colgate/hdac1 Repression of foxd3 expression is required to permit mitfa-dependent melanogenesis

Abstract

Neural crest-derived pigment cell development has been used extensively to study cell fate specification, migration, proliferation, survival and differentiation. Many of the genes and regulatory mechanisms required for pigment cell development are conserved across vertebrates. The zebrafish mutant colgate (col)/histone deacetylase1 (hdac1) has reduced numbers, delayed differentiation and decreased migration of neural crest-derived melanophores and their precursors. In hdac1(col) mutants normal numbers of premigratory neural crest cells are induced. Later, while there is only a slight reduction in the number of neural crest cells in hdac1(col) mutants, there is a severe reduction in the number of mitfa-positive melanoblasts suggesting that hdac1 is required for melanoblast specification. Concomitantly, there is a significant increase in and prolonged expression of foxd3 in neural crest cells in hdac1(col) mutants. We found that partially reducing Foxd3 expression in hdac1(col) mutants rescues mitfa expression and the melanophore defects in hdac1(col) mutants. Furthermore, we demonstrate the ability of Foxd3 to physically interact at the mitfa promoter. Because mitfa is required for melanoblast specification and development, our results suggest that hdac1 is normally required to suppress neural crest foxd3 expression thus de-repressing mitfa resulting in melanogenesis by a subset of neural crest-derived cells.

Figure 1. Melanophore development is defective in hdac1^col mutants

Lateral views of live embryos at 27 hpf (A, B) and 3 dpf (C, D). A, B: At 27 hpf in wild-type embryos melanophores are present posterior to the eye and otic vesicle and are migrating over the flank of the embryo. In hdac1^col mutants, there are fewer melanophores and most of the melanophores are located posterior to the otic vesicle (arrowhead). There are no migrating melanophores in hdac1^col mutants. C, D: By 3 dpf, melanophores are present in four stripes in wild-type; dorsal (d), lateral (l), ventral (v), and yolk (y). In hdac1^col mutants melanophore numbers do not recover. Melanophores present in hdac1^col fail to migrate and are mainly localized to the dorsal stripe and a patch of melanophores posterior to the otic vesicle (arrows, arrowhead). Melanophores that do migrate ventrally in hdac1^col mutants are present in the anterior ventral stripe over the yolk extension. Xanthophores, which give a yellowish hue to embryos, are present in the head and over the flank of wild-type embryos and hdac1^col mutants.

Figure 2. Fewer melanoblasts are specified in hdac1^col embryos

Lateral and dorsal (cranial region) views of 25 hpf embryos that are stained by in situ hybridization to reveal expression of mifta (A, B, G, H), c-kit (C, D, I, J) and dct (E, F). A, B, G, H: There are fewer mitfa-positive melanoblasts specified in hdac1^col mutants as compared to wild-type. C, D: Melanoblast specific c-kit expression in hdac1^col mutants is absent and or reduced (arrowheads), although non-melanoblast expression of c-kit in the post anal region and posterior mesoderm is equivalent to wild-type (arrows). I, J: Similarly, in the cranial region melanoblast specific c-kit expression is reduced or absent, while c-kit expression in the branchial arches is more robust albeit disorganized in hdac1^col mutants as compared to wild-type (arrowheads). E, F: There are fewer dct-positive differentiating melanoblasts in hdac1^col mutants compared to wild-type (arrowheads) and most of the dct-positive melanoblasts are located posterior to the otic vesicle in the dorsal stripe suggesting defects in migration. Also, there is a lack of dct-positive melanoblasts in the anterior head (arrow).

