Pradeep Das

French National Centre for Scientific Research, Lutetia Parisorum, Île-de-France, France

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Publications (16)219.36 Total impact

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    ABSTRACT: The role of mechanical signals in cell identity determination remains poorly explored in tissues. Furthermore, because mechanical stress is widespread, mechanical signals are difficult to uncouple from biochemical-based transduction pathways. Here we focus on the homeobox gene SHOOT MERISTEMLESS (STM), a master regulator and marker of meristematic identity in Arabidopsis. We found that STM expression is quantitatively correlated to curvature in the saddle-shaped boundary domain of the shoot apical meristem. As tissue folding reflects the presence of mechanical stress, we test and demonstrate that STM expression is induced after micromechanical perturbations. We also show that STM expression in the boundary domain is required for organ separation. While STM expression correlates with auxin depletion in this domain, auxin distribution and STM expression can also be uncoupled. STM expression and boundary identity are thus strengthened through a synergy between auxin depletion and an auxin-independent
    Full-text · Article · Dec 2015 · eLife Sciences
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    ABSTRACT: In situ mRNA hybridization is one of the most powerful techniques for analyzing patterns of gene expression. However, its usefulness is limited in complex plant tissues by the need to fix, embed and section samples before localizing the desired mRNA. Here we present a robust whole-mount in situ hybridization method that allows easy access to patterns of gene expression in intact, complex tissues, such as the inflorescence apex of Arabidopsis thaliana. The tissue is first fixed and then permeabilized by treatment with a cocktail of cell wall-digesting enzymes that has been optimized to preserve the integrity of tissue structures, while also permitting the detection of expression patterns in deep tissues. In addition to colorimetric staining, fluorimetric staining that can be imaged by confocal microscopy can also be used with this protocol, thus providing full 3D resolution. The entire procedure can take <3 d from tissue preparation to image acquisition.
    No preview · Article · Oct 2014 · Nature Protocols
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    ABSTRACT: Cell differentiation has been associated with changes in mechanical stiffness in single cell-systems, yet it is unknown whether this association remains true in a multicellular context, particularly in developing tissues. In order to address such questions, we have developed a methodology, termed quantitative tandem epifluorescence and nanoindentation (qTEN), wherein we sequentially determine cellular genetic identity with confocal microscopy, and mechanical properties with atomic force microscopy. We have applied this approach to examine cellular stiffness at the shoot apices of plants carrying a fluorescent reporter for the CLAVATA3 (CLV3) gene, which encodes a secreted glycopeptide involved in the regulation of the centrally-located stem cell zone in inflorescence and floral meristems. We found that these CLV3-expressing cells are characterized by an enhanced stiffness. Additionally, by tracking cells in young flowers before and after the onset of GFP expression, we observe that an increase in stiffness coincides with this onset. This work illustrates how qTEN can reveal the spatial and temporal dynamics of both gene expression and cell mechanics at the shoot apex, and by extension in the epidermis of any thick tissue.
    Full-text · Article · Jun 2014 · Plant physiology
  • Olivier Hamant · Pradeep Das · Agata Burian
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    ABSTRACT: Analysis of shoot meristem shape and gene expression pattern has been conducted in many species over the past decades. Recent live imaging techniques have allowed an unprecedented accumulation of data on the biology of meristematic cells, as well as a better understanding of the molecular and biophysical mechanisms behind shape changes in this tissue. Here we describe in detail how to prepare shoot apices of both Arabidopsis and tomato, in order to image them over time using a confocal microscope equipped with a long-distance water-dipping lens.
    No preview · Article · Jan 2014 · Methods in molecular biology (Clifton, N.J.)
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    ABSTRACT: How biological systems generate reproducible patterns with high precision is a central question in science. The shoot apical meristem (SAM), a specialized tissue producing plant aerial organs, is a developmental system of choice to address this question. Organs are periodically initiated at the SAM at specific spatial positions and this spatiotemporal pattern defines phyllotaxis. Accumulation of the plant hormone auxin triggers organ initiation, whereas auxin depletion around organs generates inhibitory fields that are thought to be sufficient to maintain these patterns and their dynamics. Here we show that another type of hormone-based inhibitory fields, generated directly downstream of auxin by intercellular movement of the cytokinin signalling inhibitor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), is involved in regulating phyllotactic patterns. We demonstrate that AHP6-based fields establish patterns of cytokinin signalling in the meristem that contribute to the robustness of phyllotaxis by imposing a temporal sequence on organ initiation. Our findings indicate that not one but two distinct hormone-based fields may be required for achieving temporal precision during formation of reiterative structures at the SAM, thus indicating an original mechanism for providing robustness to a dynamic developmental system.
