Publications by authors named "Robert Geisler"

56 Publications

An In Vivo Assessment of Regional Brain Temperature during Whole-Body Cooling for Neonatal Encephalopathy.

J Pediatr 2020 05 20;220:73-79.e3. Epub 2020 Feb 20.

Department of Radiology, Children's Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA; Rudi Schulte Research Institute, Santa Barbara, CA.

Objective: To assess differences in regional brain temperatures during whole-body hypothermia and test the hypothesis that brain temperature profile is nonhomogenous in infants with hypoxic-ischemic encephalopathy.

Study Design: Infants with hypoxic-ischemic encephalopathy were enrolled prospectively in this observational study. Magnetic resonance (MR) spectra of basal ganglia, thalamus, cortical gray matter, and white matter (WM) were acquired during therapeutic hypothermia. Regional brain tissue temperatures were calculated from the chemical shift difference between water signal and metabolites in the MR spectra after performing calibration measurements. Overall difference in regional temperature was analyzed by mixed-effects model; temperature among different patterns and severity of injury on MR imaging also was analyzed. Correlation between temperature and depth of brain structure was analyzed using repeated-measures correlation.

Results: In total, 53 infants were enrolled (31 girls, mean gestational age: 38.6 ± 2 weeks; mean birth weight: 3243 ± 613 g). MR spectroscopy was acquired at mean age of 2.2 ± 0.6 days. A total of 201 MR spectra were included in the analysis. The thalamus, the deepest structure (36.4 ± 2.3 mm from skull surface), was lowest in temperature (33.2 ± 0.8°C, compared with basal ganglia: 33.5 ± 0.9°C; gray matter: 33.6 ± 0.7°C; WM: 33.8 ± 0.9°C, all P < .001). Temperatures in more superficial gray matter and WM regions (depth: 21.9 ± 2.4 and 21.5 ± 2.2 mm) were greater than the rectal temperatures (33.4 ± 0.4°C, P < .03). There was a negative correlation between temperature and depth of brain structure (r = -0.36, P < .001).

Conclusions: Whole-body hypothermia was effective in cooling deep brain structures, whereas superficial structures were warmer, with temperatures significantly greater than rectal temperatures.
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http://dx.doi.org/10.1016/j.jpeds.2020.01.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265905PMC
May 2020

Abrogation of Stem Loop Binding Protein (Slbp) function leads to a failure of cells to transition from proliferation to differentiation, retinal coloboma and midline axon guidance deficits.

PLoS One 2019 29;14(1):e0211073. Epub 2019 Jan 29.

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

Through forward genetic screening for mutations affecting visual system development, we identified prominent coloboma and cell-autonomous retinal neuron differentiation, lamination and retinal axon projection defects in eisspalte (ele) mutant zebrafish. Additional axonal deficits were present, most notably at midline axon commissures. Genetic mapping and cloning of the ele mutation showed that the affected gene is slbp, which encodes a conserved RNA stem-loop binding protein involved in replication dependent histone mRNA metabolism. Cells throughout the central nervous system remained in the cell cycle in ele mutant embryos at stages when, and locations where, post-mitotic cells have differentiated in wild-type siblings. Indeed, RNAseq analysis showed down-regulation of many genes associated with neuronal differentiation. This was coincident with changes in the levels and spatial localisation of expression of various genes implicated, for instance, in axon guidance, that likely underlie specific ele phenotypes. These results suggest that many of the cell and tissue specific phenotypes in ele mutant embryos are secondary to altered expression of modules of developmental regulatory genes that characterise, or promote transitions in, cell state and require the correct function of Slbp-dependent histone and chromatin regulatory genes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0211073PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6350959PMC
October 2019

Mutation of a serine near the catalytic site of the choline acetyltransferase a gene almost completely abolishes motility of the zebrafish embryo.

PLoS One 2018 20;13(11):e0207747. Epub 2018 Nov 20.

Karlsruhe Institute of Technology (KIT), Institute of Toxicology and Genetics (ITG), Eggenstein-Leopoldshafen, Germany.

In zebrafish, the gene choline acetyltransferase a (chata) encodes one of the two ChAT orthologs responsible for the synthesis of acetylcholine. Acetylcholine (ACh) is essential for neuromuscular transmission and its impaired synthesis by ChAT can lead to neuromuscular junction disorders such as congenital myasthenic syndromes in humans. We have identified a novel mutation in the chata gene of zebrafish, chatatk64, in a collection of uncharacterised ENU-induced mutants. This mutant carries a missense mutation in the codon of a highly conserved serine changing it to an arginine (S102R). This serine is conserved among ChATs from zebrafish, rat, mice and chicken to humans. It resides within the catalytic domain and in the vicinity of the active site of the enzyme. However, it has not been reported so far to be required for enzymatic activity. Modelling of the S102R variant change in the ChAT protein crystal structure suggests that the change affects protein structure and has a direct impact on the catalytic domain of the protein which abolishes embryo motility almost completely.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0207747PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6245786PMC
April 2019

Report of Workshop on Euthanasia for Zebrafish-A Matter of Welfare and Science.

Zebrafish 2017 12 2;14(6):547-551. Epub 2017 Oct 2.

5 European Society for Fish Models in Biology and Medicine, Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT) , Karlsruhe, Germany .

