Supplementary Figure Legends
Supplementary Figure 1 - Schematic summary of experiments
We sought to determine how the developmental stage of donor retinal cells might
influence the success of transplantation. To do this we used a transgenic mouse in
which green fluorescent protein (GFP) expression is driven by the Nrl promoter. Nrl is a
rod-specific transcription factor expressed only in post-mitotic rod precursors and adult
rod photoreceptors (green). Green fluorescence is therefore developmentally regulated.
Dividing retinal progenitor cells were obtained by dissociating Nrl.gfp retinae at
embryonic day 11.5 (E11.5), prior to rod genesis. Conversely, postmitotic rod precursors
were selected by Nrl dependent green fluorescence from dissociated retinae at postnatal
days 2 to 5 (P2-5). The developmental stage of donor cells appeared critical, because
although dividing progenitor cells could survive and differentiate into photoreceptor
rosettes in the subretinal space, only post-mitotic rod precursors (but not mature rods)
had the ability to integrate into the retina and form functional connections with the host.
When using the adult rds mouse as a recipient, the transplanted cells were able
to grow outer segment discs (red), which are normally absent in this strain due to a
genetic deficiency of peripherin-2 (Prph2). In the rhodopsin knockout mouse, there was
sufficient rhodopsin expression in the transplanted cells to affect the scotopic pupil
response.
Supplementary Figure 2. Transplantation occurs via integration not cell fusion. a,
Single confocal sections, taken at the same confocal plane, through the inner segment
(the widest cytoplasmic part) (arrow) of a GFP-positive cell integrated within a CFPpositive recipient retina. Far right, cross hairs show an absence of CFP fluorescence
(red trace) at the location of the GFP-positive inner segment (green trace). b, Integrated
cells only have a single nucleus derived from the donor cell. Donor cells were prelabelled with BrdU 24-48 hrs prior to transplantation into a non-labelled host. Image
shows an integrated cell with a single nucleus that was BrdU-positive (red),
demonstrating that it originated from the donor animal. Scale bar 10 m.
Supplementary Figure 3. E11.5 cells express markers of progenitor cells. Confocal
images of dissociated E11.5 GFP-positive cells stained for the progenitor markers nestin
and Pax6 (both 1:20; Developmental Studies Hybridoma Bank) and Sox2 (1:200;
AbCam) (red). Scale bars 10 m.
Supplementary Figure 4. E11.5 cells survive and are able to differentiate in the
subretinal space of adult host retinas. a, Example of unsorted E11.5 cells from an
Nrl.gfp+/+ donor transplanted into the sub-retinal space of adult wildtype hosts, three
weeks post-injection. The cells consistently failed to integrate. However, some form
rosette-like structures in the sub-retinal space and start to express Nrl, as indicated by
GFP fluorescence. b, differentiated cells express the late photoreceptor marker,
rhodopsin (red) when arranged as rosettes. Scale bars 10 m.
Supplementary Figure 5. Transplantation into the rd mouse. Confocal projection
images of P1 GFP-positive cells transplanted into the rd mouse subretinal space. The
transplanted cells persist at 3 weeks post-transplantation but adopt variable
morphologies due to the collapse of the surrounding host ONL. Scale bar 10 m.