“All the Zebrafish News THE ZEBRAFISH That’s Fit to Print” SCIENCE MONITOR Volume 2 Issue 4 Monday, October 25, 1993 Eugene, Oregon, USA ZEBRAFISH AT THE THE PHYLOGENETIC POSITION OF ISDB MEETING THE ZEBRAFISH ( RERIO), A By M. Westerfield, Institute of Neuroscience, University of Oregon, Eugene, OR 97403 MODEL SYSTEM IN DEVELOPMENTAL Zebrafish research news was BIOLOGY: AN INVITATION TO THE reported in a number of presentations at the recent 12th Congress of the COMPARATIVE METHOD International Society of Developmen- tal Biology held in Vienna, Austria, By A. Meyer, C.H. Biermann, and G. Ortí, Department of Ecology & Evolution, State August 8-13, 1993. This issue of The University of New York, Stony Brook, NY 11794-5245, USA (Proc. R. Soc. Lond. B (1993) Monitor includes abstracts from these 252:231-236) presentations. The zebrafish, Danio (Brachydanio) rerio, has become one of the most widely As explained in the editorial on studied model systems in developmental biology. We present a DNA-based page 2, these abstracts are repro- phylogeny of zebrafish and other species of the genus Danio, and the genera duced here as part of the latest zebrafish news. They should not be Rasbora, Puntius, and Cyprinus. Homologous regions of the large (16S) cited. Reference to information from mitochondrial ribosomal RNA gene were amplified by the polymerase chain these abstracts or from any other reaction and directly sequenced. The phylogeny revealed: (i) the zebrafish, Danio articles appearing in The Monitor (Brachydanio) rerio is identical in its 16S sequence to its aquarium breeding should be made as personal communi- morph, the leopard danio; (ii) the pearl danio (Danio albolineatus) is more closely cations and then only if you have related to the zebrafish than the giant danio (Danio aequipinnatus); and (iii) received explicit permission of the species of the genus Rasbora (hetermorpha, trilineata, elegans, pauciperforata, authors. dorsiocellata) are more closely related to the than members of the genus Puntius (tetrazona, conchonius) and Cyprinus, the carp. All of these species are readily available in the aquarium trade, easily kept and bred in captivity, and CHARACTERIZATION amenable to developmental work. It is hoped that this molecular phylogeny will invite developmental biologists to use the comparative method to ask questions OF POU GENES IN about function (e.g. cellular and genetic aspects) and evolution of zebrafish ZEBRAFISH EMBRYOS developmental biology in a phylogenetic context.

By G. Hauptmann, P. Spaniol, C. Bornmann, and T. Gerster. Abt. Zellbiologie, structures in the brain express these genesis in addition to a large maternal Biozentrum der University of Basel, 4056 genes. We are currently investigating pool of RNA. Its transcript shows an Basel, SWITZERLAND how the expression patterns of the asymmetric distribution in the pro- We have identified by PCR and four brn-1-like genes differ from each spective neuroectoderm at late cDNA cloning five different POU other and how much they overlap. gastrula stages. Thereafter, the genes expressed during early embryo- Possibly, the four brn-1-like genes expression of this gene decreases genesis of zebrafish. Four of these play combinatorial roles resulting in rapidly and at 24 hours after fertiliza- genes show extended homology to the differential activation of specific cell tion, the transcript can only be found brn-1 class of POU genes previously fates in the zebrafish brain. in the extreme tip of the tail. The identified in mammals. A fifth gene we have analyzed early expression and the spatial Northern blot analysis and in situ contains a POU domain that forms arrangement of the transcript strongly hybridizations indicate that the the prototype for a novel subclass of argue that this POU gene is involved expression of these genes begins these DNA binding proteins. This in early developmental decisions shortly after gastrulation in the neural particular gene appears to be ex- determining the body plan of the tube. In the 24 h old embryo, various pressed extremely early in embryo- zebrafish. 2 THE ZEBRAFISH SCIENCE MONITOR, Monday, October 23, 1993