The increasing importance of zebrafish as a biomedical model organism is reflected by the steadily growing number of publications and laboratories working with this species. Regulatory recommendations for euthanasia as issued in Directive 2010/63/EU are, however, based on experience with fish species used for food production and do not take the small size and specific physiology of zebrafish into account. Consequently, the currently recommended methods of euthanasia in the Directive 2010/63/EU are either not applicable or may interfere with research goals. An international workshop was held in Karlsruhe, Germany, March 9, 2017, to discuss and propose alternative methods for euthanasia of zebrafish. The aim was to identify methods that adequately address the physiology of zebrafish and its use as a biomedical research model, follow the principles of the 3Rs (Replacement, Reduction, and Refinement) in animal experimentation and consider animal welfare during anesthesia and euthanasia. The results of the workshop are summarized here in the form of a white paper.
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http://dx.doi.org/10.1089/zeb.2017.1508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5706616PMC
December 2017

The Calcineurin-FoxO-MuRF1 signaling pathway regulates myofibril integrity in cardiomyocytes.

Elife 2017 08 19;6. Epub 2017 Aug 19.

Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, United States.

Altered Ca handling is often present in diseased hearts undergoing structural remodeling and functional deterioration. However, whether Ca directly regulates sarcomere structure has remained elusive. Using a zebrafish mutant, we explored the impacts of impaired Ca homeostasis on myofibril integrity. We found that the E3 ubiquitin ligase is upregulated in deficient hearts. Intriguingly, knocking down activity or inhibiting proteasome activity preserved myofibril integrity, revealing a MuRF1-mediated proteasome degradation mechanism that is activated in response to abnormal Ca homeostasis. Furthermore, we detected an accumulation of the regulator FoxO in the nuclei of -deficient cardiomyocytes. Overexpression of FoxO in wild type cardiomyocytes induced expression and caused myofibril disarray, whereas inhibiting Calcineurin activity attenuated FoxO-mediated expression and protected sarcomeres from degradation in -deficient hearts. Together, our findings reveal a novel mechanism by which Ca overload disrupts myofibril integrity by activating a Calcineurin-FoxO-MuRF1-proteosome signaling pathway.
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http://dx.doi.org/10.7554/eLife.27955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576919PMC
August 2017

Archiving of zebrafish lines can reduce animal experiments in biomedical research.

EMBO Rep 2017 01 15;18(1):1-2. Epub 2016 Dec 15.

Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.

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http://dx.doi.org/10.15252/embr.201643561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210080PMC
January 2017

Maintenance of Zebrafish Lines at the European Zebrafish Resource Center.

Zebrafish 2016 07;13 Suppl 1:S19-23

Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT) , Eggenstein-Leopoldshafen, Germany .

We have established a European Zebrafish Resource Center (EZRC) at the KIT. This center not only maintains and distributes a large number of existing mutant and transgenic zebrafish lines but also gives zebrafish researchers access to screening services and technologies such as imaging and high-throughput sequencing, provided by the Institute of Toxicology and Genetics (ITG). The EZRC maintains and distributes the stock collection of the Nüsslein-Volhard laboratory, comprising over 2000 publicly released mutations, as frozen sperm samples. Within the framework of the ZF-HEALTH EU project, the EZRC distributes over 10,000 knockout mutations from the Sanger Institute (United Kingdom), as well as over 100 mutant and transgenic lines from other sources. In this article, we detail the measures we have taken to ensure the health of our fish, including hygiene, quarantine, and veterinary inspections.
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http://dx.doi.org/10.1089/zeb.2015.1205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931740PMC
July 2016

Spermidine, but not spermine, is essential for pigment pattern formation in zebrafish.

Biol Open 2016 Jun 15;5(6):736-44. Epub 2016 Jun 15.

Max-Planck-Institut für Entwicklungsbiologie, Abteilung 3, Spemannstrasse 35, Tübingen 72076, Germany

Polyamines are small poly-cations essential for all cellular life. The main polyamines present in metazoans are putrescine, spermidine and spermine. Their exact functions are still largely unclear; however, they are involved in a wide variety of processes affecting cell growth, proliferation, apoptosis and aging. Here we identify idefix, a mutation in the zebrafish gene encoding the enzyme spermidine synthase, leading to a severe reduction in spermidine levels as shown by capillary electrophoresis-mass spectrometry. We show that spermidine, but not spermine, is essential for early development, organogenesis and colour pattern formation. Whereas in other vertebrates spermidine deficiency leads to very early embryonic lethality, maternally provided spermidine synthase in zebrafish is sufficient to rescue the early developmental defects. This allows us to uncouple them from events occurring later during colour patterning. Factors involved in the cellular interactions essential for colour patterning, likely targets for spermidine, are the gap junction components Cx41.8, Cx39.4, and Kir7.1, an inwardly rectifying potassium channel, all known to be regulated by polyamines. Thus, zebrafish provide a vertebrate model to study the in vivo effects of polyamines.
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http://dx.doi.org/10.1242/bio.018721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920196PMC
June 2016

The zebrafish reference genome sequence and its relationship to the human genome.

