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1.  Clonal Analysis of kit ligand a Functional Expression Reveals Lineage-Specific Competence to Promote Melanocyte Rescue in the Mutant Regenerating Caudal Fin 
PLoS ONE  2014;9(7):e102317.
The study of regeneration in an in vivo vertebrate system has the potential to reveal targetable genes and pathways that could improve our ability to heal and repair damaged tissue. We have developed a system for clonal labeling of discrete cell lineages and independently inducing gene expression under control of the heat shock promoter in the zebrafish caudal fin. Consequently we are able to test the affects of overexpressing a single gene in the context of regeneration within each of the nine different cell lineage classes that comprise the caudal fin. This can test which lineage is necessary or sufficient to provide gene function. As a first example to demonstrate this approach, we explored which lineages were competent to functionally express the kit ligand a protein as assessed by the local complementation of the mutation in the sparse-like (kitlgatc244b) background. We show that dermal fibroblast expression of kit ligand a robustly supports the rescue of melanocytes in the regenerating caudal fin. kit ligand a expression from skin and osteoblasts results in more modest and variable rescue of melanocytes, while lateral line expression was unable to complement the mutation.
doi:10.1371/journal.pone.0102317
PMCID: PMC4092134  PMID: 25009992
2.  Development of Translating Ribosome Affinity Purification for Zebrafish 
Genesis (New York, N.Y. : 2000)  2013;51(3):187-192.
Summary
The regulation of transcription and translation by specific cell types is essential to generate the cellular diversity that typifies complex multicellular organisms. Tagging and purification of ribosomal proteins has been shown to be an innovative and effective means of characterizing the ribosome bound transcriptome of highly specific cell populations in vivo. To test the feasibility of using translating ribosome affinity purification (TRAP) in zebrafish, we have generated both a ubiquitous TRAP line and a melanocyte-specific TRAP line using the native zebrafish rpl10a ribosomal protein. We have demonstrated the capacity to capture mRNA transcripts bound to ribosomes, and confirmed the expected enrichment of melanocyte specific genes and depletion of non-melanocyte genes when expressing the TRAP construct with a cell specific promoter. We have also generated a generic EGFP-rpl10a Tol2 plasmid construct (Tol2-zTRAP) that can be readily modified to target any additional cell populations with characterized promoters in zebrafish.
doi:10.1002/dvg.22363
PMCID: PMC3638809  PMID: 23281262
TRAP; polysome; capture; danio
3.  Bioelectric Signaling Regulates Size in Zebrafish Fins 
PLoS Genetics  2014;10(1):e1004080.
The scaling relationship between the size of an appendage or organ and that of the body as a whole is tightly regulated during animal development. If a structure grows at a different rate than the rest of the body, this process is termed allometric growth. The zebrafish another longfin (alf) mutant shows allometric growth resulting in proportionally enlarged fins and barbels. We took advantage of this mutant to study the regulation of size in vertebrates. Here, we show that alf mutants carry gain-of-function mutations in kcnk5b, a gene encoding a two-pore domain potassium (K+) channel. Electrophysiological analysis in Xenopus oocytes reveals that these mutations cause an increase in K+ conductance of the channel and lead to hyperpolarization of the cell. Further, somatic transgenesis experiments indicate that kcnk5b acts locally within the mesenchyme of fins and barbels to specify appendage size. Finally, we show that the channel requires the ability to conduct K+ ions to increase the size of these structures. Our results provide evidence for a role of bioelectric signaling through K+ channels in the regulation of allometric scaling and coordination of growth in the zebrafish.
Author Summary
The proportions of an animal can change during its lifetime. This often occurs through the phenomenon of relative growth, or allometry. In humans, for example, the head grows at a lower rate than the body resulting in a change in proportion between children and adults. The regulation of size and proportion is not well understood. We investigated fin growth in zebrafish as a model to understand this phenomenon. The mutant another longfin develops proportionally larger fins and barbels. Here, we show that another longfin mutants carry a mutation in kcnk5b, a gene coding for a potassium channel. Potassium channels control the electrical potential of cells and are known to regulate processes such as heart beat, neurotransmission and hormone secretion. We demonstrate that increased potassium channel activity can drive changes in growth in the zebrafish. Clonal analysis reveals that the channel acts directly in the fins and barbels to increase their size in a coordinated manner. Our work shows that potassium channels are involved in the determination of appendage size in zebrafish and suggests consistent with recent studies, an instructive role for bioelectrical signaling in development.
doi:10.1371/journal.pgen.1004080
PMCID: PMC3894163  PMID: 24453984
4.  Neocuproine Ablates Melanocytes in Adult Zebrafish 
Zebrafish  2008;5(4):257-264.