    No preview · Article · Dec 2013 · Nature
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    ABSTRACT: The floral organ identity factor AGAMOUS (AG) is a key regulator of Arabidopsis thaliana flower development, where it is involved in the formation of the reproductive floral organs as well as in the control of meristem determinacy. To obtain insights into how AG specifies organ fate, we determined the genes and processes acting downstream of this C function regulator during early flower development and distinguished between direct and indirect effects. To this end, we combined genome-wide localization studies, gene perturbation experiments, and computational analyses. Our results demonstrate that AG controls flower development to a large extent by controlling the expression of other genes with regulatory functions, which are involved in mediating a plethora of different developmental processes. One aspect of this function is the suppression of the leaf development program in emerging floral primordia. Using trichome initiation as an example, we demonstrate that AG inhibits an important aspect of leaf development through the direct control of key regulatory genes. A comparison of the gene expression programs controlled by AG and the B function regulators APETALA3 and PISTILLATA, respectively, showed that while they control many developmental processes in conjunction, they also have marked antagonistic, as well as independent activities.
    Full-text · Article · Jul 2013 · The Plant Cell
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    ABSTRACT: Flower patterning is determined by a complex molecular network but how this network functions remains to be elucidated. Here, we develop an integrative modeling approach that assembles heterogeneous data into a biologically coherent model to allow predictions to be made and inconsistencies among the data to be found. We use this approach to study the network underlying sepal development in the young flower of Arabidopsis thaliana. We constructed a digital atlas of gene expression and used it to build a dynamical molecular regulatory network model of sepal primordium development. This led to the construction of a coherent molecular network model for lateral organ polarity that fully recapitulates expression and interaction data. Our model predicts the existence of three novel pathways involving the HD-ZIP III genes and both cytokinin and ARGONAUTE family members. In addition, our model provides predictions on molecular interactions. In a broader context, this approach allows the extraction of biological knowledge from diverse types of data and can be used to study developmental processes in any multicellular organism.
    Full-text · Article · Dec 2011 · The Plant Cell
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    ABSTRACT: Stamens undergo a very elaborate development program that gives rise not only to many specific tissue types, but also to the male gametes. The specification of stamen identity is coordinated by a group of homeotic genes such as APETALA3 (AP3) and PISTILLATA (PI), AGAMOUS (AG) and SEPALLATA (SEP1-4) genes. Genome-wide transcriptomic comparisons between floral buds of wild-type and ap3 mutants led to the identification of the REM22 gene, which is expressed in the early stages of stamen development. This gene is member of the plant-specific B3 DNA-binding superfamily. In this work, we dissect the spatio-temporal expression pattern of REM22 during the early stages of stamen development. To this end, both in situ hybridization analyses as well as in vivo fluorescence strategies were employed. At stage 4 of flower development, REM22 is expressed exclusively in those undifferentiated cells of the floral meristem that will give rise to the stamen primordia. At stage 5, REM22 expression is restricted to the epidermal and the subepidermal layers of anther primordia. Later, this expression is confined to the middle layer and the differentiating tapetal cells. After stage 10 when all the tissues of the anther have differentiated, REM22 expression is no longer detectable. Furthermore, we examined the pREM22::GUS-GFP marker line in an inducible system where the ectopic AG function is used to promote microsporogenesis. The data support the idea that REM22 expression is a useful marker to study the early stages of stamen development.
    Full-text · Article · Aug 2011 · The International journal of developmental biology
