WUSCHEL (WUS) is a homeodomain transcription factor produced in cells of the niche/organizing center (OC) of shoot apical meristems. WUS specifies stem cell fate and also restricts its own levels by activating a negative regulator, CLAVATA3 (CLV3), in adjacent cells of the central zone (CZ). Here we show that the WUS protein, after being synthesized in cells of the OC, migrates into the CZ, where it activates CLV3 transcription by binding to its promoter elements. Using a computational model, we show that maintenance of the WUS gradient is essential to regulate stem cell number. Migration of a stem cell-inducing transcription factor into adjacent cells to activate a negative regulator, thereby restricting its own accumulation, is a theme that is unique to plant stem cell niches.Supplemental material is available for this article.Received July 5, 2011; revised version accepted September 2, 2011.Cell-cell communication between distinct cell types within stem cell niches is critical for stem cell maintenance in both plants and animals, although they differ in their niche architecture and cell behaviors (Spradling et al. 2008;Rieu and Laux 2009). In animal systems-for example, the Drosophila germline-stem cells make direct contact with the niche cells through adherens junctions and receive local signals that prevent their differentiation (Spradling et al. 2008). Oriented and asymmetric division of stem cells places one of the progeny at a distance by a cell diameter, which no longer can receive signals from the niche and differentiates; thus, precise local cell behaviors regulate stem cell number. Whereas in the shoot apical meristem (SAM) stem cell niche not all stem cells make contact with the niche, they also do not exhibit oriented and asymmetric cell divisions to regulate stem cell numbers. For example, the Arabidopsis SAM stem cell niche is a collection of ;500 cells located at the growing tip of each shoot (Reddy 2008). The CZ of the SAM harbors stem cells. The stem cell progeny that are displaced into the adjacent peripheral zone (PZ) proliferate before differentiating ( Fig. 1A; Reddy 2008). Visually, the SAM stem cell niche is a multilayered structure consisting of three clonally distinct layers of cells, and stem cells are found in each of these layers. The cells in the L1 and the L2 layers divide parallel to the surface to remain as monolayers. The cells in the L3 layer divide in random orientations to form a multilayered structure referred to as the rib meristem (RM) or the organizing center (OC), which provides stem cell-promoting cues (Rieu and Laux 2009).Previous studies have shown that WUSCHEL (WUS), a homeodomain-containing transcription factor, is both necessary and sufficient for stem cell specification (Laux et al. 1996). WUS RNA is found in a few cells of the RM/OC located just beneath the CZ ( Fig. 1A; Mayer et al. 1998). Restriction of WUS transcription to cells of the OC is critical for maintaining a constant number of stem cells, and this is mediated by the CLAVATA (CLV) signaling pathway (...

Despite the central importance of stem cells in plant growth and development, the molecular signatures associated with them have not been revealed. Shoot apical meristems (SAMs) harbor a small set of stem cells located at the tip of each plant and they are surrounded by several million differentiating cells. This imposes a major limitation in isolating pure populations of stem cells for genomic analyses. We have developed a system to isolate pure populations of distinct cell types of the SAMs, including stem cells. We have used this system to profile gene expression from 4 different cell samples of SAMs. The cell sample-specific gene expression profiling has resulted in a highresolution gene expression map to reveal gene expression networks specific to individual spatial domains of SAMs. We demonstrate that the cell sample-specific expression profiling is sensitive in identifying rare transcripts expressed in a few specific subsets of cells of SAMs. Our extensive RNA in situ analysis reveals that the expression map can be used as a predictive tool in analyzing the spatial expression patterns of genes and it has led to the identification of unique gene expression patterns within the SAMs. Furthermore, our work reveals an enrichment of DNA repair and chromatin modification pathways in stem cells suggesting that maintenance of genome stability and flexible chromatin may be crucial for stem cell function. The gene expression map should guide future reverse genetics experiments, high-resolution analyses of cell-cell communication networks and epigenetic modifications.central zone ͉ CLAVATA3 ͉ fluorescence-activated cell sorting ͉ WUSCHEL S patiotemporal regulation of transcriptional programs mediated by cell-cell communication networks is crucial in stem cell maintenance (1, 2). Understanding the complexity of transcriptional programs requires a high resolution analysis of gene expression, preferably at a cell type-specific resolution (3). Shoot apical meristems (SAMs) of higher plants represent a dynamic and interconnected network of distinct cell types. In Arabidopsis thaliana, the SAM consists of Ϸ35 stem cells, located within the central zone (CZ) (4). Stem cells are surrounded by several million differentiating cells that are part of the adjacent peripheral zone (PZ) and developing organs. The cells of the Rib-meristem (RM) that are located just beneath the CZ provide positional cues necessary for stem cell maintenance ( Fig. 1 D and E). Earlier studies have revealed molecular mechanisms involved in stem cell maintenance (5). However, gene networks that underlie the stem cell maintenance process are far from understood. The development of high resolution gene expression map of distinct cell types of SAMs could enable network analysis of pathways involved in stem cell maintenance.Expression profiling studies of specific cell types have been performed on Arabidopsis root system (3, 6), however, studies on the SAMs have been restricted to experiments of the entire tissue (7). This is because the domains of specific...

