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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Dev Biol. Author manuscript; available in PMC Jun 23, 2009.
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PMCID: PMC2700343
NIHMSID: NIHMS38749

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 hdac1col 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 hdac1col 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 hdac1col mutants. We found that partially reducing Foxd3 expression in hdac1col mutants rescues mitfa expression and the melanophore defects in hdac1col mutants. Further, 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.

Keywords: neural crest, melanophore, histone deacetylase1, foxd3, mitfa, c-kit, zebrafish

Introduction

The neural crest is an embryonic cell population that gives rise to pigment cells, craniofacial cartilage and neurons and glia of the peripheral nervous system among other cell types (LeDouarin and Kalcheim, 1999). Neural crest cell-derived pigment cell development has been studied extensively, aided by a large number of pigment mutant loci in mice as well as zebrafish. There are more than 800 coat color alleles identified in mice, corresponding to 127 genetic loci (Bennett and Lamoreux, 2003). Similarly, almost 100 pigmentation mutants have been identified in multiple genetic screens in zebrafish (Henion et al., 1996; Kelsh et al., 1996; Odenthal et al., 1996). In birds and mammals, melanocytes are the only neural crest-derived pigment cells present. In contrast, there are three types of pigment cells or chromatophores present in zebrafish. They are the black, melanin-containing melanophores, the yellow, pteridine-containing xanthophores and iridescent, purine-containing iridiphores. Henceforth, we will use the term melanophore to describe zebrafish, avian and mammalian melanocytes.

Identification of many of the genes involved in pigment cell development has revealed that, by and large, regulation of melanophore development is conserved among vertebrates. One such conserved gene is microphthalmia-associated transcription factor (mitf) which is a critical regulator of melanophore development. In humans, defects in MITF are associated with type 2a Waardenberg syndrome, which is characterized by hypopigmentation and deafness (Steingrimsson et al., 2004; Widlund and Fisher, 2003). Additionally, in humans, MITF is also found to be amplified in a fraction of malignant melanomas and can function as an oncogene (Garraway et al., 2005; Widlund and Fisher, 2003). Similar to defects in humans, the zebrafish mitfa mutant, nacre, completely lacks melanophores (Lister et al., 1999). The requirement for mitf in melanophore development is further underscored by experiments in which overexpression of mitfa in zebrafish produces ectopic pigmented cells and misexpression of Mitf in NIH/3T3 fibroblasts resulted in their conversion into a melanophore-like cell fate (Lister et al., 1999; Tachibana et al., 1996). In mice, mutations in Mitf reveal that in addition to a loss of melanophores, there are also variable defects in the eyes, osteoblasts and mast cell development depending on the severity of the mutations (reviewed by Steingrimsson et al., 2004; Widlund and Fisher, 2003). In zebrafish, there are two mitf co-orthologues, mitfa and mitfb, with mitfa being required for melanophore development (Lister et al., 1999; Lister et al., 2001). mitf regulates the expression of multiple genes within the melanophore lineage including tyrosinase, tryp1, dct, c-kit, and bcl2 (reviewed in Steingrimsson et al., 2004).

Given the central function of mitf in melanophore development, several genes and gene pathways have been shown to affect melanophore development via mitf at the transcriptional and post transcriptional level. Transcription factors and pathways that positively regulate mitf at the promoter level include CREB, sox10, pax3 and lef1/wnt/β-catenin signaling (Bertolotto et al., 1998; Bondurand et al., 2000; Dorsky et al., 2000a; Elworthy et al., 2003; Lee et al., 2000; Potterf et al., 2000; Price et al., 1998b; Saito et al., 2002; Takeda et al., 2000; Verastegui et al., 2000). Consistent with requirements of pax3 and sox10 for mitf expression, human mutations in PAX3 and SOX10 cause subtypes of Waardenburg syndromes, which overlap with hypopigmentation associated with Type 2a Waardenburg syndrome caused by defects in MITF (Pingault et al., 1998; Read and Newton, 1997; Steingrimsson et al., 2004). At the post translation level, the protoonco-receptor tyrosine kinase c-kit, via phosphorylation, affects Mitf protein activity and stability (Price et al., 1998a; Wu et al., 2000; Xu et al., 2000).

The transcription factors sox10, pax3, and lef1 positively regulate mitfa transcription yet are also expressed and required by the precursors of other neural crest derivatives. Thus, it is not presently known how sox10, pax3 and lef1 specify melanophores within the neural crest cell population. It is likely that there are additional levels of regulation of mitfa and/or other transcription factors in specifying melanogenic cell fate. A potential candidate for the regulation of melanophore specification is the Winged Helix transcription factor foxd3. foxd3 expression is induced in cells at the neural plate border and is extinguished prior to the initial expression of melanogenic sublineage-specific genes such as mitf (Kos et al., 2001; Odenthal and Nusslein-Volhard, 1998). Overexpression of foxd3 in avian embryos represses melanogenesis whereas morpholino mediated knockdown in avian neural crest cultures promotes melanogenesis (Kos et al., 2001). However, the mechanism by which foxd3 functions in melanophore development has not yet been established. Seemingly contrary to predictions from avians, in zebrafish foxd3 mutants and morphants, melanophore development is largely normal (Lister et al., 2006; Montero-Balaguer et al., 2006; Stewart et al., 2006). This apparent inconsistency is not presently understood.

In our study, we take advantage of the co1b382 mutation, that we have shown encodes histone deacetylase 1 (hdac1; Nambiar et al., 2007), in which there is a severe disruption of the specification, migration and differentiation of melanophores. We show that in hdac1col mutants there is a misregulation of foxd3 expression such that foxd3 expression is extended for a prolonged period of time in neural crest cells compared to wild-type embryos. The melanophore phenotype in hdac1col mutants, including mitfa expression, migration and overt differentiation, can be selectively rescued by a partial repression of foxd3 expression. Further, we demonstrate by EMSA assays binding of in-vitro translated Foxd3 protein to two putative Foxd3 binding sites of the mitfa promoter within a region capable of driving melanophore-specific expression (Dorsky et al., 2000) suggesting the possibility that foxd3 can represses melanophore specification and melanogenesis by directly inhibiting mitfa expression. Our results indicate that hdac1 is normally required, directly or indirectly, to repress foxd3 expression, and foxd3 repression is required to permit the induction of mitfa and subsequent mitfa-dependent melanophore development.