THE ZEBRAFISH MONTE WESTERFIELD, Editor ANALYSIS OF EARLY SCIENCE MONITOR PAT EDWARDS, Publications Coordinator DEVELOPMENT - ...an informal vehicle dedicated to communicating zebrafish news. References to information appearing in the THE ROLE OF ACTIVIN- Monitor should be made as personal communications and only if explicit permission of the authors is obtained. LIKE GENES Published at the Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403 USA J. Wittbrodt, M. Rissi, and F. Rosa, Biocenter of the University of Basel, CH- Editorial 4056 Basel, SWITZERLAND. A MATTER OF POLICY Cell-cell interactions through diffusible factors of the TGF- super- It has been our intention, since The Monitor’s inception, that this humble family are thought to be involved in publication serve as a vehicle for the rapid communication of news pertinent the specification and modification of to the Zebrafish research community. We hope that all of you will be eager cell fate in early vertebrate embryos. to send in progress reports of studies that are incomplete or not yet ready to Using medaka (Oryzias latipes) and submit to a regular journal. zebrafish (Brachydanio rerio) as “But, wait a minute” you argue. “How can I send in something I hope experimental systems, we are analyz- to publish later? Won’t its appearance in The Monitor preclude its later ing the role of homologous and newly acceptance in a more respectable place, like Nature or Cell?” “Heck, no!” the editor moans. “The Monitor, is an ‘informal’ vehicle discovered members of the TGF- dedicated to communicating news, not publishing reviewed manuscripts.” family in fish. Two new members of All references to information appearing in The Monitor should be made the DVR subfamily were discovered. as “personal communications’ and then only if you have received explicit One of these, Zac15, is conserved permission of the authors. throughout evolution and differen- Because all of us have such poor memories, we will include a statement tially expressed during embryonic of this policy in each future issue as a reminder of our charter. development. Whole-mount in situ analysis could first detect the message in the early neurula in the lining of A GENOME-WIDE SCREEN four distinct loci vary between 1/400- the prospective brain regions. Later FOR MUTATIONS 1/4000. Recessive embryonic lethal stages show expression in cells AFFECTING EMBRYONIC or visible mutations are recovered in derived from all germ layers. The DEVELOPMENT IN a two generation screen with an expression pattern in the neuroecto- average rate of 3 mutations per line. ZEBRAFISH derm suggests a role of the Zac15 Approximately half of the mutations gene in the specification of distinct By L. Solnica-Krezel, A.F. Schier, S. identified so far appear to affect regions of the CNS. Using a PCR Abdelilah, J. Malicki, S. Neuhauss, Z. specific regions or processes in Rangini, D. Stainier, D. Stemple, F. based approach, we could isolate developing embryos. We are focusing Zwartkruis, and W. Driever, CVRC, MGH- on two classes of mutants. In mu- three genes encoding activin chains East 4, 13th St., Bldg. 149, Charlestown, in medaka and zebrafish. To interfere MA 02129 USA tants of the first class formation or differentiation of the notochord and with the endogenous activins in Our studies show that mutations somitic mesoderm are affected. The transgenic , we introduced affecting specific aspects of verte- second class of mutants is character- different types of mutations into a brate development can be efficiently ized by abnormal patterning of the full-length cDNA clone encoding the identified in zebrafish (Brachydanio anterior neural tube. zebrafish activin B. While one type rerio) by genetic screens similar to of mutant allowed the analysis of the those performed in C. elegans and contribution of the zygotic expression Drosophila. We have developed of activin to axis formation, the other methods for efficient mutagenesis of type enabled us to analyze zygotic proliferating germ cells by treating and maternal contribution of activin zebrafish males with N-ethyl-N- protein to mesoderm induction and nitrosourea. The mutation rates estimated by the specific locus test at axis formation in vivo. THE ZEBRAFISH SCIENCE MONITOR, Monday, October 25, 1993 3 GENETIC