Publications by authors named "Elisabeth Busch-Nentwich"

34 Publications

NRAS melanoma tumor formation is reduced by p38-MAPK14 activation in zebrafish models and NRAS-mutated human melanoma cells.

Pigment Cell Melanoma Res 2021 03 24;34(2):150-162. Epub 2020 Sep 24.

University Hospital Zurich, University of Zurich, Zurich, Switzerland.

Oncogenic BRAF and NRAS mutations drive human melanoma initiation. We used transgenic zebrafish to model NRAS-mutant melanoma, and the rapid tumor onset allowed us to study candidate tumor suppressors. We identified P38α-MAPK14 as a potential tumor suppressor in The Cancer Genome Atlas melanoma cohort of NRAS-mutant melanomas, and overexpression significantly increased the time to tumor onset in transgenic zebrafish with NRAS-driven melanoma. Pharmacological activation of P38α-MAPK14 using anisomycin reduced in vitro viability of melanoma cultures, which we confirmed by stable overexpression of p38α. We observed that the viability of MEK inhibitor resistant melanoma cells could be reduced by combined treatment of anisomycin and MEK inhibition. Our study demonstrates that activating the p38α-MAPK14 pathway in the presence of oncogenic NRAS abrogates melanoma in vitro and in vivo. SIGNIFICANCE: The significance of our study is in the accountability of NRAS mutations in melanoma. We demonstrate here that activation of p38α-MAPK14 pathway can abrogate NRAS-mutant melanoma which is contrary to the previously published role of p38α-MAPK14 pathway in BRAF mutant melanoma. These results implicate that BRAF and NRAS-mutant melanoma may not be identical biologically. We also demonstrate the translational benefit of our study by using a small molecule compound-anisomycin (already in use for other diseases in clinical trials) to activate p38α-MAPK14 pathway.
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http://dx.doi.org/10.1111/pcmr.12925DOI Listing
March 2021

A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein.

PLoS Genet 2020 08 6;16(8):e1008941. Epub 2020 Aug 6.

Department of Embryology, Carnegie Institution for Science, Baltimore, Maryland, United States of America.

Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism. Here we report the discovery of a missense mutation in microsomal triglyceride transfer protein (Mtp), a protein that is essential for B-lp production. This mutation of a conserved glycine residue to valine (zebrafish G863V, human G865V) reduces B-lp production and results in yolk opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. However, this phenotype is milder than that of the previously reported L475P stalactite (stl) mutation. MTP transfers lipids, including triglycerides and phospholipids, to apolipoprotein B in the ER for B-lp assembly. In vitro lipid transfer assays reveal that while both MTP mutations eliminate triglyceride transfer activity, the G863V mutant protein unexpectedly retains ~80% of phospholipid transfer activity. This residual phospholipid transfer activity of the G863V mttp mutant protein is sufficient to support the secretion of small B-lps, which prevents intestinal fat malabsorption and growth defects observed in the mttpstl/stl mutant zebrafish. Modeling based on the recent crystal structure of the heterodimeric human MTP complex suggests the G865V mutation may block triglyceride entry into the lipid-binding cavity. Together, these data argue that selective inhibition of MTP triglyceride transfer activity may be a feasible therapeutic approach to treat dyslipidemia and provide structural insight for drug design. These data also highlight the power of yolk transport studies to identify proteins critical for B-lp biology.
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http://dx.doi.org/10.1371/journal.pgen.1008941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7444587PMC
August 2020

PRL3-DDX21 Transcriptional Control of Endolysosomal Genes Restricts Melanocyte Stem Cell Differentiation.

Dev Cell 2020 08 10;54(3):317-332.e9. Epub 2020 Jul 10.

MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh EH4 2XU, UK; Cancer Research UK Edinburgh Centre, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK. Electronic address:

Melanocytes, replenished throughout life by melanocyte stem cells (MSCs), play a critical role in pigmentation and melanoma. Here, we reveal a function for the metastasis-associated phosphatase of regenerating liver 3 (PRL3) in MSC regeneration. We show that PRL3 binds to the RNA helicase DDX21, thereby restricting productive transcription by RNAPII at master transcription factor (MITF)-regulated endolysosomal vesicle genes. In zebrafish, this mechanism controls premature melanoblast expansion and differentiation from MSCs. In melanoma patients, restricted transcription of this endolysosomal vesicle pathway is a hallmark of PRL3-high melanomas. Our work presents the conceptual advance that PRL3-mediated control of transcriptional elongation is a differentiation checkpoint mechanism for activated MSCs and has clinical relevance for the activity of PRL3 in regenerating tissue and cancer.
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http://dx.doi.org/10.1016/j.devcel.2020.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7435699PMC
August 2020

Identification of as a locus affecting nicotine preference in zebrafish and human smoking behaviour.

Elife 2020 03 25;9. Epub 2020 Mar 25.

School of Biological and Chemical Sciences, Queen Mary, University of London, London, United Kingdom.

