Foxd3 controls melanophore specification in the zebrafish neural crest by regulation of Mitf

Abstract

We describe a mechanistic model whereby Foxd3, a forkhead transcription factor, prevents neural crest-derived precursors from acquiring a melanophore fate. Foxd3 regulates this fate choice by repressing the mitfa promoter in a subset of neural crest cells. mitfa is only expressed in a Foxd3-negative subset of neural crest cells, and foxd3 mutants show an increase in the spatial domain of mitfa expression, thereby suggesting that Foxd3 limits the mitfa domain. Furthermore, foxd3:gfp transgenic zebrafish reveal foxd3 expression in xanthophore precursors and iridophores, but not in terminally differentiated melanophores. Luciferase experiments and embryo mRNA injections indicate Foxd3 acts directly on the mitfa promoter to negatively regulate mitfa expression. Taken together, our data suggests the presence of Foxd3 in a subset of precursors leads to mitfa repression and suppression of melanophore fate. MITF, the human mitfa ortholog, has recently been described as an oncogene and implicated in various forms of melanoma. Understanding the mechanisms that regulate mitfa and melanophore development could prove informative in the treatment and prevention of these human diseases.

Figure 1. mitfa:gfp transgenic reports mitfa positive melanoblasts

(A) Live GFP expression from 20 hpf mitfa:gfp transgenic zebrafish, streams of migratory neural crest cells arise from lateral stripes, dorsal view, anterior left, 10×. hb=hindbrain. (B) Live GFP expression from 26 hpf mitfa:gfp transgenic zebrafish, mitfa positive neural crest cells migrate ventrally (mv), lateral view, anterior left, 10×. (C,E) mitfa:gfp transgenic recapitulates mitfa expression, dorsal view, anterior left, 24 hpf Green: GFP expression, Red: mitfa mRNA. (C) 20× scale bar=20um. (E) 40× scale bar=10um. 98.4% of mitfa:gfp cells (n=311) are mitfa positive. (D,F) mitfa:gfp transgenic labels early melanoblasts, dorsal view, anterior left, 24 hpf. Green: GFP expression, Red: dct. (D) 20×. scale bar=20um. (F) 40×. scale bar=10um. Arrow points to mitfa:gfp positive cell that has yet to activate dct expression. 55% of mitfa:gfp positive cells (n=193) are dct positive.

Figure 2. mitfa expression expanded in Foxd3 mutant

mitfa mRNA expanded in head and anterior trunk of Foxd3 mutant. (Holland et al.) dorsal view, anterior left, 5×. (A-C) mitfa expression in wild-type zebrafish (D-F) mitfa expression in foxd3 -/- (sym1). (A,D) 40 hpf (B,E) 48 hpf (C,F) 60 hpf.

Figure 3. Foxd3 and mitfa expressed in separate populations of neural crest cells

(A) mitfa:gfp positive neural crest cells are mutually exclusive with Foxd3 positive cells, 18 hpf, lateral view, anterior left, 10×. (B) mitfa:gfp up-regulates in Foxd3 negative neural crest cells, 18hpf, dorsal view, anterior trunk region, 20×. (A,B) Red: Foxd3 Ab. Green: GFP expression in mitfa:gfp transgenic line (C) mitfa up-regulates in Foxd3 negative neural crest cells, 24 hpf lateral view, anterior trunk region. Red: Foxd3 Ab. Green: mitfa mRNA. 20×. (D) Cell counts of mitfa:gfp positive cells derived from 40× confocal images of migratory neural crest cells at three time points: 15-16 hpf, 16-17 hpf and 17-18 hpf. Blue line=percent of total mitfa:gfp positive cells counted which are Foxd3 negative. Yellow line=percent of total mitfa:gfp positive cells counted which are Foxd3 positive. Stage 15-16 hpf, 88% of mitfa:gfp positive cells are Foxd3 negative (583/664); 12% of mitfa:gfp positive cells are Foxd3 positive (81/664). Stage 16-17 hpf, 89% of mitfa:gfp positive cells are Foxd3 negative (497/560); 11% of mitfa:gfp positive cells are Foxd3 positive (63/495). Stage 17-18 hpf, 91% of mitfa:gfp positive cells are Foxd3 negative (448/495); 9% of mitfa:gfp positive cells are Foxd3 positive (47/495). (E) mitfa:gfp positive neural crest cells overlap with Sox10 expression, 91.8% of 250 mitfa:gfp positive cells are Sox10 positive, 20 hpf, dorsal view, anterior left, 10× (inset 20×).