Materials and methods

Animal husbandry

Adult zebrafish and embryos were raised and maintained at 28.5°C in the Ohio State University zebrafish facility. hdac1col −/− mutant embryos were obtained by pair wise matings of heterozygous adult zebrafish that were maintained in AB and WIK backgrounds. Embryos were staged according to Kimmel et al. (Kimmel et al., 1995). In order to determine the genotype of embryos before a visible phenotype was clearly apparent, genomic DNA from individual embryos was obtained and PCR was performed using chromosome 19-linked polymorphic SSLP markers that have been shown to be closely linked to the hdac1col locus (Knapik et al., 1996; Nambiar and Henion, 2004; Nambiar et al., 2007). The hdac1col +/;foxd3zdf10+/ double mutant line was created by crossing foxd3zdf10+/ (Stewart et al., 2006) heterozygous carriers, formerly known as foxd3sym1, to hdac1col +/ (b382) heterozygous carriers and double mutant embryos genotyped using the markers above and SSLP markers for the foxd3zdf10 locus (Stewart et al., 2006).

In situ hybridization

In situ hybridization was carried out on staged embryos as described previously (Thisse et al., 1993) with minor modifications. Embryos over 24 hpf were raised in 0.03 g/l 1-phenyl-2-thiourea (PTU) to prevent melanin synthesis. Probes used were tfap2a (Knight et al., 2003), c-kit (Parichy et al., 1999), crestin (Luo et al., 2001; Rubinstein et al., 2000), dct (Kelsh et al., 2000), fms and xdh (Parichy et al., 2000), foxd3 (Kelsh et al., 2000a; Odenthal and Nusslein-Volhard, 1998), snai1b (Thisse et al., 1995), sox9b (Li et al., 2002), sox10 (Dutton et al., 2001) and th (An et al., 2002).

Melanophore cell counts, statistical and image analysis

Melanophore cell counts were performed between somites 5 and 15 in the dorsal, ventral and lateral stripes under a dissecting microscope. In order to facilitate counts, embryos were first treated with epinephrine for 5 minutes to contract the melanosomes (Johnson et al., 1995; Parichy et al., 1999; Rawls and Johnson, 2000). Melanosome contraction in hdac1col mutants took between 15 and 20 min. Embryos were then fixed in 4% paraformaldehyde in PBS. Cell counts were carried out at 60 hours post fertilization (hpf), 5 days post fertilization (dpf), and 8 dpf in wild-type and mutant embryos. For each time point, between 7 and 11 embryos were counted and data analyzed using 2-way ANOVA and Bonferroni post hoc tests. Graph prism pad version 4.0 software was used to conduct statistical analysis and graph data. Embryos were examined on a Leica MZ16 stereomicroscope and photographed with a SPOT-Insight digital camera and SPOT v4.0-software, and figures were assembled using Photoshop CS2. In some figure panels, to analyze complete embryos, images of embryos that were captured in the same positions but at slightly different focal planes were fused together using Photoshop CS2.

Morpholino injections

The foxd3 morpholino is a translation blocking morpholino (Stewart et al., 2006). The sequence of the morpholino is as follows: 5′ CAC CGC GCA CTT TGC TGC TGG AGC A 3′ (Gene tools Inc.). The concentration of morpholino used in experiments was 0.25 mM. Complete phenocopy of foxd3zdf10 was achieved by injecting 20 ng of morpholino per embryo (Stewart et al., 2006). In our experiments 0.477–1.1 ng of morpholino was injected per embryo which partially knocks down Foxd3 (Supplementary figure 1). Uninjected embryos from heterozygous carrier crosses were used as controls.

EMSA assays

Electrophoretic mobility shift assays (EMSAs) were performed using in vitro translated proteins as described previously (Voronina et al., 2004). Foxd3 protein was synthesized from pCS2/Foxd3 in vitro (Lister et al., 2006) (Quick TNT Linked In Vitro Transcription/Translation Kit; Promega) with the addition of [35S]-methionine (Amersham). Proper translation was verified by SDS-PAGE and autoradiography. EMSAs were performed using annealed double-stranded oligonucleotide probes representing the predicted forkhead binding sites from the mitfa promoter (GenBank accession # AF211890), Site 1 (nucleotides 579 – 595; Site 1 wt F: 5′-ATG CTG AGA ACA AAC AAT GTT TTA TGC AG) and Site 2 (nucleotides 750 – 766; Site 2 wt F: 5′-CGT TTG GGT AAA AAA AAC AAT ATG AGA AGA). For competitions, a 100-fold molar excess of unlabelled double-stranded oligonucleotide was included. Competitors used represented the wild type or mutated Site 1 or Site 2 or the unrelated oct1 binding site, OCTA (Hinkley and Perry, 1991), Site 1 mut F: 5′-ATG CTG ATg gCA ggg gAT GTT TTA TGC AG Site 2 mut F: 5′-CGT TTG GGT Agg gAA gga gAT ATG AGA AGA (changed bases are in lower case).

Results

The zebrafish mutant colgate (co1b382) is an ENU-induced, recessive larval lethal mutation (Henion et al., 1996; Nambiar and Henion, 2004). Mutants die between 7 and 9 dpf. The co1b382 mutant was originally isolated in a screen for mutants with defects in neural crest development and was identified based on an abnormal melanophore pigment pattern (Henion et al., 1996). The co1b382 locus was recently identified as histone deacetylase1 (hdac1; Nambiar et al., 2007).

