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Zebrafish endzone regulates neural crest-derived chromatophore differentiation and morphology.

Arduini BL, Gallagher GR, Henion PD - PLoS ONE (2008)

Bottom Line: We have found that although wild-type numbers of chromatophore precursors are generated in the first day of development and migrate normally in enz mutants, the numbers of all three chromatophore cell types that ultimately develop are reduced.Further, differentiated melanophores and xanthophores subsequently lose dendricity, and iridiphores are reduced in size.Our results suggest that enz is required relatively late in the development of all three embryonic chromatophore types and is normally necessary for terminal differentiation and the maintenance of cell size and morphology.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Neurobiology, Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
The development of neural crest-derived pigment cells has been studied extensively as a model for cellular differentiation, disease and environmental adaptation. Neural crest-derived chromatophores in the zebrafish (Danio rerio) consist of three types: melanophores, xanthophores and iridiphores. We have identified the zebrafish mutant endzone (enz), that was isolated in a screen for mutants with neural crest development phenotypes, based on an abnormal melanophore pattern. We have found that although wild-type numbers of chromatophore precursors are generated in the first day of development and migrate normally in enz mutants, the numbers of all three chromatophore cell types that ultimately develop are reduced. Further, differentiated melanophores and xanthophores subsequently lose dendricity, and iridiphores are reduced in size. We demonstrate that enz function is required cell autonomously by melanophores and that the enz locus is located on chromosome 7. In addition, zebrafish enz appears to selectively regulate chromatophore development within the neural crest lineage since all other major derivatives develop normally. Our results suggest that enz is required relatively late in the development of all three embryonic chromatophore types and is normally necessary for terminal differentiation and the maintenance of cell size and morphology. Thus, although developmental regulation of different chromatophore sublineages in zebrafish is in part genetically distinct, enz provides an example of a common regulator of neural crest-derived chromatophore differentiation and morphology.

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Xanthophores are qualitatively reduced in number and size in enz mutants.(A, B) fms expression in 48 hpf wild-type (A) and enz mutant (B) embryos. Qualitatively reduced fms expression suggests that fewer xanthophores are present in enz mutants than in wild-type siblings at this stage (arrowheads in A and B). (C, D) Methylene blue-stained xanthophores appear much larger in wild-type (C) embryos than in enz mutants (D) at 3 dpf.
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pone-0002845-g006: Xanthophores are qualitatively reduced in number and size in enz mutants.(A, B) fms expression in 48 hpf wild-type (A) and enz mutant (B) embryos. Qualitatively reduced fms expression suggests that fewer xanthophores are present in enz mutants than in wild-type siblings at this stage (arrowheads in A and B). (C, D) Methylene blue-stained xanthophores appear much larger in wild-type (C) embryos than in enz mutants (D) at 3 dpf.

