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CNE_22726_sm_suppfig1.tif9359KSupporting Figure 1 (magenta-green version of Figure 4 for the assistance of color-blind readers). A small subset of the PGP9.5 and Tuj1-labeled population express markers of the cell cycle. Sections from adult, wild type mice are stained with anti-PGP9.5, Tuj1 and cell cycle markers. (A-C) Tuj1-labeled cells at the base of the neuronal layer also show heavy expression of PGP9.5 on occasion (white arrow), though the vast majority of basally situated, Tuj1-labeled cells are only very lightly labeled for PGP9.5 (black arrows). (D-F) Of the basal-most Tujl-labeled cells, a large percentage of them express PCNA, a marker of S-phase (white arrows). (G-I) The basally located, lightly labeled, PGP9.5-positive cells also co-express PCNA frequently (white arrows). (J-L) Additionally, a large number of the light PGP9.5-labeled cells co-express Ki67, a broad marker for cells in the mitotic cycle (white arrows). The arrowheads mark the basal lamina in all figures. Scale bars in all panels equal 20 μm.
CNE_22726_sm_suppfig2.tif6843KSupporting Figure 2 (magenta-green version of Figure 5 for the assistance of color-blind readers). The vast majority of the Cre-expressing cells are likely to be immediate neuronal precursors as they are only lightly labeled with PGP9.5. Sections from adult, wild type mice are stained with anti-Cre and markers of undifferentiated neuronal precursors. (A-C) A large number of basally situated, lightly labeled, PGP9.5-positive cells co-express Cre (white arrows). (D-F) In contrast, none of the SOX2-labeled GBCs label with Cre (black arrows) although Cre-only labeled cells are seen (white arrow). (G-I) Cre is generally not found at a detectable level in PCNA-labeled, cycling cells (black arrows), except rarely when a PCNA-positive cells is also lightly labeled with Cre (white arrow). Occasionally, light Cre labeling is detected in cells that also have light labeling for PCNA (tandem black and white arrows). The arrowheads mark the basal lamina in all figures. Scale bars in all panels equal 20 μm.
CNE_22726_sm_suppfig3.tif11394KSupporting Figure 3 (magenta-green version of Figure 6 for the assistance of color-blind readers). NEUROD1 is predominantly expressed prior to the onset of neuronal differentiation. Sections from adult, wild type mice are stained with anti-NeuroD1 and a range of neuronal markers. (A-C) NEUROD1-labeled GBCs always lack the mature neuronal marker NCAM (white arrows). (D-F) Lightly PGP9.5-labeled cells at the basal margin of the neuronal layer typically lack NEUROD1 (white arrows), though occasionally there is co-expression of NEUROD1 in these lightly PGP9.5-labeled cells (white arrows with asterisks). (G-I) While some NEUROD1-labeled cells lack any detectable Tuj1-reactivity (white arrows), many NEUROD1 (+) positive cells are weakly labeled with TuJ1 (white arrows with asterisks). (J-L) mAb213, a marker for atypical necklace neurons is undetectable in NEUROD1-labeled GBCs (white arrows). The arrowheads mark the basal lamina in all figures. Scale bars in all panels equal 20 μm.
CNE_22726_sm_suppfig4.tif5475KSupporting Figure 4 (magenta-green version of Figure 7 for the assistance of color-blind readers). NEUROD1 is expressed in GBCs that retain cell cycle markers and lie downstream of MASH1. Sections from adult, wild type mice are stained with anti-NEUROD1 and anti-PCNA, a marker of cell cycle progression (A-C), SOX2 (D-F) or MASH1 (G-I), as markers of GBCs. (A-C) Nearly all NEUROD1-labeled cells co-express PCNA (tandem black and white arrows), though not all PCNA-labeled cells are NEUROD1-positive (black arrows). (D-F) Roughly one-third of the NEUROD1 labeled cells co-express SOX2 (tandem black and white arrows), a marker for multiple subsets of GBCs, though some NEUROD1-labeled cells lack SOX2 (black arrows). (G-I) NEUROD1-labeled cells (white arrows) are not labeled by anti-MASH1 (black arrows), a marker for transit amplifying GBCs. The filled arrowhead marks the basal lamina in all figures. Scale bars in all panels equal 20 μm.
CNE_22726_sm_suppfig5.tif4576KSupporting Figure 5 (magenta-green version of Figure 8 for the assistance of color-blind readers). NEUROD1 is co-expressed in immediate neuronal precursors that are NEUROG1-positive. Sections from adult, wild type mice are stained with (A) anti-NeuroD1 and (B) NEUROG1, which are both reputed to be markers of immediate neuronal precursors in the OE. (C) Roughly half of the NEUROD1-labeled GBCs co-label for NEUROG1 (tandem black and white arrows), though some of the GBCs stain for only NEUROD1 (white arrows) or for only NEUROG1 versa (black arrows). The arrowheads mark the basal lamina in all figures. Scale bar equals 20 μm.

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