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Cell-Cell Interactions and Cell-Fate Specification During Morphogenesis and Patterning

Research Summary

Keiko Torii uses Arabidopsis stomatal development as a model to study how plant cells coordinate proliferation and differentiation during organ morphogenesis to generate beautiful, orderly patterns.

Development and regeneration of multicellular organisms relies on cell-cell interactions that specify cell fate. Within the context of developmental programs, individual cells receive and interpret "positional cues" and undergo proliferation and differentiation in an orderly manner. Because of the presence of cell walls, plants achieve tissue and organ morphogenesis in the absence of cell migration.

After cell division, two daughter cells will be stuck with each other (they cannot migrate away from their sisters and neighbors). Thus, plant cells face unique problems and challenges during pattern formation: how can they make fate decisions and coordinate differentiation within the context of multicellularity? I address such fundamental questions through integrated and cross-disciplinary approaches that include plant developmental genetics, systems biology, biomaterials science, and live imaging.

Coordinating Morphogenesis with Peptide Signals and Receptor Kinases
Recently, peptide signaling has emerged as a key mechanism for cell-cell communication in plants as well as in interkingdom communications between plants and microbes. Consistently, functional genomics has revealed that plants possess a large number of genes encoding small, secreted peptides, many of which form multigene families. I am interested in understanding how the same set of receptors correctly interprets closely related signals to control diverse developmental processes. For this purpose, I focus on the signaling systems mediated by ERECTA-family receptor kinases (RKs), a family of three receptors regulating multiple aspects of plant development and biotic responses.

I identified ERECTA as a leucine-rich repeat receptor kinase in the 1990s. Later, my group found that three ERECTA-family RKs—ERECTA, ERECTA-LIKE1 (ERL1), and ERL2—show synergistic interactions in regulating aboveground organ morphogenesis, including inflorescence architecture, flower patterning, and ovule and anther differentiation, through promoting cell proliferation and coordinating the timing of cell-type differentiation. To unravel how plant cells process related information coming from their neighbors and elicit specific developmental responses, we will analyze peptide ligands acting in a subset of ERECTA-family-mediated pathways.

ERECTA plays a major role for proper inflorescence growth, which involves coordinated development of several different tissues with unique functions, such as vasculature (xylem and phloem), cortex, and epidermis. How do different tissue layers consisting of unique cell types coordinate growth? We are using both classic and novel techniques in developmental biology to address this question.

Coordinating Tissue Patterning and Differentiation in Plant Epidermis
Stomata are microscopic valves on the surface of land plants for efficient gas exchange and water vapor release. Despite differences in the developmental origin of stomatal complexes across species, stomata are formed such that each stoma is surrounded by nonstomatal epidermal cells. Such coordinated spacing of stomata is critical for proper stomatal function and hence plant growth and survival, as stomatal opening and closure require rapid water and ion exchange between guard cells and neighboring nonstomatal cells. It has been proposed that stomatal precursors emit signals that inhibit neighboring cells from adopting stomatal cell fate. We have discovered ligands and receptors controlling this inhibition.

First, we found that the three ERECTA-family RKs act synergistically to enforce stomatal patterning. In the absence of all family members, plants produce massive clusters of stomata. Fine dissection of each genetic locus revealed that ERECTA restricts asymmetric divisions that create the stomatal cell lineage, while ERL1 inhibits the cell-state switch from stomatal stem cell state to differentiation state. ERECTA-family RKs act both cooperatively and antagonistically with TOO MANY MOUTHS (TMM), a receptor-like protein lacking any cytoplasmic effector domain, which appears to contribute to robust and precise signaling outputs.

Second, through expression studies, my group and colleagues discovered a family of small secretory peptides, EPIDERMAL PATTERNING FACTORS (EPFs), that are expressed in distinct steps within stomatal precursors and inhibit the asymmetric entry divisions that create stomatal cell lineages, as well as the asymmetric spacing divisions required for proper stomatal patterning. We aim to unravel the specificity and kinetics of ligand-receptor interactions, receptor dynamics, and early events in signal transduction. For this purpose, we are developing a novel receptor ectodomain biosensor platform and taking phosphoproteomic approaches. We plan to combine imaging tools and techniques to visualize the dynamics of ligand-receptor signaling.

