Spatial and temporal distribution of the traf4 genes during zebrafish development

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Abstract

The tumor necrosis factor-associated factor 4 (TRAF4) is a particular member of the TRAF protein family since it is not involved in the Tumor Necrosis Factor (TNF) and Interleukin-1 (IL-1) signaling pathways. In the present study, we cloned two zebrafish orthologs of the human traf4, traf4a and traf4b, which are the first TRAFs described in zebrafish. During embryogenesis, traf4b expression is present in a weak ubiquitous manner. In contrast, traf4a exhibits a highly specific expression pattern in the sensorial and neural cells, and the somites of embryos. This gene is tightly regulated during embryogenesis. Together, our data show that traf4 is conserved during evolution, and traf4a is the zebrafish ortholog of traf4.

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Cloning and sequencing of zebrafish traf4 orthologs

In order to isolate the cDNA encoding traf4 in the zebrafish, we screened the GenBank database and found two EST fragments of 494 and 674 bp respectively (AW455071 and AW184451), of zebrafish origin with significant sequence identity to the human homolog (52 and 73% identity between human TRAF4 protein and the predicted peptidic sequence of AW455071 and AW184451, respectively). To clone the full-length cDNA, we first generated by RT-PCR a cDNA specific probe from zebrafish gastrula-stage total

Expression pattern of traf4a and traf4b

The pattern of expression was analyzed by whole mount in situ hybridization using a digoxigenin labeled antisense RNA probe encompassing the full length traf4a and traf4b cDNA respectively.

While traf4b is expressed weakly and ubiquitously throughout embryogenesis (not shown), the expression of the traf4a gene is tightly regulated during development. Indeed, traf4a is first weakly detected at 75% epiboly in the enveloping layer (Fig. 2A). At the onset of somitogenesis (at the 3 somite stage),

Cloning of zebrafish traf4 gene

Coding sequence of human traf4 gene was compared to EST databases; two zebrafish ESTs were found: AW455071 and AW184451. AW184451 sequence was amplified from cDNA derived from zebrafish gastrula-stage total RNA, using the following primers:

  • 5′-GCAGTATTACAGTGGGCAGCGGCT-3′ (forward)

  • 5′-CACCCTGATGATTCTCGTATGCCT-3′ (reverse).

The denaturation was performed at 94 °C for 15 s, the annealing at 60 °C for 30 s and the elongation at 72 °C for 30 s; 30 cycles were used. The PCR product was purified and ligated

Acknowledgements

We thank C. Hindelang, A. Lux, S. Vicaire, I. Colas, F. Ruffenach, E. Troesh for their technical assistance. This work was supported by funds from the Institut National de la Santé et de la Recherche Médicale (INSERM), the Centre National de la Recherche Scientifique (CNRS), the Hôpital Universitaire de Strasbourg, the Association pour la Recherche sur le Cancer, the Ligue Nationale Contre le Cancer (LNCC) and the Comités du Haut-Rhin et du Bas-Rhin, and the Association Régionale pour

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