Publications by authors named "Stephen L Johnson"

77 Publications

Isotope tracing in adult zebrafish reveals alanine cycling between melanoma and liver.

Cell Metab 2021 May 11. Epub 2021 May 11.

Department of Chemistry, Washington University in St. Louis, St. Louis, MO, USA; Department of Medicine, Washington University in St. Louis, St. Louis, MO, USA; Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO, USA. Electronic address:

The cell-intrinsic nature of tumor metabolism has become increasingly well characterized. The impact that tumors have on systemic metabolism, however, has received less attention. Here, we used adult zebrafish harboring BRAF-driven melanoma to study the effect of cancer on distant tissues. By applying metabolomics and isotope tracing, we found that melanoma consume ~15 times more glucose than other tissues measured. Despite this burden, circulating glucose levels were maintained in disease animals by a tumor-liver alanine cycle. Excretion of glucose-derived alanine from tumors provided a source of carbon for hepatic gluconeogenesis and allowed tumors to remove excess nitrogen from branched-chain amino acid catabolism, which we found to be activated in zebrafish and human melanoma. Pharmacological inhibition of the tumor-liver alanine cycle in zebrafish reduced tumor burden. Our findings underscore the significance of metabolic crosstalk between tumors and distant tissues and establish the adult zebrafish as an attractive model to study such processes.
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http://dx.doi.org/10.1016/j.cmet.2021.04.014DOI Listing
May 2021

Cellular diversity of the regenerating caudal fin.

Sci Adv 2020 Aug 12;6(33):eaba2084. Epub 2020 Aug 12.

Department of Genetics, Washington University School of Medicine, St. Louis, MO 63108, USA.

Zebrafish faithfully regenerate their caudal fin after amputation. During this process, both differentiated cells and resident progenitors migrate to the wound site and undergo lineage-restricted, programmed cellular state transitions to populate the new regenerate. Until now, systematic characterizations of cells comprising the new regenerate and molecular definitions of their state transitions have been lacking. We hereby characterize the dynamics of gene regulatory programs during fin regeneration by creating single-cell transcriptome maps of both preinjury and regenerating fin tissues at 1/2/4 days post-amputation. We consistently identified epithelial, mesenchymal, and hematopoietic populations across all stages. We found common and cell type-specific cell cycle programs associated with proliferation. In addition to defining the processes of epithelial replenishment and mesenchymal differentiation, we also identified molecular signatures that could better distinguish epithelial and mesenchymal subpopulations in fish. The insights for natural cell state transitions during regeneration point to new directions for studying this regeneration model.
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http://dx.doi.org/10.1126/sciadv.aba2084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423392PMC
August 2020

Regenerating zebrafish fin epigenome is characterized by stable lineage-specific DNA methylation and dynamic chromatin accessibility.

Genome Biol 2020 02 27;21(1):52. Epub 2020 Feb 27.

Department of Genetics, Washington University School of Medicine, St. Louis, MO, 63110, USA.

Background: Zebrafish can faithfully regenerate injured fins through the formation of a blastema, a mass of proliferative cells that can grow and develop into the lost body part. After amputation, various cell types contribute to blastema formation, where each cell type retains fate restriction and exclusively contributes to regeneration of its own lineage. Epigenetic changes that are associated with lineage restriction during regeneration remain underexplored.

Results: We produce epigenome maps, including DNA methylation and chromatin accessibility, as well as transcriptomes, of osteoblasts and other cells in uninjured and regenerating fins. This effort reveals regeneration as a process of highly dynamic and orchestrated transcriptomic and chromatin accessibility changes, coupled with stably maintained lineage-specific DNA methylation. The epigenetic signatures also reveal many novel regeneration-specific enhancers, which are experimentally validated. Regulatory networks important for regeneration are constructed through integrative analysis of the epigenome map, and a knockout of a predicted upstream regulator disrupts normal regeneration, validating our prediction.

Conclusion: Our study shows that lineage-specific DNA methylation signatures are stably maintained during regeneration, and regeneration enhancers are preset as hypomethylated before injury. In contrast, chromatin accessibility is dynamically changed during regeneration. Many enhancers driving regeneration gene expression as well as upstream regulators of regeneration are identified and validated through integrative epigenome analysis.
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http://dx.doi.org/10.1186/s13059-020-1948-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7047409PMC
February 2020

GABA-A receptor and mitochondrial TSPO signaling act in parallel to regulate melanocyte stem cell quiescence in larval zebrafish.