The simplest regeneration experiments involve the ablation of a single cell type. While methods exist to ablate the melanocytes of the larval zebrafish,1,2 no convenient method exists to ablate melanocytes in adult zebrafish. Here, we show that the copper chelator neocuproine (NCP) causes fragmentation and disappearance of melanin in adult zebrafish melanocytes. Adult melanocytes expressing eGFP under the control of a melanocyte-specific promoter also lose eGFP fluorescence in the presence of NCP. We conclude that NCP causes melanocyte death. This death is independent of p53 and melanin, but can be suppressed by the addition of exogenous copper. NCP is ineffective at ablating larval melanocytes. This now provides a tool for addressing questions about stem cells and the maintenance of the adult pigment pattern in zebrafish.
doi:10.1089/zeb.2008.0540
PMCID: PMC2765050  PMID: 19133824
5.  Small Molecule Modifier Screen for kit-Dependent Functions in Zebrafish Embryonic Melanocytes 
Zebrafish  2008;5(4):279-287.
Zebrafish is gaining popularity as a vertebrate model for screening small molecules that affect specific phenotypes or genetic pathways. In this study, we present a targeted drug screen to identify drug modifiers of the melanocyte migration defect of a temperature-sensitive allele of the Kit receptor tyrosine kinase, kitts. We first test two candidate drugs, the phosphatidylinositol-3-kinase kinase inhibitor (LY294002) and the Erk/MAP kinase inhibitor (PD98059), for their effect on melanocyte migration and survival. We find that LY294002 enhances the migration defect of kitts, implicating the phosphatidylinositol-3-kinase kinase pathway in promoting kit-dependent melanocyte migration, but not survival. We then used the kitts-sensitized genetic background to screen a panel of 1280 pharmacologically active drugs to identify drug enhancers and suppressors of the kitts melanocyte migration defect. We identified three drug enhancers of migration, two of which, Papaverine and Isoliquiritigenin, specifically enhance the kitts migration defect, while 8-DPAT affected both melanocyte migration and survival. These drugs now provide additional experimental tools for investigating the mechanisms of kit-promoted melanocyte migration and survival in the zebrafish embryo.
doi:10.1089/zeb.2008.0542
PMCID: PMC2757780  PMID: 19133826
6.  Small Molecule Modifier Screen for kit-Dependent Functions in Zebrafish Embryonic Melanocytes 
Zebrafish  2008;5(4):279-287.
Abstract
Zebrafish is gaining popularity as a vertebrate model for screening small molecules that affect specific phenotypes or genetic pathways. In this study, we present a targeted drug screen to identify drug modifiers of the melanocyte migration defect of a temperature-sensitive allele of the Kit receptor tyrosine kinase, kitts. We first test two candidate drugs, the phosphatidylinositol-3-kinase kinase inhibitor (LY294002) and the Erk/MAP kinase inhibitor (PD98059), for their effect on melanocyte migration and survival. We find that LY294002 enhances the migration defect of kitts, implicating the phosphatidylinositol-3-kinase kinase pathway in promoting kit-dependent melanocyte migration, but not survival. We then used the kitts-sensitized genetic background to screen a panel of 1280 pharmacologically active drugs to identify drug enhancers and suppressors of the kitts melanocyte migration defect. We identified three drug enhancers of migration, two of which, Papaverine and Isoliquiritigenin, specifically enhance the kitts migration defect, while 8-DPAT affected both melanocyte migration and survival. These drugs now provide additional experimental tools for investigating the mechanisms of kit-promoted melanocyte migration and survival in the zebrafish embryo.
doi:10.1089/zeb.2008.0542
PMCID: PMC2757780  PMID: 19133826
7.  Neocuproine Ablates Melanocytes in Adult Zebrafish 
Zebrafish  2008;5(4):257-264.
Abstract
The simplest regeneration experiments involve the ablation of a single cell type. While methods exist to ablate the melanocytes of the larval zebrafish,1,2 no convenient method exists to ablate melanocytes in adult zebrafish. Here, we show that the copper chelator neocuproine (NCP) causes fragmentation and disappearance of melanin in adult zebrafish melanocytes. Adult melanocytes expressing eGFP under the control of a melanocyte-specific promoter also lose eGFP fluorescence in the presence of NCP. We conclude that NCP causes melanocyte death. This death is independent of p53 and melanin, but can be suppressed by the addition of exogenous copper. NCP is ineffective at ablating larval melanocytes. This now provides a tool for addressing questions about stem cells and the maintenance of the adult pigment pattern in zebrafish.
doi:10.1089/zeb.2008.0540
PMCID: PMC2765050  PMID: 19133824
8.  Gene Expression Analysis of Zebrafish Melanocytes, Iridophores, and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin 
PLoS ONE  2013;8(7):e67801.
