbonic anhydrase II. GFAP: R, S Images of a whole mount of a 5-week old GUCY1*B chicken retina that had been treated with pFM-GFAP-GW on E2 and photographed from either the photoreceptor (R) or the vitread (S) side of the whole mount. The pattern of GFP localization observed in these whole mounts suggested that the cells expressing the GFAP-GFP transgene were Müller cells. T-V Sections of the transduced retinas showed that the cell bodies of the GFP-positive cells observed in R and S were located in the INL (T). Immunostaining of these sections with an antibody against chicken carbonic anhydrase II (U) revealed that the GFP-positive cells also expressed carbonic anhydrase II (V).