Mentions: In wild-type zebrafish embryos raised at 28.5°C, neural crest-derived melanophores normally begin to differentiate at approximately 25 hpf, while xanthophores and iridiphores begin to overtly differentiate at about 42 hpf and 72 hpf, respectively [59]. By 27 hpf, primarily in anterior regions, large, stellate, dark melanophores are present in wild-type embryos. In enz mutant embryos, as in wild-type siblings, melanophores differentiate at ∼25 hpf (data not shown). At 27 hpf, enz melanophores are stellate, but pale compared to those of wild-type siblings (Figure 5A, B). After 27 hpf, enz melanophores begin to transition from the initial pale, stellate appearance to a dark, punctate form, while wild-type melanophores remain stellate and dark. The transformation of melanophores occurs in a rostro-caudal wave, and is complete by about 48 hpf (Figure 5C–F and data not shown). We hypothesized that this apparent change in cell morphology of enz melanophores might be the result of either redistribution of melanosomes within cells or of a change in cell shape. To distinguish between these two possibilities, we performed in situ hybridization of melanized 36 hpf and 48 hpf wild-type and enz mutant embryos, using the melanophore sublineage-specific riboprobes c-kit [7] and dct [23]. In wild-type embryos, c-kit and dct mRNAs are distributed throughout the cell cytoplasm, including in the processes, reflecting stellate cellular morphology (Figure 5G, H and data not shown). The distribution of dct and c-kit mRNAs is punctate in enz melanophores, similar to the distribution of melanin (Figure 5I, J and data not shown). This is consistent with a cell morphology change, rather than only relocalization of melanin or melanosomes within a stellate cell. Subsequently, we quantified the area of punctate melanophores in enz mutant embryos compared to stellate melanophores in wild-type siblings. At 2 dpf, wild-type melanophores at cephalic levels have a mean area of 282.1±19.1 µm2, while enz melanophores at similar axial levels have a mean area of 21.9±1.7 µm2 (P<0.0001, Table 3). The change in melanophore cell morphology in enz mutant embryos further suggested that the apparent absence of yellow pigmentation might be due at least in part to a reduction in size of xanthophores in these embryos. Individual xanthophores are difficult to distinguish, and even fms and xdh expression appear as diffuse staining over the dorsal aspect of the embryo, precluding the quantitative type of analysis performed on melanophores (Figure 6A, B) [11], [14], [28]. However, qualitative observations of xanthophore morphology were made using methylene blue, which is taken up specifically by xanthophores and is concentrated around active pterinosomes, the organelles that produce pteridine pigments [60]. Methylene blue staining revealed that while some xanthophores are present in enz homozygotes, these appear much smaller and less stellate than xanthophores in wild-type siblings at 3 dpf (Figure 6C, D). Similarly, iridiphores are reduced in size in enz mutant embryos compared to wild-type siblings. In contrast to melanophores and xanthophores, iridiphores in the trunk of wild-type embryos have a rounded, rather than stellate morphology at 72 hpf (see Figure 1). While this is also true in enz homozygotes, overall iridiphore cell size, as measured by area, is reduced compared to wild-type siblings. At 6 dpf, the average iridiphore area in enz mutant larvae is reduced by ∼40% compared to that in wild-type siblings (P<0.0001, data not shown). Together, these data indicate that mutations in enz similarly affect all three neural crest-derived chromatophore types with respect to cell size and also result in loss of the the typical stellate morphology of melanophores and xanthophores.


Zebrafish endzone regulates neural crest-derived chromatophore differentiation and morphology.

Arduini BL, Gallagher GR, Henion PD - PLoS ONE (2008)

Xanthophores are qualitatively reduced in number and size in enz mutants.(A, B) fms expression in 48 hpf wild-type (A) and enz mutant (B) embryos. Qualitatively reduced fms expression suggests that fewer xanthophores are present in enz mutants than in wild-type siblings at this stage (arrowheads in A and B). (C, D) Methylene blue-stained xanthophores appear much larger in wild-type (C) embryos than in enz mutants (D) at 3 dpf.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2483736&req=5

