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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.
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