Ali-Khan et al. have used gene expression analysis to elucidate signaling pathways potentially involved in the mechanism of the developmental toxicity of excess vitamin A (retinol acetate). The ability to examine simultaneously the expression of thousands of genes in a tissue of interest has emerged relatively recently. While some have characterized the use of this technology to elucidate mechanisms of toxicity as “fishing trips,” the approach can be powerful for generating hypotheses. Global gene array analysis has been used to some success in identifying developmental pathways affected by the human and murine teratogen, valproic acid (Kultima et al., 2004; Okada and Fujiwara, 2006), and has also been used for expression profiling across normal developmental stages (e.g., Bonner et al., 2003; Gheorghe et al., 2006; Wang et al., 2005). Here, the experimental model is the murine limb exposed to retinol while developing in vitro. The premise is straightforward; by examining changes in the embryonal transcriptome in response to developmental insult, one can formulate hypotheses as to the developmental pathways that are critically affected.

In practice, it is far more complex. An embryo undergoing organogenesis is a rapidly changing organism with heterogeneous tissues and cells. Highly transcriptionally active (e.g., the authors report that 40% of all genes on the arrays had detectable transcripts in control limbs), it is likely, though not well documented, that the transcriptome of the organogenesis-stage embryo is changing in a time frame of hours or even minutes. Superimpose on this moving targets the effects of a toxicant and one can quickly get into difficulty in interpreting the results of gene array experiments. To reduce these difficulties, Ali-Khan and Hales have chosen to analyze gene expression in developing limbs at a relatively early time point, after 3 h of culture with retinol. Seeking to detect the earliest transcriptional responses to insult, this approach also minimizes transcriptional differences due to diverging rates of development between treated and control limbs. The choice of the 3-h time point is reasonable—the authors report a total of 81 genes whose expression is upregulated by retinol exposure. Yet, it is still a single snapshot in time. Additional later time points might have allowed elucidation of pathogenetic processes through downstream events, and provided context for the early changes reported here.