In order to facilitate understanding of pigment cell biology, we developed a method to concomitantly purify melanocytes, iridophores, and retinal pigmented epithelium from zebrafish, and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co-enrichment in melanocytes and retinal pigmented epithelium, as well as in melanocytes and iridophores. We found 214 genes co-enriched in melanocytes and retinal pigmented epithelium, indicating the shared functions of melanin-producing cells. We found 62 genes significantly co-enriched in melanocytes and iridophores, illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine-based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate-limiting substrate for purine synthesis, phosphoribosyl pyrophosphate, thus constituting a guanine cycle. The purification procedure and expression analysis described here, along with the accompanying transcriptome-wide expression data, provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types, and will be a useful resource for further studies of pigment cell biology.
doi:10.1371/journal.pone.0067801
PMCID: PMC3706446  PMID: 23874447
9.  Collagen IX is required for the integrity of collagen II fibrils and the regulation of vascular plexus formation in Zebrafish caudal fins 
Developmental biology  2009;332(2):360-370.
Capillary plexuses form during both vasculogenesis and angiogenesis and are remodeled into mature vessel types and patterns which are delicately orchestrated with the sizes and shapes of other tissues and organs. We isolated a zebrafish mutation named prp (for persistent plexus) that causes persistent formation of vascular plexuses in the caudal fins and consequent mispatterning of bony fin rays and the fin shape. Detailed analyses revealed that the prp mutation causes a significant reduction in the size and dramatic structural defects in collagen II-rich extracellular matrices called actinotrichia of both embryonic finfolds and adult fins. prp was mapped to chromosome 19 and found to encode the zebrafish collagen9α1 (col9α1) gene which is abundantly expressed in developing finfolds. A point mutation resulting in a leucine-to-histidine change was detected in the thrombospondin domain of the col9α1 gene in prp. Morpholino-mediated knockdown of col9α1 phenocopied the prp small-finfold phenotype in wild-type embryos, and an injection of plasmids containing the col9α1 cDNA into prp embryos locally restored the finfold size. Furthermore, we found that osteoblasts in prp mutants were mispatterned apparently following the abnormal vascular plexus pattern, demonstrating that blood vessels play an important role in the patterning of bony rays in zebrafish caudal fins.
doi:10.1016/j.ydbio.2009.06.003
PMCID: PMC3690564  PMID: 19501583
Zebrafish; Vascular plexus; Collagen IX; Actinotrichia; Fin
10.  ALDH2 Mediates 5-Nitrofuran Activity in Multiple Species 
Chemistry & biology  2012;19(7):883-892.
SUMMARY
Understanding how drugs work in vivo is critical for drug design and for maximizing the potential of currently available drugs. 5-nitrofurans are a class of prodrugs widely used to treat bacterial and trypanosome infections, but despite relative specificity, 5-nitrofurans often cause serious toxic side effects in people. Here, we use yeast and zebrafish, as well as human in vitro systems, to assess the biological activity of 5-nitrofurans, and we identify a conserved interaction between aldehyde dehydrogenase (ALDH) 2 and 5-nitrofurans across these species. In addition, we show that the activity of nifurtimox, a 5-nitrofuran anti-trypanosome prodrug, is dependent on zebrafish Aldh2 and is a substrate for human ALDH2. This study reveals a conserved and biologically relevant ALDH2-5-nitrofuran interaction that may have important implications for managing the toxicity of 5-nitrofuran treatment.
doi:10.1016/j.chembiol.2012.05.017
PMCID: PMC3684953  PMID: 22840776
11.  reg6 is required for branching morphogenesis during blood vessel regeneration in zebrafish caudal fins 
Developmental biology  2003;264(1):263-274.
Postnatal neovascularization is essential for wound healing, cancer progression, and many other physiological functions. However, its genetic mechanism is largely unknown. In this report, we study neovascularization in regenerating adult zebrafish fins using transgenic fish that express EGFP in blood vessel endothelial cells. We first describe the morphogenesis of regenerating vessels in wild-type animals and then the phenotypic analysis of a genetic mutation that disrupts blood vessel regeneration. In wild-type zebrafish caudal fins, amputated blood vessels heal their ends by 24 h postamputation (hpa) and then reconnect arteries and veins via anastomosis, to resume blood flow at wound sites by 48 hpa. The truncated vessels regenerate by first growing excess vessels to form unstructured plexuses, resembling the primary capillary plexuses formed during embryonic vasculogenesis. Interestingly, this mode of vessel growth switches by 8 days postamputation (dpa) to growth without a plexus intermediate. During blood vessel regeneration, vessel remodeling begins during early plexus formation and continues until the original vasculature pattern is reestablished at ~35 dpa. Temperature-sensitive mutants for reg6 have profound defects in blood vessel regeneration. At the restrictive temperature, reg6 regenerating blood vessels first fail to make reconnections between severed arteries and veins, and then form enlarged vascular sinuses rather than branched vascular plexuses. Reciprocal temperature-shift experiments show that reg6 function is required throughout plexus formation, but not during later growth. Our results suggest that the reg6 mutation causes defects in branch formation and/or angiogenic sprouting.
