Zebrafish genomics unit

Microarray platform:

As part of the European ZF-MODELS consortium (coordinated by our group) we perform gene expression profiling in the developing zebrafish, with the aim to identify downstream targets of known regulatory genes that are not accessible to mutagenesis, to provide candidate genes for existing mutants, and to model regulatory pathways. Our facility is supporting microarray experiments done at the Department of Genetics as well as by other laboratories. We have performed screens by comparing wild type and mutant in the case of finless, sox10 and hypersensitive. Other experiments concerned circadian rhythm; regenerative processes following amputations; mutants affecting heart development; and exposure of embryos to environmental toxicants. Furthermore we are studying gene expression in the zebrafish neural crest (Georg Otto).

For rapid access to expression profiling results, we are developing a public database/browser, ZF-Espresso. It aims to integrate all publicly available expression profiling data for the zebrafish in normalized form and to make advanced tools for data mining, visualization and cross-platform validation available to the general community of zebrafish developmental biologists (Robert Geisler).

Figure 1: Comparison of gene expression in colourless (top) and wildtype (bottom) embryos on microarrays.

Mapping core facility:

In previous years a main focus of our lab has been the microsatellite mapping of zebrafish mutations and radiation hybrid mapping of zebrafish genes and BAC ends (the latter for anchoring contigs that we produced by restriction fingerprinting in support of the zebrafish genome project). Map positions of 319 mutations from the first Tübingen ENU screen have been published recently, and several have been cloned in collaborations with our lab. Approximately 8,000 radiation hybrid markers have been successfully scored in Tübingen, and we are now finalizing these data in order to publish a comprehensive map of zebrafish/human synteny. We have also collaborated on radiation hybrid mapping of the gilthead seabream (a model species for aquaculture) and medaka (Robert Geisler).

Figure 2: Mapping cross for a zebrafish mutation. In a batched segregant approach, PCR of each microsatellite marker is carried out with a pool of approximately 48 mutant F2 embryos and 48 siblings, and the products are separated on agarose gels. Differences in band intensity indicate linkage.

Sequencing core facility:

The Genome Center associated with our group operates an ABI 3730 XL sequencer for daily service sequencing for the entire institute as well as for large-scale projects of individual departments, e.g. sequencing of EST libraries. Approx. 10,000 sequencing reads are performed each month (half of them for service sequencing) (Christa Lanz).

Reverse genetics, TILLING:

A main application of sequencing in the Department of Genetics is for TILLING (i.e. targeted knock-out by resequencing a library of mutagenised zebrafish). The principle of this reverse genetic approach is to identify a mutation in a DNA sample from a mutagenised zebrafish by sequencing a portion of the gene of interest. The DNA sample originates from a fin clip of fish the testes of which were frozen. We have prepared a library of fin clips of 6,200 F1 males as part of the mutagenesis experiment that were eventually kept as frozen sperm and established protocols to amplify exons using the robot of the genomics facility. We recover the mutants by in vitro fertilisation. The library is also available to researchers from Novartis which has sponsored this project (Brigitte Walderich).

Zebrafish stock collection:

We keep and distribute mutants from the previous mutagenesis experiments. While we maintain 75 mutants in living condition, we store most of them as frozen sperm samples from testes of carrier males. We presently transfer stocks to the Zebrafish International Resource Center in Eugene, Oregon, as this center is provided with extra funding for keeping and sending stocks. Upon request, larvae are obtained by in vitro fertilisation and shipped (Hans Georg Frohnhöfer).