Melanophore development is abnormal in hdac1col mutants

In wild-type embryos at 27 hpf, differentiated (melanin granule-containing) melanophores are found just posterior to the eye and posterior to the otic vesicle (Figure 1A). By 2 dpf, these melanophores have migrated dorsally and anteriorly over the head away from their site of origin (Knight et al., 2003; Schilling and Kimmel, 1994). In the trunk of wild-type embryos at 3 dpf (Figure 1C), melanophores are organized into distinct dorsal, lateral, ventral, and yolk stripes (Kelsh et al., 1996; Schilling and Kimmel, 1994). In hdac1col mutants at 27 hpf, the total number of melanophores is reduced (Figure 1B) and those present are located just posterior to the otic vesicle. Additionally, the hdac1col mutant melanophores are hypo-pigmented compared to wild-type. Later, in hdac1col mutants, at 2 dpf and 3 dpf, the melanophores that arise in the post otic region fail to migrate and instead form a distinctive patch of cells (data not shown, see Figure 1D). At 3 dpf, most of the melanophores in the trunk fail to migrate and instead are located in the dorsal stripe. hdac1col mutants, compared to wild-type embryos, have fewer melanophores in the ventral stripe and only occasional melanophores are present in the lateral stripe. Additionally, only 0–6 melanophores migrate into the ventral stripe in the tail region in hdac1col mutants as compared to wild-type embryos, where there are numerous melanophores present. The yolk sac stripe is absent in hdac1col mutants and any individual melanophores present on the yolk are located anteriorly, close to the heart and near the post otic patch of melanophores. To quantify the melanophore phenotype, melanophore cell counts were performed in wild-type and hdac1col mutant embryos between somites 5–15 at 60 hpf, 5 dpf and 8 dpf (Table 1, A). Cell counts are a sum of the dorsal, lateral and ventral stripe melanophores. At 60 hpf, hdac1col embryos have only 59% of the number of melanophores present in wild-type embryos. The deficit in melanophore number in hdac1col mutants recovers slightly to 63% of wild-type melanophores by 5 dpf. At 8 dpf, while wild-type melanophore numbers are the same as at 5 dpf, there is a decrease in melanophore numbers to 53% of wild-type in hdac1col mutants. This indicates that between 5 and 8 dpf, some existing melanophores in hdac1col mutants die or are demelanized.

Figure 1
Melanophore development is defective in hdac1col mutants
Table 1
Reduced melanophore number and migration in hdac1col mutants compared to wild-type.

The migration of melanophores was calculated as a percentage of melanophores present in the ventral stripe to the total number of melanophores present in the dorsal, lateral and ventral stripes. In wild-type embryos at 60 hpf to 8 dpf between 41–46% of the total melanophores are present in the ventral stripe. In contrast, in hdac1col mutants, only 19–23% (Table1, B) of the total melanophores are present in the ventral stripe. The melanophore defects do not recover in hdac1col mutants. In contrast to melanophore development, xanthophore numbers and migration patterns, although initially delayed, are equivalent to wild-type embryos by 3 dpf in hdac1col mutants (Figure 1C, 1D).

hdac1col is required for melanophore specification, migration and differentiation

The reduced number of melanophores and their abnormal migration in hdac1col mutant embryos led us to examine whether there are differences in the expression of genes required for melanoblast specification, differentiation, migration and survival between wild-type and hdac1col mutants. mitfa is a critical regulator of melanophore development that is required to specify melanoblasts beginning at the 17–18 somite stage (Lister et al., 1999). In hdac1col mutants, mitfa expression is delayed by approximately 1 hour (data not shown) and later, at 20 somites (data not shown) and qualitatively at 25 hpf (Figure 2A, 2B, 2G, 2H), there are fewer mitfa-positive melanoblasts specified compared to wild-type embryos. Further, in hdac1col mutants, the expression of mitfa in the trunk does not extend as far caudal as in wild-type embryos and there are fewer melanoblasts migrating ventrally as compared to wild-type embryos. At 30 hpf (Supplementary figure 2A, 2B), there are still fewer mitfa-positive melanoblasts in hdac1col mutants compared to wild-type and most of these cells are located in the dorsal stripe and in the post-otic region, suggesting a general disruption of melanoblast migration. At 48 hpf (Figure 3A, 3B), mitfa is downregulated in most differentiating melanoblasts in wild-type embryos. In contrast, mitfa continues to be expressed in the reduced number of melanoblasts present in hdac1col mutants, suggesting a failure in the downregulation of mitfa. Additionally, mitfa-positive melanoblasts in hdac1col mutants have still not migrated at this stage and are predominantly located in the post-otic region and the dorsal stripe.

Figure 2
Fewer melanoblasts are specified in hdac1col embryos
Figure 3
Melanophore development does not recover in hdac1col mutants

We also examined the expression of c-kit, a receptor tyrosine kinase required for migration and survival of melanoblasts (Parichy et al., 1999) in wild-type and hdac1col mutants. By 25 hpf, the majority of the mitfa-positive melanoblasts also express c-kit. c-kit is expressed in migrating melanoblasts in the head and trunk regions. In addition to melanoblasts, c-kit is also expressed in the posterior notochord, near the anus, in the retina and in the branchial arches in wild-type embryos (Parichy et al., 1999). In hdac1col mutants, very few c-kit-expressing melanoblasts are present at 25 hpf (Figure 2C, 2D, 2I, 2J), 30 hpf (Supplementary figure 2C, 2D) and 36 hpf (data not shown). In addition, c-kit expression in the few c-kit-positive melanoblasts in hdac1col is also reduced when compared to the robust expression in wild-type melanoblasts. Later, by 48 hpf, the numbers of c-kit-positive melanoblasts and expression within melanoblasts does not recover to wild-type levels in hdac1col mutants (Figure 3C, 3D). In contrast to expression in melanoblasts in hdac1col mutants, c-kit expression in the branchial arches and near the anus is normal and equivalent to wild-type embryos at equivalent stages. This suggests that the reduction/absence of c-kit expression in hdac1col mutants is specific to melanoblasts.

Dopachrome tautamerase (dct) is an enzyme required for melanin synthesis and hence an indicator of melanoblast differentiation (Kelsh et al., 2000). In wild-type embryos dct is robustly expressed in differentiating melanoblasts in the head and in the trunk including migrating melanoblasts beginning at 25 hpf. In contrast, in hdac1col mutants the numbers of dct-positive melanoblasts are reduced in number and the melanoblasts are restricted in distribution to the regions posterior to the otic vesicle and in the dorsal stripe in the trunk at 25 hpf in hdac1col mutants (Figure 2E, 2F). dct expression levels in hdac1col mutant melanoblasts also appears to be reduced as compared to robust expression in wild-type melanoblasts at 25 hpf. Later at 30 hpf (Supplementary figure 2E, 2F), 36 hpf (data not shown) and 48 hpf (Figure 3E, 3F) most of the dct-positive differentiating melanoblasts are located in the dorsal stripe in the trunk and in a patch of cells just posterior to the otic vesicle, indicating a failure in migration. Finally, reduced numbers of dct-positive melanoblasts arise anterior to the otic vesicles at all stages observed in hdac1col mutants. Taken together, the mitfa, c-kit and dct expression data indicate that early melanophore specification is delayed by an hour in hdac1col mutants as compared to wild-type and there are fewer melanoblasts specified and those that are specified have defects in migration and differentiation.