PMCID: PMC3665419  PMID: 14623247
Zebrafish; Regeneration; Angiogenesis; Plexus; Branching; Anastomosis
12.  Clonal and Lineage Analysis of Melanocyte Stem Cells and Their Progeny in the Zebrafish 
The study of melanocyte biology in the zebrafish presents a highly tractable system for understanding fundamental principles of developmental biology. Melanocytes are visible in the transparent embryo and in the mature fish following metamorphosis, a physical transformation from the larval to adult form. While early developing larval melanocytes are direct derivatives of the neural crest, the remainder of melanocytes develop from unpigmented precursors, or melanocyte stem cells (MSCs). The Tol2 transposable element has facilitated the construction of stable transgenic lines that label melanocytes. In another application, integration of Tol2 constructs makes possible clonal analysis of melanocyte and MSC lineages. Drugs that block melanin synthesis, ablate melanocytes, and block establishment of MSC populations allow the interrogation of this model system for mechanisms of adult stem cell development and regulation.
doi:10.1007/978-1-61779-980-8_14
PMCID: PMC3630497  PMID: 22914941
Melanocyte; Stem cell; Clonal analysis; Lineages; Zebrafish; Tol2
13.  Functional Assessment of Human Coding Mutations Affecting Skin Pigmentation Using Zebrafish 
PLoS ONE  2012;7(10):e47398.
A major challenge in personalized medicine is the lack of a standard way to define the functional significance of the numerous nonsynonymous, single nucleotide coding variants that are present in each human individual. To begin to address this problem, we have used pigmentation as a model polygenic trait, three common human polymorphisms thought to influence pigmentation, and the zebrafish as a model system. The approach is based on the rescue of embryonic zebrafish mutant phenotypes by “humanized” zebrafish orthologous mRNA. Two hypomorphic polymorphisms, L374F in SLC45A2, and A111T in SLC24A5, have been linked to lighter skin color in Europeans. The phenotypic effect of a second coding polymorphism in SLC45A2, E272K, is unclear. None of these polymorphisms had been tested in the context of a model organism. We have confirmed that zebrafish albino fish are mutant in slc45a2; wild-type slc45a2 mRNA rescued the albino mutant phenotype. Introduction of the L374F polymorphism into albino or the A111T polymorphism into slc24a5 (golden) abolished mRNA rescue of the respective mutant phenotypes, consistent with their known contributions to European skin color. In contrast, the E272K polymorphism had no effect on phenotypic rescue. The experimental conclusion that E272K is unlikely to affect pigmentation is consistent with a lack of correlation between this polymorphism and quantitatively measured skin color in 59 East Asian humans. A survey of mutations causing human oculocutaneous albinism yielded 257 missense mutations, 82% of which are theoretically testable in zebrafish. The developed approach may be extended to other model systems and may potentially contribute to our understanding the functional relationships between DNA sequence variation, human biology, and disease.
doi:10.1371/journal.pone.0047398
PMCID: PMC3468441  PMID: 23071798
14.  A zebrafish SKIV2L2-enhancer trap line provides a useful tool for the study of peripheral sensory circuit development 
Gene expression patterns : GEP  2011;11(7):409-414.
The zebrafish is an ideal model for elucidating the cellular and molecular mechanisms that underlie development of the peripheral nervous system. A transgenic line that selectively labels all the sensory circuits would be a valuable tool for such investigations. In this study, we describe such a line: the enhancer trap zebrafish line Tg(SKIV2L2:gfp)j1775 which expresses green fluorescent protein (gfp) in the peripheral sensory ganglia. We show that this transgene marks all peripheral ganglia and sensory nerves, beginning at the time when the neurons are first extending their processes, but does not label the efferent nerves. The trapped reporter is inserted just upstream of a previously poorly described gene: lhfpl4 on LG6. The expression pattern of this gene by in situ hybridization reveals a different, but overlapping, pattern of expression compared to that of the transgene. This pattern also does not mimic that of the gene (skiv2l2), which provided the promoter element in the construct. These findings indicate that reporter expression is not dictated by an endogenous enhancer element, but instead arises through an unknown mechanism. Regardless, this reporter line should prove to be a valuable tool in the investigation of peripheral nervous system formation in the zebrafish.
doi:10.1016/j.gep.2011.06.002
PMCID: PMC3163734  PMID: 21742057
15.  Diverse mechanisms for assembly of branchiomeric nerves 
Developmental biology  2011;357(2):305-317.