An open question remains as to what coordinates cell behavior during organogenesis, permitting organs to reach their appropriate size and shape. The Arabidopsis gene STRUBBELIG (SUB) defines a receptor-mediated signaling pathway in plants. SUB encodes a putative leucine-rich repeat transmembrane receptorlike kinase. The mutant sub phenotype suggests that SUB affects the formation and shape of several organs by influencing cell morphogenesis, the orientation of the division plane, and cell proliferation. Mutational analysis suggests that the kinase domain is important for SUB function. Biochemical assays using bacterially expressed fusion proteins indicate that the SUB kinase domain lacks enzymatic phosphotransfer activity. Furthermore, transgenes encoding WT and different mutant variants of SUB were tested for their ability to rescue the mutant sub phenotype. These genetic data also indicate that SUB carries a catalytically inactive kinase domain. The SUB receptor-like kinase may therefore signal in an atypical fashion.atypical kinase ͉ flower ͉ organogenesis ͉ ovule ͉ signal transduction I t remains a salient challenge in biology to understand the coordination of cell behavior that underlies organogenesis and allows organs to develop to their correct size and shape. The task should be easier in plants as cell-division patterns are readily traced and plant cells do not move relative to each other (1). Plant organogenesis is a postembryonic event, and the aboveground organs typically originate at the periphery of the shoot apical meristem, located at the apex of the main shoot (2).Signaling involving receptor-like kinases (RLKs) constitutes an essential aspect of plant cell communication and contributes to plant-pathogen interactions, hormone signaling, and development (3-5). In Arabidopsis 417 genes are predicted to encode such proteins (6). A function is known for only a handful of these loci. A major player in meristem development is the RLK CLAVATA1 (CLV1). CLV1 participates in a feedback loop maintaining the size of the stem cell population (for reviews see refs. 7 and 8). The CLV1 extracellular domain is characterized by 21 tandem copies of a leucine-rich repeat (LRR), a motif involved in protein-protein recognition (9, 10). The LRR-RLK ERECTA (ER) plays a more direct role in oganogenesis (11)(12)(13). ER is expressed in the shoot apical meristem and young lateral organs. Plants lacking WT ER function display a more compact stature, shorter inflorescence internodes, shorter pedicels, and shorter fruits with blunted tips. The main cellular basis of the er phenotype appears to be a reduction of cortex cell numbers (14). Members of the ER family of RLKs collectively promote cell proliferation and organ development (15). In corn, CRINKLY4 (CR4) is involved in cell differentiation in the leaf epidermis and specification of the aleurone layer of the endosperm (16,17). Its extracellular domain is characterized by seven ''crinkly'' repeats and a domain containing three repeats also present in mammalian TNF receptor...

Transcriptional mechanisms that underlie the dose-dependent regulation of gene expression in animal development have been studied extensively. However, the mechanisms of dose-dependent transcriptional regulation in plant development have not been understood. In Arabidopsis shoot apical meristems, WUSCHEL (WUS), a stem cell-promoting transcription factor, accumulates at a higher level in the rib meristem and at a lower level in the central zone where it activates its own negative regulator, CLAVATA3 (CLV3). How WUS regulates CLV3 levels has not been understood. Here we show that WUS binds a group of cis-elements, cis-regulatory module, in the CLV3-regulatory region, with different affinities and conformations, consisting of monomers at lower concentration and as dimers at a higher level. By deleting cis elements, manipulating the WUSbinding affinity and the homodimerization threshold of cis elements, and manipulating WUS levels, we show that the same cis elements mediate both the activation and repression of CLV3 at lower and higher WUS levels, respectively. The concentrationdependent transcriptional discrimination provides a mechanistic framework to explain the regulation of CLV3 levels that is critical for stem cell homeostasis.WUSCHEL | cis-regulatory module | CLAVATA3 | shoot apical meristem | gene regulation