To facilitate smoking genetics research we determined whether a screen of mutagenized zebrafish for nicotine preference could predict loci affecting smoking behaviour. From 30 screened F sibling groups, where each was derived from an individual ethyl-nitrosurea mutagenized F fish, two showed increased or decreased nicotine preference. Out of 25 inactivating mutations carried by the F fish, one in the gene segregated with increased nicotine preference in heterozygous individuals. Focussed SNP analysis of the human locus in cohorts from UK (n=863) and Finland (n=1715) identified two variants associated with cigarette consumption and likelihood of cessation. Characterisation of mutant larvae and adult fish revealed decreased sensitivity to the dopaminergic and serotonergic antagonist amisulpride, known to affect startle reflex that is correlated with addiction in humans, and increased mRNA expression in mutant larvae. No effect on neuronal pathfinding was detected. These findings reveal a role for SLIT3 in development of pathways affecting responses to nicotine in zebrafish and smoking in humans.
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http://dx.doi.org/10.7554/eLife.51295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096180PMC
March 2020

Author Correction: Chemokine receptor trafficking coordinates neutrophil clustering and dispersal at wounds in zebrafish.

Nat Commun 2020 Jan 21;11(1):506. Epub 2020 Jan 21.

Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Site, Cambridge, CB2 3DY, UK.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-019-14041-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972865PMC
January 2020

Chemokine receptor trafficking coordinates neutrophil clustering and dispersal at wounds in zebrafish.

Nat Commun 2019 11 14;10(1):5166. Epub 2019 Nov 14.

Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Site, Cambridge, CB2 3DY, UK.

Immune cells congregate at specific loci to fight infections during inflammatory responses, a process that must be transient and self-resolving. Cell dispersal promotes resolution, but it remains unclear how transition from clustering to dispersal is regulated. Here we show, using quantitative live imaging in zebrafish, that differential ligand-induced trafficking of chemokine receptors such as Cxcr1 and Cxcr2 orchestrates the state of neutrophil congregation at sites of tissue damage. Through receptor mutagenesis and biosensors, we show that Cxcr1 promotes clustering at wound sites, but is promptly desensitized and internalized, which prevents excess congregation. By contrast, Cxcr2 promotes bidirectional motility and is sustained at the plasma membrane. Persistent plasma membrane residence of Cxcr2 prolongs downstream signaling and is required for sustained exploratory motion conducive to dispersal. Thus, differential trafficking of two chemokine receptors allows coordination of antagonistic cell behaviors, promoting a self-resolving migratory response.
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http://dx.doi.org/10.1038/s41467-019-13107-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856356PMC
November 2019

Common and distinct transcriptional signatures of mammalian embryonic lethality.

Nat Commun 2019 06 26;10(1):2792. Epub 2019 Jun 26.

Wellcome Sanger Institute, Wellcome Genome Campus, Cambridge, CB10 1SA, UK.

The Deciphering the Mechanisms of Developmental Disorders programme has analysed the morphological and molecular phenotypes of embryonic and perinatal lethal mouse mutant lines in order to investigate the causes of embryonic lethality. Here we show that individual whole-embryo RNA-seq of 73 mouse mutant lines (>1000 transcriptomes) identifies transcriptional events underlying embryonic lethality and associates previously uncharacterised genes with specific pathways and tissues. For example, our data suggest that Hmgxb3 is involved in DNA-damage repair and cell-cycle regulation. Further, we separate embryonic delay signatures from mutant line-specific transcriptional changes by developing a baseline mRNA expression catalogue of wild-type mice during early embryogenesis (4-36 somites). Analysis of transcription outside coding sequence identifies deregulation of repetitive elements in Morc2a mutants and a gene involved in gene-specific splicing. Collectively, this work provides a large scale resource to further our understanding of early embryonic developmental disorders.
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http://dx.doi.org/10.1038/s41467-019-10642-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594971PMC
June 2019

The gene regulatory basis of genetic compensation during neural crest induction.

PLoS Genet 2019 06 14;15(6):e1008213. Epub 2019 Jun 14.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom.

The neural crest (NC) is a vertebrate-specific cell type that contributes to a wide range of different tissues across all three germ layers. The gene regulatory network (GRN) responsible for the formation of neural crest is conserved across vertebrates. Central to the induction of the NC GRN are AP-2 and SoxE transcription factors. NC induction robustness is ensured through the ability of some of these transcription factors to compensate loss of function of gene family members. However the gene regulatory events underlying compensation are poorly understood. We have used gene knockout and RNA sequencing strategies to dissect NC induction and compensation in zebrafish. We genetically ablate the NC using double mutants of tfap2a;tfap2c or remove specific subsets of the NC with sox10 and mitfa knockouts and characterise genome-wide gene expression levels across multiple time points. We find that compensation through a single wild-type allele of tfap2c is capable of maintaining early NC induction and differentiation in the absence of tfap2a function, but many target genes have abnormal expression levels and therefore show sensitivity to the reduced tfap2 dosage. This separation of morphological and molecular phenotypes identifies a core set of genes required for early NC development. We also identify the 15 somites stage as the peak of the molecular phenotype which strongly diminishes at 24 hpf even as the morphological phenotype becomes more apparent. Using gene knockouts, we associate previously uncharacterised genes with pigment cell development and establish a role for maternal Hippo signalling in melanocyte differentiation. This work extends and refines the NC GRN while also uncovering the transcriptional basis of genetic compensation via paralogues.
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http://dx.doi.org/10.1371/journal.pgen.1008213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594659PMC
June 2019

Dicer1 is required for pigment cell and craniofacial development in zebrafish.