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    ABSTRACT: The plant hormone auxin is thought to provide positional information for patterning during development. It is still unclear, however, precisely how auxin is distributed across tissues and how the hormone is sensed in space and time. The control of gene expression in response to auxin involves a complex network of over 50 potentially interacting transcriptional activators and repressors, the auxin response factors (ARFs) and Aux/IAAs. Here, we perform a large-scale analysis of the Aux/IAA-ARF pathway in the shoot apex of Arabidopsis, where dynamic auxin-based patterning controls organogenesis. A comprehensive expression map and full interactome uncovered an unexpectedly simple distribution and structure of this pathway in the shoot apex. A mathematical model of the Aux/IAA-ARF network predicted a strong buffering capacity along with spatial differences in auxin sensitivity. We then tested and confirmed these predictions using a novel auxin signalling sensor that reports input into the signalling pathway, in conjunction with the published DR5 transcriptional output reporter. Our results provide evidence that the auxin signalling network is essential to create robust patterns at the shoot apex.
    Full-text · Article · Jul 2011 · Molecular Systems Biology
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    ABSTRACT: Quantitative information on growing organs is required to better understand morphogenesis in both plants and animals. However, detailed analyses of growth patterns at cellular resolution have remained elusive. We developed an approach, multiangle image acquisition, three-dimensional reconstruction and cell segmentation-automated lineage tracking (MARS-ALT), in which we imaged whole organs from multiple angles, computationally merged and segmented these images to provide accurate cell identification in three dimensions and automatically tracked cell lineages through multiple rounds of cell division during development. Using these methods, we quantitatively analyzed Arabidopsis thaliana flower development at cell resolution, which revealed differential growth patterns of key regions during early stages of floral morphogenesis. Lastly, using rice roots, we demonstrated that this approach is both generic and scalable.
    Full-text · Article · Jul 2010 · Nature Methods
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    ABSTRACT: The shape of an organism relies on a complex network of genetic regulations and on the homeostasis and distribution of growth factors. In parallel to the molecular control of growth, shape changes also involve major changes in structure, which by definition depend on the laws of mechanics. Thus, to understand morphogenesis, scientists have turned to interdisciplinary approaches associating biology and physics to investigate the contribution of mechanical forces in morphogenesis, sometimes re-examining theoretical concepts that were laid out by early physiologists. Major advances in the field have notably been possible thanks to the development of computer simulations and live quantitative imaging protocols in recent years. Here, we present the mechanical basis of shape changes in plants, focusing our discussion on undifferentiated tissues. How can growth be translated into a quantified geometrical output? What is the mechanical basis of cell and tissue growth? What is the contribution of mechanical forces in patterning?
    No preview · Article · May 2010 · Annual Review of Plant Biology
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    ABSTRACT: In Arabidopsis, the population of stem cells present in young flower buds is lost after the production of a fixed number of floral organs. The precisely timed repression of the stem cell identity gene WUSCHEL (WUS) by the floral homeotic protein AGAMOUS (AG) is a key part of this process. In this study, we report on the identification of a novel input into the process of floral stem cell regulation. We use genetics and chromatin immunoprecipitation assays to demonstrate that the bZIP transcription factor PERIANTHIA (PAN) plays a role in regulating stem cell fate by directly controlling AG expression and suggest that this activity is spatially restricted to the centermost region of the AG expression domain. These results suggest that the termination of floral stem cell fate is a multiply redundant process involving loci with unrelated floral patterning functions.
    Preview · Article · Jun 2009 · Development
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    ABSTRACT: Most multicellular organisms have a capacity to regenerate tissue after wounding. Few, however, have the ability to regenerate an entire new body from adult tissue. Induction of new shoot meristems from cultured root explants is a widely used, but poorly understood, process in which apical plant tissues are regenerated from adult somatic tissue through the de novo formation of shoot meristems. We characterize early patterning during de novo development of the Arabidopsis shoot meristem using fluorescent reporters of known gene and protein activities required for shoot meristem development and maintenance. We find that a small number of progenitor cells initiate development of new shoot meristems through stereotypical stages of reporter expression and activity of CUP-SHAPED COTYLEDON 2 (CUC2), WUSCHEL (WUS), PIN-FORMED 1 (PIN1), SHOOT-MERISTEMLESS (STM), FILAMENTOUS FLOWER (FIL, also known as AFO), REVOLUTA (REV), ARABIDOPSIS THALIANA MERISTEM L1 LAYER (ATML1) and CLAVATA 3 (CLV3). Furthermore, we demonstrate a functional requirement for WUS activity during de novo shoot meristem initiation. We propose that de novo shoot meristem induction is an easily accessible system for the study of patterning and self-organization in the well-studied model organism Arabidopsis.
