The family of T-Box genes have been investigated using knock out zebrafish with single knock out and multiple knock outs. Characterization of spt knock out, nt} knock out and nt} doub}e knockout embryos shows that spt protein and ntl protein function in a partially redundant manner 25. The regulation of the expression of these genes is apparently similar, as a high correlation in the expression values of spt gene and ntl gene was found, Spt protein, ntl protein and tbx6 protein might interact in three ways: (a) two factors activate target genes that neither factor can activate on its own (combinatory interaction), (b) two factors contribute to the activation of a gene in an additive manner and (c) one factor prevents activation of a gene by another factor (competitive antagonism) 40 Summarizing the studies on gene expression in zebrafish regarding the T-box 6/16 subfamily and the demonstrated increase of tbx6 mRNA due to p353-NP exposure; it is most likely that tbx6 protein is abundant when zebrafish embryos are treated with p353-NP. It is presumed that the tbx6 protein competes with spt protein and antagonized the spt protein effect, resulting in similar phenotype (deformed tail) as spt knock out zebrafishes. This postulation is supported by the fact that tbx6 protein compete effectively with ntl protein promoted expression of some T-site dependent gene transcription.

It is not known which molecular mechanisms regulate the expression of tbx6 gene. Thus far only regulation via spt/ntl proteins is described: Tbx6 gene contains six spt/ntl protein binding sites within 500 bp of the transcription site, and expression-driven by a 500 bp tbx6 gene promotor is mainly regulated by spt protein and ntl protein, spt protein exerting more influence 41. In our experiments an over expression of spt gene or ntl gene was not observed in embryos treated with p353-NP compared to the other NP isomers or the controls. The regulation of tbx6 gene in vivo is probably more complex with more regulatory elements in greater distance to the transcription site and not yet analyzed by reporter gene experiments.

It was necessary to investigate gene expression of tail formation in an early stage of embryonic development, whereas the fully developed tail was visible considerably later. The developmental stage of 48 hr was selected to visually investigate the embryos, because the visibility of the tail tip was best at this time.

It is in general not possible to investigate gene expression during migration of precursor cells of the mesoderm and phenotype of tail tip formation at the same time. It was therefore necessary to combine two experiments conducted a different time points. One could detect reasonable results for B-actin gene regulation at the time point select for investigating the gene expression. This indicates that changes in gene expression due to NP might be investigated at the selected time point. As discussed above, the observed relation of p353 NP and tbx6 gene expression is plausible as cause and effect, but this can not be proven with the shown experiments alone. With this investigation a short snapshot in embryonic gene expression was covered. Other genes related to NP toxicity might be expressed in different time frames. The combination of gene knock out experiments to analyze phenotypes induced by administration of NP may reveal new insights into the biological mechanisms of these xenobiotics. With our investigation the starting point for such future experiments is provided. Only this hypothesis driven approach shed some light on the transcriptions factors initial responsible for gene activation or gene inactivation and in conclusion for the mode of action of NP in fish embryos.

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