Biochim Biophys Acta Gene Regul Mech 2019 04 3;1862(4):472-485. Epub 2019 Mar 3.

Instituto de Biología Molecular y Celular de Rosario (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) - Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Ocampo y Esmeralda, S2000EZP Rosario, Argentina. Electronic address:

The multidomain RNase III endoribonuclease DICER is required for the generation of most functional microRNAs (miRNAs). Loss of Dicer affects developmental processes at different levels. Here, we characterized the zebrafish Dicer1 mutant, dicer1, which has a single point mutation induced by N-ethyl-N-nitrosourea mutagenesis. Heterozygous dicer1 developed normally, being phenotypically indistinguishable from wild-type siblings. Homozygous dicer1 mutants display smaller eyes, abnormal craniofacial development and aberrant pigmentation. Reduced numbers of both iridophores and melanocytes were observed in the head and ventral trunk of dicer1 homozygotes; the effect on melanocytes was stronger and detectable earlier in development. The expression of microphthalmia-associated transcription factor a (mitfa), the master gene for melanocytes differentiation, was enhanced in dicer1-depleted fish. Similarly, the expression of SRY-box containing gene 10 (sox10), required for mitfa activation, was higher in mutants than in wild types. In silico and in vivo analyses of either sox10 or mitfa 3'UTRs revealed conserved potential miRNA binding sites likely involved in the post-transcriptional regulation of both genes. Based on these findings, we propose that dicer1 participates in the gene regulatory network governing zebrafish melanocyte differentiation by controlling the expression of mitfa and sox10.
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http://dx.doi.org/10.1016/j.bbagrm.2019.02.005DOI Listing
April 2019

Compensatory growth renders Tcf7l1a dispensable for eye formation despite its requirement in eye field specification.

Elife 2019 02 19;8. Epub 2019 Feb 19.

Department of Cell and Developmental Biology, University College London, London, United Kingdom.

The vertebrate eye originates from the eye field, a domain of cells specified by a small number of transcription factors. In this study, we show that Tcf7l1a is one such transcription factor that acts cell-autonomously to specify the eye field in zebrafish. Despite the much-reduced eye field in mutants, these fish develop normal eyes revealing a striking ability of the eye to recover from a severe early phenotype. This robustness is not mediated through genetic compensation at neural plate stage; instead, the smaller optic vesicle of mutants shows delayed neurogenesis and continues to grow until it achieves approximately normal size. Although the developing eye is robust to the lack of Tcf7l1a function, it is sensitised to the effects of additional mutations. In support of this, a forward genetic screen identified mutations in , and , which give synthetically enhanced eye specification or growth phenotypes when in combination with the mutation.
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http://dx.doi.org/10.7554/eLife.40093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380838PMC
February 2019

scRNA-Seq reveals distinct stem cell populations that drive hair cell regeneration after loss of Fgf and Notch signaling.

Elife 2019 01 25;8. Epub 2019 Jan 25.

Stowers Institute for Medical Research, Kansas City, United States.

Loss of sensory hair cells leads to deafness and balance deficiencies. In contrast to mammalian hair cells, zebrafish ear and lateral line hair cells regenerate from poorly characterized support cells. Equally ill-defined is the gene regulatory network underlying the progression of support cells to differentiated hair cells. scRNA-Seq of lateral line organs uncovered five different support cell types, including quiescent and activated stem cells. Ordering of support cells along a developmental trajectory identified self-renewing cells and genes required for hair cell differentiation. scRNA-Seq analyses of mutants, in which hair cell regeneration is increased, demonstrates that Fgf and Notch signaling inhibit proliferation of support cells in parallel by inhibiting Wnt signaling. Our scRNA-Seq analyses set the foundation for mechanistic studies of sensory organ regeneration and is crucial for identifying factors to trigger hair cell production in mammals. The data is searchable and publicly accessible via a web-based interface.
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http://dx.doi.org/10.7554/eLife.44431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363392PMC
January 2019

Deep phenotyping in zebrafish reveals genetic and diet-induced adiposity changes that may inform disease risk.

J Lipid Res 2018 08 23;59(8):1536-1545. Epub 2018 May 23.

Department of Molecular Genetics and Microbiology, Duke University, Durham, NC 27710.

