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doi: 10.1242/10.1242/dev.00307

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Temporal and cellular requirements for Fms signaling during zebrafish adult pigment pattern development

Section of Integrative Biology, Section of Molecular, Cell and Developmental Biology, Institute for Cellular and Molecular Biology, University of Texas at Austin, 1 University Station C0930, Austin, TX 78712, USA

View larger version (117K): [in a new window]   Fig. 1. fms is essential for development of xanthophores and adult melanophore stripes, but is not expressed by melanophores. (A) Wild-type (strain ABUT) zebrafish exhibits several well-organized dark stripes that include melanophores with intervening light stripes that include xanthophores. (B) fmsblue mutant adult, as a representative of fms mutants, lacks xanthophores and exhibits a disorganized pattern of melanophores. (C,D) Details of wild-type and fms mutant adult pigment patterns. (C) In wild type, melanophores are abundant in dorsal and ventral melanophore stripes, and a lighter interstripe region contains numerous yellow-orange xanthophores (arrow). Horizontal line is the horizontal myoseptum. (D) In a fmsblue mutant, melanophores are reduced in number and fail to form normal stripes, and xanthophores are not present. The fish in C and D are illuminated so as to avoid reflections from iridophores throughout this region. (E) Detail of wild-type stripe margin in which melanosomes are contracted within melanophores, allowing a few xanthophores (arrows) to be discerned within the boundary of the dark melanophore stripe. Iridescent iridophores appear bluish in this image. (F,G) mRNA in situ hybridizations of zebrafish larvae during late stages of pigment pattern metamorphosis. (F) fms expression is not apparent in melanophores, but staining is observed in adjacent presumptive xanthophores (arrow). (G) In contrast, expression of the fms homologue, kit, is readily detected in melanophores (arrow). Scale bars: (A,B) 4 mm, (C,D) 60 µm, (E) 80 µm, (F,G) 120 µm.

View larger version (139K): [in a new window]   Fig. 2. Chimeras reveal cell autonomous and non-autonomous roles for fms during adult stripe development. (A,B) Bright-field (A) and fluorescence (B) micrographs of early larva (72 h) showing donor wild-type (fms+ GFP+) xanthophores over the dorsal myotomes of a fms- mutant host. (C,D) Wild-type fms- chimeras reared to adult stages (n=20) develop well-formed (C) or partial (D) melanophore stripes when donor melanophores and xanthophores are present. (E,F) Detail of wild-type fms- chimera showing organized stripes that include donor (fms+ GFP+) melanophores (large arrow) and xanthophores (small arrow), as well as host (fms- GFP-) melanophores (arrowhead). This is the same individual as in C; note the absence of GFP+ donor cells in other tissues, such as myotomes or epidermis. (G,H) Melanophore stripe morphology depends on the presence of donor wild-type pigment cells. Opposite sides of a single wild-type fms- chimera are shown in which well-defined melanophore stripes are present on the side exhibiting donor melanophores and xanthophores (arrow, G) but not on the side lacking donor pigment cells (H). (I,J) fms- wild-type chimeras reared to adult stages (n=15) developed wild-type stripes. Although donor fms- cells contributed to epidermis, nerves, bone and other derivatives, only one chimera exhibited donor (fms- GFP+) melanophores (arrow) and these were present within host melanophore stripes; donor xanthophores were not observed. Scale bars, (A,B) 30 µm, (E,F) 200 µm, (G,H) 250 µm, (I,J) 60 µm.

View larger version (67K): [in a new window]   Fig. 3. nacre mutant reveals that fms acts through the xanthophore lineage to promote melanophore stripe formation. (A,B) nacre- fms- chimeras reared to adult stages (n=29) that developed donor (nacre- fms+ GFP+) xanthophores also developed organized melanophore stripes (n=7). (B,B') Corresponding bright-field and fluorescence micrographs of the individual in A showing donor xanthophores (e.g., red arrow) adjacent to the melanophore stripe. (C) nacre- fms- chimeras, lacking xanthophores, failed to develop organized melanophore stripes (n=22). An individual exhibiting donor (nacre- fms+ GFP+) iridophores (n=6) is shown. (D,D') Corresponding bright-field and fluorescence views of the individual in C showing donor iridophores (e.g., blue arrow). Note that the orange color in some regions is due to reflections from iridophores rather than differentiated xanthophores. In contrast to melanophore arrangements, however, average melanophore densities did not differ dramatically between nacre- fms- chimeras that either developed or failed to develop donor xanthophores (means=373, 316 melanophores/mm2, s.d.=111, 69, n=4, 11, respectively; t13=1.0, P=0.3). (E,F) Development of melanophore stripes in nacre- hosts. (E) Wild-type cells transplanted to nacre- hosts develop regions of well-formed stripes. (F) fms- cells transplanted to nacre- hosts contribute to stripes resembling those formed by wild-type cells. Scale bars, (A,C) 4 mm, (B,D) 100 µm, (E,F) 800 µm.

