SummarySelf renewal is integral to the creation and maintenance of many tissues and organs including humanepithelia, muscle and blood systems. Each of these tissues establishes populations of self-renewing(stem) cells and must ensure that these stem cells divide and create new differentiated cells at theappropriate rate and in the appropriate place; not enough cells or divisions and the tissue willdeteriorate, too many divisions and tumors can form. Our long term goal is to understand themechanism(s) by which dispersed self-renewing populations are established and how their divisionand differentiation is influenced by interaction with neighboring cells. A powerful set of genetic,molecular and functional genomic tools, combined with the ability to visualize and track cell divisionsmakes Arabidopsis stomatal development an attractive system for investigating this set of questions.In our previous work we found that a trio of bHLH transcription factors and components of a MAPKpathway modulate division vs. differentiation choices at discrete stages in stomatal development.Based on data from genetic, biochemical and functional assays, we propose that activity of theearliest-acting bHLH in this trio, SPEECHLESS (SPCH), controls the asymmetric divisions that createthe self-renewing populations. We show that SPCH is a direct target of MAP kinase-dependentphosphorylation in vitro, and that phosphorylation alters the behavior of SPCH in vivo. Our specificaims in this proposal are to: (1) elucidate the molecular mechanisms by which SPCH activity isregulated (2) Create a molecular profile of stomatal lineage cells and identify and functionallycharacterize transcriptional targets of SPCH in these cells, and (3) Take advantage of SPCH-inducedphenotypes to genetically dissect the endogenous signaling network required to repress stem-celldifferentiation. Because both the bHLH class of transcriptional regulators and the MAPK pathway areuniversally conserved, these studies may contribute not only to an understanding of self-renewal, butwill contribute to our understanding of the diversity of MAPK and bHLH signaling mechanisms andresponses.