Authors:
Kerstin Howe Matthew D Clark Carlos F Torroja James Torrance Camille Berthelot Matthieu Muffato John E Collins Sean Humphray Karen McLaren Lucy Matthews Stuart McLaren Ian Sealy Mario Caccamo Carol Churcher Carol Scott Jeffrey C Barrett Romke Koch Gerd-Jörg Rauch Simon White William Chow Britt Kilian Leonor T Quintais José A Guerra-Assunção Yi Zhou Yong Gu Jennifer Yen Jan-Hinnerk Vogel Tina Eyre Seth Redmond Ruby Banerjee Jianxiang Chi Beiyuan Fu Elizabeth Langley Sean F Maguire Gavin K Laird David Lloyd Emma Kenyon Sarah Donaldson Harminder Sehra Jeff Almeida-King Jane Loveland Stephen Trevanion Matt Jones Mike Quail Dave Willey Adrienne Hunt John Burton Sarah Sims Kirsten McLay Bob Plumb Joy Davis Chris Clee Karen Oliver Richard Clark Clare Riddle David Elliot David Eliott Glen Threadgold Glenn Harden Darren Ware Sharmin Begum Beverley Mortimore Beverly Mortimer Giselle Kerry Paul Heath Benjamin Phillimore Alan Tracey Nicole Corby Matthew Dunn Christopher Johnson Jonathan Wood Susan Clark Sarah Pelan Guy Griffiths Michelle Smith Rebecca Glithero Philip Howden Nicholas Barker Christine Lloyd Christopher Stevens Joanna Harley Karen Holt Georgios Panagiotidis Jamieson Lovell Helen Beasley Carl Henderson Daria Gordon Katherine Auger Deborah Wright Joanna Collins Claire Raisen Lauren Dyer Kenric Leung Lauren Robertson Kirsty Ambridge Daniel Leongamornlert Sarah McGuire Ruth Gilderthorp Coline Griffiths Deepa Manthravadi Sarah Nichol Gary Barker Siobhan Whitehead Michael Kay Jacqueline Brown Clare Murnane Emma Gray Matthew Humphries Neil Sycamore Darren Barker David Saunders Justene Wallis Anne Babbage Sian Hammond Maryam Mashreghi-Mohammadi Lucy Barr Sancha Martin Paul Wray Andrew Ellington Nicholas Matthews Matthew Ellwood Rebecca Woodmansey Graham Clark James D Cooper James Cooper Anthony Tromans Darren Grafham Carl Skuce Richard Pandian Robert Andrews Elliot Harrison Andrew Kimberley Jane Garnett Nigel Fosker Rebekah Hall Patrick Garner Daniel Kelly Christine Bird Sophie Palmer Ines Gehring Andrea Berger Christopher M Dooley Zübeyde Ersan-Ürün Cigdem Eser Horst Geiger Maria Geisler Lena Karotki Anette Kirn Judith Konantz Martina Konantz Martina Oberländer Silke Rudolph-Geiger Mathias Teucke Christa Lanz Günter Raddatz Kazutoyo Osoegawa Baoli Zhu Amanda Rapp Sara Widaa Cordelia Langford Fengtang Yang Stephan C Schuster Nigel P Carter Jennifer Harrow Zemin Ning Javier Herrero Steve M J Searle Anton Enright Robert Geisler Ronald H A Plasterk Charles Lee Monte Westerfield Pieter J de Jong Leonard I Zon John H Postlethwait Christiane Nüsslein-Volhard Tim J P Hubbard Hugues Roest Crollius Jane Rogers Derek L Stemple

Nature 2013 Apr 17;496(7446):498-503. Epub 2013 Apr 17.

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

Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.
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http://dx.doi.org/10.1038/nature12111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3703927PMC
April 2013

Slc45a2 and V-ATPase are regulators of melanosomal pH homeostasis in zebrafish, providing a mechanism for human pigment evolution and disease.

Pigment Cell Melanoma Res 2013 Mar 20;26(2):205-17. Epub 2012 Dec 20.

Max Planck Institute for Developmental Biology, Tübingen, Germany.

We present here the positional cloning of the Danio rerio albino mutant and show that the affected gene encodes Slc45a2. The human orthologous gene has previously been shown to be involved in human skin color variation, and mutations therein have been implicated in the disease OCA4. Through ultrastructural analysis of the melanosomes in albino alleles as well as the tyrosinase-deficient mutant sandy, we add new insights into the role of Slc45a2 in the production of melanin. To gain further understanding of the role of Slc45a2 and its possible interactions with other proteins involved in melanization, we further analyzed the role of the V-ATPase as a melanosomal acidifier. We show that it is possible to rescue the melanization potential of the albino melanosomes through genetic and chemical inhibition of V-ATPase, thereby increasing internal melanosome pH.
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http://dx.doi.org/10.1111/pcmr.12053DOI Listing
March 2013

EuFishBioMed (COST Action BM0804): a European network to promote the use of small fishes in biomedical research.

Zebrafish 2012 Jun 26;9(2):90-3. Epub 2012 Apr 26.

Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany.

Small fresh water fishes such as the zebrafish (Danio rerio) have become important model organisms for biomedical research. They currently represent the best vertebrate embryo models in which it is possible to derive quantitative data on gene expression, signaling events, and cell behavior in real time in the living animal. Relevant phenotypes in fish mutants are similar to those of other vertebrate models and human diseases. They can be analyzed in great detail and much faster than in mammals. In recent years, approximately 2500 genetically distinct fish lines have been generated by European research groups alone. Their potential, including their possible use by industry, is far from being exploited. To promote zebrafish research in Europe, EuFishBioMed was founded and won support by the EU COST programme ( http://www.cost.esf.org/ ). The main objective of EuFishBioMed is to establish a platform of knowledge exchange for research on small fish models with a strong focus on widening its biomedical applications and an integration of European research efforts and resources. EuFishBioMed currently lists more than 300 member laboratories in Europe, offers funding for short-term laboratory visits, organizes and co-sponsors meetings and workshops, and has successfully lobbied for the establishment of a European Zebrafish Resource Centre. To maintain this network in the future, beyond the funding period of the COST Action, we are currently establishing the European Society for Fish Models in Biology and Medicine.
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http://dx.doi.org/10.1089/zeb.2012.0742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3371258PMC
June 2012

Zebrafish embryos as an alternative to animal experiments--a commentary on the definition of the onset of protected life stages in animal welfare regulations.