Figure 4. foxd3 expression is absent in melanophores, but present in iridophores and xanthophore precursors

(A,B) foxd3:gfp maintains expression in xanthoblasts, 27 hpf, dorsal view, anterior left. Green: anti-GFP, Red: Pax3/7 (mouse DP312 Ab). 94% of Pax3/7 xanthoblasts (n=165) are foxd3:gfp positive. (A) 20×, scale bar = 40um. (B) 40×, scale bar=7.5 um. (C,D) foxd3:gfp maintains expression in iridophores, 72 hpf, lateral view, dorsal stripe, anterior left. scale bar=25um. 88.3% of differentiated iridophores (n=342) are foxd3:gfp positive. (C) Incident light reveals 4 iridophores. (D) Green: live foxd3:gfp. (E,F) foxd3:gfp expression absent in melanophores, 72 hpf, lateral view, yolk ball, anterior left, embryo treated with PTU. Arrows: partially melanized melanophores, scale bar=90um. 5% of melanophores (n=314) express foxd3:gfp. (E) Brightfield. (F) Green: live foxd3:gfp.

Figure 5. Melb-a cell culture analysis displays Foxd3 repression of endogenous MITF expression

(A-B) Transfected melb-a (mouse melanoblast) cells, (20×). Red: anti-MITF rabbit polyclonal, Green: anti-Myc mouse monoclonal (A) Cells transfected with full-length zebrafish foxd3-myc sequence show nuclear exclusion of endogenous mouse MITF. (B) Cells transfected with DNA-binding mutant version of zebrafish foxd3-myc display co-localized staining with endogenous mouse MITF. (C) Cell counts collected from 5 separate transfection experiments. 56 melb-a cells transfected with full length foxd3 became MITF negative, 2 remained positive. 1 melb-a cell transfected with DNA-binding mutant version of zebrafish foxd3 became MITF negative, 45 transfected cells remained positive.

Figure 6. Foxd3 represses the mitfa promoter in mouse fibroblasts and mouse melanoma cells

(A,B) Luciferase assays assess Foxd3 activity on 931-bp. region of zebrafish mitfa promoter (mitfa:luciferase construct). (A) B16 mouse melanoma cells display high levels of basal mitfa:luciferase activity. Basal mitfa:luciferase activity decreased 19-fold upon co-transfection with full-length zebrafish foxd3. Basal activity not significantly altered upon co-transfection with DNA-binding mutant version of zebrafish foxd3. Negative control: snail1b failed to repress basal mitfa:luciferase activity. (B) NIH-3T3 mouse fibroblast co-transfection with zebrafish sox10 increases mitfa:luciferase activity 5 fold. Transfections with mitfa:luciferase, sox10 and foxd3 results in 7.6 fold decrease from mitfa:luciferase and sox10 activation alone. Transfections with mitfa:luciferase, sox10 and DNA-binding mutant version of foxd3 result in no significant change from mitfa:luciferase and sox10 activation. (C) NIH-3T3 mouse fibroblast luciferase assays reveal partial functionality of proposed Foxd3 binding sites on 931-bp. region of zebrafish mitfa promoter (mitfa:luciferase construct). Proposed Foxd3 binding sites (S1 and S2) have been scrambled in the mitfa:luciferase (S1,S2 mutant) construct. (C) Transfections with mitfa:luciferase, sox10 and foxd3 results in a 13 fold decrease from mitfa:luciferase and sox10 activation alone. Transfections with mitfa:luciferase, sox10 and DNA-binding mutant version of foxd3 result in no significant change from mitfa:luciferase and sox10 activation. Transfections with mitfa:luciferase (S1,S2 mutant), sox10 and foxd3 results in only a 2.5 fold decrease from mitfa:luciferase (S1,S2 mutant) and sox10 activation alone. Transfections with mitfa:luciferase (S1,S2 mutant), sox10 and DNA-binding mutant version of foxd3 result in no significant change from mitfa:luciferase (S1,S2 mutant) and sox10 activation alone. (A-C) Data are means +/- standard deviation.

Figure 7. Foxd3 represses the mitfa promoter in zebrafish embryos

(A-H) One-cell mitfa:gfp transgenic zebrafish embryos microinjected with mRNA and imaged live at 6-7 hpf, shield stage 5×. (A,C,E,G) Brightfield images.(B,D,F,H) Green: live GFP expression from mitfa:gfp. (A,B) Un-injected embryos reveal no fluorescence at shield stage (observed in 64/64 embryos). (C,D) Embryos injected with sox10 mRNA produce robust, precocious mitfa:gfp expression at shield stage (observed in 62/64 embryos). (E,F) Co-injection of full-length foxd3 with sox10 mRNA prevents mitfa:gfp expression at shield stage (observed in 47/52 embryos). (G,H) Embryos co-injected with sox10 and DNA-binding mutant version of foxd3 mRNA display a return to robust, precocious mitfa:gfp expression at shield stage (observed in 43/49 embryos).