Fig 2: Dual embryonic origin of intermediate cells. Immunostaining for melanoblast with MITF labelling (in red) and neural crest cells with P75 (in turquoise) shows at E11.5 the presence of melanoblast between the dermis and the otic vesicle and between the otic vesicle and the neural tube (Arrows). Lineage tracing of Schwann cell precursors using Plp1-YFP reporter line at E12.5 and P6. Immunostaining for melanoblast with the MITF antibody (red) and DAPI for nucleus (blue) show cells with both markers (arrowheads) or only melanoblast marker (arrows). Most intermediate cells express the glial cell marker PLP (green) and melanocyte markers DCT (in purple) at P6 except one cell that only express DCT (arrow). CVG: cochleaovestibular ganglion, NT: neural tube, OV: otic vesicle, V: vestibule.

Fig 3: A proportion of the inner ear melanocytes is derived from Schwann cell precursors and immature Schwann cells. A The scheme represents the design of the lineage-tracing experiment using Plp1CreERT2/R26YFP mice. Tamoxifen injections were administered either at E12.5, E13.5, E14.5 or E15.5, which triggered recombination and the permanent expression of fluorescent marker (YFP) in either Schwann cell precursors (marked with green “SCPs”) or immature Schwann cells (marked with magenta “iSCs”). Note that immature Schwann cells start to form around E15.5 differentiating from SCPs. However, their exact proportions and capacity to generate other cell types stay undetermined due to different opinions in the scientific community regarding the markers identifying these cells clearly. All obtained embryos were analyzed at E17.5 using immunohistochemistry (B–E), and the contribution of the SCP lineage to melanocytes was quantified and visualized in (F). B–E Pigmented melanocytes were identified using transmitted light based on the presence of pigmentation and typical cell morphology inside of the inner ear, proximally to the neuroepithelium. C–E Melanocytes co-express expected markers Sox10 and Mitf, and the lineage tracing in Plp1CreERT2/R26YFP was visualized by co-staining with the anti-GFP antibody (D). F Contribution of Schwann Cell Precursors and immature Schwann cells to melanocytes of the inner ear was assessed by performing the analysis of genetic tracing in E17.5 embryos using Plp1CreERT2/R26YFP mouse line. For every time-point, a minimum of five different embryos from three independent litters were evaluated. White square in (B) shows the area magnified in the inset, and the yellow arrowheads point at the individual pigmented melanocytes. White arrowhead and white dotted lines mark the cartilage of the inner ear in (B). Red arrowhead and the red dotted line mark neuroepithelium of the inner ear in (B). Yellow arrowheads point at individual pigmented melanocytes (in C–E) expressing one of the typical melanocytes markers: SOX10 in (C, D) or MITF in (E). Yellow-green arrowhead points at pigmented YFP+ melanocyte in (D). Mean ± SEM and n for each stage are: E12.5 (40.14 ± 1.237, n = 5), E13.5 (35.34 ± 2.115, n = 5), E14.5 (35.56 ± 2.438, n = 5) and E15.5 (23.42 ± 1.392, n = 6). Scale bars are 20 µm (B) and 10 µm (C, D)

Fig 4: Schematic of the determination of SCPs to generate Schwann cells and melanoblasts. In this model, FoxD3 and Mitf regulate each other to specify Schwann cell and melanocyte lineage from SCPs around E10.5. In the presence of a high content of FoxD3 and low content of Mitf, SCPs would be directed towards the Schwann cell lineage and in the opposite conditions SCP would be directed towards the melanocyte lineage (Mb). In a situation in which ß-catenin is activated, Mitf would be induced in SCPs to promote more efficiently the specification of melanocytes, and would also repress FoxD3 resulting in specification of fewer Schwann cells.

Fig 5: Extracutaneous melanocytes in the inner ear, the heart and the brain are recruited from Schwann cell precursors. A–I Extracutaneous melanocytes found in the heart (A–C) and the brain (D–I) of adult mice. A Overview of the heart that has been dissected in (B, C). Note that the melanocytes are found in the valves and also lining the walls of arteries and veins of the heart. D–F Melanocytes in the leptomeninges of the brain in the adult mouse are localized to the area of olfactory bulb (yellow arrowheads point at the pigmented areas or individual melanocytes). G–I Insets are magnifying the pigmented areas from (D–F). J, K Pigmented melanocytes reside in the supraorbital space of the mouse embryonic head at E17.5, proximally to the eyeball. Yellow arrowheads point at the individual melanocytes in (J). White dotted rectangle in (J) shows the area magnified in (K), 90˚ rotated. LV left ventricle, LA left atrium, RV right ventricle, RA right atrium, AO aorta, PV pulmonary valve, PA pulmonary artery, DRG dorsal root ganglion. L This scheme represents the design of the lineage-tracing experiment using Plp1CreERT2/R26YFP mice. Tamoxifen injection was administered at E11.5, which triggered recombination and the expression of the permanent fluorescent marker (YFP; visualized as a green signal) in Schwann cell precursors. The lineage tracing in Plp1CreERT2/R26YFP was visualized by co-staining with anti-GFP antibody (M–R). The embryos were analyzed at E17.5 using RNAscope® technology and immunohistochemistry (M–R), and quantified (S). The following locations were analyzed for the presence of Dct+ (and Mitf+) melanocytes: heart (M), brain (N), supraorbital region (O), skin (P), and the inner ear (Q, R). White arrowheads point at the individual melanocytes in (M–R), whereas the white dotted line shows the area magnified on the right side of each panel. For better orientation, the magnified melanocytes are numbered to distinguish the presence or absence of the YFP signal. The quantified differences in the contribution of SCPs to the extracutaneous melanocytes are visualized as a violin plot (S). The contribution of SCPs to melanocytes in different locations was calculated as a proportion of YFP+/Dct+ melanocytes from all Dct+ melanocytes. Recombination efficiency has been assessed as a proportion of YFP+/SOX10+ cells from all SOX10+ cells of the SC lineage within the nerves. For each location, three embryos (n = 3) from the independent litters were analyzed. Mean ± SEM; p-value for each location are: inner ear Dct (17.47 ± 1.784; 0.00048), heart Dct (90.11 ± 0.8285; 0.03016), brain meninges Dct (16.51 ± 10.88; 0.03228), supraorbital Dct (76.02 ± 3.041; 0.97721), skin Dct (50.47 ± 4.511; 0.05280), skin Mitf (63.78 ± 1.277; 0.03144), and the recombination efficiency in peripheral nerves (75.87 ± 1.822). Scale bars are 1 mm (A, C, D–F), 250 µm (B), 50 µm (G–J), 10 µm (K–R)