Reprod Toxicol 2012 Apr 25;33(2):128-32. Epub 2011 Jun 25.

Karlsruhe Institute of Technology, Institute of Toxicology and Genetics, Hermann-von-Helmholtz-Platz 1, D-76344 Eggenstein-Leopoldshafen, Germany.

Worldwide, the zebrafish has become a popular model for biomedical research and (eco)toxicology. Particularly the use of embryos is receiving increasing attention, since they are considered as replacement method for animal experiments. Zebrafish embryos allow the analysis of multiple endpoints ranging from acute and developmental toxicity determination to complex functional genetic and physiological analysis. Particularly the more complex endpoints require the use of post-hatched eleutheroembryo stages. According to the new EU Directive 2010/63/EU on the protection of animals used for scientific purposes, the earliest life-stages of animals are not defined as protected and, therefore, do not fall into the regulatory frameworks dealing with animal experimentation. Independent feeding is considered as the stage from which free-living larvae are subject to regulations for animal experimentation. However, despite this seemingly clear definition, large variations exist in the interpretation of this criterion by national and regional authorities. Since some assays require the use of post-hatched stages up to 120 h post fertilization, the literature and available data are reviewed in order to evaluate if this stage could still be considered as non-protected according to the regulatory criterion of independent feeding. Based on our analysis and by including criteria such as yolk consumption, feeding and swimming behavior, we conclude that zebrafish larvae can indeed be regarded as independently feeding from 120 h after fertilization. Experiments with zebrafish should thus be subject to regulations for animal experiments from 120 h after fertilization onwards.
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http://dx.doi.org/10.1016/j.reprotox.2011.06.121DOI Listing
April 2012

Fine-tuning of Hh signaling by the RNA-binding protein Quaking to control muscle development.

Development 2011 May 29;138(9):1783-94. Epub 2011 Mar 29.

Ecole Normale Supérieure, Institut de Biologie, 46 rue d'Ulm, 75005 Paris, France.

The development of the different muscles within the somite is a complex process that involves the Hedgehog (Hh) signaling pathway. To specify the proper number of muscle cells and organize them spatially and temporally, the Hh signaling pathway needs to be precisely regulated at different levels, but only a few factors external to the pathway have been described. Here, we report for the first time the role of the STAR family RNA-binding protein Quaking A (QkA) in somite muscle development. We show in zebrafish that the loss of QkA function affects fast muscle fiber maturation as well as Hh-induced muscle derivative specification and/or morphogenesis. Mosaic analysis reveals that fast fiber maturation depends on the activity of QkA in the environment of fast fiber progenitors. We further show that Hh signaling requires QkA activity for muscle development. By an in silico approach, we screened the 3'UTRs of known Hh signaling component mRNAs for the Quaking response element and found the transcription factor Gli2a, a known regulator of muscle fate development. Using destabilized GFP as a reporter, we show that the gli2a mRNA 3'UTR is a functional QkA target. Consistent with this notion, the loss of QkA function rescued slow muscle fibers in yot mutant embryos, which express a dominant-negative Gli2a isoform. Thus, our results reveal a new mechanism to ensure muscle cell fate diversity by fine-tuning of the Hh signaling pathway via RNA-binding proteins.
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http://dx.doi.org/10.1242/dev.059121DOI Listing
May 2011

The light responsive transcriptome of the zebrafish: function and regulation.

PLoS One 2011 Feb 15;6(2):e17080. Epub 2011 Feb 15.

Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany.

Most organisms possess circadian clocks that are able to anticipate the day/night cycle and are reset or "entrained" by the ambient light. In the zebrafish, many organs and even cultured cell lines are directly light responsive, allowing for direct entrainment of the clock by light. Here, we have characterized light induced gene transcription in the zebrafish at several organizational levels. Larvae, heart organ cultures and cell cultures were exposed to 1- or 3-hour light pulses, and changes in gene expression were compared with controls kept in the dark. We identified 117 light regulated genes, with the majority being induced and some repressed by light. Cluster analysis groups the genes into five major classes that show regulation at all levels of organization or in different subset combinations. The regulated genes cover a variety of functions, and the analysis of gene ontology categories reveals an enrichment of genes involved in circadian rhythms, stress response and DNA repair, consistent with the exposure to visible wavelengths of light priming cells for UV-induced damage repair. Promoter analysis of the induced genes shows an enrichment of various short sequence motifs, including E- and D-box enhancers that have previously been implicated in light regulation of the zebrafish period2 gene. Heterologous reporter constructs with sequences matching these motifs reveal light regulation of D-box elements in both cells and larvae. Morpholino-mediated knock-down studies of two homologues of the D-box binding factor Tef indicate that these are differentially involved in the cell autonomous light induction in a gene-specific manner. These findings suggest that the mechanisms involved in period2 regulation might represent a more general pathway leading to light induced gene expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017080PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3039656PMC
February 2011

Aplexone targets the HMG-CoA reductase pathway and differentially regulates arteriovenous angiogenesis.

Development 2011 Mar 9;138(6):1173-81. Epub 2011 Feb 9.

Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA 90095, USA.