    Preview · Article · Nov 2007 · Development
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    ABSTRACT: Most multicellular organisms have a capacity to regenerate tissue after wounding. Few, however, have the ability to regenerate an entire new body from adult tissue. Induction of new shoot meristems from cultured root explants is a widely used, but poorly understood, process in which apical plant tissues are regenerated from adult somatic tissue through the de novo formation of shoot meristems. We characterize early patterning during de novo development of the Arabidopsis shoot meristem using fluorescent reporters of known gene and protein activities required for shoot meristem development and maintenance. We find that a small number of progenitor cells initiate development of new shoot meristems through stereotypical stages of reporter expression and activity of CUP-SHAPED COTYLEDON 2 (CUC2), WUSCHEL (WUS), PIN-FORMED 1 (PIN1), SHOOT-MERISTEMLESS (STM), FILAMENTOUS FLOWER (FIL, also known as AFO), REVOLUTA (REV), ARABIDOPSIS THALIANA MERISTEM L1 LAYER (ATML1) and CLAVATA 3 (CLV3). Furthermore, we demonstrate a functional requirement for WUS activity during de novo shoot meristem initiation. We propose that de novo shoot meristem induction is an easily accessible system for the study of patterning and self-organization in the well-studied model organism Arabidopsis.
    No preview · Article · Oct 2007
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    ABSTRACT: Plants produce leaf and flower primordia from a specialized tissue called the shoot apical meristem (SAM). Genetic studies have identified a large number of genes that affect various aspects of primordium development including positioning, growth, and differentiation. So far, however, a detailed understanding of the spatio-temporal sequence of events leading to primordium development has not been established. We use confocal imaging of green fluorescent protein (GFP) reporter genes in living plants to monitor the expression patterns of multiple proteins and genes involved in flower primordial developmental processes. By monitoring the expression and polarity of PINFORMED1 (PIN1), the auxin efflux facilitator, and the expression of the auxin-responsive reporter DR5, we reveal stereotypical PIN1 polarity changes which, together with auxin induction experiments, suggest that cycles of auxin build-up and depletion accompany, and may direct, different stages of primordium development. Imaging of multiple GFP-protein fusions shows that these dynamics also correlate with the specification of primordial boundary domains, organ polarity axes, and the sites of floral meristem initiation. These results provide new insight into auxin transport dynamics during primordial positioning and suggest a role for auxin transport in influencing primordial cell type.
    Full-text · Article · Dec 2005 · Current Biology
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    ABSTRACT: The Arabidopsis homeotic gene AGAMOUS (AG) is necessary for the specification of reproductive organs (stamens and carpels) during the early steps of flower development. AG encodes a transcription factor of the MADS-box family that is expressed in stamen and carpel primordia. At later stages of development, AG is expressed in distinct regions of the reproductive organs. This suggests that AG might function during the maturation of stamens and carpels, as well as in their early development. However, the developmental processes that AG might control during organogenesis and the genes that are regulated by this factor are largely unknown. Here we show that microsporogenesis, the process leading to pollen formation, is induced by AG through activation of the SPOROCYTELESS gene (SPL, also known as NOZZLE,NZZ), a regulator of sporogenesis. Furthermore, we demonstrate that SPL can induce microsporogenesis in the absence of AG function, suggesting that AG controls a specific process during organogenesis by activating another regulator that performs a subset of its functions.
    Full-text · Article · Aug 2004 · Nature

Publication Stats

1k Citations
219.36 Total Impact Points

Institutions

  • 2011-2015
    • French National Centre for Scientific Research
      • Laboratoire de Biométrie et Biologie Évolutive (LBBE)
      Lutetia Parisorum, Île-de-France, France
  • 2010-2014
    • University of Lyon
      Lyons, Rhône-Alpes, France
  • 2009-2014
    • Ecole normale supérieure de Lyon
      Lyons, Rhône-Alpes, France
  • 2004-2005
    • California Institute of Technology
      • Division of Biology
      Pasadena, California, United States