Pigment Cell Melanoma Res 2020 05 11;33(3):416-425. Epub 2019 Nov 11.

Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA.

Tissue regeneration and homeostasis often require recruitment of undifferentiated precursors (adult stem cells; ASCs). While many ASCs continuously proliferate throughout the lifetime of an organism, others are recruited from a quiescent state to replenish their target tissue. A long-standing question in stem cell biology concerns how long-lived, non-dividing ASCs regulate the transition between quiescence and proliferation. We study the melanocyte stem cell (MSC) to investigate the molecular pathways that regulate ASC quiescence. Our prior work indicated that GABA-A receptor activation promotes MSC quiescence in larval zebrafish. Here, through pharmacological and genetic approaches we show that GABA-A acts through calcium signaling to maintain MSC quiescence. Unexpectedly, we identified translocator protein (TSPO), a mitochondrial membrane-associated protein that regulates mitochondrial function and metabolic homeostasis, as a parallel regulator of MSC quiescence. We found that both TSPO-specific ligands and induction of gluconeogenesis likely act in the same pathway to promote MSC activation and melanocyte production in larval zebrafish. In contrast, TSPO and gluconeogenesis appear to act in parallel to GABA-A receptor signaling to regulate MSC quiescence and vertebrate pigment patterning.
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http://dx.doi.org/10.1111/pcmr.12836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176537PMC
May 2020

Maintenance of Melanocyte Stem Cell Quiescence by GABA-A Signaling in Larval Zebrafish.

Genetics 2019 10 23;213(2):555-566. Epub 2019 Aug 23.

Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110

In larval zebrafish, melanocyte stem cells (MSCs) are quiescent, but can be recruited to regenerate the larval pigment pattern following melanocyte ablation. Through pharmacological experiments, we found that inhibition of γ-aminobutyric acid (GABA)-A receptor function, specifically the GABA-A ρ subtype, induces excessive melanocyte production in larval zebrafish. Conversely, pharmacological activation of GABA-A inhibited melanocyte regeneration. We used clustered regularly interspaced short palindromic repeats/Cas9 to generate two mutant alleles of , a subtype of GABA-A receptors. Both alleles exhibited robust melanocyte overproduction, while conditional overexpression of inhibited larval melanocyte regeneration. Our data suggest that signaling is necessary to maintain MSC quiescence and sufficient to reduce, but not eliminate, melanocyte regeneration in larval zebrafish.
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http://dx.doi.org/10.1534/genetics.119.302416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781893PMC
October 2019

Trace Phosphate Improves ZIC-pHILIC Peak Shape, Sensitivity, and Coverage for Untargeted Metabolomics.

J Proteome Res 2018 10 25;17(10):3537-3546. Epub 2018 Sep 25.

Department of Chemistry , Washington University in St. Louis , St. Louis , MO 63130 , United States.

Existing hydrophilic interaction liquid chromatography (HILIC) methods, considered individually, each exhibit poor chromatographic performance for a substantial fraction of polar metabolites. In addition to limiting metabolome coverage, such deficiencies also complicate automated data processing. Here we show that some of these analytical challenges can be addressed for the ZIC-pHILIC, a zwitterionic stationary phase commonly used in metabolomics, with the addition of trace levels of phosphate. Specifically, micromolar phosphate extended metabolome coverage by hundreds of credentialed features, improved peak shapes, and reduced peak-detection errors during informatic processing. Although the addition of high levels of phosphate (millimolar) as a HILIC mobile phase buffer has been explored previously, such concentrations interfere with mass spectrometric (MS) detection. We show that using phosphate as a trace additive at micromolar concentrations improves analysis by electrospray MS, increasing signal for a diverse set of polar standards. Given the small amount of phosphate needed, comparable chromatographic improvements were also achieved by direct addition of phosphate to the sample during reconstitution. Our results suggest that defects in ZIC-pHILIC performance are predominantly driven by electrostatic interactions, which can be modulated by phosphate. These findings constitute both a methodological improvement for untargeted metabolomics and an advance in our understanding of the mechanisms limiting HILIC coverage.
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http://dx.doi.org/10.1021/acs.jproteome.8b00487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6427830PMC
October 2018

Bioelectric-calcineurin signaling module regulates allometric growth and size of the zebrafish fin.

Sci Rep 2018 Jul 10;8(1):10391. Epub 2018 Jul 10.