Arterial and venous endothelial cells exhibit distinct molecular characteristics at early developmental stages. These lineage-specific molecular programs are instructive to the development of distinct vascular architectures and physiological conditions of arteries and veins, but their roles in angiogenesis remain unexplored. Here, we show that the caudal vein plexus in zebrafish forms by endothelial cell sprouting, migration and anastomosis, providing a venous-specific angiogenesis model. Using this model, we have identified a novel compound, aplexone, which effectively suppresses venous, but not arterial, angiogenesis. Multiple lines of evidence indicate that aplexone differentially regulates arteriovenous angiogenesis by targeting the HMG-CoA reductase (HMGCR) pathway. Treatment with aplexone affects the transcription of enzymes in the HMGCR pathway and reduces cellular cholesterol levels. Injecting mevalonate, a metabolic product of HMGCR, reverses the inhibitory effect of aplexone on venous angiogenesis. In addition, aplexone treatment inhibits protein prenylation and blocking the activity of geranylgeranyl transferase induces a venous angiogenesis phenotype resembling that observed in aplexone-treated embryos. Furthermore, endothelial cells of venous origin have higher levels of proteins requiring geranylgeranylation than arterial endothelial cells and inhibiting the activity of Rac or Rho kinase effectively reduces the migration of venous, but not arterial, endothelial cells. Taken together, our findings indicate that angiogenesis is differentially regulated by the HMGCR pathway via an arteriovenous-dependent requirement for protein prenylation in zebrafish and human endothelial cells.
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http://dx.doi.org/10.1242/dev.054049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3042872PMC
March 2011

The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left-right axis formation.

Nat Genet 2011 Jan 5;43(1):79-84. Epub 2010 Dec 5.

Department of Pediatrics, University Hospital Freiburg, Freiburg, Germany.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous autosomal recessive disorder characterized by recurrent infections of the respiratory tract associated with the abnormal function of motile cilia. Approximately half of individuals with PCD also have alterations in the left-right organization of their internal organ positioning, including situs inversus and situs ambiguous (Kartagener's syndrome). Here, we identify an uncharacterized coiled-coil domain containing a protein, CCDC40, essential for correct left-right patterning in mouse, zebrafish and human. In mouse and zebrafish, Ccdc40 is expressed in tissues that contain motile cilia, and mutations in Ccdc40 result in cilia with reduced ranges of motility. We further show that CCDC40 mutations in humans result in a variant of PCD characterized by misplacement of the central pair of microtubules and defective assembly of inner dynein arms and dynein regulatory complexes. CCDC40 localizes to motile cilia and the apical cytoplasm and is required for axonemal recruitment of CCDC39, disruption of which underlies a similar variant of PCD.
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http://dx.doi.org/10.1038/ng.727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3132183PMC
January 2011

The zebrafish mutant bumper shows a hyperproliferation of lens epithelial cells and fibre cell degeneration leading to functional blindness.

Mech Dev 2010 Apr 1;127(3-4):203-19. Epub 2010 Feb 1.

Max Planck Institute for Developmental Biology, Department of Genetics, Spemannstr. 35, D-72076 Tübingen, Germany.

The development of the eye lens is one of the classical paradigms of induction during embryonic development in vertebrates. But while there have been numerous studies aimed at discovering the genetic networks controlling early lens development, comparatively little is known about later stages, including the differentiation of secondary lens fibre cells. The analysis of mutant zebrafish isolated in forward genetic screens is an important way to investigate the roles of genes in embryogenesis. In this study we describe the zebrafish mutant bumper (bum), which shows a transient, tumour-like hyperproliferation of the lens epithelium as well as a progressively stronger defect in secondary fibre cell differentiation, which results in a significantly reduced lens size and ectopic location of the lens within the neural retina. Interestingly, the initial hyperproliferation of the lens epithelium in bum spontaneously regresses, suggesting this mutant as a valuable model to study the molecular control of tumour progression/suppression. Behavioural analyses demonstrate that, despite a morphologically normal retina, larval and adult bum(-/-) zebrafish are functionally blind. We further show that these fish have defects in their craniofacial skeleton with normal but delayed formation of the scleral ossicles within the eye, several reduced craniofacial bones resulting in an abnormal skull shape, and asymmetric ectopic bone formation within the mandible. Genetic mapping located the mutation in bum to a 4cM interval on chromosome 7 with the closest markers located at 0.2 and 0cM, respectively.
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http://dx.doi.org/10.1016/j.mod.2010.01.005DOI Listing
April 2010

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

Simplet controls cell proliferation and gene transcription during zebrafish caudal fin regeneration.

Dev Biol 2009 Jan 17;325(2):329-40. Epub 2008 Oct 17.

Max-Planck Institut für Entwicklungsbiologie, Abteilung Genetik, Spemannstrasse 35, 72076 Tübingen, Germany.

Two hallmarks of vertebrate epimorphic regeneration are a significant increase in the proliferation of normally quiescent cells and a re-activation of genes that are active during embryonic development. It is unclear what the molecular determinants are that regulate these events and how they are coordinated. Zebrafish have the ability to regenerate several compound structures by regulating cell proliferation and gene transcription. We report that fam53b/simplet (smp) regulates both cell proliferation and the transcription of specific genes. In situ hybridization and quantitative RT-PCR experiments showed that amputation of zebrafish hearts and fins resulted in strong up-regulation of the smp gene. In regenerating adult fin, smp expression remained strong in the distal mesenchyme which later expanded to the basal layers of the distal epidermis and distal tip epithelium. Morpholino knockdown of smp reduced regenerative outgrowth by decreasing cell proliferation as measured by BrdU incorporation and histone H3 phosphorylation. In addition, smp knockdown increased the expression of msxb, msxc, and shh, as well as the later formation of ectopic bone. Taken together, these data indicate a requirement for smp in fin regeneration through control of cell proliferation, the regulation of specific genes and proper bone patterning.
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http://dx.doi.org/10.1016/j.ydbio.2008.09.032DOI Listing
January 2009

Leukocyte tyrosine kinase functions in pigment cell development.

