Janus and Medusa Gal4 effector constructs for simultaneous expression of multiple subcellular labels. (A) Schematic representation of bidirectional Janus vectors J1 and J2. Upon binding of Gal4, two subcellular markers are expressed simultaneously (J1: H2B-mRFP labels the nucleus in red and GFP-DCX the microtubules in green; J2: memmRFP labels the membrane in red and H2B-CFP the nucleus in blue). (B) Schematic representation of Medusa vectors M1, M2, and M3. From each vector, the expression of three subcellular markers is activated in the presence of Gal4. M1 encodes H2B-CFP to label the nucleus in blue, memmRFP to mark the membrane in red, and Centrin2-YFP to label the centrioles of the centrosome in yellow. M2: H2B-mRFP to label the nucleus in red, GFP-DCX to label microtubules in green, and memCFP to label the membrane in blue. M3 codes for the same nuclear and membrane markers as M2, but contains EB3-GFP to label the plus-ends of microtubules. These data demonstrate that reliable coexpression of various transgenes can be achieved from Gal4-mediated multicistronic expression vectors. Images were obtained from living zebrafish embryos (24 hpf) coinjected at the one-cell stage with the respective Janus or Medusa vectors and a vector coding for Gal4. “mem“ represents a membrane localization signal, which consists of a plamitylation and myristinylation sequence of the human Lck kinase. Arrows in M1 indicate YFP-labeled centrosomes.