Project description:Plant tissue development often relies on the specification of cell type initials with stem cell-like properties. These later undergo differentiation, losing division potential and acquiring specific identities and functions. In the stomatal lineage, protodermal cells develop into guard cells (GCs) through the action of bHLH transcription factors (TFs) SPEECHLESS (SPCH), MUTE and FAMA. Existing models support that these regulators act sequentially, but recent evidence indicates that SPCH expression and function are retained in late stomatal cells. Here, we combine transcriptomic and genetic approaches to define SPCH's function during the late stomatal lineage. We show that relative levels and activities of SPCH and FAMA control GC division and expansion. Through cell type-specific TF induction and mRNA sequencing, we identify late-lineage targets of both TFs, and through genetic perturbation of these targets, we demonstrate that their precise temporal regulation is required for proper GC morphology and function. Our findings reveal a previously unrecognized role for SPCH in late stomatal development and support a revised model in which the functions of stomatal bHLHs are not strictly separated in time.
Project description:We initiated a study to investigate the transcriptional profiles associated with cell states of the stomatal lineage. A stem-cell like precursor of stomata, a meristemoid. reiterates asymmetric divisions and renews itself before differentiating into guard cells. The transient and asynchronous nature of the meristemoid has made it difficult to study its molecular characteristics. Through combinatorial use of genetic resources that either arrest or constitutively drive stomatal cell-state progressions due to loss- or gain-of-function mutations in the key transcription factor genes, SPEECHLESS, MUTE, and SCRM, we obtained seedlings highly enriched in pavement cells, meristemoids, or stomata. Here we present transcriptome and genome-wide trends in gene regulation associated with each cell state and identify molecular signatures associated with meristemoids. 12 samples are included in this study. Three biological replicates of 5-dag seedlings of speechless, scrm-D and scrm-D;mute were compared wild type seedlings for changes in gene expression.
Project description:Lineage-specific stem cells are critical for the production and maintenance of specific cell types and tissues in multicellular organisms. In higher plants, the initiation and proliferation of stomatal stem cells is controlled by the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). The stomatal stem cells and SPCH, which represent an innovation in seed plants, allow flexibility in the production of stomata, but how SPCH generates these stem cells is unclear. Here, we developed a highly sensitive chromatin immunoprecipitation (ChIP) assay and profiled the cell-type specific genome-wide targets of Arabidopsis SPCH in vivo. We found that SPCH directly controls key and novel regulators that drive cell fate and asymmetric cell divisions and enhances responsiveness to cell-cell communication. Our results provide molecular insights on how a master transcription factor generates an adult stem cell lineage that contributes to the success of land plants. Genome-wide identification of SPCH binidng sites
Project description:Stomatal abundance decrease in Arabidopsis triggered by warm-temperature is attributed to PIF4-mediated repression of SPEECHLESS (SPCH) expression. We identified the unknown developmental and transcriptional basis of this adaptive response. We analyzed stomatal lineage progression at warm- and control-T combining live-cell imaging and lineage-cell tracing with epidermal phenotyping, genetics and transcriptomics. Warm-T or PIF4 overexpression caused a fraction of stomatal precursors to lose their identity and become diverted, explaining stomatal index reduction, and hinting at developmental mechanisms underlying lineage progression. Triggering diverted precursor fate required extended exposure to warm-T, below which the process is reversible and stomatal index remains unchanged. Despite heat-induced gene reprogramming silenced key positive drivers of stomatal development as SPCH and MUTE, most lineages progressed and formed stomata. Transcriptomics revealed that warm-T shifted lineages towards uncommitted cell stages, which regained committed fates during recovery at control temperature. This indicates that stomatal development under changing temperatures occurs through partly rewired gene circuits involving alternative pathways.
Project description:Environmental factors shape the phenotypes of multicellular organisms. The production of stomata—the epidermal pores required for gas exchange in plants—is highly plastic, and provides a powerful platform to address environmental influence on cell differentiation [1-3]. Rising temperatures are already impacting plant growth, a trend expected to worsen in the near future [4]. High temperature inhibits stomatal production but the underlying mechanism is not known [5]. Here, we show that elevated temperature suppresses the expression of SPEECHLESS (SPCH), the bHLH transcription factor that serves as the master regulator of stomatal lineage initiation [6,7]. Our genetic and expression analyses indicate that the suppression of SPCH and stomatal production is mediated by the bHLH transcription factor PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), a core component of high temperature signaling [8]. Importantly, we demonstrate that upon exposure to high temperature, PIF4 accumulates in the stomatal precursors and binds to the promoter of SPCH. In addition, we find SPCH feeds back negatively to the PIF4 gene. We propose a model where the high temperature-activated PIF4 binds and represses SPCH expression to restrict stomatal production at high temperature. Our work identifies a molecular link connecting high temperature signaling and stomatal development, and reveals a direct mechanism by which production of a specific cell lineage can be controlled by a broadly-expressed environmental signaling factor.
Project description:Mouse spermatogonial stem cells (SSCs) continuously self-renew on the feeder layers in serum-free culture medium supplemented with glial cell line-derived neurotrophic factor and fibroblast growth factor 2. To identify novel nuclear proteins involved in SSC maintenance, comparative proteomic profiling of nuclear proteins was performed between self-renewing and differentiation-initiated SSCs in culture. The self-renewing SSC cultures were established from C57BL/6 mouse testes. Nuclear fractions from self-renewing SSC cultures treated with ethanol as a vehicle control (spermatogonial stem cells) and differentiation-initiated SSC cultures treated with 0.3 μM retinoic acid for 24 h (spermatogonial progenitor cells) were isolated for proteomic analysis.
Project description:Lineage-specific stem cells are critical for the production and maintenance of specific cell types and tissues in multicellular organisms. In higher plants, the initiation and proliferation of stomatal stem cells is controlled by the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). The stomatal stem cells and SPCH, which represent an innovation in seed plants, allow flexibility in the production of stomata, but how SPCH generates these stem cells is unclear. Here, we developed a highly sensitive chromatin immunoprecipitation (ChIP) assay and profiled the cell-type specific genome-wide targets of Arabidopsis SPCH in vivo. We found that SPCH directly controls key and novel regulators that drive cell fate and asymmetric cell divisions and enhances responsiveness to cell-cell communication. Our results provide molecular insights on how a master transcription factor generates an adult stem cell lineage that contributes to the success of land plants.