Department of Genetics, Harvard Medical School; Department of Orthopedic Research, Boston Children's Hospital, 300 Longwood Avenue, Boston, MA, 02115, USA.

The establishment of relative size of organs and structures is paramount for attaining final form and function of an organism. Importantly, variation in the proportions of structures frequently underlies adaptive change in morphology in evolution and maybe a common mechanism underlying selection. However, the mechanism by which growth is integrated within tissues during development to achieve proper proportionality is poorly understood. We have shown that signaling by potassium channels mediates coordinated size regulation in zebrafish fins. Recently, calcineurin inhibitors were shown to elicit changes in zebrafish fin allometry as well. Here, we identify the potassium channel kcnk5b as a key player in integrating calcineurin's growth effects, in part through regulation of the cytoplasmic C-terminus of the channel. We propose that the interaction between Kcnk5b and calcineurin acts as a signaling node to regulate allometric growth. Importantly, we find that this regulation is epistatic to inherent mechanisms instructing overall size as inhibition of calcineurin is able to bypass genetic instruction of size as seen in sof and wild-type fins, however, it is not sufficient to re-specify positional memory of size of the fin. These findings integrate classic signaling mediators such as calcineurin with ion channel function in the regulation of size and proportion during growth.
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http://dx.doi.org/10.1038/s41598-018-28450-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039437PMC
July 2018

Two complementary reversed-phase separations for comprehensive coverage of the semipolar and nonpolar metabolome.

Anal Bioanal Chem 2018 Feb 18;410(4):1287-1297. Epub 2017 Dec 18.

Department of Chemistry, Washington University in St. Louis, 1 Brookings Dr, St. Louis, MO, 63130, USA.

Although it is common in untargeted metabolomics to apply reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) methods that have been systematically optimized for lipids and central carbon metabolites, here we show that these established protocols provide poor coverage of semipolar metabolites because of inadequate retention. Our objective was to develop an RPLC approach that improved detection of these metabolites without sacrificing lipid coverage. We initially evaluated columns recently released by Waters under the CORTECS line by analyzing 47 small-molecule standards that evenly span the nonpolar and semipolar ranges. An RPLC method commonly used in untargeted metabolomics was considered a benchmarking reference. We found that highly nonpolar and semipolar metabolites cannot be reliably profiled with any single method because of retention and solubility limitations of the injection solvent. Instead, we optimized a multiplexed approach using the CORTECS T3 column to analyze semipolar compounds and the CORTECS C column to analyze lipids. Strikingly, we determined that combining these methods allowed detection of 41 of the total 47 standards, whereas our reference RPLC method detected only 10 of the 47 standards. We then applied credentialing to compare method performance at the comprehensive scale. The tandem method showed more than a fivefold increase in credentialing coverage relative to our RPLC benchmark. Our results demonstrate that comprehensive coverage of metabolites amenable to reversed-phase separation necessitates two reconstitution solvents and chromatographic methods. Thus, we suggest complementing HILIC methods with a dual T3 and C RPLC approach to increase coverage of semipolar metabolites and lipids for untargeted metabolomics. Graphical abstract Analysis of semipolar and nonpolar metabolites necessitates two reversed-phase chromatography (RPLC) methods, which extend metabolome coverage more than fivefold for untargeted profiling. HILIC hydrophilic interaction liquid chromatography.
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http://dx.doi.org/10.1007/s00216-017-0768-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5776055PMC
February 2018

Dynein/dynactin is necessary for anterograde transport of mRNA in oligodendrocytes and for myelination in vivo.

Proc Natl Acad Sci U S A 2017 10 12;114(43):E9153-E9162. Epub 2017 Oct 12.

Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110;