In contrast to delayed differentiation and migration of melanophores and their precursors, in hdac1col mutants there is no delay in the expression of xanthine dehydrogenase (xdh) (Supplementary figure 2I, 2J, Figure 3I, 3J), an enzyme that is expressed in differentiating xanthoblasts (Parichy et al., 2000). Also, fms, a receptor tyrosine kinase orthrologue of c-kit, required for xanthoblast survival and migration is expressed robustly in hdac1col mutants and is equivalent to wild-type embryos at all stages analyzed (Supplementary figure 2G, 2H, Figure 3G, 3H) (Parichy et al., 2000). Thus, normal hdac1 function is selectively required for melanogenesis and is not required for xanthophore differentiation and migration.

Neural crest cell induction and the migration of non-melanogenic neural crest cells are unaffected in hdac1col mutants

Since there is a reduction of melanophores and their precursors during development, we decided to analyze the expression of genes expressed by premigratory and migratory neural crest cells to discern if there is a similar reduction in neural crest cells in hdac1col mutants. At the 3 and 6-somite stages, there is no difference in the neural crest cell expression of tfap2a, sox9b, sox10 and snai1b between wild-type and hdac1col mutant embryos (Figure 4A–D, data not shown). Later, at the 14–15- somite stage there is no difference in the expression of tfap2a, sox10, foxd3 and ctn between hdac1col mutants and wild-type embryos (Figure 4E– L). The early expression of tfap2a, sox10, snail1b, sox9b, foxd3 and ctn indicated to us that equivalent numbers of neural crest cells are induced in hdac1col mutants and wild-type embryos. By 24 hpf, there is a slight decrease in the number of cells expressing ctn and sox10 in the migrating trunk neural crest in hdac1col mutants (Figure 4M–P). There is also a slight delay in neural crest cell migration in the trunk in hdac1col mutants. However, relative to the reduction in mitfa in hdac1col mutants there are many more ctn and sox10 positive neural crest cells present. In cranial neural crest cells, there is no difference in the expression of sox10 and ctn between wild-type and hdac1col mutants at 24 hpf, except in the region of the branchial arches where there are fewer ctn positive cells migrating out of the post otic region (data not shown). Overall, however, general neural crest migration in the trunk is much more robust than the migration of melanoblasts suggesting that non-melanoblast neural crest cells successfully migrate, albeit after a brief delay.

Figure 4
Neural crest induction and migration of non melanogenic cells are largely unaffected in hdac1col mutants

Prolonged delay in the down regulation of foxd3 in hdac1col mutants

Defects in melanophore development are first detectable at the level of mitfa expression in hdac1col mutants. Therefore, we looked at the expression of the transcription factors tfap2a, foxd3, sna1b, sox9a/b and sox10 which are generally required earlier than mitfa in neural crest cell development. After 24 hpf, sox10 expression is downregulated in differentiating neural crest cell derivatives in wild-type embryos. However, in hdac1col mutants there is a delay in extinguishing sox10 expression and sox10 continues to be expressed robustly in neural crest cells as late as 36 hpf and 52 hpf (unpublished data, Figure 5A, 5B). In addition to sox10, foxd3 is the only other transcription factor in which there is a delay in downregulation in hdac1col mutants compared to wild-type expression (Figure 5C–5J). Beginning at approximately 16 hpf, foxd3 expression is extinguished in the premigratory neural crest in wild-type embryos in a rostral to caudal fashion. By 24 hpf, foxd3 is only expressed in cranial satellite glia and in the premigratory neural crest at the tip of the tail (Figure 5C). At 30 hpf, foxd3 is no longer expressed in the neural crest, but continues to be expressed in cranial satellite glia (Kelsh et al., 2000). In contrast, at 24 hpf in hdac1col mutants, foxd3 continues to be expressed in trunk neural crest cells, in addition to premigratory neural crest cells in the tail region (Figure 5D). In the cranial region, there is an increase in foxd3-positive cells in hdac1col mutants compared to wild-type embryos (Figure 5E, 5F). The location of the foxd3-positive cranial neural crest cells in the area of the cranial satellite glia suggests that there are more cranial satellite glial cells in hdac1col mutants as compared to wild-type embryos. However, as the domain of foxd3 expression is expanded in hdac1col mutants relative to wild-type embryos, it is possible that some of the foxd3 positive cells could also be undifferentiated neural crest cells in addition to cranial satellite glia. At 30 hpf (data not shown) and 36 hpf (Figure 5G, 5H), foxd3 continues to be expressed in the premigratory neural crest cells in the tail, while in wild-type embryos, foxd3 is no longer expressed. Finally, at 48 hpf, foxd3 is no longer expressed in the premigratory neural crest cells in the tail and the number of foxd3 positive presumptive cranial satellite glia in hdac1col embryos is greater than in wild-type (data not shown, Figure 5I, 5J). Thus, overall, there is a significant delay in the downregulation of foxd3 expression in neural crest cells in hdac1col mutants.

Figure 5
foxd3expression is prolonged in the premigratory neural crest and increased numbers of cranial satellite glia are present inhdac1colmutants

Reduction of foxd3 expression in hdac1col mutants rescues melanophore numbers and migration

The prolonged expression of foxd3 in trunk neural crest cells and reduced expression of mitfa, raised the possibility that continued expression of foxd3, which can function as a transcriptional repressor (Pohl and Knochel, 2001), could result in the repression of melanogenesis in hdac1col mutants (Kos et al., 2001). To test this hypothesis, we designed two strategies to reduce foxd3 expression in hdac1col −/− mutants. First, we knocked down FoxD3 protein using a translation blocking morpholino (Stewart et al., 2006). The second approach was to generate hdac1col −/−; foxd3zdf10−/− double mutants.