The regional distribution of adipose tissues is implicated in a wide range of diseases. For example, proportional increases in visceral adipose tissue increase the risk for insulin resistance, diabetes, and CVD. Zebrafish offer a tractable model system by which to obtain unbiased and quantitative phenotypic information on regional adiposity, and deep phenotyping can explore complex disease-related adiposity traits. To facilitate deep phenotyping of zebrafish adiposity traits, we used pairwise correlations between 67 adiposity traits to generate stage-specific adiposity profiles that describe changing adiposity patterns and relationships during growth. Linear discriminant analysis classified individual fish according to an adiposity profile with 87.5% accuracy. Deep phenotyping of eight previously uncharacterized zebrafish mutants identified as a novel gene that alters adipose distribution. When we applied deep phenotyping to identify changes in adiposity during diet manipulations, zebrafish that underwent food restriction and refeeding had widespread adiposity changes when compared with continuously fed, equivalently sized control animals. In particular, internal adipose tissues (e.g., visceral adipose) exhibited a reduced capacity to replenish lipid following food restriction. Together, these results in zebrafish establish a new deep phenotyping technique as an unbiased and quantitative method to help uncover new relationships between genotype, diet, and adiposity.
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http://dx.doi.org/10.1194/jlr.D084525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071777PMC
August 2018

The age of heterozygous mutant parents influences the adult phenotype of their offspring irrespective of genotype in zebrafish.

Wellcome Open Res 2017 4;2:77. Epub 2017 Sep 4.

Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, UK.

Background: Mutations in proteins involved in telomere maintenance lead to a range of human diseases, including dyskeratosis congenita, idiopathic pulmonary fibrosis and cancer. Telomerase functions to add telomeric repeats back onto the ends of chromosomes, however non-canonical roles of components of telomerase have recently been suggested.

Methods: Here we use a zebrafish telomerase mutant which harbours a nonsense mutation in to investigate the adult phenotypes of fish derived from heterozygous parents of different ages. Furthermore we use whole genome sequencing data to estimate average telomere lengths.

Results: We show that homozygous offspring from older heterozygotes exhibit signs of body wasting at a younger age than those of younger parents, and that offspring of older heterozygous parents weigh less irrespective of genotype. We also demonstrate that homozygous mutant fish have a male sex bias, and that clutches from older parents also have a male sex bias in the heterozygous and wild-type populations. Telomere length analysis reveals that the telomeres of younger heterozygous parents are shorter than those of older heterozygous parents.

Conclusions: These data indicate that the phenotypes observed in offspring from older parents cannot be explained by telomere length. Instead we propose that Tert functions outside of telomere length maintenance in an age-dependent manner to influence the adult phenotypes of the next generation.
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http://dx.doi.org/10.12688/wellcomeopenres.12530.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5840683PMC
September 2017

Placentation defects are highly prevalent in embryonic lethal mouse mutants.

Nature 2018 03 14;555(7697):463-468. Epub 2018 Mar 14.

The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK.

Large-scale phenotyping efforts have demonstrated that approximately 25-30% of mouse gene knockouts cause intrauterine lethality. Analysis of these mutants has largely focused on the embryo and not the placenta, despite the crucial role of this extraembryonic organ for developmental progression. Here we screened 103 embryonic lethal and sub-viable mouse knockout lines from the Deciphering the Mechanisms of Developmental Disorders program for placental phenotypes. We found that 68% of knockout lines that are lethal at or after mid-gestation exhibited placental dysmorphologies. Early lethality (embryonic days 9.5-14.5) is almost always associated with severe placental malformations. Placental defects correlate strongly with abnormal brain, heart and vascular development. Analysis of mutant trophoblast stem cells and conditional knockouts suggests that a considerable number of factors that cause embryonic lethality when ablated have primary gene function in trophoblast cells. Our data highlight the hugely under-appreciated importance of placental defects in contributing to abnormal embryo development and suggest key molecular nodes that govern placenta formation.
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http://dx.doi.org/10.1038/nature26002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866719PMC
March 2018

mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay.

PLoS Genet 2017 11 21;13(11):e1007105. Epub 2017 Nov 21.

Carnegie Institution for Science, Department of Embryology, Baltimore, Maryland, United States of America.

As model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, a low frequency of abnormal phenotypes is being observed in lines with mutations predicted to lead to deleterious effects on the encoded protein. In zebrafish, this low frequency is in part explained by compensation by genes of redundant or similar function, often resulting from the additional round of teleost-specific whole genome duplication within vertebrates. Here we offer additional explanations for the low frequency of mutant phenotypes. We analyzed mRNA processing in seven zebrafish lines with mutations expected to disrupt gene function, generated by CRISPR/Cas9 or ENU mutagenesis methods. Five of the seven lines showed evidence of altered mRNA processing: one through a skipped exon that did not lead to a frame shift, one through nonsense-associated splicing that did not lead to a frame shift, and three through the use of cryptic splice sites. These results highlight the need for a methodical analysis of the mRNA produced in mutant lines before making conclusions or embarking on studies that assume loss of function as a result of a given genomic change. Furthermore, recognition of the types of adaptations that can occur may inform the strategies of mutant generation.
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http://dx.doi.org/10.1371/journal.pgen.1007105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716581PMC
November 2017

A high-resolution mRNA expression time course of embryonic development in zebrafish.

Elife 2017 11 16;6. Epub 2017 Nov 16.

Wellcome Trust Sanger Institute, Hinxton, United Kingdom.

