My project is build of two sub-projects, each applying different approaches in order to identify and characterize factors important in the specification of stem cells during early plant embryogenesis.
The first sub-project is about functional characterization of downstream factors of the transcription factor MONOPTEROS. When this transcription factor is mutated, the plant is not able to make a root, due to misspecification of a few cells in the first stages of development. Using microarray techniques we have identified a number of genes that are misregulated in the mutant background and by systematically characterizing these downstream targets we aim to dissect which are important for the drastic phenotype of this mutant. This will also give us insights into the requirements for normal embryo development.
The second sub-project (together with Joakim Palovaraa and Shunsuke Saiga) applies a high-throughput method to achieve a similar goal. Using a method called INTACT we isolate individual cell types in a certain stage of development, the stages where the first stem cells are established. By subsequent transcriptional profiling we will be able to reconstruct an atlas of expressed genes in separate cell types, through development. When looking at the differences between the stage prior to stem cell specification and right after (or during) stem cell specification, we will be able to find factors that are important for this specification. We will next apply a reverse genetics approach in order to functionally analyze these factors.
PCR, DNA isolation, gel electrophoresis, RNA isolation, Q-RT-PCR, INTACT, microarray, immunoprecipitation, (confocal) microscopy.