View larger version (104K): [in a new window]   Fig. 4. Isolation of a temperature sensitive fms allele. (A) fms174A cDNA exhibits a tyr his substitution within the second immunoglobulin-like domain. Grey, untranslated regions. Green, signal sequence and transmembrane domain. Red, split kinase domains. (B) Primer extension analysis for genotyping fms174A nucleotide substitution. (Upper trace) Wild-type (or fms1, fmsblue) alleles result in the addition of 3 nucleotides (nt) to the extension primer. The peak at +0 nt represents excess extension primer without added nucleotides. (Lower trace) The fms174A allele results in addition of 2 nt to the extension primer; shown is a chromatogram for a heterozygous fms174A/fms+ individual. (C-F) Homozygous fms174A individuals reared at 24°C (C,D) or 33°C (E,F). (C,D) At 24°C, hatchling larvae exhibit normal numbers of xanthophores (here evidenced by the yellow cast to the flank; C); adults exhibit melanophore stripes indistinguishable from wild-type (D). (E,F) At 33°C, hatchling larvae lack xanthophores (E) and adults both lack xanthophores and exhibit a severe disruption of melanophore stripes (F), resembling that seen in fms1 or fmsblue (Fig. 1B). (G-I) Molecular marker analyses reveal that fms174A conditionally affects the development of xanthophore precursors, as revealed by distributions of cells expressing the xanthophore lineage markers fms (G), gch (H), and xdh (I). (Upper panels) Presumptive xanthophore precursors are abundant over the myotomes of 60 h embryos at 24°C. (Lower panels) Presumptive xanthophore precursors are absent from over the myotomes of embryos reared at 33°C. Scale bars, (C,E) 600 µm, (D,F) 2 mm, (G-I) 40 µm.

View larger version (25K): [in a new window]   Fig. 5. Temperature shift experiments reveal temporal requirements for Fms activity in promoting xanthophore development and melanophore stripe formation in homozygous fms174A mutants. (A,B) Mean densities (±95% confidence intervals) of xanthophores present in the adult pigment patterns of individuals following temperature shift at the sizes indicated. Orange line in A and B indicates the mean density of xanthophores in control individuals reared at 24°C throughout development; xanthophores were absent in control individuals reared at 33°C throughout development. n, sample sizes for each size class. Note that only midpoints of size classes are indicated, and that ranges of sizes per class vary (1 mm per class at sizes <8 mm, when rapid changes occur during pigment pattern metamorphosis; 2 mm per class at sizes 8 mm, reflecting slower changes at late metamorphic and juvenile stages; DMP and JMT, manuscript in preparation). (A) Temperature up-shift ablates xanthophores through middle stages of pigment pattern metamorphosis though residual xanthophores persist in individuals shifted during late pigment pattern metamorphosis or beyond. Mean densities of xanthophores were significantly reduced in upshifted individuals as compared to sibling controls left at 24°C (F1,148=500.0, P<0.0001). (B) Temperature down-shift allows substantial xanthophore recovery through middle stages of pigment pattern metamorphosis, but less marked recovery during later metamorphic and juvenile stages (see text for details). Mean densities of xanthophores for downshifted individuals were significantly greater overall than sibling controls left at 33°C (F1,74=9.27, P<0.005). (C) Melanophore organization is correlated with xanthophore density. Reduced variation in nearest neighbor distances between melanophores is associated with increased xanthophore densities in both temperature upshift (red points) and downshift (green points) experiments. Red diamond indicates the mean for individuals completely lacking xanthophores at 33°C (upshift and control, pooled); green diamond, the mean for control individuals reared exclusively at 24°C. Note that variability in melanophore nearest neighbor distances is increased among individuals with partially disrupted stripes, whereas the most severe phenotypes at 33°C have somewhat lower coefficients of variation, reflecting a more uniform dispersion of melanophores once xanthophores and stripes have been lost. Regression shown includes only individuals with partial xanthophore deficits compared to controls. Individual and pooled values shown are based on 98,643 melanophores, 47,067 xanthophores. (D) Complete melanophore stripes are more common when Fms activity is provided by temperature downshift prior to late metamorphic stages. Shown are percentages of individuals downshifted at different sizes that exhibited complete melanophore stripes (as defined by 1 200 µm gap per side). Thus, individuals shifted at sizes 8 mm SL typically exhibited more broken stripe patterns (2=65.2, d.f.=13, P<0.0001). The orange line indicates the percentage of individuals with complete stripes among controls reared at 24°C.

