Verification of EGFP expression in melanocytes.

In all panels, representative images of the same zebrafish embryo at 35 hours post fertilization (hpf) are shown. This embryo had been injected with the control construct pmitfa_EGFP, which drives expression of green fluorescent protein under the control of the zebrafish mitfa promoter. (A) The brightfield image shows individual darkly pigmented melanocytes. (B) EGFP expression was confirmed by antibody staining with an anti-EGFP antibody resulting in green fluorescence. (C) Superimposition of the brightfield image showing melanocytes with the corresponding fluorescent image showing EGFP positive cells. (D-F) Magnifications of the areas indicated by the dashed yellow square. An individual EGFP-positive melanocyte is indicated by arrows.

Reduction of melanocytes in zebrafish embryos expressing bovine TWIST2 at 35 hpf.

(A) Representative images of zebrafish embryos injected with either a control construct (pmitfa_EGFP) or a construct driving the expression of bovine TWIST2 under control of the zebrafish mitfa promoter (pmitfa_btaTWIST2_EGFP). Melanocyte counts for head and trunk are given in brackets. Scale bars correspond to 1 mm. (B) Melanocytes in the head and trunk were counted in three experiments (controls: n = 14, 16, 19; TWIST2: n = 17, 16, 19). (C) The experiments were repeated with another set of constructs that additionally had CFP as a reporter for transgenesis. In transgenic animals, cerulean fluorescence in the eyes becomes visible at ~4–5 dpf. (D) Melanocyte counts in two replicate experiments at 35 hpf from zebrafish injected with the constructs pmitfa_EGFP_cryst_CFP and pmitfa_TWIST2_EGFP_cryst_CFP. If (B) and (D) are taken together the TWIST2 overexpressing embryos had significantly fewer melanocytes in the head in 4 out of 5 experiments and in the trunk in 3 out of 5 experiments.