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First published online December 1, 2003
doi: 10.1242/10.1242/dev.00872

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Mitf expression is sufficient to direct differentiation of medaka blastula derived stem cells to melanocytes

Julia Béjar, Yunhan Hong^* and Manfred Schartl

Physiological Chemistry I, Biocenter of the University Würzburg, D-97074 Würzburg, Germany

View larger version (88K): [in a new window]   Fig. 1. Transfection of Mitf into ES-like cell and fibroblast cell lines of medaka. (A) Non transfected MES-1 cells. (B) Bright field view of A; (C) MES-1 cells 4 days after transfection with XmaMitf and GFP vectors (arrow shows a faintly pigmented cell). (D) Bright field view of C. (E) Terminally differentiated melanocyte seven days after the transfection of MES-2 cells. (F) Dark field showing GFP expression. (G) Melanocytes in MES-1 transfected cells 12 days after the transfection (arrows indicate melanocytes). (H) Pigmentation in OLF cells transfected with XmaMitf (arrow indicates pigmented cell). Scale bars: 100 µm.

View larger version (16K): [in a new window]   Fig. 2. Number of melanocytes observed in a defined area of the culture dishes after transfection of the different cell lines with XmaMitf. Absolute numbers are given because non transfected cells continue proliferating as usual, thus a percentage value of melanocytes does not really reflect the increase in the number of differentiated cells observed over time.

View larger version (128K): [in a new window]   Fig. 3. Melanocytes differentiated from XmaMitf transfected MES-1 cells and treated with norepinephrine (NE). (A) Before adding NE. (B) Thirty minutes after, the melanosomes start to aggregate. (C) Two hours later, the melanosomes are completely aggregated around the nucleus. Scale bar: 100 µm.

View larger version (100K): [in a new window]   Fig. 4. Expression assays of MES-1 transfected cells. (A) Transfected with XmaMitf. (B) Transfected with the mutant XmaMitf. nt, non-transfected cells; -, no cDNA (negative control); C, positive control. cDNA from medaka skin probed for tyrosinase, Tyrp1, Dct, Kit, Fms, Aim1, Ednrb, Tbx2, P53, OlaMitf and Actin. cDNA from 5-day-old medaka embryos probed for Erbb3, Sox10, Ap2, Snail2, Ncam and enolase. cDNA from adult muscle for Myf5 and for XmaMitf, MES-1 cells transfected with the XmaMitf construct.

View larger version (72K): [in a new window]   Fig. 5. Expression assays of OLF cells after transfection with XmaMitf. Controls are as for the MES cells (see Fig. 4).