View larger version (139K): [in a new window]   Fig. 6. Curtailing Fms activity eliminates xanthophores and perturbs melanophore stripes thoughout development. (A-C) Examples of fms174A individuals reared at 24°C to the sizes indicated (upper panels) then shifted to 33°C until an adult pigment pattern had formed (lower panels). (A) Larva shifted during early pigment pattern metamorphosis (7.6 mm SL) loses xanthophores and fails to develop normal adult stripes (14.3 mm SL, A') after 28 days at 33°C. (B) Larva shifted during middle stages of pigment pattern metamorphosis (8.9 mm SL) loses xanthophores and initial melanophore stripes degenerate (15.6 mm SL, B') after 28 days at 33°C. (C) Individual that has already attained a juvenile pigment pattern (13.5 mm SL) retains some xanthophores and a partial stripe pattern with more variably spaced melanophores (14.9 mm SL, C') after 14 days at 33°C. (Insets) Higher magnification views of boxed regions showing absence of xanthophores (A',B') or residual xanthophores (C' arrow). (D-I) Prolonged rearing at 33°C results in a complete loss of xanthophores Shown are sequential images of the same region on a representative fms174A individual that had developed a juvenile pattern of melanophore stripes (18 mm SL) at 24°C (D), with times after shifting to 33°C of (E) 3 days, (F) 6 days, (G) 8 days, (H) 12 days and (I) 20 days. (Upper images) Low magnification showing melanophore distributions. (Lower images) Higher magnification showing depletion of xanthophores (arrow). (Inset) in G, high magnification showing melanophore debris indicated by arrow. Scale bars, (A,B) 1 mm, (C) 2 mm, (A'-C') 500 µm, (D-I) 250 µm.

View larger version (107K): [in a new window]   Fig. 7. Chromatoblast and chromatophore death following Fms inactivation. (A) Two hours after shifting fms174A mutants from 24°C to 33°C, an increase in TUNEL+ cells is observed in neural crest migratory pathways. Shown are superimposed fluorescence and bright-field images of TUNEL+ cells (arrow) adjacent to the dorsal neural tube during the stages of neural crest cell migration in a 28 h embryo. nt, neural tube. e, epidermis. m, myotome. B,C,D,E,F are brightfield images; B' and D' are corresponding fluorescence images of B and D. (B) In juvenile fish shifted from 24°C to 33°C, unpigmented TUNEL+ cells (arrow B') occur in the dermis where fms-expressing cells are found. s, scale. (C) Extensive chromatophore debris can be identified in the skin after shifting adult fish from 24°C to 33°C. Shown is a whole mount region of the caudal fin, with orange and black debris from xanthophores and melanophores, respectively. c, capillary. r, fin ray. (D) Extrusions in the superficial epidermis contain orange pigment. (D') Autofluorescence reveals presence of xanthophore-derived pteridine pigments. (E) Extrusion from the fin epidermis (arrow) contains debris of both xanthophores and melanophores. (F) Extrusion containing xanthophore-derived pigment on the fin of a wild-type adult. Scale bars: (A-C) 40 µm, (D,E) 20 µm.

View larger version (17K): [in a new window]   Fig. 8. Quantitative analyses of cell death when Fms activity is curtailed. (A) Mean (+95% confidence intervals) numbers of TUNEL+ cells observed in trunk neural crest migratory pathways of 24-28 h embryos (n=128) maintained either at 24°C, transferred during these stages of neural crest migration from 24°C to 33°C for 2-3 hours, or maintained at 33°C. A dramatic increase in TUNEL+ cells occurs in fms174A homozygous embryos exposed to the 33°C restrictive temperature, as compared to fms174A homozygotes at 24°C or heterozygotes at either temperature (genotype x temperature interaction for square root-transformed data: F2,122=10.0, P<0.0001). (B) Mean numbers (+95% confidence intervals) of xanthophore pigment-containing exclusion bodies in caudal fins of adults (n=14) maintained either at 24°C or transferred from 24°C to 33°C for 3 days. A sharp increase in the numbers of such exclusion bodies occurs in fms174A individuals transferred to 33°C as compared to fms174A maintained at 24°C or wild-type individuals transferred from 24°C to 33°C (square root-transformed data, F2,11=28.04, P<0.0001).