ANALYSIS OF laboratories are developing the tools GENETICS OF AXIS EMBRYONIC PATTERN for a genetic analysis in this organ- DEVELOPMENT IN ism. It remains to be seen whether FORMATION IN this approach will be as fruitful as it ZEBRAFISH DROSOPHILA AND THE has been in Drosophila. By C.B. Kimmel, M.E. Halpern, R.K. Ho, K. ZEBRAFISH, Hatta, A.E. Melby, C. Walker, and *B. Trevarrow, Institute of Neuroscience, BRACHYDANIO RERIO University of Oregon, Eugene, OR 97403 BLASTEMA CELLS OF USA. *Department of Biology, California By C. Nüsslein-Volhard, Max-Planck-Institut Institute of Technology, Pasadena, CA 91125 für Entwicklungsbiologie, Tübingen, REGENERATING USA GERMANY ZEBRAFISH FINS We have used zygotic lethal The systematic searches for mutations to analyze notochord and mutants affecting embryonic pattern EXPRESS MSX AND floor plate development. ntl mutants in Drosophila and the subsequent DISTAL-LESS GENES lack a notochord, cyc mutants lack a molecular analysis led to the identifi- 1 2 By M.-A. Akimenko , S. Johnson , M. floor plate, and both tissues are cation of a large number of important 1 2 1 Ekker , and M. Westerfield . Loeb Institute affected in the trunk and tail of flh regulatory proteins. Their interac- and Depts. of Medicine & Anatomy, 2 mutants. Mosaic analysis and tions in several pathways have been University of Ottawa, CANADA. Institute epistatic relationships between cyc established, controlling the progres- of Neuroscience, University of Oregon, Eugene, OR 97403-1254 USA and ntl suggest that floor plate sive subdivisions and differentiation development depends on early of the initially uniform egg cell. This Members of the msx and distal- inductive signaling of ectoderm by approach has led to the fact that less (dlx) families of homeobox genes dorsal mesoderm. Signaling appears Drosophila is now in many respects are transiently expressed during to require flh+ but not ntl+ function the best understood system in biology. development of the zebrafish primor- in the trunk. Neither function is How much of the knowledge from dial fin fold, which gives rise to the required in the head, and both are studying Drosophila can be applied unpaired fins. Transcripts of some of required in the tail. to vertebrate organisms is an open the genes are also observed in the question. As the body organization of pectoral fin buds. However, 3 days a vertebrate and an is very after fertilization, msx and dlx DEVELOPMENT OF different, a number of pattern- expression in fins falls below levels IDENTIFIED forming mechanisms that do not have detectable by in situ hybridization. a parallel in Drosophila are expected We have assayed for the presence of MOTONEURONS IN to take place. Systematic mutational msx and dlx transcripts during fin EMBRYONIC approaches were not yet feasible in a regeneration. We observe msxB, ZEBRAFISH vertebrate and much of our knowl- msxC, msxD, and dlx3 transcripts edge about controlling genes comes during regeneration of both paired By J.S. Eisen, Institute of Neuroscience, and unpaired fins. MsxD transcripts University of Oregon, Eugene, OR 97403 from a surprising degree of homology USA between Drosophila genes and are detected in the distal-most layer of vertebrate genes. Homology ap- cells of the blastema in contrast to We are interested in how neurons proaches, however, a priori are msxB and msxC which are expressed acquire their identities and express restricted to conserved, and exclude in more proximal blastema cells. cell-specific characteristics, such as novel, structures, and conservation of Amputation performed in different shape and synaptic connectivity. To structure is not always accompanied planes along the proximo-distal axis address these issues, we have exam- by conservation of function. In recent of the fin suggests that msxB expres- ined the development of a set of years, a small freshwater fish, the sion correlates with the growth rate of individually identified motoneurons in zebrafish, has attracted the interest of the regenerate. These results suggest the embryonic zebrafish. This set experimental embryologists and a role for msx and dlx genes in the consists of 3-4 bilaterally symmetric geneticists. It has many good quali- mechanisms