We have produced an mRNA expression time course of zebrafish development across 18 time points from 1 cell to 5 days post-fertilisation sampling individual and pools of embryos. Using poly(A) pulldown stranded RNA-seq and a 3' end transcript counting method we characterise temporal expression profiles of 23,642 genes. We identify temporal and functional transcript co-variance that associates 5024 unnamed genes with distinct developmental time points. Specifically, a class of over 100 previously uncharacterised zinc finger domain containing genes, located on the long arm of chromosome 4, is expressed in a sharp peak during zygotic genome activation. In addition, the data reveal new genes and transcripts, differential use of exons and previously unidentified 3' ends across development, new primary microRNAs and temporal divergence of gene paralogues generated in the teleost genome duplication. To make this dataset a useful baseline reference, the data can be browsed and downloaded at Expression Atlas and Ensembl.
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http://dx.doi.org/10.7554/eLife.30860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5690287PMC
November 2017

Genetic Screen for Postembryonic Development in the Zebrafish (): Dominant Mutations Affecting Adult Form.

Genetics 2017 10 23;207(2):609-623. Epub 2017 Aug 23.

Department of Orthopedic Research, Boston Children's Hospital, Massachusetts 02115

Large-scale forward genetic screens have been instrumental for identifying genes that regulate development, homeostasis, and regeneration, as well as the mechanisms of disease. The zebrafish, , is an established genetic and developmental model used in genetic screens to uncover genes necessary for early development. However, the regulation of postembryonic development has received less attention as these screens are more labor intensive and require extensive resources. The lack of systematic interrogation of late development leaves large aspects of the genetic regulation of adult form and physiology unresolved. To understand the genetic control of postembryonic development, we performed a dominant screen for phenotypes affecting the adult zebrafish. In our screen, we identified 72 adult viable mutants showing changes in the shape of the skeleton as well as defects in pigmentation. For efficient mapping of these mutants and mutation identification, we devised a new mapping strategy based on identification of mutant-specific haplotypes. Using this method in combination with a candidate gene approach, we were able to identify linked mutations for 22 out of 25 mutants analyzed. Broadly, our mutational analysis suggests that there are key genes and pathways associated with late development. Many of these pathways are shared with humans and are affected in various disease conditions, suggesting constraint in the genetic pathways that can lead to change in adult form. Taken together, these results show that dominant screens are a feasible and productive means to identify mutations that can further our understanding of gene function during postembryonic development and in disease.
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http://dx.doi.org/10.1534/genetics.117.300187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5629327PMC
October 2017

Loss of the chromatin modifier Kdm2aa causes BrafV600E-independent spontaneous melanoma in zebrafish.

PLoS Genet 2017 Aug 14;13(8):e1006959. Epub 2017 Aug 14.

Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom.

KDM2A is a histone demethylase associated with transcriptional silencing, however very little is known about its in vivo role in development and disease. Here we demonstrate that loss of the orthologue kdm2aa in zebrafish causes widespread transcriptional disruption and leads to spontaneous melanomas at a high frequency. Fish homozygous for two independent premature stop codon alleles show reduced growth and survival, a strong male sex bias, and homozygous females exhibit a progressive oogenesis defect. kdm2aa mutant fish also develop melanomas from early adulthood onwards which are independent from mutations in braf and other common oncogenes and tumour suppressors as revealed by deep whole exome sequencing. In addition to effects on translation and DNA replication gene expression, high-replicate RNA-seq in morphologically normal individuals demonstrates a stable regulatory response of epigenetic modifiers and the specific de-repression of a group of zinc finger genes residing in constitutive heterochromatin. Together our data reveal a complex role for Kdm2aa in regulating normal mRNA levels and carcinogenesis. These findings establish kdm2aa mutants as the first single gene knockout model of melanoma biology.
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http://dx.doi.org/10.1371/journal.pgen.1006959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570503PMC
August 2017

Integration of Tmc1/2 into the mechanotransduction complex in zebrafish hair cells is regulated by Transmembrane O-methyltransferase (Tomt).

Elife 2017 05 23;6. Epub 2017 May 23.

Oregon Hearing Research Center and the Vollum Institute, Oregon Health and Science University, Portland, United States.

Transmembrane O-methyltransferase (/) is responsible for non-syndromic deafness DFNB63. However, the specific defects that lead to hearing loss have not been described. Using a zebrafish model of DFNB63, we show that the auditory and vestibular phenotypes are due to a lack of mechanotransduction (MET) in Tomt-deficient hair cells. GFP-tagged Tomt is enriched in the Golgi of hair cells, suggesting that Tomt might regulate the trafficking of other MET components to the hair bundle. We found that Tmc1/2 proteins are specifically excluded from the hair bundle in mutants, whereas other MET complex proteins can still localize to the bundle. Furthermore, mouse TOMT and TMC1 can directly interact in HEK 293 cells, and this interaction is modulated by His183 in TOMT. Thus, we propose a model of MET complex assembly where Tomt and the Tmcs interact within the secretory pathway to traffic Tmc proteins to the hair bundle.
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http://dx.doi.org/10.7554/eLife.28474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5462536PMC
May 2017

KDM2A integrates DNA and histone modification signals through a CXXC/PHD module and direct interaction with HP1.