View larger version (173K): [in a new window]   Fig. 9. Temperature downshift experiments reveal xanthophore recovery and pattern regulation after Fms activation. (A-C) Examples of fms174A homozygotes reared at 33°C to the size indicated (upper panels) then shifted to 24°C until an adult pigment pattern had formed (lower panels). (A) Larva shifted during early pigment pattern metamorphosis (6.5 mm SL) recovered xanthophores and a wild-type pattern of adult melanophore stripes (15.9 mm SL, A') after 34 days at 24°C. (B) Larva shifted during middle stages of pigment pattern metamorphosis (8.1 mm SL) recovered xanthophores and normal adult melanophore stripes (17.3 mm SL, B') after 34 days at 24°C. (C) Individual shifted when pigment pattern metamorphosis was essentially completed (12.3 mm SL) has recovered some xanthophores but not a normally organized stripe pattern (14.1 mm SL, C') after 14 days at 24°C. (Insets) Higher magnification views of boxed regions showing xanthophores. (D-I) Individuals reared initially at 33°C through late larval stages can regulate xanthophores and stripes after several weeks to months following shift to 24°C. Shown are sequential images of the same region on a representative fms174A individual (starting 12 mm SL) at 2 days (D), 10 days (E), 16 days (F), 22 days (G), 27 days (H), and 40 days (I) after temperature downshift. Note increasingly organized and spread melanophores as xanthophores populate the flank. (Upper images) Low magnification showing melanophore distributions. (Lower images) Higher magnification of boxed regions showing recovery of xanthophores (arrow). (J-L) Perturbation of fin patterning when Fms is activated late in development. (J) Normal horizontal stripes form in the caudal fin of fms174A individuals reared at 24°C. Xanthophore stripes on the fin extend caudally from stripes on the body (arrow). (K) Xanthophores and stripes are absent in the fins of fms174A individuals reared at 33°C. (L) Xanthophore recovery accompanied by stripe reorientation in fms174A when Fms is activated only late in development. Scale bars: (A,B) 1 mm, (C) 2 mm, (A'-C') 500 µm, (D-I) 250 µm, (J-L) 1 mm.

View larger version (70K): [in a new window]   Fig. 10. Pattern regulation in fms174A/fmsblue transheterozygotes. (A) Normal adult melanophore stripes develop in fms174A/fmsblue individuals reared at 24°C. (B) Xanthophores and melanophore stripes are recovered in fms174A/fmsblue individuals reared 35°C prior to feeding, and at 24°C thereafter. (C) More frequent breaks in adult melanophore stripes occur in fms174A/fmsblue individuals reared at 33°C prior to hatching, as compared to fms174A/fmsblue reared at 24°C prior to hatching, or fms174A/+ or fmsblue/+ heterozygotes reared at either temperature (temperature x genotype interaction for square root-transformed data: F1,110=6.33, P<0.05). Shown are unilateral mean numbers of breaks in melanophore stripes per individual (±95% confidence intervals). Scale bar: 500 µm.

View larger version (22K): [in a new window]   Fig. 11. Model for pigment pattern metamorphosis in zebrafish. (A) Throughout metamorphosis new pigment cells appear from undifferentiated stem cells (see text for references). These cells (white) may be specified for one or another cell fate, or they may be pluripotent. Recruitment of stem cells to the xanthophore lineage (yellow cells, left) requires fms; in the absence of Fms activity these cells die, fail to advance through stages of xanthophore differentiation, or both. Stem cells also are recruited to melanophore fates (grey cells, right) under the influence of ednrb1, mitfa and kit. Although gene expression analyses reveal fms expression at early stages in some of these cells, a cell autonomous role for fms in promoting the development of early stages in the melanophore lineage has yet to be documented. (B) Terminal differentiation of chromatophores depends on genes encoding pigment synthesis enzymes that are likely to differ between xanthophores (e.g., gch; xdh) and melanophores (e.g., dopachrome tautomerase, dct; tyrosinase, tyr). During these stages, xanthoblasts express and require fms (F). A parallel requirement for kit is observed for fin melanoblasts, and likely body melanoblasts that also express kit (K). (C) During middle stages of pigment pattern metamorphosis and possibly prior to the terminal differentiation of chromatophores, fms-dependent cells of the xanthophore lineage influence kit-dependent cells of the melanophore lineage to form stripes. Although this interaction promotes melanophore competence for stripe formation, the directionality of these stripes depends on additional cues, possibly including initial asymmetries in chromatoblast or stem cell distributions, or other features of the extracellular environment. In the absence of Fms activity, xanthophores are not recruited and do not influence melanophore stripe formation. (D) During late stages of pigment pattern metamorphosis extending through adult life, fms-dependent xanthophores (or their precursors) contribute to maintaining melanophore stripes. In the absence of Fms activity, xanthophores die and melanophore stripes degenerate.

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