Dependence of CLV3 expression on WUS and STM. A, GUS-stained heart stage CLV3::GUS embryo. B, CLV3 expression in a bent cotyledon stage embryo, detected by in situ hybridization with a CLV3 probe. C, CLV3 expression in a CLV3::GUS inflorescence meristem, detected by in situ hybridization with a GUS probe. D, GUS-stained mature CLV3::GUS embryo; compare with B. E, GUS-stained wild-type seedling 10 d after germination (d.a.g.). F, Wild-type seedling 10 d.a.g. The first leaf pair is visible. G, GUS-stained mature CLV3::GUS/wus-1 embryo. CLV3 expression is not detectable. H, GUS-stained CLV3::GUS/wus-1 seedling 10 d.a.g. I, wus-1 seedling 10 d.a.g. J, GUS-stained mature CLV3::GUS/stm-11 embryo showing CLV3 expression (arrow). K, GUS-stained CLV3::GUS/stm-11 seedling 10 d.a.g. showing CLV3 expression. L, An stm-11 seedling 10 d.a.g. has formed cotyledons, but no SAM is visible. M, Wild-type CLV3::GUS axillary meristem 21 d.a.g. GUS RNA is detected by in situ hybridization. N, GUS-stained CLV3::GUS/wus-1 axillary meristem 21 d.a.g. O, GUS-stained CLV3::GUS/stm-11 axillary meristem 21 d.a.g. Scale bars in F, I, and L = 1 mm; in all other figures, scale bars = 20 μm.

Induction of CLV3 expression by STM. A, STM-GR seedling 24 d.a.g., induced with Dex at 4 d.a.g.; a dense array of leaves with new meristems is formed. B, Scanning electron micrograph of a seedling, as in A. C, GUS-stained CLV3::GUS/STM-GR seedling, as in A; ectopic meristems are initiated on the adaxial leaf surface, GUS signal is found in the center of these meristems (arrow). D, Section through a leaf of a STM-GR seedling, as in A. Expression of CLV3 is detected by RNA in situ hybridization in the central zone of ectopic meristems (arrow). E, Section through the SAM of a STM-GR seedling, as in A. WUS expression, detected by RNA in situ hybridization, is found in the inner regions of the meristem, similar to wild type. F, Section through an axillary meristem of a STM-GR seedling, as in A. The domain of WUS expression resembles the expression in the SAM. E and F, No ectopic expression of WUS is observed. G, CLV3::GUS/STM-GR/wus-1 seedling 24 d.a.g.; Dex induction commenced 7 d.a.g. when the seedlings could be phenotypically identified as wus mutants. H, GUS-stained seedling as in G; GUS signal was detected in ectopic stipules that occasionally formed on the leaf margins (arrow). Note the absence of ectopic meristems. L, Leaf. Scale bars in A through C, G, and H = 500 μm; in D through F, scale bars = 20 μm.

Induction of CLV3 expression by WUS. A, GUS-stained CLV3::GUS/WUS-GR seedling 2 d.a.g., induced with Dex during germination. B, CLV3::GUS/WUS-GR seedling 24 d.a.g., induced with Dex at 4 d.a.g. C, GUS-stained seedling, as in B. D, GUS-stained CLV3::GUS/WUS-GR seedling 28 d.a.g., induced with Dex at 4 d.a.g. The cotyledons have been removed and GUS staining is visible in the leaf axils. E, Longitudinal section through a GUS-stained leaf, as in D; strong GUS expression is found in the apical cell layers of the laterally expanded axillary meristem. F, CLV3::GUS/WUS-GR/STM-GR seedling 24 d.a.g., induced with Dex 4 d.a.g. (compare with B). G, GUS-stained seedling, as in F; GUS expression is found in leaves. H, Section through a GUS-stained primary leaf of G, viewed with dark-field illumination. GUS signal (red) is found in all leaf cells. Scale bars in A, E, and H = 20 μm; in B through D, F, and G, scale bars = 1 mm.

Cell-autonomous induction of CLV3 by WUS in the stem cell domain. A, CLV3::WUS seedling 28 d.a.g.; a large apical dome is formed between the cotyledons, but organs are missing. B, In situ detection of WUS RNA in sections through the apical dome of a CLV3::WUS seedling. WUS RNA is found only in three apical cell layers, but not in the deeper regions of the meristem. D, High magnification view of the outer cell layers in B. C, As in B, but detection of CLV3 RNA. CLV3 and WUS are expressed in the same cells. E, High magnification of C. Scale bars in A and B = 500 μm; scale bars in D = 10 μm.