Fig. 3. Regeneration gives rise to all cells layers of the retina. Rows: de, normal retinal development; td, retinas that arose from transdifferention; cr, retinas that regenerated from the CB/CMZ. Immunohistochemistry using antibodies against Brn3a (ganglion cells) and Napa73 (NFL) are shown in the first column (A1-I1). Immunohistochemistry using antibodies against Pax6 (ganglion cells, amacrine cells and horizontal cells) and Chx10 (bipolar cells) are shown in column 2 (A2-I2). Immunohistochemistry using antibodies against visinin (photoreceptors) and Mitf (RPE) are shown in column 3 (A3-I3). Sections in column 4 (A4-I4) were stained for vimentin to detect Müller glia. Rows A, B and C show eyes at E7, D, E and F at E11 and G, H and I at E15. (Row A) At E7, during eye development, ganglion cells are starting to differentiate and an NFL is visible (A1), Pax6 is mostly expressed in the region where ganglion cells are present while Chx10 is expressed through a large part of the retina (A2). Photoreceptors have started to differentiate and Mitf expression in the RPE is noticeable (A3). At this stage Müller glia are also present (A4). (Row B) At E7 transdifferentiating retina shows some Napa73 staining but no Brn3a (B1). Pax6 is expressed throughout the retina but its expression is very prominent in the ganglion cells. There are low levels of Chx-10 at this stage (B2). There is no detectable Mitf expression in the retina that arises from transdifferentiation and visinin is expressed in the presumptive photoreceptor region. (B3). Vimentin expression is limited to Müller glias that span the retina (B4). (Row C) Retina that regenerates from the CB/CMZ shows a similar pattern of cell markers as the retina that arises from transdifferentiation. There is no Brn3a expression but there is some Napa73 staining (C1), and Pax6 is expressed throughout the retina, while there is a very low level of Chx10 expression (C2). However, visinin is not present and Mitf is expressed in the intact RPE (C3). Müller glia are present at this stage (C4). (Row D) By E11, developing retina has a clearly defined NFL, GCL (D1), INL (D2) and ONL (D3). Mitf continues to be expressed in the RPE (D3) and Müller glia are present through out the retina (D4). (Row E) Transdifferentiation results in all differentiated cell types by E11. Ganglion cells (E1), amacrine cells, bipolar cells, horizontal cells (E2), photoreceptors (E3) and Müller glia (E4) are present. The orientation of this layer, however, is flipped (E1-E4). (Row F) Regeneration from the CB/CMZ also results in production of all differentiated cell types by E11. The NFL, GCL (F1), INL (F2), and ONL (F3) have all the cells types seen in a normal retina. Here, the RPE is still intact and expresses Mitf (F3). Müller glia span the retina (F4). The orientation of this layer is similar to that of developing retina. These expression patterns continue to be maintained at E15 in the developing eye (G1-G4), the transdifferentiated retina (H1-H4) and the retina that regenerated from the CB/CMZ (I1-I4). Scale bar: 100 µm.