Sequoia trees continue development for more than a thousand years without "forgetting" the program or running out of cells, due to the robust regulatory network of plant meristems. How does this work?

Plants have the remarkable ability to give rise to new organs throughout their life which can last for up to several hundred years. This is due to stem cells in the apical meristems (for reading up on this subject: Aichinger et al. 2012). A major focus in our laboratory is to learn what makes stem cells so special and how they and their differentiating daughter cells are regulated. Stems cell are defined as cells that are able to give rise to two types of daughter cells, those that remain pluripotent and self-renew the stem cell pool and those that differentiate to generate organs and tissues (for review: Laux 2003). For us, a fascinating question is "what is the molecular basis of pluripotency". We are studying stem cells in a genetically tractable system, the shoot and root meristems of the model plant Arabidopsis.

Our current goal is to understand the signaling mechanism by which stem cells are specified and the signals and the molecular features that determine cellular pluripotency. For this purpose we combine molecular genetics, biochemistry, live imaging approaches, and system biology.

Molecular characterization of stem cells in the root apical meristem

The root stem cell niche has an invariant architecture that allows tracing cells and offers a number of experimental opportunities to address signaling mechanisms including live imaging and fluorescent activated cell sorting to analyze molecular profiles of individual cell types and differentiation stages. Our experimental system is WOX5 signaling. WOX5 encodes a homeodomaine transcription factor, a homolog to WUS (see below),  which is expressed in the quiescent center of the root meristem from where it is signals to maintain the surrounding cells pluripotent (Sarkar et al., 2007).

Left: Wildtype root meristem. Signaling from the QC (blue) maintains the underlying layer of columella stem cells (CSC, unstained) in a pluripotent state. Divisions of the CSC generate rapidly differentiation root cap cells (violett by Lugol staining for starch, individual starch grains can be recognized).

Right: wox5 knockout mutants lack QC derived signaling and CSCs are missing. Cells underneath the QC are differentiated (contain starch grains).

Isolated stem cells from the Arabidopsis root meristem expressing a GFP protein after fluorescent activated cell sorting. These cells are subsequently being used for expression profiling via microarrays and chromatin  characterization by Chromatin Immuno Precipitation (ChIP).

Our currrent work explores the spatiotemporal dynamics of WOX5 triggered signaling combining transcriptome, histone code, and proteomic approaches. To accomplish this goal, we isolate specific cells from the niche by fluorescent cell sorting. To understand the changes that cells undergo when they progress from a pluripotent stem cell towards a differentiated cell, we explore the gene expression program by microarrays and the chromating organization by Chromatin Immuno Precipitation (ChIP)S. To follow signaling and signal response in space and in time, we using live imaging of fluorescent signaling reporter genes during cell fate changes.

Network regulating stem cells in the shoot apical meristem

Top view of an Arabidopsis inflorescence. The stem cells of the inflorescence meristem (middle) and the surrounding floral meristems are shown in blue color

The stem cells in shoot meristems have the capacity to give rise not only to cells for one tissue but to provide the cells from which all aboveground organs are formed, such as leaves, branches and flowers. The fate of each individual daughter cell is determined by its relative position. Those that stay at the most apical position of the shoot meristem, that is in a “stem cell niche”, renew the stem cell population, whereas those that are displaced from this position differentiate.

We found that cells, termed organizing center (OC) underneath the stem cells express the WUSCHEL (WUS) homeobox gene  (Mayer et al., 1998), which results in a signal to maintain the overlying stem cells in a pluripotent state.

The stem cells express the signal peptide CLV3  to signal back and restrict the size of the OC by repressing WUS transcription (Schoof et al., 2000). By this negative feedback loop, the size of the stem cell pool is thus dynamically maintained.

In our recent work, we investigate stem cells can be stably maintained within the rapidly dividing shoot meristem. We found that a mikro RNA acts as a signal from the surface layer (the L1) to enable the stem cell to respond to WUS (Knauer et al 2013).

The shoot meristem stem cell population is maintained by a regulatory feedback loop between stem cells and underlying organizing center (Schoof et al., 2000)

Stem cell initiation in the embryo

The shoot meristem (blue, expressing CLV3) appears during embryogenesis. Its development depends on signals from the vasculature (yellow) expressing the small micro RNA producing protein ZLL.

How is such a comlex system as the shoot meristem set up at the first place? Morphologically, the shoot meristem can not be seen before mid embryo stages where it is in the cleft between the cotyledon primordia (image). Why are stem cells established there and not elsewhere in the embryo? We found that it is the underlying vascular tissue that provides essential signals. As one important part, we showed that the ZWILLE (ZLL) gene, encoding an ARGONAUTE protein that binds small micro RNAs Moussian et al., 1998) blocks stem cell precursors from differentiation (Tucker et al 2008). In our current work we investigate the mechanisms of how this signal pathway links stem cells to the vasculature, and what the roles of miRNAs and plant hormones play in this process (Mallory et al 2009).