20 ng of the foxd3 morpholino per embryo used in this study can phenocopy the complete loss of sympathetic neurons in foxd3zdf10mutants, the foxd3 mutant allele formerly known as foxd3sym1 (Stewart et al., 2006). In our study, a 20-fold dilution of between 0.56–1.1 ng of foxd3 morpholino per embryo was used to partially knockdown Foxd3. The injection of 0.56–1.1 ng of morpholino per wild-type embryo results in the partial reduction in the number of tyrosine hydroxylase-positive sympathetic neurons at 56 hpf (Supplementary figure 1A–D). Partial knockdown of Foxd3 resulted in an initial delay in melanophore differentiation, as is the case in both hdac1col mutants and foxd3zdf10 mutants. In contrast, by 2 dpf (data not shown) and later at 3 dpf (Figure 6A–D), there is an increase in melanophore number and many more melanophores are migrating over the anterior head, over the yolk and in the tail of hdac1col/foxd3 mutant/morphant embryos. In hdac1col embryos most of the melanophores in the trunk fail to migrate and are located in the dorsal stripe. As a result the dorsal stripe is at least 3–5 melanophores wide in hdac1col compared to the 2–3 melanophore wide dorsal stripe in wild-type (Figure 6E, 6F). Partial knock down of Foxd3 via morhpolino injection, resulted in the migration of melanophores from the dorsal stripe of the hdac1col/foxd3 mutant/morphant embryos into the ventral stripe and over the yolk sac. The resulting dorsal stripe in hdac1col/foxd3 mutant/morphants is reduced to the wild-type 2–3 melanophore width (Figure 6G, 6H).

Figure 6
Repression of Foxd3 rescues melanogenesis in hdac1col mutants

To score for the rescue of melanophore number and migration, the following 3 minimum criteria were established based on the hdac1col mutant phenotype. First, there must be migration of melanophores into the anterior head, since very few melanophores are found migrating over the head in hdac1col mutants (Figure 6B, 6D, arrowhead). Second, in the trunk, there must be an increase in the migration of melanophores over the yolk sac ( 15 melanophores), compared to mutants in which melanophores are only rarely present over the posterior yolk sac and in the yolk extension region (Figure 6B, 6D). Finally, in the ventral stripe of the tail, there are usually between 0 and 6 melanophores present in hdac1col embryos (Figure 6B, 6D). Therefore, a rescued mutant must have more than 6 melanophores present. Based on these criteria, 84% of the hdac1col embryos injected with foxd3 morpholino showed rescue of the melanophore phenotype (n= 72, Table 2 a). We quantified the rescue of melanophore number and migration by performing melanophore cell counts at 60 hpf in somites 5 through 15 of the trunk in wild-type/foxd3 morphant and hdac1col/foxd3 mutant/morphant embryos (Table 3). Melanophore counts are a total of pigmented melanophores present in the dorsal, ventral and lateral stripes. There is a statistically significant increase in melanophore numbers from 68±10.7 to 86±11.1 in hdac1col/foxd3 mutant/morphants as compared to uninjected hdac1col −/− mutants (P< 0.05). Additionally, wild-type and hdac1col −/− embryos injected with foxd3 morpholino have statistically equivalent number of melanophores in the trunk showing a rescue of the melanophore phenotype at 60 hpf (92±17.1 vs. 86.3±11.13 melanophores, P>0.05). However, wild-type/foxd3 morphants have fewer melanophores as compared to uninjected wild-type embryos (92±17.1 vs. 109± 8.7 melanophores, P <0.05) at 60 hpf. This is consistent with the delay in melanophore development in foxd3zdf10 mutants (Stewart et al. 2006). In addition to the rescue of melanophore numbers, there is a rescue in the migration of melanophores into the ventral stripe in hdac1col/foxd3 mutant/morphant embryos as compared to uninjected hdac1col −/− embryos (Table 3). The percentage of melanophores that migrate into the ventral stripe is statistically the same between wild-type uninjected, wild-type/foxd3 morphants and hdac1col/foxd3 mutant/morphant embryos at 60 hpf.

Table 2
Rescue of melanophore number and migration in hdac1col mutants at 3 dpf by reducing foxd3 levels genetically, by morpholino mediated translational interference, or both.
Table 3
Rescue of melanophore number and migration at 60 hpf in hdac1col mutants by low dose of foxd3 morpholino injection.

We then reduced foxd3 expression genetically by mating hdac1col +/; foxd3zdf10+/ double mutant heterozygous carriers. There are 4 distinct phenotypes observed: wild-type, foxd3zdf10 mutants, hdac1col mutants and hdac1col; foxd3zdf10 double mutants (Figure 7A–D). In a heterozygous cross between double mutant hdac1col+/; foxd3zdf10+/ heterozygous carriers, the following ratio of phenotypes was obtained wild-type: hdac1col: foxd3zdf10: hdac1col;foxd3zdf10::152:48:52:17 confirming a Mendelian 2 factor ratio of 9:3:3:1. Loss of hdac1col and foxd3 function in hdac1col −/−; foxd3zdf10 −/− double mutants, resulted in the reduction of the total number of melanophores at all stages observed, compared to foxd3zdf10 −/− or hdac1col −/− single mutants or wild-type embryos. This indicates a genetic interaction between hdac1col and foxd3zdf10 in melanophore development which is only revealed when both genes are simultaneously knocked out. In separate genetic incrosses of hdac1col +/; foxd3zdf10+/ double heterozygous carriers, we observed that of the mutant embryos which are phenotypically hdac1col, 10 % (n= 80) have a rescue of melanophore number and migration (Figure 7E). Based on the hypothesis that foxd3 represses melanogenesis we would predict that the rescued hdac1col mutants should also be heterozygous for foxd3. Genotyping the rescued hdac1col mutants for foxd3 allele status with closely linked polymorphic SSLP marker z5294 revealed that all rescued hdac1col mutants were also foxd3zdf10+/ (Table 2b). Additionally, although not all completely satisfying the rescue criteria, 42.5 % (n= 80) of hdac1col mutant embryos derived form a double heterozygous cross (hdac1col +/; foxd3zdf10+/) have more melanophores present, qualitatively and a greater migration of these melanophores over the head and into the lateral and ventral stripes, especially in the tail region as compared to more severe melanophore number and migration defects in hdac1col −/− mutants obtained from a hdac1col +/ only heterozygous incross. This is significant as we would expect 50% of hdac1col mutants from a hdac1col+/; foxd3zdf10+/ double heterozygous incross to also be heterozygous for foxd3zdf10. Partial morpholoino-mediated knock down of foxd3 function in hdac1col mutants produced a more efficient (>80% vs. 10%) rescue of melanophore number and migration as compared to hdac1col +/; foxd3zdf10+/ double mutant analysis. It is possible that the level of Foxd3 present within the neural crest cells is important for its function to repress melanogenesis and that in many of the hdac1col −/−; foxd3zdf10+/− mutants Foxd3 levels are sufficiently high to prevent complete rescue of the melanophore phenotype. Therefore, we reasoned then that by still further reducing Foxd3 using foxd3 morpholino in the hdac1col −/−; foxd3zdf10+/−, or hdac1col −/−; foxd3zdf10+/+ mutants would enhance the melanophore rescue phenotype. This was observed, wherein 62.5 % (n= 80, Table 2b, Supplementary figure 3A–D) of phenotypically hdac1col mutants obtained from a hdac1col +/; foxd3zdf10+/ double heterozygous incross injected with 0.477 ng of foxd3 morpholino have a complete rescue of melanophore number and migration. In contrast, reducing Foxd3 using morpholino in hdac1col −/−; foxd3zdf10 −/− double mutants which already completely lack functional Foxd3 has no effect on melanophore recovery. Taken together, the morpholino and double mutant approaches indicate that foxd3 represses melanogenesis in hdac1col mutants and this repression can be eliminated by partially reducing Foxd3 expression. Also, a complete loss of foxd3 in hdac1col mutants results in more severe melanophore defects as compared to relatively milder defects in single hdac1col −/−or foxd3zdf10 −/− mutants indicating a genetic interaction between hdac1 and foxd3 in melanophore development.

