A Molecular Framework for the Embryonic Initiation of Shoot Meristem Stem Cells  Zhongjuan Zhang, Elise Tucker, Marita Hermann, Thomas Laux  Developmental Cell  Volume 40, Issue 3, Pages 264-277.e4 (February 2017) DOI: 10.1016/j.devcel.2017.01.002 Copyright © 2017 Elsevier Inc. Terms and Conditions

Developmental Cell 2017 40, 264-277. e4DOI: (10. 1016/j. devcel. 2017 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 The WOX2 Module Is Required for Initiation of CLV3 Expression (A–C) CLV3 in situ hybridization in wild-type (WT) transition (A), torpedo (B), and bent cotyledon (C) embryos. Arrowheads indicate CLV3 expression. (D–I) Expression of pCLV3:er-CFP in WT and segregating wus-1/+ embryos at the heart (D and E), and torpedo stages in frontal (F and G) and sagittal (H and I) planes. (J–O) Expression of pCLV3:er-tdTomato in early heart (J, L, and N) and early torpedo stage (K, M, and O) embryos of WT, wox1235, and pWOX2:rPHV wox1235 (rPHV wox1235). Arrowhead in (M) indicates very weak pCLV3:er-tdTomato expression. (P–U) Phenotypes of 10-day-old seedlings: WT; WT-like (two cotyledons with shoot apical meristem, SAM); scot + SAM (single cotyledon with SAM); scot − SAM (single cotyledon without SAM); pin + SAM (pin-like shoot with SAM). (V) Box plot of pCLV3:er-tdTomato expression levels in embryos with two cotyledons. 18 ≤ n ≤ 28. wox, wox1235; rPHV, pWOX2:rPHV. a, b, and c indicate significance categories (p < 0.01, ANOVA combined with Tukey’s HSD test). Scale bars, 20 μm (A–O) and 1 mm (P–U). See also Figure S1 and Tables S1–S3. Developmental Cell 2017 40, 264-277.e4DOI: (10.1016/j.devcel.2017.01.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 The WOX2 Module Promotes Central and Inhibits Peripheral Cell Identity in Globular Embryos (A–F) Embryo phenotypes. Cell walls are shown in red (pWOX2:tdTomato-LTI6b), and nuclei in green (pWOX2:H2B-GFP). Arrowheads denote missing protodermal divisions in (D) and premature horizontal cell divisions in (E). The boundary between the apical and basal domains is outlined with dashed lines. (G–I) In situ hybridization of AHP6 mRNA in globular embryos. (J–P) pDPA4:NLS-YFP3 expression in WT (J and J′, serial images of the same embryo; J, periphery; J′, median plane; L, front view; M, sagittal view) and wox1235 embryos (K, median plane; N–P, front views) at globular (J–K) and early heart stages (L–P). %, percentage of embryos with the shown phenotype. Scale bars, 20 μm. Developmental Cell 2017 40, 264-277.e4DOI: (10.1016/j.devcel.2017.01.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Elevated Auxin Activity Mediates Stem Cell Defects in wox1235 Embryos (A–G) DR5:GFP expression in early globular (A–C) and transition stage embryos (D–G). (H–M) pPIN1:PIN1-GFP expression in early globular (H–J) and early heart stage embryos (K–M). (F) + (G) is 58.1% (n = 43); (L) + (M) is 60% (n = 70); remaining wox1235 embryos display WT-like expression. Arrowheads and asterisks in (K) to (M) indicate the central domain and lens-shaped cells, respectively. (N) pWOX2:YUC1 (YUC1) increases stem cell defects in wox1235 (wox) embryos. pCLV3:er-tdTomato (pCLV3) was used as stem cell marker. In seedlings, the shoot meristem was scored morphologically. n = 20 for each embryo stage and 111 ≤ n ≤ 162 for seedlings. (O) pWOX2:iaaL (iaaL) significantly increases the frequency of wox1235 (wox) seedlings with shoot meristem. WT-like, scot/pin + SAM, and scot/pin-SAM are as shown in Figures 1Q, 1(R + S), and 1(T + U), respectively. The same applies to (N) and Figures 6A, 7C, and S5E. Three transgenic lines are shown. 