Our selected publications on stem cells in meristems

Knauer, S., Holt, A. L., Rubio-Somoza, I., Tucker, E. J., Hinze, A., Pisch, M., Javelle, M., Timmermans, M. C., Tucker, M. R. and Laux, T. (2013). A Protodermal miR394 Signal Defines a Region of Stem Cell Competence in the Arabidopsis Shoot Meristem. Developmental Cell 24,125-132

Aichinger, E., Kornet, N., Friedrich, T., and Laux, T. (2012). Plant Stem Cell Niches. Annu Rev Plant Biol. 63, 615-636.

Katsir, L., Davies, K. A., Bergmann, D. C. and Laux, T. (2011). Peptide signaling in plant development. Curr Biol 21, R356-64.

Graf, P., Dolzblasz, A., Wurschum, T., Lenhard, M., Pfreundt, U., and Laux, T. (2010). MGOUN1 Encodes an Arabidopsis Type IB DNA Topoisomerase Required in Stem Cell Regulation and to Maintain Developmentally Regulated Gene Silencing. Plant Cell 22, 716-728

Mallory, A. C., Hinze, A., Tucker, M. R., Bouché, N., Gasciolli, V., Elmayan, V., Lauressergues, D., Jauvion, V., Vaucheret, H.,, and Laux, T. (2009). Redundant and specific roles of the ARGONAUTE (AGO) proteins AGO1 and ZLL in development and small RNA-directed gene silencing. PLoS Genet. 5, e1000646.

Tucker, M. R., Hinze, A., Tucker, E. J., Takada, S., Jurgens, G., and Laux, T. (2008). Vascular signalling mediated by ZWILLE potentiates WUSCHEL function during shoot meristem stem cell development in the Arabidopsis embryo. Development. 135, 2839-2843.

Sarkar, A., Luijten, M., Miyashima, S., Lenhard, M., Hashimoto, T., Nakajima, K., Scheres, B., Heidstra, R. and Laux, T. (2007). Conserved factors regulate signaling in Arabidopsis shoot and root stem cell organizers. Nature 446, 811-814.

Würschum, T., R. Gross-Hardt, and T. Laux (2006). APETALA2 Regulates the Stem Cell Niche in the Arabidopsis Shoot Meristem. Plant Cell 18: 295-307.
 

Bäurle, I. and Laux, T. (2005). Regulation of WUSCHEL transcription in the Arabidopsis shoot meristem. Plant Cell 17, 2271-2280

Laux, T. (2003) The Stem Cell Concept in Plants: A Matter of Debate. Cell 113, 281-283.

Lenhard, M., and Laux, T. (2003). Stem cell homeostasis in the Arabidopsis shoot meristem is regulated by intercellular movement of CLAVATA3 and its sequestration by CLAVATA1. Development 130, 3163-3173.

Moussian, B., Haecker, A. and Laux, T. (2003). ZWILLE buffers meristem stability in Arabidopsis thaliana. Dev Genes Evol 213, 534-40.

Gross-Hardt, R., Lenhard, M., and Laux, T. (2002). WUSCHEL signaling functions in interregional communication during Arabidopsis ovule development. Genes Dev 16, 1129-1138.

Lenhard, M., Jürgens, G., and Laux, T. (2002). The WUSCHEL and SHOOTMERISTEMLESS genes fulfil complementary roles in Arabidopsis shoot meristem regulation. Development 129, 3195-206.

Lenhard, M., Bohnert, A., Jürgens, G. and Laux, T. (2001). Termination of stem cell maintenance in Arabidopsis floral meristems by interactions between WUSCHEL and AGAMOUS. Cell 105, 805-814.

Schoof, H., Lenhard, M., Haecker, A., Mayer, K.F.M., Jürgens, G., and Laux, T. (2000). The stem cell population of Arabidopsis shoot meristems is maintained by a regulatory loop between the CLAVATA and WUSCHEL genes. Cell 100, 635-644.

Mayer, K.F.X, Schoof, H., Haecker, A., Lenhard, M., Jürgens, G. and Laux, T. (1998). Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem. Cell 95, 805-815.

Moussian, B., Schoof, H., Haecker, A., Jürgens, G. and Laux, T. (1998). Role of the ZLL gene in the regulation of central shoot meristem cell fate during Arabidopsis embryogenesis. EMBO J. 17, 1799-1809.

Endrizzi, K., Moussian, B., Haecker, A., Levin, J. and Laux, T. (1996). The SHOOT MERISTEMLESS gene is required for maintenance of undifferentiated cells in Arabidopsis shoot and floral meristems and acts at a different regulatory level than the meristem genes WUSCHEL and ZWILLE. Plant J. 10, 967-979.

Laux, T., Mayer, K. F. X., Berger, J. and Jürgens, G. (1996). The WUSCHEL gene is required for shoot and floral meristem integrity in Arabidopsis. Development 122, 87-96.