Fig. 2

ATHB8 and MP Expression Domains and Levels in Leaf Development. First leaves 2, 3, and 4 DAG. Column 1: schematics of leaves — imaged in columns 2–5 — illustrating onset of ATHB8 expression (red) — imaged in column 2 — associated with formation of midvein (2 DAG), first loop (3 DAG), or second loop (4 DAG) [16, 22, 44]. Magenta: epidermis; increasingly darker gray: progressively older ATHB8 expression domains. Columns 2–5: confocal laser scanning microscopy. Column 2: ATHB8::nCFP expression. Column 3: MP::MP:YFP expression; dashed magenta outline: MP::MP:YFP-expressing epidermal nuclei. Column 4: autofluorescence. Column 5: overlays of images in columns 2–4; red: ATHB8::nCFP expression; green: MP::MP:YFP expression; blue: autofluorescence. Column 6: ATHB8::nCFP and MP::MP:YFP expression levels (mean ± SE) in nuclei flanking ATHB8::nCFP-expressing nuclei (positions “-2”, “-1”, “+1”, and “+2”) relative to ATHB8::nCFP and MP::MP:YFP expression levels in nuclei co-expressing ATHB8::nCFP (position “0”) during formation of midvein (top), first loop (middle), or second loop (bottom). Difference between ATHB8::nCFP expression levels in nuclei at position -2, -1, +1, or +2 and ATHB8::nCFP expression levels in nuclei at position 0, and between MP::MP:YFP expression levels in nuclei at position -2, -1, +1, or +2 and MP::MP:YFP expression levels in nuclei at position 0 was significant at P < 0.001 (***) by One-Way ANOVA and Tukey’s Pairwise test. ATHB8::nCFP sample sizes: 35 (2 DAG), 33 (3 DAG), or 32 (4 DAG) leaves; position -2: 29 (2 DAG), 43 (3 DAG), or 49 (4 DAG) nuclei; position -1: 57 (2 DAG), 70 (3 DAG), or 66 (4 DAG) nuclei; position 0: 63 (2 DAG), 73 (3 DAG), or 69 (4 DAG) nuclei; position +1: 52 (2 DAG), 46 (3 DAG), or 58 (4 DAG) nuclei; position +2: 23 (2 DAG), 19 (3 DAG), or 37 (4 DAG) nuclei. MP::MP:YFP sample sizes: 35 (2 DAG), 33 (3 DAG), or 32 (4 DAG) leaves; position -2: 30 (2 DAG), 45 (3 DAG), or 50 (4 DAG) nuclei; position -1: 63 (2 DAG), 72 (3 DAG), or 67 (4 DAG) nuclei; position 0: 70 (2 DAG), 75 (3 DAG), or 70 (4 DAG) nuclei; position +1: 58 (2 DAG), 47 (3 DAG), or 59 (4 DAG) nuclei; position +2: 24 (2 DAG), 19 (3 DAG), or 38 (4 DAG) nuclei. Column 7: Local background levels (mean ± SE) in CFP and YFP confocal channels — measured in image regions containing no features of interest as in [14, 100] — relative to ATHB8::nCFP and MP::MP:YFP expression levels in nuclei co-expressing ATHB8::nCFP (position “0”) during formation of midvein (top), first loop (middle), or second loop (bottom). Scale bars (shown, for simplicity, only in column 2): 5 μm