PLoS Genet 2008 Mar 7;4(3):e1000026. Epub 2008 Mar 7.

Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, United Kingdom.

A fundamental problem in developmental biology concerns how multipotent precursors choose specific fates. Neural crest cells (NCCs) are multipotent, yet the mechanisms driving specific fate choices remain incompletely understood. Sox10 is required for specification of neural cells and melanocytes from NCCs. Like sox10 mutants, zebrafish shady mutants lack iridophores; we have proposed that sox10 and shady are required for iridophore specification from NCCs. We show using diverse approaches that shady encodes zebrafish leukocyte tyrosine kinase (Ltk). Cell transplantation studies show that Ltk acts cell-autonomously within the iridophore lineage. Consistent with this, ltk is expressed in a subset of NCCs, before becoming restricted to the iridophore lineage. Marker analysis reveals a primary defect in iridophore specification in ltk mutants. We saw no evidence for a fate-shift of neural crest cells into other pigment cell fates and some NCCs were subsequently lost by apoptosis. These features are also characteristic of the neural crest cell phenotype in sox10 mutants, leading us to examine iridophores in sox10 mutants. As expected, sox10 mutants largely lacked iridophore markers at late stages. In addition, sox10 mutants unexpectedly showed more ltk-expressing cells than wild-type siblings. These cells remained in a premigratory position and expressed sox10 but not the earliest neural crest markers and may represent multipotent, but partially-restricted, progenitors. In summary, we have discovered a novel signalling pathway in NCC development and demonstrate fate specification of iridophores as the first identified role for Ltk.
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http://dx.doi.org/10.1371/journal.pgen.1000026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2265441PMC
March 2008

Investigator profile. An interview with Robert Geisler, Ph.D. Interview by Vicki Glaser.

Authors:
Robert Geisler

Zebrafish 2005 ;2(4):237-41

Max-Planck-Institut für Entwicklungsbiologie, Tübingen, Germany.

Robert Geisler, Ph.D., is Group Leader at the Max-Planck-Institut für Entwicklungsbiologie, in Tübingen, Germany. His research focus is on genome mapping and gene expression profiling in the zebrafish. He completed a Bachelor's degree in physiological chemistry and a doctorate in genetics at Tübingen University. Dr. Geisler pursued a postdoctoral fellowship in zebrafish genomics in the laboratory of Pascal Haffter, at MPI für Entwicklungsbiologie, where he developed methods and software for large-scale PCR-based mapping of zebrafish mutations and genes. He has been Scientific Advisor of the ZFIN database project since 2001.
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http://dx.doi.org/10.1089/zeb.2005.2.237DOI Listing
April 2008

The zebrafish mutant lbk/vam6 resembles human multisystemic disorders caused by aberrant trafficking of endosomal vesicles.

Development 2008 Jan 12;135(2):387-99. Epub 2007 Dec 12.

Swiss Federal Institute of Technology, Department of Biology, and Brain Research Institute of the University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.

The trafficking of intracellular vesicles is essential for a number of cellular processes and defects in this process have been implicated in a wide range of human diseases. We identify the zebrafish mutant lbk as a novel model for such disorders. lbk displays hypopigmentation of skin melanocytes and the retinal pigment epithelium (RPE), an absence of iridophore reflections, defects in internal organs (liver, intestine) as well as functional defects in vision and the innate immune system (macrophages). Positional cloning, an allele screen, rescue experiments and morpholino knock-down reveal a mutation in the zebrafish orthologue of the vam6/vps39 gene. Vam6p is part of the HOPS complex, which is essential for vesicle tethering and fusion. Affected cells in the lbk RPE, liver, intestine and macrophages display increased numbers and enlarged intracellular vesicles. Physiological and behavioural analyses reveal severe defects in visual ability in lbk mutants. The present study provides the first phenotypic description of a lack of vam6 gene function in a multicellular organism. lbk shares many of the characteristics of human diseases and suggests a novel disease gene for pathologies associated with defective vesicle transport, including the arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome, the Hermansky-Pudlak syndrome, the Chediak-Higashi syndrome and the Griscelli syndrome.
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http://dx.doi.org/10.1242/dev.006098DOI Listing
January 2008

Radiation hybrid maps of Medaka chromosomes LG 12, 17, and 22.

DNA Res 2007 Jun 25;14(3):135-40. Epub 2007 Jun 25.

The Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.