Figure 7
Genetically reducing foxd3 rescues melanophore development in hdac1col −/−; foxd3zdf10+/mutants

Foxd3 may repress melanogenesis at the the level of mitfa

The observation that melanoblast-specific expression of both mitfa as well as c-kit is significantly reduced in hdac1col mutants raised the question as to whether foxd3 represses melanogenesis at the level of either or both mitfa and c-kit. To address this question we analyzed hdac1col/foxd3 mutant/morphants for recovery of mitfa and/or c-kit expression. 84% of hdac1col/foxd3 mutant/morphants have a recovery of melanophore number and migration at 3 dpf. We performed in situ hybridization for mitfa and c-kit in hdac1col/foxd3 mutant/morphant and wild-type/foxd3 morphant embryos with uninjected wild-type and hdac1col mutants as controls. At 32 hpf, there is an increase in the number of mitfa- positive melanoblasts in hdac1col/foxd3 mutant/morphants as compared to uninjected hdac1col mutant embryos (Figure 8A–D). In wild-type/foxd3 morphant embryos, although there is no difference in the number of mitfa-positive melanoblasts, qualitatively, there is a slight decrease in the number of mitfa-positive melanoblasts migrating as compared to uninjected wild-type embryos. However, mitfa continues to be expressed robustly in melanoblasts in wild-type/foxd3 morphants as well as hdac1col/foxd3 mutant/morphants, when compared to uninjected wild-type or hdac1col mutants. Next, we analyzed c-kit expression at 32 hpf in hdac1col/foxd3 mutant morphants and wild-type/foxd3 morphants. In hdac1col/foxd3 mutant/morphants, c-kit expression does not recover and is similar to uninjected hdac1col mutants (Figure 8E–H). In hdac1col/foxd3 mutant/morphants embryos the rescue of the differentiated black pigmented melanophore number and migration is only clearly visible by 2 dpf. Therefore, we also analyzed c-kit expression at 48 hpf in hdac1col/foxd3 mutant/morphants and wild-type/foxd3 morphants. At 48 hpf, qualitatively there is a slight increase in c-kit expression levels in hdac1col/foxd3 mutant morphant embryos when compared to uninjected hdac1col mutants (data not shown). However, c-kit expression in hdac1col/foxd3 mutant/morphant melanoblasts remains significantly reduced compared to wild-type or wild-type/foxd3 mutant/morphants. This indicates that foxd3 initially genetically represses melanogenesis at the level of mitfa and not c-kit in hdac1col mutants. This data also correlates with the expression pattern of foxd3 in wild-type embryos where at 16–18 s stages foxd3 is switched off in a rostral-caudal fashion in the premigratory neural crest, while mitfa starts to be expressed around the same time in a rostral-caudal fashion. In hdac1col mutants, foxd3 fails to be downregulated and mitfa expression is repressed.

Figure 8
Foxd3 negatively regulates mitfa

Analysis of an 836 bp promoter region of mitfa which is sufficient to drive melanoblast-specific expression of mitfa (Dorsky et al., 2000) contains two putative foxd3 binding sites. The predicted Foxd3 binding sites are located overlapping the ATG transcriptional start site and 171 bp upstream of the translation start site of mitfa. The −171 bp site is present clustered between two functional sox10 (S1 and S3) binding sites in the mitfa promoter region (Elworthy et al., 2003). We decided to test whether Foxd3 can bind directly to the mitfa promoter. We used electrophoretic mobility shift assays (EMSA) to investigate the interaction of Foxd3 with the predicted forkhead sites in the mitfa promoter. We found that in vitro translated (IVT) Foxd3 was capable of interacting with probes spanning either predicted forkhead site to form complexes exhibiting retarded electrophoretic mobility (Figure 9). We observed competition for formation of the complex using an excess of unlabeled wild type competitors. No competition was observed when we added an excess of mutated or nonspecific (OCTA) competitors. These results indicate that Foxd3 can specifically interact with both predicted forkhead binding sites in the mitfa promoter.

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

Discussion

Our analysis of the hdac1col mutant has revealed that hdac1 is indirectly required for melanogenesis during zebrafish neural crest development. We found that expression of the transcription factor foxd3 fails to be extinguished in neural crest cells of hdac1col mutants in a temporally appropriate manner and that concomitantly, mitfa expression and melanogenesis are suppressed. We also found that Foxd3 is capable of physically interacting at the mitfa promoter, raising the possibility that foxd3 acts as a repressor of mitfa expression. Together, our results suggest that hdac1 function is required to repress foxd3 expression thereby permitting the expression of mitfa and melanophore specification of a subset of neural crest cells during development. Thus, we identify hdac1 as a regulator of neural crest development and identify a mechanism by which foxd3 is capable of repressing melanogenesis.

