Regulation of the shoot apical meristem (SAM) is important for plant architectural development because it is the source of shoot growth and origin of organ development. In Arabidopsis thaliana, maintenance of the stem cell population at the SAM is accomplished by a negative feedback loop. Signaling downstream of the hormone cytokinin is positively reinforced by the WUSCHEL (WUS) gene, which represses a cytokinin signaling inhibitor. WUS also promotes its own inhibition in the form of CLAVATA (CLV) signaling, which ultimately keeps WUS and cytokinin signaling in check. While these mechanisms are understood to some extent in Arabidopsis, the SAM of the moss Physcomitrella patens is not as well understood, nor is it identical to that of Arabidopsis. In this moss species, WUSCHEL-LIKE HOMEOBOX (WOX) genes, genes similar to WUS in Arabidopsis, are not active in the regulation of SAM development. Cytokinin and CLV signaling, however, interact in some form to regulate this growth, conserving the antagonistic nature of CLV and cytokinin signaling, which respectively inhibit and induce stem cell identity in the SAM. We hypothesized that CLV signaling directly (interacting with some of the components of the cytokinin pathway) or indirectly (interacting on a target shared with the cytokinin pathway) affects cytokinin signaling in Physcomitrella patens SAM development and regulation. There are a few moss homologs of receptor-kinases in Arabidopsis SAMs that are involved in CLAVATA3 signaling. The mutants of these receptors produce ectopic stem cells (cells that are out of place). We stained CLV pathway mutant and wild type Physcomitrella with propidium iodide and used confocal microscopy to capture 3D image stacks of the meristems at varying stages of development in each genotype. For each genotype, we quantified the number of ectopic stem cells to evaluate the severity of the phenotype. We assayed the phenotypes at several developmental stages to determine the stage that best shows the phenotype. Finally, that developmental stage was imaged in moss shoots grown with varying concentrations of cytokinin (10nM, or 100nM) added to the media. We saw that as cytokinin concentrations increased, wild type and clv1 mutants displayed increased stem size and number of apical cells, replicating or enhancing the CLV mutant phenotypes, respectively. These results support our hypothesis that CLV and cytokinin signaling are antagonistic regulators of similar downstream processes including stem cell formation. In addition, rpk2 appeared to have additive effects when combined with clv mutants, suggesting that rpk2 may be in a separate but parallel pathway to CLV. Altogether, this helps us understand the development of other organs, like fruit, in other species of plants that similarly rely on cytokinin and CLV signaling to regulate organ size.
I arrived without much expectation. I was aware that any lab could entail different needs and circumstances, which allowed me to be open and learn as much as possible. Academically, I learned about plant development (specifically the shoot apical meristem) and the most recent concepts and theories on plant development through papers and seminars. It taught me to view new ideas/theories presented in my courses as tools and starting points for expansion rather than immutable exam material. This will enhance my learning to become more than simply memory. Professionally, I learned new techniques and concepts that were used in the lab. There are limited resources at our disposal. Consequently, we had to improvise equipment to reach our goals, at times by using substances or tools in ways not originally intended. It kept our techniques economically efficient and practical. In addition, I practiced using a confocal microscope and an accompanying software to analyze 3D images of moss cells. I became familiar with the non-scientific duties imbedded in research, too. Being exposed to the nature and logistics of funding and collaborations among different institutions gave me an honest view of what entails from a career in research. Personally, I have come to appreciate plant research and the diversity of topics it reaches, like animal behavior and bioinformatics. Also, my writing skills have improved, and I will apply them into my career to improve even more along the way.