39 ≤ n ≤ 51. a, b, and c indicate the significance categories (p < 0.01, chi-square test). Scale bars, 20 μm. See also Figure S2. Developmental Cell 2017 40, 264-277.e4DOI: (10.1016/j.devcel.2017.01.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 The WOX2 Module Promotes HD-ZIP III Expression (A–F) In situ hybridization of PHB mRNA in globular (A and D), transition (B and E), and torpedo (C and F) stage embryo. Cells strongly expressing PHB in globular embryos are outlined. Arrowheads indicate the region with reduced signal. (G) In situ hybridization of ZPR3 mRNA in WT globular embryo. (H–J) Expression of pZPR3:NLS-GFP3 in globular embryos. Scale bars, 20 μm. See also Figure S2. Developmental Cell 2017 40, 264-277.e4DOI: (10.1016/j.devcel.2017.01.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 The WOX2 Module Regulates PHV and MIR166B Transcription (A–D) Expression levels of pPHV:NLS-GFP3 (A and B) and pMIR166B:erGFP expression (C and D) in globular embryos. Dashed line in (C) and (D) indicates the boundary between apical and basal domains. (E) Box plot shows the fluorescence intensity of pMIR166B:erGFP in WT (n = 31) and wox1235 (wox, n = 26) embryos. (F) Domains measured in (E). ap, apical domain. ba, basal domain. sus, suspensor. (G–J) pRPS5a:sGFP expression in globular embryos. (K) Box plot shows the fluorescence intensity of pRPS5a:sGFP in WT, wox1235 (wox), and pWOX2:MIM165/166 (MIM). 19 ≤ n ≤ 35. (L) Domains measured in (K). a and b indicate the significantly associated categories (in E, p < 0.001, Student’s t test; in (K), p < 0.01, ANOVA combined with Tukey’s HSD test). Scale bars, 20 μm. (G) and (I) are fluorescence images, and (H) and (J) fluorescence merged with differential interference contrast images. See also Figures S3 and S4. Developmental Cell 2017 40, 264-277.e4DOI: (10.1016/j.devcel.2017.01.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Restored Expression of HD-ZIP III and IPT1 Suppress wox1235 Stem Cell Defects (A) Percentage of seedling phenotypes. 49 ≤ n ≤ 660. rPHV and wox indicate pWOX2:rPHV and wox1235, respectively. (B–E) Expression pattern of pAHP6:NLS-GFP3. Arrowheads indicate regions with reduced expression. Scale bars, 20 μm (F) Embryos with various DR5:GFP expression patterns. The categories WT-like, Em pro (embryo proper), and whole Em (whole embryo) are as shown in Figures 3A–3C. 24 ≤ n ≤ 32. a, b, and c indicate the significantly associated categories (p < 0.001, chi-square test). See also Figures S4 and S5. Developmental Cell 2017 40, 264-277.e4DOI: (10.1016/j.devcel.2017.01.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 The WOX2 Module Promotes Stem Cell Initiation Partially through Cytokinin (A) Relative expression of IPT1. Error bars denote ±SD. (B) pWOX2:IPT1 restores pCLV3:er-tdTomato (pCLV3) expression at the heart stage wox1235 embryos. 18 ≤ n ≤ 27. (C) Seedling phenotypes after expression of cytokinin related genes. 134 ≤ n ≤ 354. (D) Schematic shows the WOX2 module network in stem cell initiation. CKX7, (pWOX2:CKX7); IPT1, (pWOX2:IPT1); rPHV, (pWOX2:rPHV); wox, (wox1235). Black arrows indicate interactions found in this study. a, b, and c indicate the significantly associated categories (in A and B, p < 0.05, ANOVA combined with Tukey’s HSD test; in C, p < 0.05, chi-square test). See also Figures S4 and S5. Developmental Cell 2017 40, 264-277.e4DOI: (10.1016/j.devcel.2017.01.002) Copyright © 2017 Elsevier Inc. Terms and Conditions