The Medaka is an excellent genetic system for studies of vertebrate development and disease and environmental and evolutionary biology studies. To facilitate the mapping of markers or the cloning of affected genes in Medaka mutants identified by forward-genetic screens, we have established a panel of whole-genome radiation hybrids (RHs) and RH maps for three Medaka chromosomes. RH mapping is useful, since markers to be mapped need not be polymorphic and one can establish the order of markers that are difficult to resolve by genetic mapping owing to low genetic recombination rates. RHs were generated by fusing the irradiated donor, OLF-136 Medaka cell line, with the host B78 mouse melanoma cells. Of 290 initial RH clones, we selected 93 on the basis of high retention of fragments of the Medaka genome to establish a panel that allows genotyping in the 96-well format. RH maps for linkage groups 12, 17, and 22 were generated using 159 markers. The average retention for the three chromosomes was 19% and the average break point frequency was approximately 33 kb/cR. We estimate the potential resolution of the RH panel to be approximately 186 kb, which is high enough for integrating RH data with bacterial artificial chromosome clones. Thus, this first RH panel will be a useful tool for mapping mutated genes in Medaka.
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http://dx.doi.org/10.1093/dnares/dsm012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2779899PMC
June 2007

The UCS factor Steif/Unc-45b interacts with the heat shock protein Hsp90a during myofibrillogenesis.

Dev Biol 2007 Aug 18;308(1):133-43. Epub 2007 May 18.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 rue Laurent Fries, 67404 Illkirch Cedex, CU de Strasbourg, France.

Contraction of muscles is mediated by highly organized arrays of myosin motor proteins. We report here the characterization of a mutation of a UCS gene named steif/unc-45b that is required for the formation of ordered myofibrils in both the skeletal and cardiac muscles of zebrafish. We show that Steif/Unc-45b interacts with the chaperone Hsp90a in vitro. The two genes are co-expressed in the skeletal musculature and knockdown of Hsp90a leads to impaired myofibril formation in the same manner as lack of Steif/Unc-45b activity. Transcripts of both genes are up-regulated in steif mutants suggesting co-regulation of the two genes. Our data indicate a requirement of Steif/unc-45b and Hsp90a for the assembly of the contractile apparatus in the vertebrate skeletal musculature.
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http://dx.doi.org/10.1016/j.ydbio.2007.05.014DOI Listing
August 2007

Cohesin-dependent regulation of Runx genes.

Development 2007 Jul 13;134(14):2639-49. Epub 2007 Jun 13.

Department of Molecular Medicine and Pathology, School of Medical Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand.

Runx transcription factors determine cell fate in many lineages. Maintaining balanced levels of Runx proteins is crucial, as deregulated expression leads to cancers and developmental disorders. We conducted a forward genetic screen in zebrafish for positive regulators of runx1 that yielded the cohesin subunit rad21. Zebrafish embryos lacking Rad21, or cohesin subunit Smc3, fail to express runx3 and lose hematopoietic runx1 expression in early embryonic development. Failure to develop differentiated blood cells in rad21 mutants is partially rescued by microinjection of runx1 mRNA. Significantly, monoallelic loss of rad21 caused a reduction in the transcription of runx1 and of the proneural genes ascl1a and ascl1b, indicating that downstream genes are sensitive to Rad21 dose. Changes in gene expression were observed in a reduced cohesin background in which cell division was able to proceed, indicating that cohesin might have a function in transcription that is separable from its mitotic role. Cohesin is a protein complex essential for sister chromatid cohesion and DNA repair that also appears to be essential for normal development through as yet unknown mechanisms. Our findings provide evidence for a novel role for cohesin in development, and indicate potential for monoallelic loss of cohesin subunits to alter gene expression.
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http://dx.doi.org/10.1242/dev.002485DOI Listing
July 2007

The zebrafish udu gene encodes a novel nuclear factor and is essential for primitive erythroid cell development.

Blood 2007 Jul 16;110(1):99-106. Epub 2007 Mar 16.

Laboratory of Molecular & Developmental Immunology, Institute of Molecular and Cell Biology, Proteos, Singapore.

Hematopoiesis is a complex process which gives rise to all blood lineages in the course of an organism's lifespan. However, the underlying molecular mechanism governing this process is not fully understood. Here we report the isolation and detailed study of a newly identified zebrafish ugly duckling (Udu) mutant allele, Udu(sq1). We show that loss-of-function mutation in the udu gene disrupts primitive erythroid cell proliferation and differentiation in a cell-autonomous manner, resulting in red blood cell (RBC) hypoplasia. Positional cloning reveals that the Udu gene encodes a novel factor that contains 2 paired amphipathic alpha-helix-like (PAH-L) repeats and a putative SANT-L (SW13, ADA2, N-Cor, and TFIIIB-like) domain. We further show that the Udu protein is predominantly localized in the nucleus and deletion of the putative SANT-L domain abolishes its function. Our study indicates that the Udu protein is very likely to function as a transcription modulator essential for the proliferation and differentiation of erythroid lineage.
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http://dx.doi.org/10.1182/blood-2006-11-059204DOI Listing
July 2007

Differential gene expression as a toxicant-sensitive endpoint in zebrafish embryos and larvae.

Aquat Toxicol 2007 Mar 12;81(4):355-64. Epub 2007 Jan 12.

Helmholtz Centre for Environmental Research - UFZ, Department of Cell Toxicology, Permoserstrasse 15, 04318 Leipzig, Germany.

