From stem cells to differentiated tissues

To understand stem cell function we have focused on the cells responsible for generating two distinct lineages in the root, the endodermis and cortex. Their shared stem cell divides asymmetrically to regenerate itself, while the other daughter undergoes a second asymmetric division to produce the first cells of these two lineages. A mutant that fails to undergo this second division led us to the SHORT-ROOT (SHR) gene. We showed that SHR protein is synthesized in the internal vascular tissue then moves to the adjacent cell layer where it interacts with SCARECROW (SCR), a member of the same family of plant-specific transcription factors. The interaction of SCR and SHR initiates binding of both factors to the SCR promoter producing a positive feedback loop. Both factors also bind to the promoter of CYCLIN-D6 (CYCD6), a component of the cell cycle machinery that is specifically expressed in the stem cell daughter prior to the second asymmetric division. The activation of SCR by SHR followed by joint activation of CYCD6 produces a feed-forward loop. CYCD6 and its associated kinase are required to phosphorylate Retinoblastoma Related (RBR), which binds and inactivates SCR. Mathematical modeling of these interactions indicated that as SHR concentration increases, there is a sudden jump in SCR concentration producing a bi-stable switch. Positive feedback and feed-forward loops are common motifs in gene regulatory networks and have been hypothesized to operate in various biological contexts, but their functionality has rarely been tested. Our research aims to experimentally test hypotheses as to how gene regulatory networks function.

The acquisition of cell identity has been described as a series of irreversible switches in which the stages include competence, commitment and terminal. Because the longitudinal axis of the root serves as a developmental time line, we can localize critical developmental transitions to specific regions of the root. By identifying genes downstream of SHR and SCR we identified a family of zinc-finger transcription factors called the BIRDs as key regulators of endodermal and cortex identity in addition to controlling asymmetric division of the stem cells. Transcriptome analysis revealed that expression of individual BIRD genes allowed for partial rescue of endodermal and cortex identities in a shr mutant. Moreover, in the absence of two of the BIRDs and SCR, the stem cells for endodermis and cortex disappear shortly after germination. Maintaining stem cells over long periods of time is of particular importance in plants, which can continue to grow for hundreds of years.

A second approach was to ask what regulates the signature differentiated attribute of the endodermis, production of a waterproof intercellular band known as the Casparian strip. From a mutant screen of plants containing a GFP tagged component of the Casparian strip we identified a transcription factor, MYB36 that is required to activate expression of genes involved in Casparian strip formation.  Strikingly, MYB36 is a direct target of SCR, suggesting that there are relatively few steps between the regulatory network that controls stem cell divisions and the network that controls terminal differentiation.