Nucleic Acids Res 2017 02;45(3):1114-1129

MRC Clinical Sciences Centre (CSC), Du Cane Road, London, UK.

Functional genomic elements are marked by characteristic DNA and histone modification signatures. How combinatorial chromatin modification states are recognized by epigenetic reader proteins and how this is linked to their biological function is largely unknown. Here we provide a detailed molecular analysis of chromatin recognition by the lysine demethylase KDM2A. Using biochemical approaches we identify a nucleosome interaction module within KDM2A consisting of a CXXC type zinc finger, a PHD domain and a newly identified Heterochromatin Protein 1 (HP1) interaction motif that mediates direct binding between KDM2A and HP1. This nucleosome interaction module enables KDM2A to decode nucleosomal H3K9me3 modification in addition to CpG methylation signals. The multivalent engagement with DNA and HP1 results in a nucleosome binding circuit in which KDM2A can be recruited to H3K9me3-modified chromatin through HP1, and HP1 can be recruited to unmodified chromatin by KDM2A. A KDM2A mutant deficient in HP1-binding is inactive in an in vivo overexpression assay in zebrafish embryos demonstrating that the HP1 interaction is essential for KDM2A function. Our results reveal a complex regulation of chromatin binding for both KDM2A and HP1 that is modulated by DNA- and H3K9-methylation, and suggest a direct role for KDM2A in chromatin silencing.
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http://dx.doi.org/10.1093/nar/gkw979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5388433PMC
February 2017

Report of a Meeting on Contemporary Topics in Zebrafish Husbandry and Care.

Zebrafish 2016 12 18;13(6):584-589. Epub 2016 Aug 18.

1 Research Animals Department , RSPCA, Southwater, United Kingdom .

A meeting on Contemporary Topics in Zebrafish Husbandry and Care was held in the United Kingdom in 2014, with the aim of providing a discussion forum for researchers, animal technologists, and veterinarians from academia and industry to share good practice and exchange ideas. Presentation topics included protocols for optimal larval rearing, implementing the 3Rs (replacement, reduction, and refinement) in large-scale colony management, and environmental enrichment. The audience also participated in a survey of current practice relating to practical husbandry, cryopreservation, and the provision of enrichment.
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http://dx.doi.org/10.1089/zeb.2016.1324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5124746PMC
December 2016

Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish.

BMC Genomics 2016 Mar 24;17:259. Epub 2016 Mar 24.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

Background: The CRISPR/Cas9 system is a prokaryotic immune system that infers resistance to foreign genetic material and is a sort of 'adaptive immunity'. It has been adapted to enable high throughput genome editing and has revolutionised the generation of targeted mutations.

Results: We have developed a scalable analysis pipeline to identify CRISPR/Cas9 induced mutations in hundreds of samples using next generation sequencing (NGS) of amplicons. We have used this system to investigate the best way to screen mosaic Zebrafish founder individuals for germline transmission of induced mutations. Screening sperm samples from potential founders provides much better information on germline transmission rates and crucially the sequence of the particular insertions/deletions (indels) that will be transmitted. This enables us to combine screening with archiving to create a library of cryopreserved samples carrying known mutations. It also allows us to design efficient genotyping assays, making identifying F1 carriers straightforward.

Conclusions: The methods described will streamline the production of large numbers of knockout alleles in selected genes for phenotypic analysis, complementing existing efforts using random mutagenesis.
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http://dx.doi.org/10.1186/s12864-016-2563-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806435PMC
March 2016

High-throughput and quantitative genome-wide messenger RNA sequencing for molecular phenotyping.

BMC Genomics 2015 Aug 5;16:578. Epub 2015 Aug 5.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, UK.

Background: We present a genome-wide messenger RNA (mRNA) sequencing technique that converts small amounts of RNA from many samples into molecular phenotypes. It encompasses all steps from sample preparation to sequence analysis and is applicable to baseline profiling or perturbation measurements.

Results: Multiplex sequencing of transcript 3' ends identifies differential transcript abundance independent of gene annotation. We show that increasing biological replicate number while maintaining the total amount of sequencing identifies more differentially abundant transcripts.

Conclusions: This method can be implemented on polyadenylated RNA from any organism with an annotated reference genome and in any laboratory with access to Illumina sequencing.
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http://dx.doi.org/10.1186/s12864-015-1788-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524448PMC
August 2015

Zebrafish models for nemaline myopathy reveal a spectrum of nemaline bodies contributing to reduced muscle function.

Acta Neuropathol 2015 Sep 1;130(3):389-406. Epub 2015 May 1.

School of Biological Sciences, Monash University, Melbourne, VIC, Australia.