The zebrafish (Danio rerio) embryo toxicity test (DarT) is under consideration as an alternative to the acute fish toxicity test. Microscopically visible developmental disorders or death are the endpoints used to report on toxicity in DarT. These endpoints are easily observed. They, however, rarely reveal mechanisms leading to a toxic effect and are relatively insensitive compared to chronic toxic effects. We hypothesized that, by using gene expression profiles as an additional endpoint, it may be possible to increase the sensitivity and predictive value of DarT. Therefore, as a proof of principle, we exposed zebrafish embryos to the reference compound 3,4-dichloroaniline (3,4-DCA) and analyzed gene expression patterns with a 14k oligonucleotide array. Important stress response genes not included in the microarray were additionally quantified by reverse transcriptase polymerase chain reaction. Six genes involved in biotransformation (cyp1a, ahr2), stress response (nfe212, maft, hmox1) and cell cycle control (fzr1) were significantly regulated. With the exception of fzr1, these genes proved to be differentially expressed in post hatch life stages as well. The identified genes point toward an aryl hydrocarbon receptor-mediated response. Differential gene expression in embryos exposed for 48 h was observed at 3,4-DCA concentrations as low as 0.78 microM, which is more than 10-fold below the concentrations that elicited visible toxic effects. Upon exposure for 5 days, differential expression was detected at concentrations as low as 0.22 microM of 3,4-DCA, which was close to the lowest observed effect concentration (0.11 microM) in the 30-day early life stage test. This study therefore indicates that gene expression analysis in DarT is able to reveal mechanistic information and may also be exploited for the development of replacement methods for chronic fish tests.
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http://dx.doi.org/10.1016/j.aquatox.2006.12.013DOI Listing
March 2007

A gene-based radiation hybrid map of the gilthead sea bream Sparus aurata refines and exploits conserved synteny with Tetraodon nigroviridis.

BMC Genomics 2007 Feb 7;8:44. Epub 2007 Feb 7.

Institute of Marine Biology and Genetics, Hellenic Center for Marine Research, Crete, Greece.

Background: Comparative teleost studies are of great interest since they are important in aquaculture and in evolutionary issues. Comparing genomes of fully sequenced model fish species with those of farmed fish species through comparative mapping offers shortcuts for quantitative trait loci (QTL) detections and for studying genome evolution through the identification of regions of conserved synteny in teleosts. Here a comparative mapping study is presented by radiation hybrid (RH) mapping genes of the gilthead sea bream Sparus aurata, a non-model teleost fish of commercial and evolutionary interest, as it represents the worldwide distributed species-rich family of Sparidae.

Results: An additional 74 microsatellite markers and 428 gene-based markers appropriate for comparative mapping studies were mapped on the existing RH map of Sparus aurata. The anchoring of the RH map to the genetic linkage map resulted in 24 groups matching the karyotype of Sparus aurata. Homologous sequences to Tetraodon were identified for 301 of the gene-based markers positioned on the RH map of Sparus aurata. Comparison between Sparus aurata RH groups and Tetraodon chromosomes (karyotype of Tetraodon consists of 21 chromosomes) in this study reveals an unambiguous one-to-one relationship suggesting that three Tetraodon chromosomes correspond to six Sparus aurata radiation hybrid groups. The exploitation of this conserved synteny relationship is furthermore demonstrated by in silico mapping of gilthead sea bream expressed sequence tags (EST) that give a significant similarity hit to Tetraodon.

Conclusion: The addition of primarily gene-based markers increased substantially the density of the existing RH map and facilitated comparative analysis. The anchoring of this gene-based radiation hybrid map to the genome maps of model species broadened the pool of candidate genes that mainly control growth, disease resistance, sex determination and reversal, reproduction as well as environmental tolerance in this species, all traits of great importance for QTL mapping and marker assisted selection. Furthermore this comparative mapping approach will facilitate to give insights into chromosome evolution and into the genetic make up of the gilthead sea bream.
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http://dx.doi.org/10.1186/1471-2164-8-44DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1805437PMC
February 2007

Large-scale mapping of mutations affecting zebrafish development.

BMC Genomics 2007 Jan 9;8:11. Epub 2007 Jan 9.

Department 3--Genetics, Max-Planck-Institut für Entwicklungsbiologie, Spemannstr, 35/III, 72076 Tübingen, Germany.

Background: Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers.

Results: We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM.

Conclusion: By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations.
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http://dx.doi.org/10.1186/1471-2164-8-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1781435PMC
January 2007

Learning from small fry: the zebrafish as a genetic model organism for aquaculture fish species.

Mar Biotechnol (NY) 2006 Jul-Aug;8(4):329-45. Epub 2006 Apr 25.

Department of Genetics, Max-Planck-Institute for Developmental Biology, D-72076, Tübingen, Germany.

In recent years, the zebrafish has become one of the most prominent vertebrate model organisms used to study the genetics underlying development, normal body function, and disease. The growing interest in zebrafish research was paralleled by an increase in tools and methods available to study zebrafish. While zebrafish research initially centered on mutagenesis screens (forward genetics), recent years saw the establishment of reverse genetic methods (morpholino knock-down, TILLING). In addition, increasingly sophisticated protocols for generating transgenic zebrafish have been developed and microarrays are now available to characterize gene expression on a near genome-wide scale. The identification of loci underlying specific traits is aided by genetic, physical, and radiation hybrid maps of the zebrafish genome and the zebrafish genome project. As genomic resources for aquacultural species are increasingly being generated, a meaningful interaction between zebrafish and aquacultural research now appears to be possible and beneficial for both sides. In particular, research on nutrition and growth, stress, and disease resistance in the zebrafish can be expected to produce results applicable to aquacultural fish, for example, by improving husbandry and formulated feeds. Forward and reverse genetics approaches in the zebrafish, together with the known conservation of synteny between the species, offer the potential to identify and verify candidate genes for quantitative trait loci (QTLs) to be used in marker-assisted breeding. Moreover, some technologies from the zebrafish field such as TILLING may be directly transferable to aquacultural research and production.
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http://dx.doi.org/10.1007/s10126-006-5139-0DOI Listing
October 2007