Maintaining and Differentiating Stem Cells in the Stomatal Lineage
Intercellular signals mediated by the above-mentioned peptide signals and receptors instruct cells to be or not to be stomata, perhaps acting on the key transcriptional networks driving stomatal differentiation. We identified a set of basic helix-loop-helix (bHLH) transcription factors that act in a sequential and combinatorial manner to specify a series of cell-state transitions leading to stomatal differentiation. SPEECHLESS and MUTE are sister bHLH proteins specifying the initiation and termination, respectively, of the stomatal precursor, called a meristemoid, which is capable of reiterating asymmetric divisions while renewing itself. These bHLH proteins are both necessary and sufficient for cell-state specification, thus acting as "master regulatory" proteins of stomata. We also found heterodimeric partners of these bHLH proteins, SCREAM (SCRM) and SCRM2, which trigger initiation, proliferation, and differentiation of stomatal cell lineages in a dosage-dependent manner. SCRM is identical to ICE1, a key upstream transcription factor for cold-induced transcription and freezing tolerance. Thus, SCRM/ICE1 may define a molecular intersection of developmental and environmental signaling pathways.

Self-renewing ability via reiterative asymmetric divisions is the key characteristic of stem cells, and understanding its molecular basis is of central importance in developmental biology and regenerative medicine. We are taking advantage of our mutant resources and the molecular markers developed in my laboratory to perform genome-wide approaches to identify molecular signatures associated with the meristemoid (stomatal stem cell) state. A pivotal element of this approach is the synthetic combination of loss of function in MUTE and gain of function in SCRM, which results in a striking epidermis solely composed of meristemoids and their sisters. Through a simple comparison of transcriptomes, we were able to identify components of asymmetric cell division.

The transition from stomatal stem cell (meristemoid) to differentiation state occurs in the absence of cell division. I aim to further unravel the molecular events of genome reprogramming from the proliferation to differentiation states of stomatal precursor cells, a transition triggered by the activity of MUTE. These studies will provide knowledge of stem cell function and differentiation.

Visualizing Dynamic Cell-Cell Interactions Leading to Pattern Formation
Understanding the mechanism that creates asymmetry among a population of seemingly uniform, undifferentiated cells may provide critical insights into pattern formation, tissue differentiation, and regeneration. To understand the in vivo, spatiotemporal dynamics of cell-cell communication that patterns stomata, we are developing a real-time imaging system to visualize the regulatory dynamics and lateral inhibition during the entire process of stomatal differentiation. The flat surface of developing cotyledons provides excellent accessibility for high-resolution real-time imaging with relatively simple experimental setups. Our system enables us to visualize the whole process of epidermal differentiation (from germinating embryos to fully differentiated seedlings) under confocal laser scanning microscopy while monitoring in vivo expression dynamics of fluorescent protein-tagged stomatal regulatory proteins.

Visualizing expression of known regulators of stomatal patterning in the wild-type background has already provided insight into the dynamics of cell-cell interaction leading to stomatal differentiation. We are expanding our system with optical techniques (e.g., gene induction, laser ablation) and peptide applications to visualize the in situ, real-time response of plant epidermis when regulatory circuits are manipulated.

Stomatal patterning is influenced by various environmental cues, such as temperature, light, drought, and CO2 concentration. Understanding how these environmental signals impinge on stomatal differentiation will bring fundamental insights into plant growth and survival in a changing environment. I plan to expand my imaging system to include environmental perturbations and directly monitor spatiotemporal dynamics of cell-cell signals and cell-fate determinants under different environmental regimes. For example, I could mimic past and predicted world temperature and CO2 levels to test the potential robustness of the stomatal system. This interdisciplinary approach would foster the translation of my fundamental research of plant development into guidance for conservation decisions in the field.

As of May 30, 2012

Scientist Profile

University of Washington
Developmental Biology, Plant Biology