The mechanism of action by which hdac1col affects the expression of foxd3 within the neural crest is not known. However, it is known that the acetylation and deacetylation and other post-translational modifications of histone tails are important in the regulation of chromatin structure, which in turn plays an important role in regulating eukaryotic gene expression (Jenuwein and Allis, 2001; Rice and Allis, 2001; Strahl and Allis, 2000). Transcriptionally active chromatin usually has highly acetylated histone cores and transcriptionally inactive chromatin is associated with deacetylated histones (Ahringer, 2000; Allfrey, 1966). Histone acetylases (HATs) and histone deacetylases (HDACs) are required to control the acetylation and deactylation of histones (Marks et al., 2003; Roth et al., 2001). This suggests the possibility that the deacetylation activity of HDAC1 is necessary to extinguish foxd3 expression, in response to an unknown signal, and that the disruption of HDAC1 function in hdac1col mutants results in the failure to extinguish foxd3 expression, resulting in the repression of melanogenesis. However, in addition to regulating transcription via controlling chromatin conformation, HATs and HDACs are also known to regulate several non-histone proteins via acetylation and deactylation. Changing the acetylation status on proteins has been shown to regulate protein stability, protein-protein interactions, sub-cellular localization of proteins and also regulate DNA binding (Minucci and Pelicci, 2006) suggesting that gene regulation by HDACs including HDAC1 is likely to be more complex than just histone acetylation states. Thus, whether hdac1 directly regulates foxd3 expression and the mechanism by which this regulation occurs will require further investigation.

Our finding that repressing foxd3 expression in hdac1col mutants rescues mitfa expression and melanogenesis can be thought of as an experimental recapitulation of normal development. Specifically, in wild-type zebrafish embryos, the termination of neural crest foxd3 expression is temporally and spatially contemporaneous with the induction of mitfa expression and melanophore specification. Our finding that Foxd3 is capable of interacting at the mitfa promoter in vitro indicates the possibility that this interaction could be direct in vivo. Consistent with this possibility, it has been reported that overexpression of foxd3 in melanoma cell lines repressed the expression of endogenous mitf (Lister et al., 2005; Thomas and Erickson, 2006). Also, in chick it has been reported that Foxd3 can repress luciferase expression driven by the chick Mitf promoter and that Foxd3 can physically interact with the chick Mitf promoter (Thomas and Erickson, 2006). However, a more rigorous analysis of this interaction will be required to determine whether it occurs directly in zebrafish embryos. In any case, it is important to emphasize that the termination of foxd3 expression is permissive, not instructive, for melanogenesis. This is indicated by the fact that most if not all premigratory neural crest cells express foxd3 whereas only a subset of the neural crest cell population develop as melanophores. Instructive cues likely include pax3, CREB, sox10 and lef1 based on their demonstrated ability to positively regulate mitf (Bertolotto et al., 1998; Bondurand et al., 2000; Dorsky et al., 2000; Elworthy et al., 2003; Lee et al., 2000; Potterf et al., 2000; Price et al., 1998b; Saito et al., 2002; Takeda et al., 2000; Verastegui et al., 2000). However, as these factors are more broadly expressed in the neural crest population than mitfa, it remains to be determined what factors limit the induction of mitfa within the neural crest cell population. Nevertheless, our results strongly suggest that the termination of foxd3 expression during normal development, mediated at least in part by hdac1, is required for melanogenesis to occur (Figure 10).

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

The effects of the hdac1col mutation on neural crest sox10 expression may be informative. Briefly, we found that, like foxd3, sox10 expression is inappropriately extended in hdac1col mutants. In fact, the expression of both transcription factors is expanded among cranial neural crest cells and appears to result in an expansion of cranial satellite glial numbers. This consequence is consistent with the requirement for both sox10 and foxd3 in zebrafish for cranial satellite glial development (Dutton et al., 2001; Kelsh and Eisen, 2000; Kelsh et al., 2000; Stewart et al., 2006). In contrast, the extension of neural crest sox10 expression in hdac1col mutants might be predicted to increase melanogenesis given the requirement for sox10 for melanogenesis (Dutton et al., 2001; Elworthy et al., 2003; Kelsh and Eisen, 2000) and the direct positive regulation of mitfa by sox10 (Elworthy et al., 2003). However, the fact that melanogenesis remains suppressed in hdac1col mutants suggests that sox10 is incapable of promoting mitfa expression when foxd3 is expressed, further indicating a requirement for foxd3 expression termination for melanogenesis via mitfa. Consistent with our data, it has been reported that foxd3 represses sox10 mediated transcriptional activation of luciferase driven by the 836 bp endogenous zebrafish mitfa promoter (Lister et. al., 2005). Although this suggests a hierarchical relationship, more sensitive means of quantifying expression than in situ hybridization will be required to detail the in vivo regulation of mitfa by sox10 and foxd3.

The melanophore phenotype rescue of hdac1col mutants by reducing foxd3 expression levels is somewhat surprising given the apparent lack of a concomitant rescue of c-kit expression by melanogenic neural crest cells. C-kit has been shown to be required for melanophore migration and survival in zebrafish (Parichy et al., 1999). Thus, the rescue of melanophore migration in rescued hdac1col mutants in which c-kit remains suppressed is puzzling. It is formally possible that repression of foxd3 in hdac1col mutants results in the expression or overexpression of an unknown factor or factors that facilitate melanophore and melanophore precursor migration in the absence of c-kit. Instead, we suggest that this apparent quandary is most likely due to the relatively insensitive in situ hybridization method of detecting gene expression. For example, in the zebrafish c-kit mutant sparse (spa), melanophore migration fails, there is extensive melanophore cell death, and a patch of melanophores forms behind the ear (Parichy et al.., 1999). While these phenotypes are also characteristic of hdac1col mutants, they are less severe. For example, some melanophores migrate and fewer die in hdac1col mutants than in spa mutants suggesting the presence of at least low levels of c-kit expression in hdac1col mutant melanophores. Thus, we speculate that c-kit expression in rescued hdac1col mutants is elevated to levels permitting extensive migration and survival of melanophores but at expression levels still below that which can be readily detectable by in situ hybridization. Thus, we suggest that the principle consequence on melanogenesis of foxd3 expression is the suppression of melanophore specification due to the repression of mitfa.