Oligodendrocytes in the central nervous system produce myelin, a lipid-rich, multilamellar sheath that surrounds axons and promotes the rapid propagation of action potentials. A critical component of myelin is myelin basic protein (MBP), expression of which requires anterograde mRNA transport followed by local translation at the developing myelin sheath. Although the anterograde motor kinesin KIF1B is involved in mRNA transport in zebrafish, it is not entirely clear how transport is regulated. From a forward genetic screen for myelination defects in zebrafish, we identified a mutation in , which encodes the Arp11 subunit of dynactin, a critical activator of the retrograde motor dynein. Both the mutation and pharmacological dynein inhibition in zebrafish result in failure to properly distribute mRNA in oligodendrocytes, indicating a paradoxical role for the retrograde dynein/dynactin complex in anterograde mRNA transport. To address the molecular mechanism underlying this observation, we biochemically isolated reporter-tagged mRNA granules from primary cultured mammalian oligodendrocytes to show that they indeed associate with the retrograde motor complex. Next, we used live-cell imaging to show that acute pharmacological dynein inhibition quickly arrests mRNA transport in both directions. Chronic pharmacological dynein inhibition also abrogates mRNA distribution and dramatically decreases MBP protein levels. Thus, these cell culture and whole animal studies demonstrate a role for the retrograde dynein/dynactin motor complex in anterograde mRNA transport and myelination in vivo.
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http://dx.doi.org/10.1073/pnas.1711088114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664533PMC
October 2017

Whole Genome Sequencing-Based Mapping and Candidate Identification of Mutations from Fixed Zebrafish Tissue.

G3 (Bethesda) 2017 10 5;7(10):3415-3425. Epub 2017 Oct 5.

Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri 63110

As forward genetic screens in zebrafish become more common, the number of mutants that cannot be identified by gross morphology or through transgenic approaches, such as many nervous system defects, has also increased. Screening for these difficult-to-visualize phenotypes demands techniques such as whole-mount hybridization (WISH) or antibody staining, which require tissue fixation. To date, fixed tissue has not been amenable for generating libraries for whole genome sequencing (WGS). Here, we describe a method for using genomic DNA from fixed tissue and a bioinformatics suite for WGS-based mapping of zebrafish mutants. We tested our protocol using two known zebrafish mutant alleles, and , both of which cause myelin defects. As further proof of concept we mapped a novel mutation, , identified in a zebrafish WISH screen for myelination defects. We linked to chromosome 1 and identified a candidate nonsense mutation in the () gene. Importantly, mutants phenocopy previously described mutants, and knockdown of in wild-type animals produced similar defects, demonstrating that disrupts Together, these data show that our mapping protocol can map and identify causative lesions in mutant screens that require tissue fixation for phenotypic analysis.
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http://dx.doi.org/10.1534/g3.117.300212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633390PMC
October 2017

Cx43 suppresses evx1 expression to regulate joint initiation in the regenerating fin.

Dev Dyn 2017 09 12;246(9):691-699. Epub 2017 Jul 12.

Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania.

Background: How joints are correctly positioned in the vertebrate skeleton remains poorly understood. From our studies on the regenerating fin, we have evidence that the gap junction protein Cx43 suppresses joint formation by suppressing the expression of the evx1 transcription factor. Joint morphogenesis proceeds through at least two discrete stages. First, cells that will produce the joint condense in a single row on the bone matrix ("initiation"). Second, these cells separate coincident with articulation of the bone matrix. We propose that Cx43 activity is transiently reduced prior to joint initiation.

Results: We first define the timing of joint initiation with respect to regeneration. We next correlate reduced cx43 expression and increased evx1 expression with initiation. Through manipulation of cx43 expression, we demonstrate that Cx43 negatively influences evx1 expression and joint formation. We further demonstrate that Cx43 activity in the dermal fibroblasts is required to rescue joint formation in the cx43 mutant, short fin .

Conclusions: We conclude that Cx43 activity in the dermal fibroblasts influences the expression of evx1, and therefore the differentiation of the precursor cells that give rise to the joint-forming osteoblasts. Developmental Dynamics 246:691-699, 2017. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/dvdy.24531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5548607PMC
September 2017

Three-Dimensional Printed Fish Graders: A Tool to Rapidly and Reliably Size Select Zebrafish.

Zebrafish 2017 06 13;14(3):280-283. Epub 2017 Mar 13.

Department of Genetics, Washington University in St. Louis , St. Louis, Missouri.

Research into adult zebrafish often requires fish to be of a specific size. Currently, fish must be individually measured to achieve this goal. Here, we design and utilize fish graders to quickly sort fish by width. We characterize graders individually for the length of fish they discriminate between and we also analyze graders in pairs to define the range of lengths for a retained population of fish. We note that a 1 mm increase of fish width increases fish length by 6.2-7.2 mm. We provide the schematics to print a series of eight retention widths, and note that graders of any desired retention width can easily be printed by slightly modifying our design files.
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http://dx.doi.org/10.1089/zeb.2016.1414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5466055PMC
June 2017

Lactate metabolism is associated with mammalian mitochondria.

