To access our recently published plant single cell RNA-Sequencing browser click here.

The formation of stable, precisely defined boundaries between two distinct cell fates is a fundamental feature of plant and animal development. Such cell fate boundaries coordinate the differentiation and growth of the tissue or organ. In this regard, development of flat leaf architecture poses an unusual and mechanistically challenging problem; namely, how to create a stable adaxial-abaxial (top-bottom) boundary within the plane of a long and wide, but shallow, structure? Adaxial-abaxial polarity is based on an intricate gene regulatory network with several highly conserved transcription factors that promote either adaxial or abaxial fate at its core. These are expressed in complementary domains on the top and bottom side of the developing organ, respectively. We have shown that the positional information needed to define these domains is provided in part by small RNAs that reminiscent to classical morphogens generate sharply defined domains of target gene expression through an intrinsic, threshold-based readout of their mobility gradients (Skopelitis et al., 2017).

We have established time-lapse confocal imaging of the vegetative shoot apical meristem. Together with microsurgical and genetic manipulations, this allows us to link patterns of gene expression emerging in space and time at the meristem to successive cell fate decisions. In addition, we use a combination of theoretical and experimental approaches to examine the network properties needed to maintain the adaxial-abaxial boundary uniformly positioned during organogenesis.

Fig. 1: Cell fate analysis at the vegetative shoot apical meristem. (A) The abaxial determinant KAN1 accumulates at meristem-organ boundaries and on the bottom side of leaf primordia. (B) Time-lapse confocal imaging of the meristem allows us to link patterns of gene expression to cell fate decisions.

An obvious advantage of small RNAs in development is that their mobility through plasmodesmata is regulated via mechanisms distinct from those controlling the movement of proteins (Skopelitis et al., 2018). Small RNA mobility is gated at individual cell-cell interfaces to generate directional movement that limits long-distance trafficking and patterns their activity within stem cell niches. We now seek to understand how small RNAs move, what gates their mobility at plasmodesmata, and how the threshold-based readout of mobile small RNA gradients is realized? This information will be used to understand the interplay of signals that coordinate the many patterning processes occurring in close spatial and temporal vicinity within plant stem cell niches.

Fig. 2: Gradients of mobile small RNAs have morphogen-like patterning activities. Mobility of miRNAs from their site of biogenesis in the bottom epidermis (A) yields a miRNA gradient (B) that through a threshold-based read-out establishes an on-off pattern of target gene expression (C,D).

Fig. 3: Synthetic system to study miRNA mobility. In seedlings expressing fluorescent reporters (A), artificial miRNAs targeting these reporters are expressed in a regulated often tissue-specific manner (B) to arrive at the parameters and mechanisms of miRNA mobility (C).

High-throughput single cell RNA-Sequencing (scRNA-Seq) provides unprecedented power in understanding gene expression in complex tissues. We recently generated a scRNA-Seq expression atlas of the Arabidopsis root that provides an unparalleled spatiotemporal perspective of root cell differentiation (Denyer et al., 2019; Ma et al., 2020). We currently use this and related single cell approaches to resolve how cell fates emerge and how these orchestrate the differentiation of specialized cell types in both the root and shoot stem cell niches.

To access our publicly available plant single cell RNA-Sequencing browser click here.

Fig. 4: t-SNE plot of 4,727 profiled Arabidopsis root cells. The cluster map shows that cells of the niche and meristem form a central core from which cells of different identities and progressive maturity, radiate in contrasting directions.