Personnel:

Genome mapping:

• Dr. Robert Geisler                                           Project leader

• Ines Gehring                                                    Technician

Gene expression:

• Dr. Georg W. Otto                                            Postdoc

• Christopher Dooley                                        PhD student
• Martina Konantz                                              PhD student
• Horst Geiger                                                     Technician

Sequencing:

• Dr. Christa Lanz                                              Staff scientist

• Andrea Belkacemi                                          Technician

Stock collection:

• Dr. Hans Georg Frohnhöfer                          Research assistant

Tilling library:

• Dr. Brigitte Walderich                                     Fish facility manager

• Ieda Santana-Stamm                                     Sperm freezing

• Christian Weiler                                              Sperm freezing
  
  

Collaborators:

• Dr. Makoto Furutani-Seiki                             ERATO, Kyoto, Japan (medaka)
  
• Dr. Sean Humphray                                       The Wellcome Trust Sanger Institute, Hinxton, UK (fingerprinting)
  
• Dr. Georgios Kotoulas                                   Institute of Marine Biology of Crete, Heraklion, Greece (seabream)
  
• Dr. Mitch Levesque                                        Nüsslein-Volhard group (TILLING)
• Prof. Dr. Ronald Plasterk                               Hubrecht Laboratory, Utrecht, Netherlands (fingerprinting)
  
• Dr. Stefan Scholz                                            UFZ, Leipzig (expression profiling)
  
• Prof. Dr. Herman Spaink                               Leiden University, Netherlands (expression profiling)
  
• Dr. Yi Zhou                                                       Children’s Hospital Boston, USA (RH mapping)
  
  

Key publications: (click here for a complete list)

• Geisler, R., Rauch, G.-J., Geiger-Rudolph, S., Albrecht, A., van Bebber, F., Berger, A., Busch-Nentwich, E., Dahm, R., Dekens, M. P., Dooley, C., Elli, A. F., Gehring, I., Geiger, H., Geisler, M., Glaser, S., Holley, S., Huber, M., Kerr, A., Kirn, A., Knirsch, M., Konantz, M., Küchler, A. M., Maderspacher, F., Neuhauss, S.C., Nicolson, T, Ober, E. A., Praeg, E., Ray, R., Rentzsch, B., Rick, J. M., Rief, E., Schauerte, H.E., Schepp, C.P., Schönberger, U., Schonthaler, H. B., Seiler, C., Sidi, S., Söllner, C., Wehner, A., Weiler, C. and Nüsslein-Volhard, C. (2007). Large-scale mapping of mutations affecting zebrafish development. BMC Genomics 8, 11.
  
• Sarropoulou, E., Franch, R., Louro, B., Power, D.M., Bargelloni, L., Magoulas, A., Senger, F., Tsalavouta, M., Patarnello, T., Galibert, F., Kotoulas, G. and Geisler, R. (2007). A gene-based radiation hybrid map of the gilthead sea bream Sparus aurata refines and exploits conserved synteny with Tetraodon nigroviridis. BMC Genomics 8, 44.
• Schönthaler, H.B., Lampert, J.M., von Lintig, J., Schwarz, H., Geisler, R. and Neuhauss, S.C. (2005). A mutation in the silver gene leads to defects in melanosome biogenesis and alterations in the visual system in the zebrafish mutant fading vision. Dev Biol 284, 421-36.
• Wolff, C., Roy, S., Lewis, K.E., Schauerte, H., Rauch, J., Kirn, A., Weiler, C., Geisler, R., Haffter, P. and Ingham, P. (2004). Iguana encodes a novel zinc-finger protein with coiled-coil domains essential for Hedgehog signal transduction in the zebrafish embryo. Genes Dev 13, 1565-76.
  
• Geisler, R. (2002). Mapping and cloning. In Zebrafish, A Practical Approach, C. Nüsslein-Volhard and R. Dahm, eds. (Oxford: Oxford University Press), 175-212.
• Wienholds, E., Schulte-Merker, S., Walderich, B. and Plasterk, R. (2002): Target-selected inactivation of the zebrafish rag1 gene. Science 297, 99-102.

Links:

Tübingen Map of the Zebrafish Genome (with an online form for RH data submission)

ZF-Espresso (microarray database)

ZF-MODELS Project

Zebrafish Genome Fingerprinting Project (Sanger Institute)

Vorlesung "Genomik von Modellorganismen"

Support:

European Commission : EU Contract LSHG-CT-2003-503496