Our data suggests a similar function of foxd3 in repressing melanogenesis in zebrafish and chick systems, in contrast to earlier studies in which there appeared to be differences. In zebrafish, loss of foxd3 in foxd3zdf10 mutants generally does not affect neural crest induction and does not affect melanophore development, although there is a slight delay in differentiation (Stewart et al., 2006). Additionally, in zebrafish, loss of function of foxd3 suggests that foxd3 is required in premigratory and migratory neural crest for subsets of neural crest derivatives which include neurons and glia of the peripheral nervous system, and posterior elements of the pharyngeal skeleton (Lister et al., 1999; Montero-Balaguer et al., 2006; Stewart et al., 2006). In chick, electporation of foxd3 into the neural tube induces a neural crest cell-like fate (Cheung et al., 2005; Dottori et al., 2001; Kos et al., 2001). In addition, foxd3 overexpression prevents the migration of neural crest cells onto the dorso-lateral pathway and most cells migrate along the medial pathway. Neural crest cells that migrate on the dorsa-lateral pathway in avian embryos are exclusively melanophores and never express foxd3, while those that migrate along the medial pathway form neurons and glia and initially express foxd3, which is then extinguished in late migrating neural crest cells (Dottori et al., 2001; Kos et al., 2001). Further, knock down of Foxd3 via morpholinos in avian neural crest cultures increases the numbers of melanophores without affecting proliferation, indicating that foxd3 represses melanogenesis (Kos et al., 2001). Conflicting results obtained in chick and zebrafish argue for differences between the chick and zebrafish systems. However, our data reconciles differences between these two systems by defining gain of function requirements of foxd3 in zebrafish. In this study, we demonstrate that as a result of temporally abnormal foxd3 expression after it has normally been extinguished in neural crest cells, fewer melanophores are specified. This abnormal gain of function of foxd3 in the hdac1col mutants along with a loss of function in foxd3zdf10 mutants and knock down via morpholino suggests that while foxd3 is not necessary to specify melanophores, it can repress specification when present. Reconciliation between the apparent induction of neural crest-like cells in chick in response to foxd3 misexpression and the absence of this effect in zebrafish remains to be investigated.

Supplementary Material

01

Supplementary figure 1. Injection of 0.56 ng of foxd3 morpholino per embryo only partially phenocopies the foxd3zdf10 mutant sympathetic neuron defect.

A–D: side views of 56 hpf wild-type embryos labeled by in situ hybridization expression for tyrosine hydroxylase. foxd3 is required for sympathetic neuron development and loss of foxd3 in foxd3zdf10 mutants or by knock down with high doses (20 ng/embryo) of translational blocking morpholino results in the complete loss of th-positive sympathetic neurons. A: wild-type uninjected control embryo in which there are two clear stripes of th-positive sympathetic neurons in the anterior trunk region (arrowheads). B–D: All embryos shown are from the same treated clutch. Reducing Foxd3 partially (0.56 ng/embryo) decreases the number of th-positive sympathetic neurons, although rare wild-type morpholino treated embryos (D) have an absence of sympathetics (arrowheads). A–D: In contrast to differences in th-positive sympathetic neuron expression, th expression in the hind-brain (arrows) and locus coeruleus (white arrowheads) is similar between control uninjected and wild-type/foxd3 morphants.

02

Supplementary figure 2. Melanoblast differentiation and migration does not recover at 30 hpf.

All panels are lateral views of 30 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 30 hpf, the number of melanoblasts that express mitfa does not recover in hdac1col mutants. Also, most of the mitfa-positive melanoblasts fail to migrate and are located in the dorsal stripe and in the post otic region in hdac1col mutants. In contrast, wild-type melanoblasts have migrated into the ventral stripe and over the head. C, D: Additionally, melanoblast-specific c-kit expression in hdac1col is still absent and/or reduced (arrowhead), although non-melanoblast expression of c-kit in the post anal region and posterior mesoderm is equivalent to wild-type (arrows). E, F: There are fewer dct-positive differentiating melanoblasts in hdac1col mutants as compared to wild-type (arrowheads) and most of the dct-positive melanoblasts are located posterior to the otic vesicle and in the dorsal stripe. A few dct-positive melanoblasts are migrating in the anterior trunk in hdac1col mutants, although, dct is not expressed as robustly compared to expression in wild-type melanoblasts. G, H: In contrast to c-kit-positive melanoblasts, there are many more fms-positive differentiating xanthoblasts in hdac1col mutants compared to wild-type. I, J: although reduced in number when compared to wild-type, xdh-positive differentiating xanthoblasts are more numerous in the cranial region (arrows) and trunk (arrowheads) as compared to differentiating dct-positive melanoblasts in hdac1col mutants.

03

Supplementary figure 3. Further repression of Foxd3 in hdac1col−/−; foxd3zdf10+/ and hdac1col−/−; foxd3zdf10+/+ mutants increases the rate of rescue of melanogenesis.

A–D: side views of live images of hdac1col embryos at 3.5 dpf obtained from hdac1col+/; foxd3zdf10+/ heterozygous mutant incrosses in which either Foxd3 is further reduced with 0.477ng of foxd3 morpholino (mo) or are uninjected controls. A: At 3.5 dpf 10 % of hdac1col embryos obtained from a double mutant hdac1col+/; foxd3zdf10+/ heterozygous incross have increased number of melanophores which migrate over the head, into the ventral stripe (v) in the tail and onto the yolk sac areas (y). The genotype of the rescued mutants was identified as hdac1col −/−; foxd3zdf10+/. B: hdac1col −/−; foxd3zdf10−/− double homozygous mutants have fewer melanophores than hdac1col −/− single mutants and most of the melanophores are present in the dorsal (d) stripe. C, D: Reducing Foxd3 via morpholino rescues the melanophore defect in hdac1col −/−; foxd3zdf10+/ and hdac1col −/−; foxd3zdf10+/+ mutants (62.5 % of all hdac1col mutants) but not in hdac1col −/−; foxd3zdf10−/− double homozygous mutants. A–D: hdac1col −/−; foxd3zdf10+/, hdac1col −/−; foxd3zdf10+/+ and hdac1col−/−; foxd3zdf10−/− mutants have the same eye defect as hdac1col −/− single mutants and knock down of Foxd3 has no effect on the eye phenotype (arrows)

Acknowledgments

We thank our colleagues for numerous reagents employed in this study. This work was supported by NIH grant EY015480 to H. E–H. and NIH grant GM076505 to P. D. H. with additional support from NIH grant P30-NS045758.

Footnotes

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