Fig 2: Endothelial-mesenchymal transformation (EMT) of RCECs using specific endothelial-mesenchymal transformation medium (SEMTM). (A) Immunohistochemistry of the fibroblastic markers a-SMA and vimentin were characteristic of fibroblasts in EMT-RCECs compared to the hexogonal morphology of Fresh RCECs (Scale bar upper panel = 100 µm, Scale bar middle and bottom panel = 50 µm). (B) RT-PCR showed that both Fresh RCECs and EMT-RCECs expressed a-Sma. Original gel is shown in Supplemental Fig. S2. However, qRT-PCR in the lower panel shows a-Sma was significantly upregulated in EMT-RCECs. (C) Western blots showed that Fresh RCECs and EMT-RCECs expressed Vimentin, Atp1a1, a marker of differentiated endothelial cells, and b-actin. Original gel and blots are shown in Supplemental Figs S3, 4. Semiquantitative analysis shows significant upregulation of the protein levels of Vimentin (p = 0.048) and downregulation of the protein level of Atp1a1 (p = 0.0043). Data expressed as mean ± SD of three replicate experiments. Student’s t test (*p < 0.05, **p < 0.01). (n = 3). RCECs: rabbit corneal endothelial cells, a-SMA: smooth muscle a-actin. Atp1a1: Na,K-ATPase a-subunit.

Fig 3: Utricle and ampulla at W14 of development and adult ampulla. (A) H&E overview of utricle (Utr), ampulla (Amp), and saccule (Sacc). (B) Detail of A: showing ampulla and epithelial fold with dark cell epithelium (arrows). (C) Irregular laminin staining profile of the basement membrane of the transitional zone and dark cell epithelium near the ampulla. (D) Consecutive immunostained section with melanocytes underlying the transitional zone and dark cell epithelium near the ampulla. (E) Detail of A: showing the epithelial fold containing dark cell epithelium with subepithelial connective tissue. (F) Detail of A: showing the dark cell epithelium in the utricular roof. (G) Dark cell area of an adult ampulla. Green: MLANA (B and D–G) or laminin (C); red: ATP1A1; and blue: DAPI. Scale bars: 20 µm (E, F and G), 50 µm (C and D), and 200 µm (A and B) [Color figure can be viewed at wileyonlinelibrary.com]

Fig 4: Utricle and ampullae at W10 of development. (A) H&E overview of ampulla (Amp) and utricle (Utr). (B) Melanocytes migrating through an epithelial fold separating the endolymphatic duct and the common lumen of the utricle and saccule. (C) Overview of ampulla and utricle. (D) A small number of melanocytes underlying the ampullar transitional zone. (E) Detail of C: melanocytes underneath the dark cell epithelium opposite to the ampullar crista. (F) Detail of C: melanocytes underneath the dark cell epithelium opposite the utricular macula. (G) Detail of C: melanocytes in between the ampulla and utricle. Green: MLANA; red: ATP1A1; and blue: DAPI. Scale bars: 20 µm (B, C, D, E, F, and G) and 100 µm (A) [Color figure can be viewed at wileyonlinelibrary.com]

Fig 5: Melanocytes located around the vestibular organs at W7 of development. (A) H&E overview showing a transected semicircular canal (SCC), the utricle (Utr), and the saccule (Sacc). (B) H&E overview of an ampulla (Amp). (C) Melanocytes stained with MLANA underneath the transitional epithelium of the ampulla. (D) H&E overview of a transection of an SCC. (E) Melanocytes in the future periotic mesenchyme surrounding the SCC. (F) Detail of A: melanocytes within an epithelial fold near the utricular macula. (G) Detail of A: melanocyte underneath the dark cell epithelium in the utricular roof opposite to the macula. (H) Higher magnifications of melanocytes from panels E and G, showing short dendritic processes and their spindle-shaped morphology. No expression of Na+/K+-ATPase a1 was observed (E, F, and G). Green: MLANA; red: ATP1A1; and blue: DAPI. Scale bars: 5 µm (H), 20 µm (B, C, D, E, F, and G), and 50 µm (A) [Color figure can be viewed at wileyonlinelibrary.com]