My long-term goal is to understand how cells are generated from a stem cell population and how they become patterned and integrated to form a functioning organ. I became interested in developmental biology as a graduate student with former HHMI investigator Philip Leder at Harvard Medical School. There, I worked on cells that were part of the hematopoietic lineage and was struck by the number of different cells in this lineage and by the difficulty of following the progression from stem cell to fully differentiated tissue. I was drawn to plants, in part, because of the relatively small number of cell types in most organs.
While characterizing genes expressed in the root during my postdoc with Nam-Hai Chua (Rockefeller University), I realized what an ideal system it is for studying organogenesis. Root tissues are organized as concentric cylinders around a central vascular tissue. A stem cell population located near the tip of the root gives rise to all the cells in the root, and because there is no cell movement, each cell file contains all of the different developmental stages. Thus, the organization of the root reduces what is normally a four-dimensional problem (three spatial dimensions and time) to a two-dimensional problem, with cell types along the radial axis and developmental time along the longitudinal axis.
When I started my independent lab, we performed genetic screens for abnormal root development in the model plant Arabidopsis. One of the first mutants we identified, short-root (shr), turned out to have abnormal radial patterning, in that it was missing one of the concentric cylinders of tissues. We then identified a second mutant, scarecrow (scr), which was also missing a cell layer. We cloned both of the mutated genes and found that they encoded transcription factors that were members of the same plant-specific family.
A major turning point in our research was when we discovered that the SHR protein is able to move from the vascular tissue where it is produced to the adjacent stem cells and endodermis where it acts. In those cells, it physically interacts with SCR and together they turn on a suite of genes including a specific component of the cell cycle machinery. This component, a D-type cyclin, regulates the asymmetric division of the stem cell to form the two cell layers of endodermis and cortex. We thus were able to identify a direct link between key developmental regulators and the core cell cycle machinery. More recently, we identified a set of zinc-finger transcription factors that are regulated by SHR and SCR and are involved in orchestrating asymmetric cell divisions, specifying cell lineages and maintaining a specific stem cell population.
In addition to starting from the stem cells in the tip of the root, we are also working our way backward from fully differentiated tissue, higher up in the root. Using a genetic approach we identified a mutant in which the differentiated signature of the endodermis, the Casparian Strip, is defective. The mutation was in a transcription factor of the Myb family, MYB36. We showed that this gene has a dual function of activating targets involved in Casparian Strip formation as well as repressing genes that control cellular proliferation. In turn, MYB36 is regulated by SCR, so we have now elucidated some of the gene regulatory networks that result in the progression from asymmetric division of the stem cell to fully differentiated tissue.
The advent of technology to sample gene expression across the entire genome, coupled with the simplicity of root organization, provided an opportunity to generate a comprehensive expression map of an organ in all cell types and developmental stages. To accomplish this, we used cell sorting of GFP (green fluorescent protein)-marked cell populations and microdissection of individual roots, followed by microarray analysis, to generate a transcriptome map for all cell types and developmental stages. We then analyzed the effects of abiotic stresses on gene expression at cell-type-specific resolution and found that there were dramatic responses that were highly specific. We have used the same approach to sample proteins, small RNAs, and metabolites in individual cell types. More recently, we used it to map epigenomic differences between cell types in the root.
The primary functions of the root are to acquire water and nutrients and to anchor the plant. To perform these functions requires branching out, which is mediated through the formation of new lateral roots from internal cell layers. The remarkably even placement of lateral roots along the primary root of Arabidopsis was previously thought to involve random induction and lateral inhibition. We were able to show that, in fact, there is a periodic process that marks the positions of future lateral roots. This process depends upon a wave of gene expression that occurs near the root tip and moves up the root. Analysis of microarrays performed on adjacent pieces of individual roots allowed us to identify a large number of genes whose expression oscillates. We determined that some of these genes are involved in the periodic formation of lateral roots and that the wave-like gene expression process has similarities to the wave-like process underlying somitogenesis in vertebrates.
Once branch roots are formed, they grow out and down to generate the three-dimensional physical network known as root system architecture. To date, little is known about the genes that control formation of root system architecture, in part because it is hard to determine the phenotype of root systems when growing in soil. To address this issue, we developed an imaging platform in which plants are grown in translucent media in cylinders that are rotated through 360 degrees. Images are automatically acquired at specific angles, three-dimensional reconstructions are generated, and traits are measured. We have used this platform to identify root quantitative trait loci (QTL) from a rice recombinant inbred population. Our long-term goal is to identify the genes that control root system architecture, which could lead to enhanced root traits that allow better nutrient acquisition and drought tolerance.
Grants from the National Institutes of Health, the National Science Foundation, and the Defense Advanced Research Projects Agency (DARPA) provide partial support for these projects.
As of April 7, 2016