of dedifferentiation and motoneurons that are segmentally ties of an experimental system, such redifferentiation necessary for fin iterated along the embryonic axis. as transparent eggs and embryos and regeneration. Supported by the Analysis of mutations affecting a speedy development. We and other MRC, the NCIC, and the NIH. specific tissue types suggests that the 4 THE ZEBRAFISH SCIENCE MONITOR, Monday, October 23, 1993 segmental organization of these EXPRESSION OF THE 24 h after fertilization was observed motoneurons is influenced by interac- XENOPUS HOMEOGENE in whole-mounts and in sections. It tions with the paraxial mesoderm. appeared to be exclusively restricted Transplantation of single motoneu- XHOX3 IN ZEBRAFISH to posterior mesoderm tissue. rons suggests that their individual EGGS CAUSES A Our results on the zebrafish identities depend on their spinal cord DISRUPTION OF embryo are surprisingly similar to positions. Motoneurons in different those obtained in Xenopus after spinal cord positions seem to have no ANTERIOR-POSTERIOR XHOX3endogenous gene analysis (2). influence on one another’s axonal AXIS This evidence supports the hypothesis of a high conservation of pathway choices, and proper synaptic 1 2 3 By O. Barro , A. Ruiz i Altaba , J.S. Joly , the mechanism implicated in embry- connectivity appears to be the result C. Joly1, H. Condamine3, and H. of cell-specific pathway recognition. Boulekbache1, 1Laboratoire de biologie du onic development involving the Interactions between two motoneu- développement Université Paris 7, 2 place XHOX3 gene. rons that form an equivalence pair Jussieu, 75251 Paris cedex 05, FRANCE. 1) Scharf, S.R. and J.C. Gerhart, 2Howard Hughes Medical Institute, determine which one of them will Dev. Biol. 99:75-87, 1983. Columbia University, New York, NY 10032. 2) Ruiz i Altaba, A. and D.A. survive. 3Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris cedex, FRANCE. Melton, Cell 57:317-326, 1989. DNA UPTAKE FROM THE The ectopic expression in the YOLK INTO THE zebrafish of the Xenopus homeogene SCALES IN THE BLASTOMERES IN EARLY XHOX3 was carried out by microin- ZEBRAFISH, DEVELOPMENTAL jection of synthetic XHOX3 mRNA (with an eve homeobox) into BRACHYDANIO: CELL STAGES OF FISH uncleaved embryos and resulted in ORIENTATION AND EMBRYOS various degrees of anterior-posterior ANISOTROPIC CELL axis disruption. By T. Papp, F. Erdélyi, A. Ádám, and L. AGGREGATIONS MAY The phenotypic variants observed Orbán, Institute for Molecular Genetics, DETERMINE SCALE Agricultural Biotechnology Center, Gödöllõ, mainly show anomalies in neural tube P.O.B. 170, H-2101 HUNGARY development, going to mi- ORIENTATION crophthalmia to acephali, and could Various gene constructs injected By K. Nübler-Jung, Biologie Institut I, have been classed according to an into the yolk of fish eggs were Alberstr. 21a, D-7800 Freiburg, GERMANY. index of axis deficiency (IAD) (1). A transiently expressed in the early How do polarized structures (e.g embryo. The expression of the - dose-dependent effect of injected XHOX3 mRNA was evident; from hairs or scales) adopt their specific galactosidase gene was first detected orientations in integuments? in the blastomeres and syncytial layer 5pg to 10pg of mRNA into each egg, we observed a major change from We find that the ripple patterns on 5 hours after fertilization. At 24 our finger tips, as well as the various hours postfertilization -galactosidase prim 5 stage embryos with a normal orientations of hairs and scales in staining was observed in the whole phenotype, to an acephalic pheno- vertebrates resemble polarity patterns embryo including the yolk. An type. The specificity of exogenous intensive flow from the yolk towards XHOX3 protein effects was con- seen in . This may indicate the blastomeres was revealed in the trolled by the observation of a normal similar orienting mechanisms in earliest stages of development of phenotype for the majority (91.8%) insects and vertebrates. zebrafish and African catfish eggs by of embryos injected with XHOX3 In the zebrafish, Brachydanio, injection with FITC-dextran and transcripts with a deleted HTH motif dermal and epidermal cells together labeled plasmids. Our observations