Figure 3. Melanophore development does not recover in hdac1^col mutants

All panels are lateral views of 48 hpf embryos that are stained by in situ hybridization to reveal expression of mifta (A, B), c-kit (C, D), dct (E, F), fms (G, H) and xdh (I, J). A, B: By 48 hpf mitfa expression is switched off in differentiating melanoblasts in wild-type however in hdac1^col mutants, mitfa continues to be expressed robustly in melanoblasts. A, B, E, and F: There are still reduced numbers of mitfa and dct expressing melanoblasts in hdac1^col mutants as compared to wild-type. Additionally, there is still a migration defect as most of the mitfa and dct expressing melanoblasts are located at their site of origin in the post-oitc region (arrowhead) and in the dorsal stripe (arrow). C, D: In contrast to levels of mitfa and dct expression in melanoblasts in hdac1^col mutants, c-kit expression in melanoblasts does not recover to wild-type levels (arrowheads). G–J: Unlike melanophore development, xanthophore number and migration recovers by 48 hpf, where there are numerous migrating fms-positive and xdh-positive xanthoblasts in wild-type and hdac1^col mutants.

Figure 4. Neural crest induction and migration of non melanogenic cells are largely unaffected in hdac1^col mutants

In situ hybridizations for tfap2a (A, E, F), sox9b (B), sox10 (C, G, H, O, P), sna1b (D), foxd3 (I, J) and ctn (K, L, M, N) between 3 somites (s) and 24 hpf. A–D: Dorsal views of representative gene expression in 3 somite embryos. Neural crest induction is normal and there is no difference in the expression of tfap2a, sox9b, sox10 and sna1b between wild-type and hdac1^col mutants. E–L: lateral views of 15 somite embryos. At the 15 s stage there is no difference in the expression and number of cranial and trunk neural crest cells (arrowheads) expressing tfap2a (E, F), sox10 (G, H), foxd3 (I, J), and ctn (K, L) between wild-type and hdac1^col mutants. M–P: lateral views of 24 hpf embryos. Later by 24 hpf there is a slight reduction in the number of trunk neural crest cells expressing ctn and sox10 in hdac1^col mutants compared to wild-type. However, the overall migration of trunk neural crest cells in hdac1^col mutants is largely unaffected though slightly delayed (arrows, arrowheads).

Figure 5. foxd3expression is prolonged in the premigratory neural crest and increased numbers of cranial satellite glia are present inhdac1^colmutants

In situ hybridizations of sox10 (A, B) and foxd3 (C–J) between 24 hpf and 52 hpf. A, B: lateral views of 52 hpf embryos. At 52 hpf sox10 continues to be expressed robustly in the dorsal stripe in hdac1^col mutants, while expression in wild-type is absent to faint (arrowheads). C, D: lateral views of 24 hpf embryos. There is a prolonged expression of foxd3 in the premigratory neural crest cells at 24 hpf in hdac1^col mutants compared to wild-type (D, arrowheads). In wild-type, foxd3-positive neural crest cells are mostly present at the tip of the tail (C, arrowhead). In addition to increased numbers of neural crest cells expressing foxd3, foxd3 expression is also increased in the somites in hdac1^col mutants when compared to wild-type. E, F: Flat mounts of the cranial region of 24 hpf embryos indicates that there is an increase in the number of cranial satellite glia in the pre-otic and post-otic placodes (arrowheads) in hdac1^col mutants as compared to wild-type. In contrast, pineal gland foxd3 expression (arrows) in hdac1^col mutants is slightly reduced compared to wild-type. G, H: lateral views of the tail region at 36 hpf. While foxd3 is not expressed in the premigratory neural crest cells in wild-type, it is still expressed in this population in hdac1^col mutants (arrowhead). I, J: lateral views of the head at 48 hpf. The number of foxd3-positive cranial satellite glia associated with the cranial ganglia is much larger than those present in wild-type embryos (black arrow and white arrowheads).

Figure 6. Repression of Foxd3 rescues melanogenesis in hdac1^col mutants

Images of live embryos at 3 dpf, A–D lateral views, E–H dorsal views. A, B: There are fewer melanophores present in hdac1^col mutants at 3 dpf compared to wild-type embryos. Melanophores present in hdac1^col mutants fail to migrate and are present at the site of origin in the post otic region, in the dorsal (d) stripe and in the anterior ventral stripe. In wild-type embryos, melanophores migrate to form four stripes; dorsal (d), lateral (l), ventral (v) and yolk (y). C, D: Partial knock down of Foxd3 in hdac1^col mutants increases melanophore numbers and the melanophores migrate over the head (arrowhead), into the ventral stripe and over the yolk. E, F: In control uninjected hdac1^col mutants the dorsal stripe (d) is usually 3–5 melanophores wide (arrowhead), in comparison the wild-type dorsal stripe is 2–3 melanophores wide (arrowhead). G, H: Partially reducing Foxd3 with a translation blocking morpholino (mo) in hdac1^col mutants increases the migration of melanophores into the ventral stripe and over the yolk, the resulting a dorsal stripe in hdac1^col/foxd3 mo mutant/morphants is only 2–3 melanophores wide (arrowhead).