Nemaline myopathy is characterized by muscle weakness and the presence of rod-like (nemaline) bodies. The genetic etiology of nemaline myopathy is becoming increasingly understood with mutations in ten genes now known to cause the disease. Despite this, the mechanism by which skeletal muscle weakness occurs remains elusive, with previous studies showing no correlation between the frequency of nemaline bodies and disease severity. To investigate the formation of nemaline bodies and their role in pathogenesis, we generated overexpression and loss-of-function zebrafish models for skeletal muscle α-actin (ACTA1) and nebulin (NEB). We identify three distinct types of nemaline bodies and visualize their formation in vivo, demonstrating these nemaline bodies not only exhibit different subcellular origins, but also have distinct pathological consequences within the skeletal muscle. One subtype is highly dynamic and upon breakdown leads to the accumulation of cytoplasmic actin contributing to muscle weakness. Examination of a Neb-deficient model suggests this mechanism may be common in nemaline myopathy. Another subtype results from a reduction of actin and forms a more stable cytoplasmic body. In contrast, the final type originates at the Z-disk and is associated with myofibrillar disorganization. Analysis of zebrafish and muscle biopsies from ACTA1 nemaline myopathy patients demonstrates that nemaline bodies also possess a different protein signature. In addition, we show that the ACTA1(D286G) mutation causes impaired actin incorporation and localization in the sarcomere. Together these data provide a novel examination of nemaline body origins and dynamics in vivo and identifies pathological changes that correlate with muscle weakness.
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http://dx.doi.org/10.1007/s00401-015-1430-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4541704PMC
September 2015

Multi-allelic phenotyping--a systematic approach for the simultaneous analysis of multiple induced mutations.

Methods 2013 Aug 23;62(3):197-206. Epub 2013 Apr 23.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

The zebrafish mutation project (ZMP) aims to generate a loss of function allele for every protein-coding gene, but importantly to also characterise the phenotypes of these alleles during the first five days of development. Such a large-scale screen requires a systematic approach both to identifying phenotypes, and also to linking those phenotypes to specific mutations. This phenotyping pipeline simultaneously assesses the consequences of multiple alleles in a two-step process. First, mutations that do not produce a visible phenotype during the first five days of development are identified, while a second round of phenotyping focuses on detailed analysis of those alleles that are suspected to cause a phenotype. Allele-specific PCR single nucleotide polymorphism (SNP) assays are used to genotype F2 parents and individual F3 fry for mutations known to be present in the F1 founder. With this method specific phenotypes can be linked to induced mutations. In addition a method is described for cryopreserving sperm samples of mutagenised males and their subsequent use for in vitro fertilisation to generate F2 families for phenotyping. Ultimately this approach will lead to the functional annotation of the zebrafish genome, which will deepen our understanding of gene function in development and disease.
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http://dx.doi.org/10.1016/j.ymeth.2013.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3770900PMC
August 2013

A systematic genome-wide analysis of zebrafish protein-coding gene function.

Nature 2013 Apr 17;496(7446):494-7. Epub 2013 Apr 17.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at a rapid rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in model vertebrate organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes, this number falls considerably short of the more than 22,000 mouse protein-coding genes. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning, insertional mutagenesis, antisense morpholino oligonucleotides, targeted re-sequencing, and zinc finger and TAL endonucleases have made substantial contributions to our understanding of the biological activity of vertebrate genes, but again the number of genes studied falls well short of the more than 26,000 zebrafish protein-coding genes. Importantly, for both mice and zebrafish, none of these strategies are particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Here we describe an active project that aims to identify and phenotype the disruptive mutations in every zebrafish protein-coding gene, using a well-annotated zebrafish reference genome sequence, high-throughput sequencing and efficient chemical mutagenesis. So far we have identified potentially disruptive mutations in more than 38% of all known zebrafish protein-coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1,000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis.
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http://dx.doi.org/10.1038/nature11992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3743023PMC
April 2013

Xirp proteins mark injured skeletal muscle in zebrafish.

PLoS One 2012 15;7(2):e31041. Epub 2012 Feb 15.

Max Delbrück Center (MDC) for Molecular Medicine, Berlin, Germany.

Myocellular regeneration in vertebrates involves the proliferation of activated progenitor or dedifferentiated myogenic cells that have the potential to replenish lost tissue. In comparison little is known about cellular repair mechanisms within myocellular tissue in response to small injuries caused by biomechanical or cellular stress. Using a microarray analysis for genes upregulated upon myocellular injury, we identified zebrafish Xin-actin-binding repeat-containing protein1 (Xirp1) as a marker for wounded skeletal muscle cells. By combining laser-induced micro-injury with proliferation analyses, we found that Xirp1 and Xirp2a localize to nascent myofibrils within wounded skeletal muscle cells and that the repair of injuries does not involve cell proliferation or Pax7(+) cells. Through the use of Xirp1 and Xirp2a as markers, myocellular injury can now be detected, even though functional studies indicate that these proteins are not essential in this process. Previous work in chicken has implicated Xirps in cardiac looping morphogenesis. However, we found that zebrafish cardiac morphogenesis is normal in the absence of Xirp expression, and animals deficient for cardiac Xirp expression are adult viable. Although the functional involvement of Xirps in developmental and repair processes currently remains enigmatic, our findings demonstrate that skeletal muscle harbours a rapid, cell-proliferation-independent response to injury which has now become accessible to detailed molecular and cellular characterizations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0031041PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3280289PMC
June 2012

The zebrafish dystrophic mutant softy maintains muscle fibre viability despite basement membrane rupture and muscle detachment.

