Fig. 3. Prolongation of the transient enhancer activity of the ASE of Lefty2 by addition of Nkx2-binding sequences. (A,B) Structures of two lacZ transgenes are shown at the top. Red circles and blue ovals indicate Foxh1- and Nkx2-binding sequences, respectively. The partially blue circles and boxes indicate the hsp68 promoter and lacZ, respectively. The construct in A includes the Lefty2 ASE, which contains two Foxh1-binding sequences. The construct in B contains the Lefty2 ASE plus seven tandem repeats of the Nkx2-binding sequence derived from the Pitx2 ASE. X-gal staining patterns of E8.2 mouse embryos harboring each transgene are shown below. Both lacZ constructs gave rise to typical asymmetric staining in left LPM. (C-H) Transverse sections of X-gal-stained transgenic embryos at E10.5. Expression of lacZ is shown for the common atrial chamber (C,D), foregut (E,F), and gut dorsal mesentery (G,H). Black arrows indicate the domains where asymmetric X-gal staining was detected. Encircled + or - signs indicate whether the enhancer was active or inactive, respectively. The Lefty2 ASE construct did not give rise to asymmetric staining (C,E,G), whereas the N⁷-Lefty2 ASE construct did so in the common cardinal vein, lung bud and gut dorsal mesentery (D,F,H). Scale bars: 200 µm.