Thisse, B., Pflumio, S., Fürthauer, M., Loppin, B., Heyer, V., Degrave, A., Woehl, R., Lux, A., Steffan, T., Charbonnier, X.Q. and Thisse, C. (2001) Expression of the zebrafish genome during embryogenesis (NIH R01 RR15402). ZFIN Direct Data Submission. . (http://zfin.org).
We perform a large scale in situ hybridization screen to characterize genes expressed in a spatially regulated manner during zebrafish embryogenesis. cDNAs collected from various zebrafish cDNA libraries are used as templates for the synthesis of digoxygenin labeled antisense RNA probes. These probes are subsequently used to analyze the expression pattern of the corresponding gene at different developmental stages (from gastrula to hatching). cDNAs associated with a spatially restricted expression pattern are sequenced at their 5' and 3' ends to provide information about the encoded protein product and to map the corresponding gene genetically. For each clone analyzed, we provide 5' and 3' sequences of the cDNA (deposited in Genbank), the result of BLAST analysis (against protein databases: SwissProt and translated EMBL) as well as a description of the corresponding expression pattern at six different stages (gastrula, early somitogenesis, middle of somitogenesis, 24h, 36h and 48h) and a set of annotated pictures. These data are deposited in the ZFIN database and cDNA clones are publically available upon request at the Zebrafish International Resource Center . This work provides hundreds of specific cell or tissue markers to analyze mutant phenotypes and to help identify candidates for mutant loci or downstream targets of regulatory genes. This project allows the description of zebrafish embryonic development in terms of gene expression and will eventually establish a "molecular anatomy" of the developing embryo.
Embryos from AB/TU fish (a strain generated from crosses of two wild-type lines, AB and TU) are collected, dechorionated by pronase treatment, allowed to develop at 28.5°C until the appropriate stage and then fixed by incubation over night in 4% paraformaldehyde at 4°C. Embryos older than 24h (hours post fertilization) are incubated in 0.3x Danieau medium supplemented with 1-phenyl-2-thiourea (PTU, 0.003%) to prevent accumulation of pigment. After fixation, embryos are dehydrated and stored at -20°C in 100% methanol prior to in situ hybridization.
The in situ hybridization is performed according to Thisse, C. and Thisse, B. (1998). High resolution whole-mount in situ hybridization. Zebrafish Science Monitor, vol 5. Eugene: University of Oregon Press.
The labeling reaction is monitored under a dissecting microscope and the reaction is stopped with 1x PBS at pH 5.5. Embryos are then mounted in 100% glycerol and incubated at least 24 hours in the dark at room temperature prior to observation. Embryos are mounted under a coverslip in 100% glycerol. Pictures are taken using a color CCD camera (Roper Scientific, Coolsnap) mounted on a dissecting microscope (Leica, M420) or on a compound microscope (Leica, DM RA2HC or Nikon, FXA).
cDNAs obtained from various zebrafish cDNA libraries are used as templates for the preparation of digoxygenin labeled antisense RNA probes. Plasmid DNA is purified, linearized using appropriate restriction enzymes and antisense RNA probe is synthetized according to the in situ hybridization protocol. cDNAs associated with a gene whose expression is spatially restricted are sequenced at their 5' and 3' ends. The 5' sequence is then used to perform a blast analysis against protein sequence databases (SwissProt and translated EMBL).
Every expression pattern and sequence has been analyzed twice before release in ZFIN.
cDNA libraries used:
Segmentation stages: lambda ZAP oligo-dT primed cDNA library constructed by B. Riggleman.
Random primed: lambda ZAP random primed cDNA library made from RNA purified from a pool of embryos collected between 18h and 48h. Constructed by C. Fromental and J.M. Garnier (IGBMC, Strasbourg, France).
Gastrula stages: lambda ZAP oligo-dT primed cDNA library constructed by T. Lepage (Villefranche/mer, France)
26 Somite stage: oligo-dT primed cDNA library prepared in pSPORT1 vector by M. Clark (Max Planck Institute, Berlin)
Shield stage: oligo-dT primed cDNA library prepared in pSPORT1 vector by M. Clark (Max Planck Institute, Berlin).
Adult kidney: Oligo dT cDNA library prepared in pBK-CMV vector Constructed by by L. Zon (HHMI, Children's Hospital of Boston)
Acknowledging this work: All clones, information, and images used from this work should cite this summary and first description of this work posted in ZFIN: (Thisse, B, Pfumio, S., Fürthauer, M., Loppin B., Heyer, V., Degrave, A., Woehl, R., Lux, A., Steffan, T., Charbonnier, X.Q. and Thisse, C. Expression of the zebrafish genome during embryogenesis. ZFIN on-line publication, 2001). This work is supported by grant RR15402-01 from the NIH. Please acknowledge this grant number in all publications resulting from the materials we are providing to you.
If you have any questions or comments on this project, please contact CB Thisse.