ABSTRACT: The definition of cell identity is a central problem in biology. Single-cell RNA-seq provides a wealth of information regarding the developmental state of individual cells. However, better methods are needed to map the identity of single cells, especially during identity transitions. We have developed a quantitative classification method that is robust to expression noise and can detect primary and chimeric identities from single-cell RNA sequencing profiles. The method uses existing transcriptome repositories of grouped cell-types to define a set of optimal cell-identity markers, which are then used to define a cell identity metric. This metric accurately classified diverse cell identities in Arabidopsis root tips and human glioblastoma cells. We demonstrate the strength of the approach to resolve a dynamic developmental process by analyzing the identity of single cells captured from regenerating Arabidopsis roots following removal of their stem-cell-niche. We discover that, apart from new niche formation at the vicinity of the cut site, cells that are distant from the injury site also undergo a transient, partial collapse of identity during the regeneration and reorganization of the root, demonstrating the usefulness of a quantitative cell identity metric.
Project description:The definition of cell identity is a central problem in biology. Single-cell RNA-seq provides a wealth of information regarding the developmental state of individual cells. However, better methods are needed to map the identity of single cells, especially during identity transitions. We have developed a quantitative classification method that is robust to expression noise and can detect primary and chimeric identities from single-cell RNA sequencing profiles. The method uses existing transcriptome repositories of grouped cell-types to define a set of optimal cell-identity markers, which are then used to define a cell identity metric. This metric accurately classified diverse cell identities in Arabidopsis root tips and human glioblastoma cells. We demonstrate the strength of the approach to resolve a dynamic developmental process by analyzing the identity of single cells captured from regenerating Arabidopsis roots following removal of their stem-cell-niche. We discover that, apart from new niche formation at the vicinity of the cut site, cells that are distant from the injury site also undergo a transient, partial collapse of identity during the regeneration and reorganization of the root, demonstrating the usefulness of a quantitative cell identity metric. Overall design: 4 Technical replicates (pooled-and-split single QC cells), 23 cells from the QC, 3 cells from the Stele, and 4 cells from the stele, 16h following root tip decapitation
Project description:Plant roots can regenerate after complete excision of their tip, including the stem cell niche, but it is not clear what developmental program mediates such repair. Here, we use a combination of lineage tracing, single-cell RNA-seq, and marker analysis to test different models of tissue reassembly. We show that rapid cell-identity transitions lead to the formation of a new stem cell niche from multiple remnant tissues. The transcriptome of regenerating cells prior to stem cell activation resembled that of the embryonic root progenitor, and regeneration defects were more severe in embryonic versus adult root mutants. Furthermore, the signaling domains of the hormones auxin and cytokinin mirrored their embryonic dynamics, and manipulation of both hormones altered the position of new tissues and stem cell niche markers. Our findings suggest that plant organ regeneration resembles the developmental stages of embryonic patterning and is guided by spatial information laid down by complementary hormone domains. 215 single cells isolated from marked stele tissue (either using WOL or AHP6 promoters), before, at 3h, 16h and 46h post root tip decapitation
Project description:We used fluorescence activated cell sorting (FACS) to isolate the different cell populations in the Arabidopsis root stem cell niche
Project description:Detached Arabidopsis leaves can regenerate adventitious roots, providing a platform to study de novo root regeneration (DNRR). We performed single-cell RNA-seq analysis and revealed that regeneration primarily originated from vascular stem-cell organizer within procambium, followed by step-by-step changes of transcriptome in cell fate transition, including gradually erasing the vascular stem-cell organizer identity and recruiting the root development program.
Project description:This model is from the article:
The influence of cytokinin-auxin cross-regulation on cell-fate determination in Arabidopsis thaliana root development
Muraro D, Byrne H, King J, Voss U, Kieber J, Bennett M.
J Theor Biol.2011 Aug 21;283(1):152-67.
PMID: 21640126,
Abstract:
Root growth and development in Arabidopsis thaliana are sustained by a specialised zone termed the meristem, which contains a population of dividing and differentiating cells that are functionally analogous to a stem cell niche in animals. The hormones auxin and cytokinin control meristem size antagonistically. Local accumulation of auxin promotes cell division and the initiation of a lateral root primordium. By contrast, high cytokinin concentrations disrupt the regular pattern of divisions that characterises lateral root development, and promote differentiation. The way in which the hormones interact is controlled by a genetic regulatory network. In this paper, we propose a deterministic mathematical model to describe this network and present model simulations that reproduce the experimentally observed effects of cytokinin on the expression of auxin regulated genes. We show how auxin response genes and auxin efflux transporters may be affected by the presence of cytokinin. We also analyse and compare the responses of the hormones auxin and cytokinin to changes in their supply with the responses obtained by genetic mutations of SHY2, which encodes a protein that plays a key role in balancing cytokinin and auxin regulation of meristem size. We show that although shy2 mutations can qualitatively reproduce the effect of varying auxin and cytokinin supply on their response genes, some elements of the network respond differently to changes in hormonal supply and to genetic mutations, implying a different, general response of the network. We conclude that an analysis based on the ratio between these two hormones may be misleading and that a mathematical model can serve as a useful tool for stimulate further experimental work by predicting the response of the network to changes in hormone levels and to other genetic mutations.