Nat Chem Biol 2016 Nov 12;12(11):937-943. Epub 2016 Sep 12.

Department of Chemistry, Washington University, St. Louis, Missouri, USA.

It is well established that lactate secreted by fermenting cells can be oxidized or used as a gluconeogenic substrate by other cells and tissues. It is generally assumed, however, that within the fermenting cell itself, lactate is produced to replenish NAD and then is secreted. Here we explore the possibility that cytosolic lactate is metabolized by the mitochondria of fermenting mammalian cells. We found that fermenting HeLa and H460 cells utilize exogenous lactate carbon to synthesize a large percentage of their lipids. Using high-resolution mass spectrometry, we found that both C and 2-H labels from enriched lactate enter the mitochondria. The lactate dehydrogenase (LDH) inhibitor oxamate decreased respiration of isolated mitochondria incubated in lactate, but not of isolated mitochondria incubated in pyruvate. Additionally, transmission electron microscopy (TEM) showed that LDHB localizes to the mitochondria. Taken together, our results demonstrate a link between lactate metabolism and the mitochondria of fermenting mammalian cells.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5069139PMC
http://dx.doi.org/10.1038/nchembio.2172DOI Listing
November 2016

Exogenous Fatty Acids Are the Preferred Source of Membrane Lipids in Proliferating Fibroblasts.

Cell Chem Biol 2016 04 31;23(4):483-93. Epub 2016 Mar 31.

Department of Chemistry, Washington University, St. Louis, MO 63130, USA; Department of Medicine, Washington University, St. Louis, MO 63110, USA. Electronic address:

Cellular proliferation requires the formation of new membranes. It is often assumed that the lipids needed for these membranes are synthesized mostly de novo. Here, we show that proliferating fibroblasts prefer to take up palmitate from the extracellular environment over synthesizing it de novo. Relative to quiescent fibroblasts, proliferating fibroblasts increase their uptake of palmitate, decrease fatty acid degradation, and instead direct more palmitate to membrane lipids. When exogenous palmitate is provided in the culture media at physiological concentrations, de novo synthesis accounts for only a minor fraction of intracellular palmitate in proliferating fibroblasts as well as proliferating HeLa and H460 cells. Blocking fatty acid uptake decreased the proliferation rate of fibroblasts, HeLa, and H460 cells, while supplementing media with exogenous palmitate resulted in decreased glucose uptake and rendered cells less sensitive to glycolytic inhibition. Our results suggest that cells scavenging exogenous lipids may be less susceptible to drugs targeting glycolysis and de novo lipid synthesis.
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http://dx.doi.org/10.1016/j.chembiol.2016.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5510604PMC
April 2016

Bar Coding MS(2) Spectra for Metabolite Identification.

Anal Chem 2016 Mar 11;88(5):2538-42. Epub 2016 Feb 11.

Department of Chemistry, Washington University in St. Louis , St. Louis, Missouri 63130, United States.

Metabolite identifications are most frequently achieved in untargeted metabolomics by matching precursor mass and full, high-resolution MS(2) spectra to metabolite databases and standards. Here we considered an alternative approach for establishing metabolite identifications that does not rely on full, high-resolution MS(2) spectra. First, we select mass-to-charge regions containing the most informative metabolite fragments and designate them as bins. We then translate each metabolite fragmentation pattern into a binary code by assigning 1's to bins containing fragments and 0's to bins without fragments. With 20 bins, this binary-code system is capable of distinguishing 96% of the compounds in the METLIN MS(2) library. A major advantage of the approach is that it extends untargeted metabolomics to low-resolution triple quadrupole (QqQ) instruments, which are typically less expensive and more robust than other types of mass spectrometers. We demonstrate a method of acquiring MS(2) data in which the third quadrupole of a QqQ instrument cycles over 20 wide isolation windows (coinciding with the location and width of our bins) for each precursor mass selected by the first quadrupole. Operating the QqQ instrument in this mode yields diagnostic bar codes for each precursor mass that can be matched to the bar codes of metabolite standards. Furthermore, our data suggest that using low-resolution bar codes enables QqQ instruments to make MS(2)-based identifications in untargeted metabolomics with a specificity and sensitivity that is competitive to high-resolution time-of-flight technologies.
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http://dx.doi.org/10.1021/acs.analchem.5b04925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869618PMC
March 2016

Ibuprofen slows migration and inhibits bowel colonization by enteric nervous system precursors in zebrafish, chick and mouse.