The formation of branchiomeric nerves (cranial nerves V, VII, IX and X) from their sensory, motor and glial components is poorly understood. The current model for cranial nerve formation is based on the Vth nerve, in which sensory afferents are formed first and must enter the hindbrain in order for the motor efferents to exit. Using transgenic zebrafish lines to discriminate between motor neurons, sensory neurons and peripheral glia, we show that this model does not apply to the remaining three branchiomeric nerves. For these nerves, the motor efferents form prior to the sensory afferents, and their pathfinding show no dependence on sensory axons, as ablation of cranial sensory neurons by ngn1 knockdown had no effect. In contrast, the sensory limbs of the IXth and Xth nerves (but not the Vth or VIIth) were misrouted in gli1 mutants, which lack hindbrain bmn, suggesting that the motor efferents are crucial for appropriate sensory axon projection in some branchiomeric nerves. For all four nerves, peripheral glia were the intermediate component added and had a critical role in nerve integrity but not in axon guidance, as foxd3 null mutants lacking peripheral glia exhibited defasciculation of gVII, gIX, and gX axons. The bmn efferents were unaffected in these mutants. These data demonstrate that multiple mechanisms underlie formation of the four branchiomeric nerves. For the Vth, sensory axons initiate nerve formation, for the VIIth the sensory and motor limbs are independent, and for the IXth/Xth motor axons initiate formation. In all cases the glia are patterned by the initiating set of axons and are needed to maintain axon fasciculation. These results reveal that coordinated interactions between the three neural cell types in branchiomeric nerves differ according to their axial position.
doi:10.1016/j.ydbio.2011.06.044
PMCID: PMC3164235  PMID: 21777575
zebrafish; sensory neurons; branchiomotor neurons; peripheral glia
16.  ALDH2 Mediates 5-Nitrofuran Activity in Multiple Species 
Chemistry & Biology  2012;19(7):883-892.
Summary
Understanding how drugs work in vivo is critical for drug design and for maximizing the potential of currently available drugs. 5-nitrofurans are a class of prodrugs widely used to treat bacterial and trypanosome infections, but despite relative specificity, 5-nitrofurans often cause serious toxic side effects in people. Here, we use yeast and zebrafish, as well as human in vitro systems, to assess the biological activity of 5-nitrofurans, and we identify a conserved interaction between aldehyde dehydrogenase (ALDH) 2 and 5-nitrofurans across these species. In addition, we show that the activity of nifurtimox, a 5-nitrofuran anti-trypanosome prodrug, is dependent on zebrafish Aldh2 and is a substrate for human ALDH2. This study reveals a conserved and biologically relevant ALDH2-5-nitrofuran interaction that may have important implications for managing the toxicity of 5-nitrofuran treatment.
Graphical Abstract
Highlights
► Zebrafish provide a viable assay for the biological toxicity of 5-nitrofurans ► ALDH2 inhibitors prevent 5-nitrofuran toxicity in zebrafish and yeast ► Genetic dependence on ALDH2 for 5-nitrofuran toxicity in zebrafish and yeast systems ► 5-Nitrofurans bind to and are substrates of human ALDH2
5-nitrofurans are antibiotics activated by pathogen specific enzymes, however, less is known about what happens in the host. Zhou et al. identify aldehyde dehydrogenase 2 as a 5-nitrofuran activating enzyme that has implications for managing some of the toxicity associated with 5-nitrofuran treatment.
doi:10.1016/j.chembiol.2012.05.017
PMCID: PMC3684953  PMID: 22840776
17.  Fate restriction in the growing and regenerating zebrafish fin 
Developmental cell  2011;20(5):725-732.
