Fig. 3. Multiple regions of the Nrv2 extracellular domain are required for function. (A) A chimera containing the entire Nrv2 extracellular domain and the Nrv3IT domains (Nrv2ABCD) rescues tracheal tube size and paracellular barrier function (A1 and see Fig. 2F1), is targeted to the lateral membrane and SJ (A2), and restores proper Coracle localization (green) and levels (A3,4). (B-G) By contrast, chimeras missing any region fail to restore tracheal tube size (data not shown) and barrier function (B-G, image 1). All chimeras were expressed (B2' and C-G image 2), with Nrv2A (C2), Nrv2CD (D2) and Nrv2BCD (E2) being targeted to the SJ and supporting SJ localization of Coracle at WT or near-WT levels (C3,D3,E3 and C4,D4,E4) indicating these chimeras nonetheless have some SJ-organizing activity. An anti-Nrv3 antibody recognizing the Nrv3I domain (red) was used to detect the chimeras because there is no expression of Nrv3 in nrv2/+ salivary glands [compare B2 (no chimera) and B2' (UAS Nrv2AB)]. Scale bars: in A1, 10 µm for A-G image 1; in A4, 5 µm for A-G images 2-4 and B2'.