GREEN FLUORESCENT PROTEIN IN THE SEA-URCHIN - NEW EXPERIMENTAL APPROACHES TO TRANSCRIPTIONAL REGULATORY ANALYSIS IN EMBRYOS AND LARVAE

The use of Green Fluorescent Protein (GFP) as a reporter for expressio n transgenes opens the way to several new experimental strategies for the study of gene regulation in sea urchin development, A GFP coding s equence was associated with three different previously studied cis-reg ulatory systems, viz those of the SM50 gene, expressed in skeletogenic mesenchyme, the CyIIa gene, expressed in archenteron, skeletogenic an d secondary mesenchyme, and the Endo16 gene, expressed in vegetal plat e, archenteron and midgut, We demonstrate that the sensitivity with wh ich expression can be detected is equal to or greater than that of who le-mount in situ hybridization applied to detection of CAT mRNA synthe sized under the control of the same cis-regulatory systems, However, i n addition to the important feature that it can be visualized nondestr uctively in living embryos, GFP has other advantages, First, it freely diffuses even within fine cytoplasmic cables, and thus reveals connec tions between cells, which in sea urchin embryos is particularly usefu l for observations on regulatory systems that operate in the syncytial skeletogenic mesenchyme, Second, GFP expression can be dramatically v isualized in postembryonic larval tissues, This brings postembryonic l arval developmental processes for the first time within the easy range of gene transfer analyses, Third, GFP permits identification and segr egation of embryos in which the clonal incorporation of injected DNA h as occurred in any particular desired region of the embryo, Thus, we s how explicitly that, as expected, GFP transgenes are incorporated in t he same nuclei together with other transgenes with which they are co-i njected.