pone-0002845-g006: Xanthophores are qualitatively reduced in number and size in enz mutants.(A, B) fms expression in 48 hpf wild-type (A) and enz mutant (B) embryos. Qualitatively reduced fms expression suggests that fewer xanthophores are present in enz mutants than in wild-type siblings at this stage (arrowheads in A and B). (C, D) Methylene blue-stained xanthophores appear much larger in wild-type (C) embryos than in enz mutants (D) at 3 dpf.
Mentions: In wild-type zebrafish embryos raised at 28.5°C, neural crest-derived melanophores normally begin to differentiate at approximately 25 hpf, while xanthophores and iridiphores begin to overtly differentiate at about 42 hpf and 72 hpf, respectively [59]. By 27 hpf, primarily in anterior regions, large, stellate, dark melanophores are present in wild-type embryos. In enz mutant embryos, as in wild-type siblings, melanophores differentiate at ∼25 hpf (data not shown). At 27 hpf, enz melanophores are stellate, but pale compared to those of wild-type siblings (Figure 5A, B). After 27 hpf, enz melanophores begin to transition from the initial pale, stellate appearance to a dark, punctate form, while wild-type melanophores remain stellate and dark. The transformation of melanophores occurs in a rostro-caudal wave, and is complete by about 48 hpf (Figure 5C–F and data not shown). We hypothesized that this apparent change in cell morphology of enz melanophores might be the result of either redistribution of melanosomes within cells or of a change in cell shape. To distinguish between these two possibilities, we performed in situ hybridization of melanized 36 hpf and 48 hpf wild-type and enz mutant embryos, using the melanophore sublineage-specific riboprobes c-kit [7] and dct [23]. In wild-type embryos, c-kit and dct mRNAs are distributed throughout the cell cytoplasm, including in the processes, reflecting stellate cellular morphology (Figure 5G, H and data not shown). The distribution of dct and c-kit mRNAs is punctate in enz melanophores, similar to the distribution of melanin (Figure 5I, J and data not shown). This is consistent with a cell morphology change, rather than only relocalization of melanin or melanosomes within a stellate cell. Subsequently, we quantified the area of punctate melanophores in enz mutant embryos compared to stellate melanophores in wild-type siblings. At 2 dpf, wild-type melanophores at cephalic levels have a mean area of 282.1±19.1 µm2, while enz melanophores at similar axial levels have a mean area of 21.9±1.7 µm2 (P<0.0001, Table 3). The change in melanophore cell morphology in enz mutant embryos further suggested that the apparent absence of yellow pigmentation might be due at least in part to a reduction in size of xanthophores in these embryos. Individual xanthophores are difficult to distinguish, and even fms and xdh expression appear as diffuse staining over the dorsal aspect of the embryo, precluding the quantitative type of analysis performed on melanophores (Figure 6A, B) [11], [14], [28]. However, qualitative observations of xanthophore morphology were made using methylene blue, which is taken up specifically by xanthophores and is concentrated around active pterinosomes, the organelles that produce pteridine pigments [60]. Methylene blue staining revealed that while some xanthophores are present in enz homozygotes, these appear much smaller and less stellate than xanthophores in wild-type siblings at 3 dpf (Figure 6C, D). Similarly, iridiphores are reduced in size in enz mutant embryos compared to wild-type siblings. In contrast to melanophores and xanthophores, iridiphores in the trunk of wild-type embryos have a rounded, rather than stellate morphology at 72 hpf (see Figure 1). While this is also true in enz homozygotes, overall iridiphore cell size, as measured by area, is reduced compared to wild-type siblings. At 6 dpf, the average iridiphore area in enz mutant larvae is reduced by ∼40% compared to that in wild-type siblings (P<0.0001, data not shown). Together, these data indicate that mutations in enz similarly affect all three neural crest-derived chromatophore types with respect to cell size and also result in loss of the the typical stellate morphology of melanophores and xanthophores.

Bottom Line: We have found that although wild-type numbers of chromatophore precursors are generated in the first day of development and migrate normally in enz mutants, the numbers of all three chromatophore cell types that ultimately develop are reduced.Further, differentiated melanophores and xanthophores subsequently lose dendricity, and iridiphores are reduced in size.Our results suggest that enz is required relatively late in the development of all three embryonic chromatophore types and is normally necessary for terminal differentiation and the maintenance of cell size and morphology.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Neurobiology, Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
The development of neural crest-derived pigment cells has been studied extensively as a model for cellular differentiation, disease and environmental adaptation. Neural crest-derived chromatophores in the zebrafish (Danio rerio) consist of three types: melanophores, xanthophores and iridiphores. We have identified the zebrafish mutant endzone (enz), that was isolated in a screen for mutants with neural crest development phenotypes, based on an abnormal melanophore pattern. We have found that although wild-type numbers of chromatophore precursors are generated in the first day of development and migrate normally in enz mutants, the numbers of all three chromatophore cell types that ultimately develop are reduced. Further, differentiated melanophores and xanthophores subsequently lose dendricity, and iridiphores are reduced in size. We demonstrate that enz function is required cell autonomously by melanophores and that the enz locus is located on chromosome 7. In addition, zebrafish enz appears to selectively regulate chromatophore development within the neural crest lineage since all other major derivatives develop normally. Our results suggest that enz is required relatively late in the development of all three embryonic chromatophore types and is normally necessary for terminal differentiation and the maintenance of cell size and morphology. Thus, although developmental regulation of different chromatophore sublineages in zebrafish is in part genetically distinct, enz provides an example of a common regulator of neural crest-derived chromatophore differentiation and morphology.

Show MeSH
Related in: MedlinePlus