Dev Biol 2016 Jan 14;409(2):473-88. Epub 2015 Nov 14.

Department of Pediatrics, The Children's Hospital of Philadelphia Research Institute and the Perelman School of Medicine at the University of Pennsylvania, Abramson Research Center, 3615 Civic Center Blvd, Philadelphia, PA 19104, USA. Electronic address:

Hirschsprung Disease (HSCR) is a potentially deadly birth defect characterized by the absence of the enteric nervous system (ENS) in distal bowel. Although HSCR has clear genetic causes, no HSCR-associated mutation is 100% penetrant, suggesting gene-gene and gene-environment interactions determine HSCR occurrence. To test the hypothesis that certain medicines might alter HSCR risk we treated zebrafish with medications commonly used during early human pregnancy and discovered that ibuprofen caused HSCR-like absence of enteric neurons in distal bowel. Using fetal CF-1 mouse gut slice cultures, we found that ibuprofen treated enteric neural crest-derived cells (ENCDC) had reduced migration, fewer lamellipodia and lower levels of active RAC1/CDC42. Additionally, inhibiting ROCK, a RHOA effector and known RAC1 antagonist, reversed ibuprofen effects on migrating mouse ENCDC in culture. Ibuprofen also inhibited colonization of Ret+/- mouse bowel by ENCDC in vivo and dramatically reduced bowel colonization by chick ENCDC in culture. Interestingly, ibuprofen did not affect ENCDC migration until after at least three hours of exposure. Furthermore, mice deficient in Ptgs1 (COX 1) and Ptgs2 (COX 2) had normal bowel colonization by ENCDC and normal ENCDC migration in vitro suggesting COX-independent effects. Consistent with selective and strain specific effects on ENCDC, ibuprofen did not affect migration of gut mesenchymal cells, NIH3T3, or WT C57BL/6 ENCDC, and did not affect dorsal root ganglion cell precursor migration in zebrafish. Thus, ibuprofen inhibits ENCDC migration in vitro and bowel colonization by ENCDC in vivo in zebrafish, mouse and chick, but there are cell type and strain specific responses. These data raise concern that ibuprofen may increase Hirschsprung disease risk in some genetically susceptible children.
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http://dx.doi.org/10.1016/j.ydbio.2015.09.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862364PMC
January 2016

Cyp27c1 Red-Shifts the Spectral Sensitivity of Photoreceptors by Converting Vitamin A1 into A2.

Curr Biol 2015 Dec 5;25(23):3048-57. Epub 2015 Nov 5.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

Some vertebrate species have evolved means of extending their visual sensitivity beyond the range of human vision. One mechanism of enhancing sensitivity to long-wavelength light is to replace the 11-cis retinal chromophore in photopigments with 11-cis 3,4-didehydroretinal. Despite over a century of research on this topic, the enzymatic basis of this perceptual switch remains unknown. Here, we show that a cytochrome P450 family member, Cyp27c1, mediates this switch by converting vitamin A1 (the precursor of 11-cis retinal) into vitamin A2 (the precursor of 11-cis 3,4-didehydroretinal). Knockout of cyp27c1 in zebrafish abrogates production of vitamin A2, eliminating the animal's ability to red-shift its photoreceptor spectral sensitivity and reducing its ability to see and respond to near-infrared light. Thus, the expression of a single enzyme mediates dynamic spectral tuning of the entire visual system by controlling the balance of vitamin A1 and A2 in the eye.
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http://dx.doi.org/10.1016/j.cub.2015.10.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910640PMC
December 2015

Developmental enhancers revealed by extensive DNA methylome maps of zebrafish early embryos.

Nat Commun 2015 Feb 20;6:6315. Epub 2015 Feb 20.

1] Department of Genetics, Washington University School of Medicine, St Louis, Missouri 63108, USA [2] Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St Louis, Missouri 63108, USA.