Summary
We use transposon-based clonal analysis to identify the lineage classes that make the adult zebrafish caudal fin. We identify 9 distinct lineage classes, including epidermis, melanocyte/xanthophore, iridophore, intra-ray glia, lateral line, osteoblast, dermal fibroblast, vascular endothelium, and resident blood. These lineage classes argue for distinct progenitors, or organ founding stem cells (FSCs), for each lineage, which retain fate restriction throughout growth of the fin. Thus, distinct FSCs exist for the 4 neuroectoderm lineages, and dermal fibroblasts are not progenitors for fin ray osteoblasts; however, artery and vein cells derive from a shared lineage in the fin. Transdifferentiation of cells or lineages in the regeneration blastema is often postulated. However, our studies of single progenitors or FSC reveal no transfating or transdifferentiation between these lineages in the regenerating fin. This result shows that, the same as in growth, lineages retain fate restriction when passed through the regeneration blastema.
doi:10.1016/j.devcel.2011.04.013
PMCID: PMC3096007  PMID: 21571228
18.  mitfa is required at multiple stages of melanocyte differentiation but not to establish the melanocyte stem cell 
Developmental biology  2010;350(2):405-413.
The mitfa gene encodes a zebrafish ortholog of the microphthalmia-associated transcription factor (Mitf) which, like its counterparts in other species, is absolutely required for development of neural crest melanocytes. In order to evaluate mitfa’s role in different stages of melanocyte development, we have identified hypomorphic alleles of mitfa, including two alleles that are temperature-sensitive for melanocyte development. Molecular analysis revealed that the mitffh53ts results from a single base pair change producing an asparagine to tyrosine amino acid substitution in the DNA-binding domain, and the mitfavc7 ts allele is a mutation in a splice donor site that reduces the level of correctly-spliced transcripts. Splicing in the mitfavc7 allele does not itself appear to be temperature-dependent. A third, hypomorphic allele, mitfaz25 results in an isoleucine to phenylalanine substitution in the first helix domain of the protein. Temperature upshift experiments with mitfafh53ts show that mitfa is required at several stages of melanocyte differentiation, including for expression of the early melanoblast marker dct, again for progression from dct expression to differentiation, and again for maintenance of dendritic form following differentiation. mitfafh53ts mutants recover melanocytes within 2–3 days when downshifted at all stages of larval development. However, when melanocyte stem cells (MSCs) are ablated by early treatment with the erbB3 inhibitor AG1478, melanocyte recovery is lost by 48 hours. This result indicates first that the MSC is established at the restrictive temperature, and that melanoblasts die or lose the ability to recover after being held at the restrictive temperature for approximately one day.
doi:10.1016/j.ydbio.2010.12.004
PMCID: PMC3040983  PMID: 21146516
melanocyte; MITF; zebrafish; neural crest
19.  Oxytocin Receptor (OXTR) Polymorphisms and Attachment in Human Infants 
Ordinary variations in human infants’ attachment behaviors – their proclivity to seek and accept comfort from caregivers – are associated with a wide range of individual differences in psychological functioning in adults. The current investigation examined variation in the oxytocin receptor (OXTR) gene as one possible source of these variations in infant attachment. One hundred seventy-six infants (77 Caucasian, 99 non-Caucasian) were classified as securely or insecurely attached based on their behavior in the Strange Situation (Ainsworth et al., 1978). The A allele of OXTR rs2254298 was associated with attachment security in the non-Caucasian infants (p < 0.005). These findings underscore the importance of oxytocin in the development of human social behavior and support its role in social stress-regulation and the development of trust.
doi:10.3389/fpsyg.2011.00200
PMCID: PMC3161247  PMID: 21904531
attachment; trust; development; oxytocin; polymorphisms
20.  Lineage Relationship of Direct-Developing Melanocytes and Melanocyte Stem Cells in the Zebrafish 
PLoS ONE  2011;6(6):e21010.
Previous research in zebrafish has demonstrated that embryonic and larval regeneration melanocytes are derived from separate lineages. The embryonic melanocytes that establish the larval pigment pattern do not require regulative melanocyte stem cell (MSC) precursors, and are termed direct-developing melanocytes. In contrast, the larval regeneration melanocytes that restore the pigment pattern after ablation develop from MSC precursors. Here, we explore whether embryonic melanocytes and MSCs share bipotent progenitors. Furthermore, we explore when fate segregation of embryonic melanocytes and MSCs occurs in zebrafish development. In order to achieve this, we develop and apply a novel lineage tracing method. We first demonstrate that Tol2-mediated genomic integration of reporter constructs from plasmids injected at the 1–2 cell stage occurs most frequently after the midblastula transition but prior to shield stage, between 3 and 6 hours post-fertilization. This previously uncharacterized timing of Tol2-mediated genomic integration establishes Tol2-mediated transposition as a means for conducting lineage tracing in zebrafish. Combining the Tol2-mediated lineage tracing strategy with a melanocyte regeneration assay previously developed in our lab, we find that embryonic melanocytes and larval regeneration melanocytes are derived from progenitors that contribute to both lineages. We estimate 50–60 such bipotent melanogenic progenitors to be present in the shield-stage embryo. Furthermore, our examination of direct-developing and MSC-restricted lineages suggests that these are segregated from bipotent precursors after the shield stage, but prior to the end of convergence and extension. Following this early fate segregation, we estimate approximately 100 embryonic melanocyte and 90 MSC-restricted lineages are generated to establish or regenerate the zebrafish larval pigment pattern, respectively. Thus, the dual strategies of direct-development and MSC-derived development are established in the early gastrula, via fate segregation of the two lineages.
