Retinoic acid-induced truncation of zebrafish (Danio rerio) embryos Sean Anderson Biology 024: Embryology Swarthmore College, Swarthmore, PA Spring 2004 See Also: Ligas, Adam Objective The purpose of this experiment is to examine the effects of treating zebrafish (Danio rerio) embryos with retinoic acid according to gross morphology and to examine the extent to which the magnitude of the effects is dependent on the concentration of retinoic acid with which the embryos are treated. Introduction Retinoic acid (RA) is a highly teratogenic derivative of vitamin A that is known to influence Hox gene expression. RA-mediated gene activation is important for normal vertebrate development; RA acts as a posteriorizing signal in many developing systems, including mammals, and is also involved in limb formation (Gilbert, 2003). When embryonic exposure is higher than normal, however, developmental anomalies occur. Exposure of the human fetus to 13-cis-retinoic acid results in a characteristic pattern of anomalies, including absent or defective ears, absent or small jaws, cleft palate, aortic arch abnormalities, thymic deficiencies, and abnormalities of the central nervous system. Similar anomalies are observed in other mammals. In mice, for example, embryonic exposure to retinoic acid results in axial truncation and causes a dramatic reduction in the sizes of the first and second pharyngeal arches, which normally form the jaw, ear, and other facial bones (Gilbert, 2003; Ligas, 2000). The truncated embryo exhibits a posterior region having the characteristics of the anterior region of an embryo that had developed normally, including a posterior extension of the ribcage. At very high concentrations, the cells do not differentiate to form the posterior of the embryo at all (Ligas, 2000). Retinoic acid disrupts development by altering the expression of Hox genes, causing the re-specification of the anterior–posterior axis and inhibition of neural crest cell migration from the cranial region of the neural tube (Gilbert, 2003). Retinoic acid cannot bind directly to genes, so in order to affect gene expression, the retinoic acid molecule needs to bind to specific retinoic acid receptors (RAR). After binding, the receptor becomes an active transcription factor. The retinoic acid-bound RARs have at least two modes of action, one of which is to bind to their DNA enhancer sequences and activate particular genes that are not usually activated in these cells. These genes include certain homeotic genes that specify the anterior-posterior position along the body axis. In this way, they can cause homeotic transformations, generally converting anterior structures into more posterior structures (Gilbert, 2003). In the embryo, there is a gradient of retinoic acid from the anterior end to the posterior end. Excess RA results in a posterior region having a higher than normal level of RA, and so more anterior Hox genes are expressed in typically posterior regions (Ligas, 2000). Zebrafish embryos have been shown to exhibit truncation similar to that observed in mice embryos (Ligas, 2000). In order to further examine the effect of retinoic acid on zebrafish embryos and to determine whether the magnitude of such effects is concentration dependent, the embryos will be treated with different concentrations of retinoic acid and allowed to continue development. Development will later be examined at different times according to gross morphology. Download Lab Handout