Development 2009 Oct;136(19):3367-76

The Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia.

The skeletal muscle basement membrane fulfils several crucial functions during development and in the mature myotome and defects in its composition underlie certain forms of muscular dystrophy. A major component of this extracellular structure is the laminin polymer, which assembles into a resilient meshwork that protects the sarcolemma during contraction. Here we describe a zebrafish mutant, softy, which displays severe embryonic muscle degeneration as a result of initial basement membrane failure. The softy phenotype is caused by a mutation in the lamb2 gene, identifying laminin beta2 as an essential component of this basement membrane. Uniquely, softy homozygotes are able to recover and survive to adulthood despite the loss of myofibre adhesion. We identify the formation of ectopic, stable basement membrane attachments as a novel means by which detached fibres are able to maintain viability. This demonstration of a muscular dystrophy model possessing innate fibre viability following muscle detachment suggests basement membrane augmentation as a therapeutic strategy to inhibit myofibre loss.
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http://dx.doi.org/10.1242/dev.034561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2739150PMC
October 2009

Organ-specific requirements for Hdac1 in liver and pancreas formation.

Dev Biol 2008 Oct 21;322(2):237-50. Epub 2008 Jul 21.

National Institute for Medical Research, Division of Developmental Biology, The Ridgeway, Mill Hill, London, UK.

Liver, pancreas and lung originate from the presumptive foregut in temporal and spatial proximity. This requires precisely orchestrated transcriptional activation and repression of organ-specific gene expression within the same cell. Here, we show distinct roles for the chromatin remodelling factor and transcriptional repressor Histone deacetylase 1 (Hdac1) in endodermal organogenesis in zebrafish. Loss of Hdac1 causes defects in timely liver specification and in subsequent differentiation. Mosaic analyses reveal a cell-autonomous requirement for hdac1 within the hepatic endoderm. Our studies further reveal specific functions for Hdac1 in pancreas development. Loss of hdac1 causes the formation of ectopic endocrine clusters anteriorly to the main islet, as well as defects in exocrine pancreas specification and differentiation. In addition, we observe defects in extrahepatopancreatic duct formation and morphogenesis. Finally, loss of hdac1 results in an expansion of the foregut endoderm in the domain from which the liver and pancreas originate. Our genetic studies demonstrate that Hdac1 is crucial for regulating distinct steps in endodermal organogenesis. This suggests a model in which Hdac1 may directly or indirectly restrict foregut fates while promoting hepatic and exocrine pancreatic specification and differentiation, as well as pancreatic endocrine islet morphogenesis. These findings establish zebrafish as a tractable system to investigate chromatin remodelling factor functions in controlling gene expression programmes in vertebrate endodermal organogenesis.
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http://dx.doi.org/10.1016/j.ydbio.2008.06.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3710974PMC
October 2008

Vesicular glutamate transporter 3 is required for synaptic transmission in zebrafish hair cells.

J Neurosci 2008 Feb;28(9):2110-8

Howard Hughes Medical Institute, Oregon Hearing Research Center and Vollum Institute, Oregon Health & Science University, Portland, Oregon 97239, USA.

Hair cells detect sound and movement and transmit this information via specialized ribbon synapses. Here we report that asteroid, a gene identified in an ethylnitrosourea mutagenesis screen of zebrafish larvae for auditory/vestibular mutants, encodes vesicular glutamate transporter 3 (Vglut3). A splice site mutation in exon 2 of vglut3 results in a severe truncation of the predicted protein product and morpholinos directed against the vglut3 ATG start site or the affected splice junction replicate the asteroid phenotype. In situ hybridization shows that vglut3 is exclusively expressed in hair cells of the ear and lateral line organ. A second transporter gene, vglut1, is also expressed in zebrafish hair cells, but the level of vglut1 mRNA is not increased in the absence of Vglut3. Antibodies against Vglut3 label the basal end of hair cells and labeling is not present in asteroid/vglut3 mutants. Based on the localization of Vglut3 in hair cells, we suspected that the lack of vestibulo-ocular and acoustic startle reflexes in asteroid/vglut3 mutants was attributable to a defect in synaptic transmission in hair cells. In support of this notion, action currents in postsynaptic acousticolateralis neurons are absent in asteroid/vglut3 mutants. At the ultrastructural level, mutant asteroid/vglut3 hair cells show a decrease in the number of ribbon-associated synaptic vesicles, indicating a role for Vglut3 in synaptic vesicle biogenesis and/or tethering to the ribbon body. Lack of postsynaptic action currents in the mutants suggests that the remaining hair-cell synaptic vesicles contain insufficient levels of glutamate for generation of action potentials in first-order neurons.
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http://dx.doi.org/10.1523/JNEUROSCI.5230-07.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6671858PMC
February 2008
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