homeobox. Controls included the orient the scale anlagen. Dermal cells suggest that DNA injected into the injection of lacZ mRNA and the (like insect epidermal cells) orient yolk is transferred to the blastomeres immunostaining of mes-metencepha- along the dorsoventral body axis and by this flow. Microinjection into the lon, using the 4D9 antibody. thereby may determine the axis of the yolk has some advantages over the The nuclear localization of scale, while epidermal cells accumu- conventional microinjection method: “XHOX3-like” protein was deter- late at the posterior rim of the lower mortality rate and higher mined using an antibody against the anlagen. Interactions between dermis occurrence of transiently expressing NH2 terminal part of the protein. and epidermis thus seem to determine embryos. The distribution of positive nuclei at the orientation of scales. THE ZEBRAFISH SCIENCE MONITOR, Monday, October 25, 1993 5 STRUCTURE AND zebrafish spermatogonia were estab- damesoderm. These results suggest lished through the use of the chemical that the induction of Isl-1 in the COMPOSITION OF THE mutagen ethylnitrosourea. Mutation primary neurons is dependent on ZEBRAFISH EGG rates range from 0.1 to 0.3% for an signaling spreading in the plane of CHORION individual locus, similar to those used ectoderm. Isl-1 positive cells will be in genetic screens in Drosophila and the first neurons to send axons which By D. Bonsignorio, S. Raisoni, C. Lora C. elegans. will pioneer the major axonal tracts. Lamia, and F. Cotteli. Department of We performed a small-scale Biology, University of Milano, Milano, classical F2 diploid screen. Most of ITALY the mutants we isolated have specific COMBINATORIAL The zebrafish egg is completely defects, but the abnormalities arise EXPRESSION OF THREE surrounded by an amorphous thick late in embryonic development and do DISTAL-LESS GENES envelope usually called the chorion. not appear to affect embryonic During oogenesis, this coat is as- pattern formation. We have only a DURING ZEBRAFISH sembled between the egg and the small number of mutants displaying EMBRYONIC follicle cells. It is multi-layered and early embryonic defects. Our large- DEVELOPMENT made up by a fibrillar material scale saturation screen is currently in embedded in an amorphous matrix as progress. By M. Ekker1, M.-A. Akimenko1, and M. in other fish. In zebrafish, the Westerfield2, 1Loeb Institute and chorion is made up by at least 4 Departments of Medicine and Anatomy, major layers. Isolated and purified University of Ottawa, CANADA. 2Institute chorions have been analyzed by SDS- ZEBRAFISH of Neuroscience, University of Oregon, PAGE under reducing and non- HOMEODOMAIN Eugene, OR 97403-1210 USA reducing conditions. A reproducible PROTEIN ISL-1 IS pattern of polipeptides with molecular We have isolated three zebrafish weights ranging from 30 to 125 KDa EXPRESSED IN genes, dlx2, dlx3 and dlx4, with was revealed. PRIMARY NEURONES homeoboxes related to that of the Using various lectins, we have Drosophila distal-less gene. Al- investigated the presence of glycopro- PIONEERING AXONAL though the overall patterns of expres- teins and we have detected the most TRACTS sion of the three genes during embry- representative carbohydrates. onic development are distinct, we By V. Korzh, S. Thor, and T. Edlund, observe remarkable similarities in the Department of Microbiology, University of expression of two or three dlx genes MUTAGENESIS AND Umeå, SWEDEN in specific regions of the embryos. SCREEN FOR Isl-1 was initially isolated as a For example, a subset of cells in the factor binding to the insulin gene ventral forebrain expresses dlx2 and EMBRYONIC MUTANTS enhancer. Isl-1 expression is regu- dlx4 starting at 16 h after fertiliza- IN THE ZEBRAFISH, lated by the notochord/floor plate tion. Cells of the olfactory placodes BRACHYDANIO RERIO inducing signals. We have shown or their precursors express dlx3 and using anti-rat Isl-1 antibody staining dlx4. Similarly, dlx3 and dlx4 By M. Mullins, M. Hammerschmidt, P. on whole-mounted and sectioned transcripts are present in the primor- Haffter, and C. Nüsslein-Volhard, Max- embryos of zebrafish that Isl-1 dial fin fold and in a subset of cells in