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Fig. 4. Mitf and Chx10 function together in a dose-dependent antagonistic fashion to regulate retinal cell identity. (A) An unstained control Chx10^or-J/or-J retinal section at P0 (labeled J/J above the column of panels) shows pigmentation only in the RPE monolayer (arrow) and in the ciliary margin (square bracket). The NR (arrowhead) is unpigmented. (B) An enlargement of (A) displays the pigmented RPE (arrow) and non-pigmented NR (arrowhead). The broken white line indicates the edge of the NR. (C) NR-MITF/+;Chx10^or-J/or-J littermates have a normal RPE (arrow), greatly expanded ciliary margin (square bracket) and a pigmented monolayer (PML) instead of a NR (arrowhead). (D) An enlargement of (C) shows the pigmented RPE (arrow) and the PML (arrowhead). (E) The Chx10^or-J/or-J NR (arrowhead) expresses the neural-specific cell-adhesion molecule NCAM, while the RPE does not express NCAM (arrow). (F) The PML also expresses NCAM (arrowhead); the RPE does not (arrow). (G) The Chx10^or-J/or-J NR expresses Pax6 (arrowhead), while the RPE does not (arrow). (H) The nuclei of the PML contains the neuroretinal marker Pax6 (arrowhead) in contrast to the RPE, which lacks Pax6 (arrow). Pax6 subcellular localization changes compared with that in a Chx10^or-J/or-J NR (G), which we verified with a second independent Pax6 antibody (data not shown). The mechanism mediating the differential localization is unknown, and its significance is unclear. (I) In situ hybridization of a Chx10^or-J/or-J eye at E11.5 shows normal neuroretinal Rax expression (arrowhead). (J) A NR-MITF/+;Chx10^or-J/or-J littermate also expresses Rax in the NR. Normal Rax expression (arrowhead) at E11.5 in NR-MITF-1, n=5/5. (K) A Chx10^or-J/or-J animal expresses Dct in the RPE (arrow) and presumptive ciliary margin (asterisk). (L) An identical Dct expression pattern is seen in NR-MITF/+;Chx10^or-J/or-J littermates. Normal Dct or Tyr (not shown) expression at E11.5 in NR-MITF-1, n=5/5. (M) An unstained P0 coronal section of an Mitf^mi/+ eye (labeled mi/+) showing RPE pigmentation. The pigmentation present in the NR (asterisk) is an artifact of the dissection; it is RPE tissue that has adhered to the NR. (N) RPE-CHX10/+;Mitf^mi/+ littermates have a dramatic decrease in pigmentation in the RPE (arrow). (O) Hematoxylin and Eosin-stained P0 coronal section of an Mitf^mi/+ eye has a normal RPE (arrow). (P) A higher magnification view of (O) shows the RPE. (Q) RPE-CHX10/+;Mitf^mi/+ littermates have a morphological change in the dorsal RPE; the RPE monolayer has become a thickened, multicellular structure (arrow). (R) An enlargement of Q illustrates the thickened multicellular dorsal RPE (arrow). (S) The Mitf^mi/+ NR expresses the homeodomain protein Pax6 (arrowhead), while the RPE expresses very low levels of Pax6 (arrow). (T) Pax6 is present in the thickened RPE (arrow) as well as the NR (arrowhead) in RPE-CHX10/+;Mitf^mi/+ mice, suggesting that this RPE structure is a NRLL. (U) Both the NR (arrowhead) and the NRLL (arrow) in RPE-CHX10/+;Mitf^mi/+ mice express Rax mRNA. (V) The NRLL in RPE-CHX10/+;Mitf^mi/+ mice expresses mouse Chx10 (arrow), as does the NR (arrowhead). (W) Graphical representation of the percentage frequency of the PML phenotype in different NR MITF/+;Chx10^or-J/or-J transgenic lines. The numbers within the bars indicate the number of individuals examined for each transgenic line. NR-MITF-1, n=14/19; NR-MITF-2, n=4/4. (X) Graphical representation of the percent frequency of changes in RPE phenotype in the RPE-CHX10/+;Mitf^mi/+ transgenic lines. The depigmentation and NRLL phenotypes are shown. Numbers within the bars indicate the number of individuals tested. RPE-CHX10-1, depigmentation, n=6/13; four animals with no pigment were tested for the presence of an NRLL: n=2/4 (denoted by the asterisk). RPE-CHX10-2, both depigmentation and NRLL phenotypes: n=1/5. Scale bars: 50 µm in A,C; 25 µm in B,D-L,S,T; 80 µm in M,N,O,Q; 12.5 µm in P,R,U,V.

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