DEV01571 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. Ectopic expression of MITF in the NR or CHX10 in the RPE is not sufficient to induce changes in cell identity. (A) Schematic of the NR-MITF construct. Expression of the full-length human MITF (M isoform) cDNA was driven by 6 kb of the human PAX6-NRE and the b-globin gene promoter. (B) Schematic of the RPE-CHX10 construct. Full-length human CHX10 expression was directed by 3.3 kb of the mouse Dct promoter/enhancer. (C) NR-MITF/+ mice (three independent lines) express human MITF mRNA from at least E10.5 (arrowhead).  Expression is highest in the periphery of the NR; however, one line expressed MITF throughout the NR (data not shown). (D) Human MITF is expressed in the nuclei (arrowhead) of the E10.5 NR in NR-MITF/+ mice. At this time, mouse Mitf protein is detected in the RPE (arrow). (E) At E12.5 RPE-CHX10/+ mice (three independent lines) express nuclear human CHX10 protein in the dorsal RPE (arrow), similar to the expression pattern of Dct. These mice also express mouse Chx10 protein in the NR (arrowhead). Human CHX10 is detectable in the RPE from at least E10.5 in RPE-CHX10/+ mice (data not shown). (F) Hematoxylin and Eosin staining of a wild-type E15.5 eye. (G) A Hematoxylin and Eosin-stained NR-MITF/+ E15.5 eye appears normal when compared with its non-transgenic littermate shown in F (three independent lines). (H) Hematoxylin and Eosin staining of a wild-type eye at birth. (I) An RPE-CHX10/+ P0 eye stained with Hematoxylin and Eosin appears phenotypically identical to its wild-type littermate shown in H (three independent lines). Scale bars: 25 mm in C,E; 12.5 mm in D; 40 mm in F,G; 80 mm in H,I.

• Supplemental Figure 2 -

  Fig. S2. The PML cells in NR-MITF/+;Chx10^or-J/or-J animals appear to be a mixed RPE/neuroretinal cell type, while NRLL cells in RPE-CHX10/+;Mitf^mi/+ individuals are neuroretinal. Further marker analysis was performed to supplement the data shown in Fig. 4. (A) The Chx10^or-J/or-J NR at birth (arrowhead) expresses the neuroretinal-specific membrane protein Notch1 (outlining the neuroretinal cells), while the RPE does not express Notch1 (arrow). The extent of the RPE monolayer is indicated by the bracket in the top left-hand corner. (B) The PML in NR-MITF/+;Chx10^or-J/or-J littermates does not express Notch1 (arrowhead); neither does the RPE (arrow). Background Notch1 staining is seen in the lens (asterisk). (C) The Chx10^or-J/or-J NR expresses Islet1 (arrowhead), representing differentiating neurons, while the RPE does not (arrow). (D) Neither the PML (arrowhead), nor the RPE (arrow) contain Islet1. (E) In situ hybridization of a Chx10^or-J/or-J eye at E16.5 shows normal neuroretinal Math5 expression (arrowhead). E16.5 was chosen because of the delay in development of the Chx10^or-J/or-J eye. (F) A NR-MITF/+;Chx10^or-J/or-J littermate also expresses Math5 in the NR (arrowhead), suggesting that the NR is properly specified. (G) The Mitf^mi/+ NR at birth (arrowhead) expresses the neuroretinal-specific membrane protein Notch1, while the RPE does not (arrow). (H) Both the NR (arrowhead) and NRLL (arrow) in RPE-CHX10/+;Mitf^mi/+ littermates express Notch1. The asterisk highlights an artefact of torn NRLL. (I) Islet1 is present in differentiating neurons of the ganglion cell layer and inner nuclear layer of the Mitf^mi/+ NR (arrowhead), but not in the RPE (arrow). (J) In RPE-CHX10/+;Mitf^mi/+ littermates, both the NR (arrowhead) and NRLL (arrow) express Islet1 protein. The asterisk highlights the same artefact as discussed in H. Scale bars: 12.5 mm in A-D,G-J; 40 mm in E,F.

• Supplemental Figure 3 -

  Fig. S3. The NR-to-RPE transdifferentiation observed in NR-MITF/+;Chx10^or-J/or-J animals occurs progressively in development from E11.5 to P0 and appears to proceed in a peripheral to central manner, associated with both a decrease in the size and thickness of the NR and an increase in pigmentation. (A) H and E staining of an E14.5 NR-MITF/+;Chx10^or-J/or-J eye showing the thin NR especially at the periphery (arrowhead). (B) A similar panel showing pigment in the RPE of the NR-MITF/+;Chx10^or-J/or-J animals (arrow) and some pigmentation in the peripheral NR (arrowhead). (C) At E16.5 the NR in NR-MITF/+;Chx10^or-J/or-J animals is shrinking, especially in the ciliary margin area (arrowhead). (D) The pigmentation in an NR-MITF/+;Chx10^or-J/or-J eye appears to expand from the cilary margin region into the NR (arrowhead). (E) In an E18.5 NR-MITF/+;Chx10^or-J/or-J eye, the NR is forming a PML (arrowhead). (F) The PML in this E18.5 NR-MITF/+;Chx10^or-J/or-J animal is more highly pigmented in the peripheral area (arrowhead) than the central region. Scale bars: 25 mm in A-D; 32 mm in E,F.