Dynamics of Regulatory Networks

Organ formation involves stem cell divisions, patterning and differentiation, all of which are fundamental aspects of developmental biology. Stem cells frequently undergo asymmetric cell divisions, their progeny take on new fates to specify cell types and progress down a pathway toward end-stage differentiation. In contrast to animal organogenesis, plant organs are formed primarily after embryogenesis. In the case of the root, initial patterning takes place during early embryonic development. This pattern is then propagated by cells that form the root apical meristem. This reservoir of stem cells in the meristem is considered to be the source of all post-embryonic root development. Fundamental questions, then, are how cell division is regulated in stem cells and how cell specification and differentiation are controlled in their progeny to generate distinct tissues within this organ.

The Arabidopsis root provides an excellent system to address these questions. Because plant cells don’t move and because the initials (which are the equivalent of animal stem cells) divide in a stereotypic fashion, the progeny of the initials are found in contiguous cell files. Thus, the root offers unambiguous, spatially oriented lineages from stem cells to their mature differentiated progeny.

In transverse sections, the outer four layers of the root (from outside to inside: epidermis, cortex, endodermis and pericycle) have rotational symmetry and form the root radial pattern. The pericycle together with the vascular tissue internal to it, are referred to as the stele. As in animal cells, the plant stem cell niche defines the region that maintains the stem cells, undifferentiated cells with the capacity for self-renewal. Four sets of initials surround the quiescent center (QC), which are less mitotically active cells required for stem cell maintenance. The radial pattern is generated through asymmetric cell divisions of the initials. A well-characterized example is the division process that gives rise to the two ground tissue layers, cortex and endodermis. The cortex/endodermal initial (CEI) divides first in an anticlinal (transverse) orientation. This division regenerates the initial and produces a daughter cell (CEID), which subsequently divides in the periclinal (longitudinal) orientation. This second division produces the first cells of the endodermal and cortex lineages. As new cells form, they are added into the files of their older counterparts pushing the root tip downwards into the soil. Further along the longitudinal axis they elongate and differentiate.

Our analysis of root development began nearly 20 years ago with a genetic screen for abnormal root morphogenesis. Among mutants with shorter roots we focused on two with abnormal radial patterns. Both short root (shr) and scarecrow (scr) are missing a cell layer between the epidermis and stele. The use of cell-type specific markers revealed that the remaining “mutant layer” in shr has characteristics of cortex, while the mutant layer in scr has characteristics of both cortex and endodermis. We isolated the genes and showed that they both belong to the plant specific GRAS family of putative transcription factors (TFs).

Later we showed that SHR protein is able to move from the stele, where it is synthesized to the adjacent cells including the QC, CEI, CEID and the endodermal lineage. Several TFs had been documented to move, but SHR was the first case in which there was a clear developmental consequence of movement. What was striking in the case of SHR movement was that it was tightly regulated. We found that SHR interacts with SCR in the endodermis (and in the CEI, CEID and QC) and SCR sequesters SHR in the nucleus. Moreover, SHR and SCR together bind to the SCR promoter to activate its transcription. Thus, a positive feedback loop is formed that rapidly increases SCR protein levels ensuring a tight control over SHR movement. The SHR-SCR complex directly regulates the cell cycle through activation of CYCLIN D6 (CYCD6). Another cell cycle factor, RETINOBLASTOMA-RELATED (RBR) binds SCR and inhibits SHR-SCR activity. RBR activity in turn can be inhibited by CYCD6. In addition, a family of C2H2 zinc finger TFs are also direct targets of SHR and SCR and regulate both the asymmetric cell division and the steps toward cellular differentiation. We are currently working to characterize the regulatory networks that link the stem cells in the root to their differentiated progeny.