DNA methylation undergoes dynamic changes during development and cell differentiation. Recent genome-wide studies discovered that tissue-specific differentially methylated regions (DMRs) often overlap tissue-specific distal cis-regulatory elements. However, developmental DNA methylation dynamics of the majority of the genomic CpGs outside gene promoters and CpG islands has not been extensively characterized. Here, we generate and compare comprehensive DNA methylome maps of zebrafish developing embryos. From these maps, we identify thousands of developmental stage-specific DMRs (dsDMRs) across zebrafish developmental stages. The dsDMRs contain evolutionarily conserved sequences, are associated with developmental genes and are marked with active enhancer histone posttranslational modifications. Their methylation pattern correlates much stronger than promoter methylation with expression of putative target genes. When tested in vivo using a transgenic zebrafish assay, 20 out of 20 selected candidate dsDMRs exhibit functional enhancer activities. Our data suggest that developmental enhancers are a major target of DNA methylation changes during embryogenesis.
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http://dx.doi.org/10.1038/ncomms7315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4339225PMC
February 2015

Temperature-sensitive splicing of mitfa by an intron mutation in zebrafish.

Pigment Cell Melanoma Res 2015 Mar 29;28(2):229-32. Epub 2014 Dec 29.

MRC IGMM, MRC Human Genetics Unit, University of Edinburgh Cancer Research UK Centre, Edinburgh, UK.

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http://dx.doi.org/10.1111/pcmr.12336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4333074PMC
March 2015

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 10;9(7):e102317. Epub 2014 Jul 10.

Washington University School of Medicine, Department of Genetics, St. Louis, Missouri, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102317PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092134PMC
November 2015

After the feature presentation: technologies bridging untargeted metabolomics and biology.

Curr Opin Biotechnol 2014 Aug 6;28:143-8. Epub 2014 May 6.

Department of Genetics, Washington University School of Medicine, St. Louis, MO, United States; Department of Chemistry, Washington University, St. Louis, MO, United States; Department of Medicine, Washington University School of Medicine, St. Louis, MO, United States. Electronic address:

Liquid chromatography/mass spectrometry-based untargeted metabolomics is now an established experimental approach that is being broadly applied by many laboratories worldwide. Interpreting untargeted metabolomic data, however, remains a challenge and limits the translation of results into biologically relevant conclusions. Here we review emerging technologies that can be applied after untargeted profiling to extend biological interpretation of metabolomic data. These technologies include advances in bioinformatic software that enable identification of isotopes and adducts, comprehensive pathway mapping, deconvolution of MS(2) data, and tracking of isotopically labeled compounds. There are also opportunities to gain additional biological insight by complementing the metabolomic analysis of homogenized samples with recently developed technologies for metabolite imaging of intact tissues. To maximize the value of these emerging technologies, a unified workflow is discussed that builds on the traditional untargeted metabolomic pipeline. Particularly when integrated together, the combination of the advances highlighted in this review helps transform lists of masses and fold changes characteristic of untargeted profiling results into structures, absolute concentrations, pathway fluxes, and localization patterns that are typically needed to understand biology.
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http://dx.doi.org/10.1016/j.copbio.2014.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4111999PMC
August 2014

Bioelectric signaling regulates size in zebrafish fins.

PLoS Genet 2014 Jan 16;10(1):e1004080. Epub 2014 Jan 16.

Orthopedic Research Laboratories, Boston Children's Hospital; Department of Genetics, Harvard Medical School, Enders, Massachusetts, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.1004080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3894163PMC
January 2014

Loss of col8a1a function during zebrafish embryogenesis results in congenital vertebral malformations.

Dev Biol 2014 Feb 11;386(1):72-85. Epub 2013 Dec 11.

Department of Developmental Biology, Washington University School of Medicine, Saint Louis, MO 63110, USA; Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.

Congenital vertebral malformations (CVM) occur in 1 in 1000 live births and in many cases can cause spinal deformities, such as scoliosis, and result in disability and distress of affected individuals. Many severe forms of the disease, such as spondylocostal dystostosis, are recessive monogenic traits affecting somitogenesis, however the etiologies of the majority of CVM cases remain undetermined. Here we demonstrate that morphological defects of the notochord in zebrafish can generate congenital-type spine defects. We characterize three recessive zebrafish leviathan/col8a1a mutant alleles ((m531, vu41, vu105)) that disrupt collagen type VIII alpha1a (col8a1a), and cause folding of the embryonic notochord and consequently adult vertebral column malformations. Furthermore, we provide evidence that a transient loss of col8a1a function or inhibition of Lysyl oxidases with drugs during embryogenesis was sufficient to generate vertebral fusions and scoliosis in the adult spine. Using periodic imaging of individual zebrafish, we correlate focal notochord defects of the embryo with vertebral malformations (VM) in the adult. Finally, we show that bends and kinks in the notochord can lead to aberrant apposition of osteoblasts normally confined to well-segmented areas of the developing vertebral bodies. Our results afford a novel mechanism for the formation of VM, independent of defects of somitogenesis, resulting from aberrant bone deposition at regions of misshapen notochord tissue.
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http://dx.doi.org/10.1016/j.ydbio.2013.11.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3938106PMC
February 2014

Novel role for carbamoyl phosphate synthetase 2 in cranial sensory circuit formation.