doi:10.1371/journal.pone.0021010
PMCID: PMC3116864  PMID: 21698209
21.  SOX10 directly modulates ERBB3 transcription via an intronic neural crest enhancer 
Background
The ERBB3 gene is essential for the proper development of the neural crest (NC) and its derivative populations such as Schwann cells. As with all cell fate decisions, transcriptional regulatory control plays a significant role in the progressive restriction and specification of NC derived lineages during development. However, little is known about the sequences mediating transcriptional regulation of ERBB3 or the factors that bind them.
Results
In this study we identified three transcriptional enhancers at the ERBB3 locus and evaluated their regulatory potential in vitro in NC-derived cell types and in vivo in transgenic zebrafish. One enhancer, termed ERBB3_MCS6, which lies within the first intron of ERBB3, directs the highest reporter expression in vitro and also demonstrates epigenetic marks consistent with enhancer activity. We identify a consensus SOX10 binding site within ERBB3_MCS6 and demonstrate, in vitro, its necessity and sufficiency for the activity of this enhancer. Additionally, we demonstrate that transcription from the endogenous Erbb3 locus is dependent on Sox10. Further we demonstrate in vitro that Sox10 physically interacts with that ERBB3_MCS6. Consistent with its in vitro activity, we also show that ERBB3_MCS6 drives reporter expression in NC cells and a subset of its derivative lineages in vivo in zebrafish in a manner consistent with erbb3b expression. We also demonstrate, using morpholino analysis, that Sox10 is necessary for ERBB3_MCS6 expression in vivo in zebrafish.
Conclusions
Taken collectively, our data suggest that ERBB3 may be directly regulated by SOX10, and that this control may in part be facilitated by ERBB3_MCS6.
doi:10.1186/1471-213X-11-40
PMCID: PMC3124416  PMID: 21672228
22.  Differential Contribution of Direct-Developing and Stem Cell-Derived Melanocytes to the Zebrafish Larval Pigment Pattern 
Developmental biology  2009;337(2):425-431.
The extent of adult stem cell involvement in embryonic growth is often unclear, as reliable markers or assays for whether a cell is derived from an adult stem cell, such as the Melanocyte Stem Cell (MSC), are typically not available. We have previously shown that two lineages of melanocytes can contribute to the larval zebrafish pigment pattern. The embryo first develops an ontogenetic pattern that is largely composed of ErbB-independent, direct-developing melanocytes. This population can be replaced during regeneration by an ErbB-dependent MSC-derived population following melanocyte ablation. In this study, we developed a melanocyte differentiation assay, used together with drugs that ablate the MSC, to investigate whether MSC-derived melanocytes contribute to the ontogenetic pattern. We found that essentially all melanocytes that develop before 3 dpf arise from the ErbB-independent, direct-developing population. Similarly, late-developing (after 3 dpf) melanocytes of the head are also ErbB independent. In contrast, the melanocytes that develop after three days postfertilization in the lateral and dorsal stripe are sensitive to ErbB inhibitor, indicating that they are derived from the MSC. We show that melanocyte regeneration mutants kitj1e99 and skiv2l2j24e1, that are grossly normal for the overall ontogenetic pattern, also lack the MSC-derived contribution to the lateral stripe. This result suggests that the underlying regeneration defect of these mutations is a defect in MSC regulation. We suggest that the regulative functions of the MSC may serve quality control roles during larval development, in addition to its established roles in larval regeneration and growth and homeostasis in the adult.
doi:10.1016/j.ydbio.2009.11.019
PMCID: PMC2812685  PMID: 19931238
23.  A gain of function mutation causing skeletal overgrowth in the rapunzel mutant 
Developmental biology  2009;334(1):224-234.