Planck-Institut für Entwicklungsbiologie, immunoreactivity is selectively the otic vesicle. All three dlx genes Spemannstrasse 35/III, D-7400 Tübingen 1, expressed in the primary neurones are expressed in cells of the visceral GERMANY (primary motor neurons, Rohon- arches and their primordia. dlx2 We are investigating the embry- Beard cells, primary interneurons, alone is expressed during gastrula- onic development of the zebrafish neurons of the trigeminal ganglia and tion. Based on these results, we through the isolation and character- anterior group of cells) in the spinal suggest that combinatorial expression ization of mutations in zygotically- cord and brain. Isl-1 expression is of the dlx2, dlx3 and dlx4 genes is expressed genes involved in embry- initiated at the very beginning of part of a new type of homeobox gene onic pattern formation, morphogen- neurulation (10 hpf). Induction of code which could be important in esis, and differentiation. We have Isl-1 expression in all these cell specifying pattern formation or cell developed the technology to make groups takes place almost simulta- fate determination in multiple regions practical a large-scale screen. High neously and before the notochord of the embryo. Supported by the rates of point mutagenesis of differentiates from the axial chor- MRC, the NCIC, and the NIH. 6 THE ZEBRAFISH SCIENCE MONITOR, Monday, October 23, 1993 ZEBRAFISH RAR’S AND RXR’S: EVOLUTIONARY CONSERVATION OF STRUCTURE AND Meeting on FUNCTION Zebrafish Development and Genetics By M. Petkovich, J. White, B. Jones, and M. Cold Spring Harbor Laboratory Boffa, Cancer Research Labs, Queen’s April 27 - May 1, 1994 University, Kingston, Ontario, CANADA K7L 3N6. Organized by: Retinoic acid (RA) is an impor- Wolfgang Driever, Massachusetts General Hospital tant signaling molecule in vertebrate Judith Eisen, University of Oregon pattern formation. The effects of RA are due largely to regulation of gene David Grunwald, University of Utah transcription, mediated by 2 classes Charles Kimmel, University of Oregon of nuclear receptors - retinoic acid receptors (RAR- , RAR- , RAR- ) This is the first open-invitation meeting dedicated to research on the and retinoid X receptors (RXR- , zebrafish. The meeting will cover a broad range of topics. Anticipated RXR- , RXR- ). We have been using sessions and chairpersons include: zebrafish as a developmental model to study the role of retinoic acid in Gastrulation, body patterning, and morphogenesis: C. vertebrate development and have Nusslein-Volhard and R. Ho isolated cDNA’s which closely Determination of cell fate: N. Holder and M.-A. Akimenko correspond in sequence to their mouse Development of the nervous system: J. Campos-Ortega and J. and human receptors. Zebrafish Kuwada RAR’s (zfRAR- , - , - ) and RXR’s Growth control: M. Schartl (zfRXR- , - , - ) are also functionally Organogenesis: M. Fishman conserved and form RAR/RXR Genetics and Genomics: M. Westerfield heterodimers, and exhibit similar Gene transfer, gene expression, new methodologies: N. Hopkins target gene specific activation com- pared to their mouse counterparts. Submitted abstracts will be considered for oral or poster Developmental patterns of zebrafish presentations. Abstract deadline is Feb. 9, 1994. For further RAR and RXR expression are also information and registration materials, contact conserved. For example, by whole mount in situ hybridization, zebrafish Meetings Coordinator RAR- expression is detectable in Cold Spring Harbor Laboratory head and tail bud mesenchyme, and at later stages, is in the mesenchyme of 1 Bungtown Road the developing fin bud. Thus, the Cold Spring Harbor, New York 11724-2213 zebrafish retinoid signal transduction TEL:516-367-8346 system is highly conserved, strongly FAX:516-367-8845 supporting the relevance of the zebrafish model to study the develop- Everyone who receives an individual copy of the Zebrafish Science mental role of retinoic acid. Monitor should be receiving registration materials directly from Cold Spring Harbor Laboratory. THE ZEBRAFISH SCIENCE MONITOR, Monday, October 25, 1993 7 8 THE ZEBRAFISH SCIENCE MONITOR, Monday, October 23, 1993

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