Project description:Plant roots can regenerate after complete excision of their tip, including the stem cell niche, but it is not clear what developmental program mediates such repair. Here, we use a combination of lineage tracing, single-cell RNA-seq, and marker analysis to test different models of tissue reassembly. We show that rapid cell-identity transitions lead to the formation of a new stem cell niche from multiple remnant tissues. The transcriptome of regenerating cells prior to stem cell activation resembled that of the embryonic root progenitor, and regeneration defects were more severe in embryonic versus adult root mutants. Furthermore, the signaling domains of the hormones auxin and cytokinin mirrored their embryonic dynamics, and manipulation of both hormones altered the position of new tissues and stem cell niche markers. Our findings suggest that plant organ regeneration resembles the developmental stages of embryonic patterning and is guided by spatial information laid down by complementary hormone domains.
Project description:Roots are fundamental organs for plant development and response to their environment: they anchor the plant to its growth substrate, uptake nutrients and water vital to plant growth, and can sense and respond to a variety of biotic and abiotic stresses. The architecture of root systems and their growth are known to be strongly affected by the environmental conditions found in the soil. However, the acquisition of cell identities at the root meristem is still mainly viewed as ontogenetically driven, where a small number of stem cells generate all the cell types through stereotyped divisions followed by differentiation, along a simple developmental trajectory. The extent to which environmental cues precisely shape and affect these developmental trajectories remains an open question. We used single-cell RNA-seq, combined with spatial mapping, to deeply explore the trajectories of cell states at the tip of Arabidopsis roots, known to contain multiple developing lineages. Surprisingly, we found that most lineage trajectories exhibit a stereotyped bifid topology with two developmental trajectories rather than one. The formation of one of the trajectories is driven by a strong and specific activation of genes involved in the responses to various environmental stimuli, that affects only of a subset of the cells in multiple cell types simultaneously, revealing another layer of patterning of cell identities in the root that is independent of cell ontogeny. We demonstrate the robustness of this environmentally-responsive transcriptional state by showing that it is present in a mutant where cell type identities are greatly perturbed, as well as in different Arabidopsis ecotypes. We also show that the root can adapt the proportion of cells that acquire this particular state in response to environmental signals such as nutrient availability. The discovery of this transcriptional signature further highlights the adaptive potential of plant development.
Project description:Transcriptional programs that regulate development are exquisitely controlled in space and time. Elucidating these programs that underlie development is essential to understanding the acquisition of cell and tissue identity. We present microarray expression profiles of a high resolution set of developmental time points within a single Arabidopsis root, and a comprehensive map of nearly all root cell-types. These cell-type specific transcriptional signatures often predict novel cellular functions. A computational pipeline identified dominant expression patterns that demonstrate transcriptional connections between disparate cell types. Dominant expression patterns along the rootâs longitudinal axis do not strictly correlate with previously defined developmental zones, and in many cases, expression fluctuation along this axis was observed. Both robust co-regulation of gene expression and potential phasing of gene expression were identified between individual roots. Methods that combine these two sets of profiles demonstrate transcriptionally rich and complex programs that define Arabidopsis root development in both space and time. We used microarrays to profile expression of nearly all cell types in the Arabidopsis root, and to profile at high resolution, developmental time points along the root's longitudinal axis. Experiment Overall Design: Microarray expression profiles of 8 new GFP marked lines with 2-3 replicates were used to augment existing microarray expression profiles of the root. RNA isolated from 13 cross sections along a single root's longitudinal axis were also profiled by microarray analysis. An independent root with 12 sections were used as a biological replicate.
Project description:The RETINOBLASTOMA–RELATED (RBR) is a key regulator of cell proliferation and differentiation in plants, and plays an important role in maintenance of the stem cell niche in the root. We used microarray analysis to characterize the transcriptional response of Arabidopsis thaliana root tips from rRBr mutant (7 samples) against Col-0 wild type (6 samples) after 4, 6 and 10 das.
Project description:We combined transcriptomic profiling of auxin related mutants with genetic and biochemical approaches and live-cell imaging techniques of Arabidopsis roots to understand the role of auxin-driven gibberellin level changes during root development, particularly root cell elongation. We show that auxin negatively regulates the level of gibberellin in root elongation zone. Auxin signalling steers the expression of gibberellin deactivating enzymes - GIBBERELLIN 2-OXIDASES (GA2OX) exclusively in root elongation zone. Interestingly, GA2OX8 expression is high in tissues with elevated auxin levels, such as vasculature or stem cell niche, fitting with the observed effect of auxin on gibberellin level. Here we show that GA2OX enzymes are negative regulators of root cell elongation. Gibberellin decrease caused by GA2OX8 overexpression inhibits root cell elongation. In contrast, roots missing GA2OX genes elongate faster. These findings indicate that GA2OX8 enzymes represent an integration core of auxin and gibberellin signalling pathway in root elongation zone, vascular development and regulation of stem cell niche. Our results enhance understanding of complex mechanisms controlling root cell elongation.