Mechanisms that regulate the growth and form of the vertebrate skeleton are largely unknown. The zebrafish mutant rapunzel has heterozygous defects in bone development, resulting in skeletal overgrowth, thus identification of the genetic lesion underlying rapunzel might provide insight into the molecular basis of skeletogenesis. In this report, we demonstrate that the rapunzel mutant results from a missense mutation in the previously uncharacterized rpz gene. This conclusion is supported by genetic mapping, identification of a missense mutation in rapunzelc14 in a highly conserved region of the rpz gene, and suppression of the rapunzel homozygous embryonic phenotype with morpholino knockdown of rpz. In addition, rpz transcripts are identified in regions correlating with the homozygous embryonic phenotype (head, pectoral fin buds, somites and fin fold). This report provides the first gene identification for a mutation affecting segment number in the zebrafish fin and development of both the fin ray (dermal) and the axial skeleton.
doi:10.1016/j.ydbio.2009.07.025
PMCID: PMC2756807  PMID: 19632218
zebrafish; fin; bone; skeleton; growth; overgrowth
24.  Melanocyte regeneration reveals mechanisms of adult stem cell regulation 
Utilization of adult stem cells in regenerative therapies may require a thorough understanding of the mechanisms that establish, recruit and renew the stem cell, promote the differentiation of its daughters, or how the stem cell is repressed by its target tissue. Regeneration of melanocytes in the regenerating zebrafish caudal fin, or following larval melanocyte-specific ablation, or recruitment of new melanocytes during pigment pattern metamorphosis each provide evidence for melanocyte stem cells (MSCs) that support the melanocyte pigment pattern. We discuss the mechanisms of MSC regulation provided from analysis of normal or mutant regeneration in each of these systems, including the implications drawn from evidence that regeneration does not simply recapitulate ontogenetic development. These results suggest that analysis of melanocyte regeneration in zebrafish will provide a fine scale dissection of mechanisms establishing or regulating adult stem cells.
doi:10.1016/j.semcdb.2008.09.007
PMCID: PMC2677296  PMID: 18950723
Regeneration; Melanocyte Stem Cell (MSC); zebrafish; kit
25.  Basonuclin-2 Requirements for Zebrafish Adult Pigment Pattern Development and Female Fertility 
PLoS Genetics  2009;5(11):e1000744.
Relatively little is known about the generation of adult form. One complex adult trait that is particularly amenable to genetic and experimental analysis is the zebrafish pigment pattern, which undergoes extensive remodeling during post-embryonic development to form adult stripes. These stripes result from the arrangement of three classes of neural crest-derived pigment cells, or chromatophores: melanophores, xanthophores, and iridophores. Here, we analyze the zebrafish bonaparte mutant, which has a normal early pigment pattern but exhibits a severe disruption to the adult stripe pattern. We show that the bonaparte mutant phenotype arises from mutations in basonuclin-2 (bnc2), encoding a highly conserved, nuclear-localized zinc finger protein of unknown function. We show that bnc2 acts non-autonomously to the melanophore lineage and is expressed by hypodermal cells adjacent to chromatophores during adult pigment pattern formation. In bonaparte (bnc2) mutants, all three types of chromatophores differentiate but then are lost by extrusion through the skin. We further show that while bnc2 promotes the development of two genetically distinct populations of melanophores in the body stripes, chromatophores of the fins and scales remain unaffected in bonaparte mutants, though a requirement of fin chromatophores for bnc2 is revealed in the absence of kit and colony stimulating factor-1 receptor activity. Finally, we find that bonaparte (bnc2) mutants exhibit dysmorphic ovaries correlating with infertility and bnc2 is expressed in somatic ovarian cells, whereas the related gene, bnc1, is expressed within oocytes; and we find that both bnc2 and bnc1 are expressed abundantly within the central nervous system. These findings identify bnc2 as an important mediator of adult pigment pattern formation and identify bonaparte mutants as an animal model for dissecting bnc2 functions.
Author Summary
The pigment patterns of animals are some of the most distinctive traits and serve as useful models for understanding the development of adult form more generally. In zebrafish, horizontal stripes result from the arrangements of several classes of pigment cells. Here, we used a mutational approach to identify a critical new gene required for stripe development, basonuclin-2, which encodes a highly conserved zinc finger protein that localizes to the nucleus. In contrast to other genes known in this species so far, which act within the pigment cells during pattern formation, we show that basonuclin-2 is expressed and functions in the extracellular environment in which the pigment cells reside. Without basonuclin-2, pigment cells die and, literally, fall off the fish. However, the effects of basonuclin-2 also extend beyond pigmentation, as female mutants have ovarian defects and are infertile, and the gene is expressed widely in the central nervous system, hinting at functions there as well. Our study thus reveals a critical component of the “canvas” on which these stunning pigment patterns are painted, and provides a new model for dissecting basonuclin-2 roles in the development of adult form and function.
doi:10.1371/journal.pgen.1000744
PMCID: PMC2776513  PMID: 19956727

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