Figure 2.

Acquisition of cell identity and polarity in vein formation. Arabidopsis first-leaf primordia. PIN1:GFP marker throughout. Sample orientation and all in-figure information as in Figure 1. (Red arrowhead) Convergence point; (red asterisk) bipolar cell; (m) midvein; (ULD#, LLD#) subdomains of vein loop number (l#) as defined in Figure 7. (A) Schematic PED pattern of 6-DAG first leaf depicting the positions of images shown in B–Z. Early vein ontogenies are temporally connected in Figure 7 and summarized in Supplementary Movie S1. (B–G) Apical convergence points and midvein PED formation in young leaf primordia. Largely epidermal PIN1 expression in barely visible 1-DAG leaf primordia in B. Note that the cell at each primordium tip is flanked by cells of opposite polarities; furthermore, PIN1 expression beneath the tip epidermal cell in the left primordium (arrow). (C,E) Pronounced subepidermal PIN1 expression in 2-DAG primordia. LUT signal intensity monitor in D and E (bottom) visualize the epidermal polarity orientation toward the primordium tip, which is associated with a general tip-directed polarity in the epidermal layer at this stage (F). (F,G) Tangential abaxial (F) and median (G) longitudinal optical sections of the same leaf primordium. Note the apical PIN1:GFP polarity in epidermal cells (F) associated with basal PIN1:GFP polarity in the midvein (G). (H–N) First (second-order vein) loop initiation at lateral convergence point. Convergence point formation at primordium flank (H) (enlarged in I, signal quantified in J) followed by the formation of a broad subepidermal LLD1 domain (K) (enlarged in L, smaller pinhole in M to visualize convergence point) that becomes connected distally to the midvein by the formation of a gradually extending ULD1 (N). Note the basal PIN1 polarity in both LLD1 and ULD1 (N,O). (O–U) Completion of ULD1 formation. Integration of LLD1 and ULD1 (O,P), associated with a stretch of opposite polarity distal to a bipolar cell (P) (enlarged in Q, intensity quantified in R). (S–U) Persistence of bipolar cell in fully integrated first loop. (V–AB) Higher-order veins. (V) Initiation of third-order vein PEDs in median optical section (top) is not associated with any epidermal PIN1 expression (tangential abaxial optical section, bottom). (W–Y) Initiation of third-order freely ending PED in the area enclosed by the first loop. Enlargement in X and intensity monitored in Y to visualize basal polarity both in the first loop and in the newly formed third-order PED. (Z–AB) “Connected” third-order PEDs. PED connecting the first and second loops (Z) comprises a bipolar cell bridging two stretches of opposite polarities, each directed toward the adjacent pre-existing PED. Enlargement (AA) and intensity monitor (AB) to visualize opposite polarities and bipolar cell. (B–E,I–U,W–AB) GFP fluorescence. (F–H,V) GFP fluorescence and transmitted light. (I,L,Q,T,X,AA) Details of the areas boxed in H, K, P, S, W, and Z, respectively. (D,E [bottom],J,R,U,Y,AB) LUT images of C, E (top), I, Q, T, X, and AA, respectively (LUT palette and corresponding gray values shown in D only). Note that PIN1:GFP polarity is not visible in lower-order veins in V because of the large pinhole settings used to visualize PIN1:GFP polarity in higher-order veins. Veins are outlined by blue lines in V, W, and Z. Bars: B–H,K–P,S–AB, 10 μm; I,J,Q,R, 5 μm. Further stages and higher-resolution images are shown in Supplementary Figures S2 and S3.