Int J Dev Neurosci 2014 Apr 23;33:41-8. Epub 2013 Nov 23.

Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, 1402 S. Grand Blvd, St. Louis, MO 63104, USA. Electronic address:

In zebrafish, cranial sensory circuits form by 4 days post-fertilization. We used a forward genetic screen to identify genes involved in the formation of these circuits. In one mutant allele, sl23, axons arising from the epibranchial sensory ganglia do not form their stereotypical terminal fields in the hindbrain. These embryos also had small eyes and deformed jaws, suggesting a pleiotropic effect. Using positional cloning, a 20-nucleotide deletion in the carbamoyl-phosphate-synthetase2-aspartate-transcarbamylase-dihydroorotase (cad) gene was found. Injection of a CAD morpholino phenocopied the mutant and mutants were rescued by injection of cad RNA. Cad activity is required for pyrimidine biosynthesis, and thus is a prerequisite for nucleic acid production and UDP-dependent protein glycosylation. Perturbation of nucleic acid biosynthesis can result in cell death. sl23 mutants did not exhibit elevated cell death, or gross morphological changes, in their hindbrains. To determine if defective protein glycosylation was involved in the aberrant targeting of sensory axons, we treated wild type embryos with tunicamycin, which blocks N-linked protein glycosylation. Interference with glycosylation via tunicamycin treatment mimicked the sl23 phenotype. Loss of cad reveals a critical role for protein glycosylation in cranial sensory circuit formation.
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http://dx.doi.org/10.1016/j.ijdevneu.2013.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3944931PMC
April 2014

Gene expression analysis of zebrafish melanocytes, iridophores, and retinal pigmented epithelium reveals indicators of biological function and developmental origin.

PLoS One 2013 9;8(7):e67801. Epub 2013 Jul 9.

Department of Genetics, Washington University, St. Louis, Missouri, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0067801PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706446PMC
February 2014

Kit signaling is involved in melanocyte stem cell fate decisions in zebrafish embryos.

Development 2013 Mar 30;140(5):996-1002. Epub 2013 Jan 30.

Department of Genetics, Washington University Medical School, St Louis, MO 63130, USA.

Adult stem cells are crucial for growth, homeostasis and repair of adult animals. The melanocyte stem cell (MSC) and melanocyte regeneration is an attractive model for studying regulation of adult stem cells. The process of melanocyte regeneration can be divided into establishment of the MSC, recruitment of the MSC to produce committed daughter cells, and the proliferation, differentiation and survival of these daughter cells. Reduction of Kit signaling results in dose-dependent reduction of melanocytes during larval regeneration. Here, we use clonal analysis techniques to develop assays to distinguish roles for these processes during zebrafish larval melanocyte regeneration. We use these clonal assays to investigate which processes are affected by the reduction in Kit signaling. We show that the regeneration defect in kita mutants is not due to defects in MSC recruitment or in the proliferation, differentiation or survival of the daughter cells, but is instead due to a defect in stem cell establishment. Our analysis suggests that the kit MSC establishment defect results from inappropriate differentiation of the MSC lineage.
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http://dx.doi.org/10.1242/dev.088112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3583038PMC
March 2013

Development of translating ribosome affinity purification for zebrafish.

Genesis 2013 Mar 26;51(3):187-92. Epub 2013 Feb 26.

Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA.

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.
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http://dx.doi.org/10.1002/dvg.22363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3638809PMC
March 2013

Functional assessment of human coding mutations affecting skin pigmentation using zebrafish.

PLoS One 2012 10;7(10):e47398. Epub 2012 Oct 10.

Jake Gittlen Cancer Research Foundation, Penn State Hershey College of Medicine, Hershey, PA, USA.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047398PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3468441PMC
February 2013

Clonal and lineage analysis of melanocyte stem cells and their progeny in the zebrafish.

Methods Mol Biol 2012 ;916:181-95

Department of Genetics, Washington School of Medicine, St. Louis, MO, USA.

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.
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http://dx.doi.org/10.1007/978-1-61779-980-8_14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3630497PMC
March 2013