Figure 7. Genetically reducing foxd3 rescues melanophore development in hdac1^{col −/−}; foxd3^zdf10+/−mutants

A–E: Live images with lateral views of 3.5 dpf embryos. Embryos obtained from a heterozygous incross of hdac1^{col +/−}; foxd3^zdf10+/− carriers gave rise to 4 distinct phenotypes. A: wild-type that have melanophores present in 4 distinct stripes; dorsal, lateral, ventral and yolk. B: foxd3^zdf10 mutants in which the melanophore migration patterns has largely recovered to resemble the wild-type. C: hdac1^{col −/−}; foxd3^zdf10+/+ and hdac1^{col −/−}; foxd3^zdf10+/− mutants whose melanophore phenotype resembles single hdac1^{col −/−} homozygous mutant phenotype. D: hdac1^{col −/−}; foxd3^zdf10−/− double mutants in which there is a reduction in melanophore numbers as well as reduced migration into the lateral, ventral and yolk stripe locations. E: 10% of phenotypically hdac1^col mutants that are hdac1^{col −/−}; foxd3^zdf10+/− display a rescue of melanophore number as well as migration into the lateral, ventral and yolk stripes.

Figure 8. Foxd3 negatively regulates mitfa

A–D: lateral views of mitfa in situ hybridizations of 32 hpf embryos. B, D: there is an increase in the number of mitfa-positive melanoblasts in hdac1^col/foxd3 mutant/morphant embryos as compared to uninjected hdac1^col mutants. A, C: in wild-type/foxd3 morphant embryos mitfa expression in melanoblasts is more robust than uninjected controls. E–H: lateral views of c-kit in situ hybridizations in 32 hpf embryos. F, H: there is no rescue of c-kit expression in melanoblasts in hdac1^col/foxd3 mutant morphants when compared to uninjected hdac1^col mutants at 32 hpf. E, G: Qualitatively, there are fewer c-kit positive melanoblasts in foxd3 morpholino treated wild-type embryos as compared to uninjected wild-type embryos. E–H: In contrast to melanoblast expression, there is no difference in c-kit expression in the post anal region between wild-type, hdac1^col mutants, wt/foxd3 wild-type/morphants, and hdac1^col/foxd3 mutant/morphant embryos (arrows).

Figure 9. Foxd3 can physically interact with predicted forkhead binding sites in the mitfa promoter

Electrophoretic mobility shift assay (EMSA) using synthetic Foxd3 protein, radiolabelled conserved region probes spanning the predicted forkhead binding sites (Site 1 and Site 2), and unlabelled wild type or mutated site 1 or site 2 or unrelated (OCTA) oligonucleotide competitors. Arrows denote specific bands observed with Foxd3 protein. wt – wild-type site1 or site 2 competitors; Mut: Mutated site 1 or site 2 competitors; NS – nonspecific (OCTA) competitor; FP – free probe (arrowhead).

Figure 10. A model for the regulation of the initiation of melanogenesis

A. Expression of foxd3 prohibits the expression of mitfa and the initiation of melanogenesis by premigratory neural crest cells. Foxd3 is depicted as acting at the mitfa promoter, which can occur in vitro, but it is not known if this is the case in vivo. B. Prior to migration Hdac1 is required, directly or indirectly, to repress neural crest foxd3 expression. Repression of foxd3 expression permits transcriptional activation of mitfa by Sox10, Lef1 and other (O) regulators, resulting in the initiation of melanogenesis by a subpopulation of neural crest cells.