1. Volume 24, Issue 2, Pages 125-132 (January 2013)
A Protodermal miR394 Signal Defines a Region of Stem Cell Competence in the Arabidopsis Shoot Meristem 
Steffen Knauer, Anna L. Holt, Ignacio Rubio-Somoza, Elise J. Tucker, Annika Hinze, Melanie Pisch, Marie Javelle, Marja C. Timmermans, Matthew R. Tucker, Thomas Laux 
Developmental Cell 
Volume 24, Issue 2, Pages (January 2013)
DOI: /j.devcel
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Figure 1
The enh146 ago10-1 Double Mutant Terminates Stem Cell Maintenance in the Shoot Meristem
(A–C) Seedlings of the indicated genotypes at 14 days postgermination (dpg). The functional shoot meristem in (B) and (C) produces rosette leaves (rl), in contrast to (A), where the shoot meristem has terminated in a central leaf-like structure (arrow).
(D–G) Expression of pCLV3:GFPer is detected in ago10-1 embryos from early heart stage onward (D and E). In enh146 ago10-1, expression is initiated correctly (F), but is discontinued at the bent cotyledon stage (G). Insets in (E) and (G) show higher magnifications.
(H–M) gWUS-GFP3 (H and I) expression and endogenous WUS mRNA (J) in ago10-1 embryos compared to the expanded expression domains in enh146 ago10-1 embryos (K–M) from the torpedo stage onward. Insets in (I and J) and (L and M) show higher magnification of the embryonic shoot apices. co, cotyledon; cp, cotyledonary primordium; SP, shoot apical meristem primordium. Embryo images are in the median plane.
Scale bars, 2.5 mm in (A)–(C) and 20 μm in (D)–(M). See also Tables S1 and S2.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 2
miR394 Is Essential for Shoot Meristem Formation and Targets the Stem Cell Inhibitor LCR
(A) Shoot meristem phenotypes of the indicated genotypes at 14 dpg (n > 500/genotype). The average of at least two independent transgenic lines is shown, including SD.
(B) Northern blot analysis reveals reduced levels of miR394 in the mir394b-1 mutant and no detectable amounts in pRPS5A:MIM394 lines. U6 RNA was used as loading control.
(C) Gene structure of LCR with the miR394 recognition site (red box) and the sequence of the miR394-resistant LCR-4m. The T-DNA insertion site of lcr-1 is indicated. Light gray boxes, untranslated regions; dark gray boxes, coding sequence; black box, F box motif.
(D) Shoot meristem phenotypes of the indicated genotypes at 14 dpg (n > 500/genotype). The average of at least two independent transgenic lines is shown, including the SD.
(E) Expression of the miR394 resistant LCR-4m from the endogenous promoter causes shoot meristem termination in ago10-1 background (left image), which is completely repressed by coexpression of an accordingly modified miR394-4m version (middle image). LCR-4m causes shoot meristem termination in the wild-type background when expressed from the 35S promoter (right image).
(F) lcr-1 completely rescues a functional shoot meristem in the mir394b-1 ago10-1 background. Inset in the middle image shows the enlarged view of the filamentous structure in place of the shoot meristem. SAM, shoot apical meristem. Scale bars, 2.5 mm.
See also Figure S1.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

4. Figure 3
miR394 Acts Non-Cell-Autonomously in Shoot Meristem Formation by Triggering RNAi in the Distal Cell Layers of the Embryo Apex
(A) pMIR394B:NLS-YFP3 reporter gene expression pattern during wild-type embryogenesis.
(B) In situ hybridization of Col wild-type embryos with anti-miR394 locked nucleic acid probe shows miR394 in cell layers subjacent to the protoderm (indicated by dotted line). From the torpedo stage onward, a weak signal is detected in the vasculature and the basal embryo pole (yellow arrowheads). Insets show complete embryos.
(C) Complete shoot meristem rescue in mir394b-1 ago10-1 is obtained by expressing MIR394B from promoters providing expression in the protoderm (pPDF2, pMIR394B, and pYUC4), but not by expression in the L1 of the adjacent cotyledonary primordia (pAS2), as shown by a linked pOp:YFPer operator.
(D) Ubiquitous accumulation of a miR394-resistant green fluorescent protein (GFP) sensor (r-GFP).
(E) miR394-sensitive GFP (s-GFP) accumulation is reduced in several apical layers compared to (D).
(F) In situ hybridization with a GFP antisense probe in a line expressing a s-GFP under the control of pRPS5A. Signal is absent in several apical cell layers and, from torpedo stage onward, also in the provasculature (arrowheads).
(G) Sense control.
(H) GFP antisense probe in embryos with ubiquitous expression of r-GFP reveals a stronger signal in apical layers compared to the heart stage embryo in (F).
(I and J) Downregulation of s-GFP is suppressed by pRPS5A:MIM394 (I) and in ago1-1 embryos (J).
(A), (C)–(E), (I), and (J) show overlays of confocal and Nomarski images. cp, cotyledonary primordium; SP, shoot apical meristem primordium. Scale bars, 20 μm. See also Figure S2.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

5. Figure 4
miR394-Triggered RNAi of LCR in Subepidermal Target Cells Is Crucial for Stem Cell Maintenance
(A) Shoot meristem phenotypes of seedlings with tissue-specific expression of LCR-4 m or MIM394 transgenes. The average of at least two independent transgenic lines is shown, error bars represent SD (n > 500/line). Control is without driver construct.
(B) Expression patterns of the tandem pOp:YFPer reporter for the indicated promoters. pYUC1∗ displays abnormal promoter activity in individual transformants (compare to pYUC1). Higher magnification highlighting the differences between pUFO-, pYUC1∗-, pZLL/AGO10-, and pMIR394B-driven expression domains in the shoot apex is shown at the bottom. The stem cell layers are indicated by dashed lines.
(C) Model for the role of miRNA394 in stem cell regulation. miR394 molecules (black dots) supplied by the protoderm (outlined in green) repress LCR in subtending cells, which enables WUS to regulate stem cell identity and CLV3 expression. Blue color, stem cells. cp, cotyledonary primordium; SP, shoot apical meristem primordium. Scale bars, 20 μm.
See also Figure S3.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions