Research

Our lab has currently two main research focus, with specific case-studies for each
 

  • nuclear organization dynamics during cellular reprogramming: somatic-to-reproductive fate transition; light-induced physiological transition

The nucleus is more than a genetic container. This organelle is the chief orchestra of cellular processes by controlling and fine-tuning gene expression in response to developmental and environmental cues. We are interested in the functional organization of the plant nucleus and its dynamics, particularly in relation to cell fate changes during developmental or physiological transitions. We use a combination of genetic, molecular, cell biology and cytogenetics approaches to describe nuclear organization dynamics at a microscopic and sub-microscopic scale, with the aim to decipher the functional role of these events on cellular reprogramming. We established protocols for high-resolution, 3D quantitative analyses of chromatin composition and organization at the single-cell level in whole-mount plant tissues.
 

  • contribution of organ growth to germline fate differentiation in the plant ovule

Organ shape is contributed by coordinated tissue growth and cellular differentiation involving an interplay between mechanical constraints and gene regulation (Whitewoods and Coen 2017, Johnson and Lenhard 2011). We are interested in understanding the contribution of growth constraints in the plant ovule to the differentiation of the spore mother cells at the somatic-to-reproductive cell fate transition. We deploy high-resolution imaging and 3D image processing approaches to quantify tissue growth in 3D at the cellular level.

 

Check our ongoing projects and innovative protocols, tutorials and animations.

 

Our research is supported by:

Tthe University of Zurich, the Swiss National Science Foundation (SNSF), the SystemsX.ch program, the Zurich-Basel Plant Science Center/Syngenta PhD program, the Swiss Commission for Technology and Innovation (CTI/KTI), the Baugarten Stiftung, the Velux Stiftung and the Ricola Stiftung.

 

NEW!

indepth


 

book cover

 

 

  • Book: Plant Chromatin Dynamics. Methods, Protocols and Technical review. Edited by Marian Bemer and Célia Baroux, Springer NY


 

 

 

 

  • Protocols: 3D cell-based segmentation for tissue morphodynamics studies, spatial analysis of gene position following 3D FISH, nanoscale chromatin distribution analyses from T.E.M. images [Link].


 

Ongoing projects


 

  1. Mechanisms and role of linker histones dynamics in plant reproduction [Link]
  2. Identification of nuclear architects influencing in vitro plant regeneration [Link]
  3. Functional organization of the plant nucleus: spatial gene positioning relative to gene expression [Link]
  4. Nanoscale organization of the plant chromatin [Link]
  5. Light-induced chromatin dynamics [Link]
  6. Spatial, nuclear organization of TE families in the monocot Brachypodium [Link]
  7. Cellular morphodynamics shaping Arabidopsis ovule primordia and underlying germline fate acquisition: the IMAGO project [Link]

 

Open projects

 

  8.  Endosperm chromatin dynamics in intra- and inter-specific hybridization [Link]

  9.  Functional characterization of atypical H1-related variants Link [Link]

 

Protocols and video tutorials


 

Protocols Panel

 

  • Efficient and Rapid Isolation of early-stage Embryos from Arabidopsis thaliana Seeds (JOVE)
  • An efficient method for quantitative, single-cell analysis of Chromatin Modification and Nuclear Architecture in Whole-Mount Ovules in Arabidopsis (video tutorial)
  • Video Tutorial: Quantification of Chromatin Modifications in whole-mount plant tissue (Link)
  • High-resolution, live-reporter imaging of reproductive tissues using lightsheet microscopy (contact us for more information)
  • Analysis of 3D Cellular Organization of Fixed Plant Tissues Using a User-guided Platform for Image Segmentation (Link)
  • Transmission Electron Microscopy Imaging to Analyze Chromatin Density Distribution at the Nanoscale Level (Link)
  • Automated 3D Gene Position Analysis Using a Customized Imaris Plugin: XTFISHInsideNucleus (Link)
  • Technical Review: Microscopy and Image Processing Tools to Analyze Plant Chromatin: Practical Considerations (Link)
  • Cell-Type Specific Chromatin Analysis in Whole-Mount Plant Tissues by Immunostaining (Link)

 

Animations

 

  • Arabidopis embryo development (morph animation)
  • Arabidopsis embryo sac (animated 3D reconstruction, powered by Imaris, Bitplane AG)
  • 3D segmentation and cell identity labeling for morphodynamic studies of Arabidopsis ovule primordia.
    Animation powered by Imaris, Bitplane AG (animation)
  • Lightsheet microscopy imaging and Multiview angle reconstruction of an Arabidopsis carpel stained with the mPS-PI method (Truernit et al, Plant Cell 2008). Animation powered by Imaris (animation)
  • A 3D travel through the plant cells - animation for the Scientifica public exhibition 2015 (animation)

 

 

PROJECT DETAILS

1. Mechanisms and Role of linker histones dynamics in plant reproduction

Jasmin Schubert, Kinga Rutowicz, Célia Baroux
Collaboration: Maciej Kotliński, Prof. Andrzej Jermanowski (IBB, University of Warsaw, Poland)
Funding: SNF project 31003A_149974 (2014-2017)

The differentiation of spore mother cells (SMCs) marks the somatic-to-reproductive fate transition in plants. Meiosis entails the formation of the haploid multicellular gametophytes, from which the gametes are derived, and during which epigenetic reprogramming takes place. We have shown that in the Arabidopsis both male and female SMC differentiation are accompanied by large-scale chromatin reprogramming characterized by chromatin decondensation, reduction in heterochromatin, depletion of linker histones, changes in core histone variants and in histone modification landscapes (She et al, Development 2013; Front Pl Sci 2015). Genetic analysis indicated that these events contribute to establishing postmeiotic competence, ie permissive o the development of the pluripotent gametophyte (She et al., 2013). In this study, we found that the eviction of linker histones H1.1 and H1.2 is a precocious event contributed a proteasome-mediated degradation (She et al., 2013). We are now focusing on elucidating the mechanisms of H1 dynamics by engineering conditional degradation-resistant versions and manipulating candidate chaperones. These genetic perturbation tools, together with developmental and molecular profiling analyses will allow elucidating the immediate or long-term function of H1 dynamics.

Linker histones’ dynamics during female and male sporogenesis in Arabidopsis thaliana.
Linker histones’ dynamics during female and male sporogenesis in Arabidopsis thaliana.

H1.1-GFP, H1.2-GFP (green), FM4-64 (left panel, red) or, chloroplasts (red, right panel).
From She et al. Development 2013, She and Baroux. Frontiers in Plant Sciences 2015.

 

 

2. Identification of nuclear architects influencing in vitro plant regeneration

Kinga Rutowicz, Célia Baroux
Collaborations: Rene Holtackers, Prof. Lucas Pelkmans (IMS, University of Zurich)
Funding: Sciex project 13.229 (2014-2015), Plant Science Fellowship and Forschungskredit to KR

Following the observation that H1’s are rapidly lost at the onset of cell fate transition in MMC differentiation (see 1), we asked whether H1 eviction underlies other cellular reprogramming events. Cellular transdifferentiation upon protoplast release and culturing represents a suitable, well characterized system to follow and manipulate chromatin reprogramming. Arabidopsis protoplast cells derived from leaf tissue were shown to undergo drastic nuclear reorganization during trans-differentiation, including reduction in heterochromatin, dispersion of centromeric repeats, redistribution of histone modifications (Tessadori et al, J Cell Sci. 2007;120:1200-8). Consistently, we observed rapid a loss/reduction of H1-GFP signals in leaf protoplasts during the de-differentiation phase. We are undertaking a genetic screen, using conditional amiRNA-based silencing against candidate genes in a dual reporter background line to identify factors controlling H1 eviction and heterochromatin decondensation. Together with the group of Prof. Lukas Pelkmans, we are optimizing a semi-automated, microscopy-based phenotyping and nuclei classification workflow.

Figure 3

 

 

3. Functional organization of the plant nucleus: spatial gene positioning relative to gene expression

Mariamawit Ashenafi, Johan Jaenisch, Célia Baroux
Project partners: Dr. Peter Majer (Bitplane AG), Prof. Ueli Grossniklaus (IPMB, University of Zurich), Prof. Reinhard Furrer (Institute for Mathematics, University of Zurich)
Funding: SNF- SystemsX.ch  IPhD project “Functional Organization of the Plant Nucleus“  (2014-2017)

The nucleus is more than a genome-packaging organelle. Decades of studies, essentially performed in yeast and animal cells, revealed several key organizing principles of the interphase nucleus that influence nuclear functions. Notably, gene positioning relative to the nuclear periphery in yeast or to chromosome territories in metazoans influences transcription (Lanctot, Cheutin et al. 2007). In plants by contrast, little is known although a few recent studies indicate that gene re-positioning, in response to developmental or environmental cues, correlates with their activity (Costa and Shaw 2006; Feng, Qiu et al. 2014). Furthermore in mammals, distinct spatial positioning of parental alleles relative to the nuclear periphery contributes to differential expression of imprinted loci (Gribnau, Hochedlinger et al. 2003). The project aims at a benchmark study of the functional organization of the plant nucleus. We are investigating the spatial organization of the transcriptional compartment in leaf nuclei using Fluorescent In situ Hybridization (FISH) and immunolabeling of active isoforms of PolII, in intact, 3D nuclei. For the purpose of the study, customized pluggins written in Python were developed to batch-process the images in Imaris (Bitplane), and a specific workflow for statistical modeling of signal distribution positions is being developed

Figure4

 

 

4. Nanoscale organization of the plant chromatin

Célia Baroux
Collaborations: Dr Lusik Cherkezeyan (Image processing, Backman's Biophotonics Laboratory at Northwestern University, USA), Tohnyui Fabrice, Christophe Ringli and Andreas Kaech (TEM images, IPMB and ZMB, University of Zurich), Jana Doehner (GSD imaging, ZMB, University of Zurich)

Chromatin organization in plant cells is classically described at four main levels: biochemical (histone variant composition), molecular (epigenome), microscopic (distribution of histone modification) and probabilistic (mapping of possible spatial interactions domains). The ultrastrucutural, nanoscale level of characterization remains, however, elusive. Yet, recent development in image processing offers the possibility to interpret chromatin density distribution from TEM images and inform on the nanoscale degree of organization. Notably, the periodic arrangement of nucleosomal arrays can be interpreted from the length scales derived from spatial autocorrelation analyses (Cherkeseyan et al, 2014, “Nanoscale changes in chromatin organization represent the initial steps of tumorigenesis: a transmission electron microscopy study”, Biomed Central Cancer, 14(189)). In addition, the advent of super-resolution microscopy enables to further investigate the nucleosomal-level degree of chromatin compaction and arrangement. We are establishing the expertise and streamlining these imaging and computational processing to decipher the role of candidate chromatin proteins and cellular differentiation in fine-scale chromatin organization.

Figure5

 

 

5. Light-induced chromatin dynamics

Open position in Baroux and Barneche lab! Contact us!
Collaborative project together with Dr Fredy Barneche (IBENS, Paris, France) [Link]
Funding: Velux Stiftung, Ricola Stiftung

Seedlings exposed to light for the first time, following germination, undergo a rapid reprogramming of gene expression. The lab of Dr Barneche showed that this event is contemporary to massive chromatin reorganisation (Bourbousse et al. 2015) and found a candidate chromatin remodeler central to this process. In this collaborative project, we will elucidate the molecular and structural variations of the chromatin rapidly induced by light perception, aiming for unprecedented levels of details using state-of-art molecular profiling and super-resolution microscopy imaging.

Bourbousse C, Mestiri I, Zabulon G, Bourge M, Formiggini F, Koini MA, Brown SC, Fransz P, Bowler C, Barneche F. (2015) Light signaling controls nuclear architecture reorganization during seedling establishment. PNAS 112(21):E2836-44

 

 

6. Spatial, nuclear organization of TE families in the monocot Brachypodium


Michele Wyler, Célia Baroux and Anne Roulin (IPMB, University of Zurich)
Plant Science Center-Sygenta joint doctoral funding

In collaboration with Dr Anne Roulin we will analyse the spatial distribution and local chromatin organization of TE families associated with selection in a scheme of local adaptation in Brachypodium. See also: Link.

 

 

7. Cellular morphodynamics during germline fate acquisition in plant ovules

Ethel Mendocilla-Sato, Nuno Pires, Daphné Autran, Célia Baroux
Collaborations: Dr Daphné Autran (IRD Montpellier, France), Bitplane (R&D Zurich, CH), Center for Microscopy Imaging, University of Zurich
Funding: CTI project 16997.1 PFLS-LS (2014-2017), SNF/ANR Bilateral project 2016-2019
IMAGO consortium: D. Autran, D. Grimanelli (IRD Montpellier, France); C. Godin (INRIA, France); O. Hamant, A. Bouaoud (ENS Lyon). Link

In plants, the germline is established late in development -by contrast to animals- during a critical lifestyle transition where plants switch from a vegetative to a reproductive effort. The differentiation of spore mother cells (SMC) is stereotypical in the Arabidopsis ovule: the female SMC differentiates in the upmost, central and subepidermic position of the digit-shaped ovule primordium. SMC differentiation and unicity is controlled by epigenetic and cell signaling mechanisms (Olmedo-Montfil et al Nature 2010; Lieber et al Curr Biol 2011). SMC differentiation is also concomitant to ovule primordium growth. To better understand the ontogeny of the ovule primordia and how growth constraints influences SMC identiy, we aim to establish an atlas of cell lineages and morphodynamic events (cell division index, growth volume and directionality, cell neigborhod) in normally developing versus (genetically or environmentally) perturbed ovules. We use high-resolution imaging of cellular boundaries and cellular markers in whole-mount ovules, using confocal laser scanning and lightsheet microscopy followed by membrane-based image segmentation.

Figure6

 

 

OPEN PROJECTS

The following project is open to candidates contributing  their own fellowship.

8. Endosperm chromatin dynamics in intra and inter-specific hybridization

Célia Baroux, Stefan Wyder, Ueli Grossniklaus, Jordi Romero, Claudia Köhler

Reproductive success relies on the endosperm, an extra-embryonic nurse tissue produced at fertilization. The dosage of paternally vs maternally-derived products defines a fine balance critically influencing its development, where alteration can result in seed abortion and hybridization failure (reviewed in Lafon-Placette and Köhler, Mol Ecol 25, 2016). We discovered a specific heterochromatin fraction (ESI) which responds to ploidy alterations in a parent-of-origin-dependent manner (Baroux et al., 2007) as well as to interspecific hybridization (Baroux, unpublished). One aim is to elucidate the mechanisms and targets of maternal ESI heterochromatin in relation to dosage regulation of parental genomes’ expression in the endosperm. Another aim is to determine genetic loci associated with ESI and possibly responding to parental dosage. We indeed identified a handful of genes enriched in a histone mark enriched at ESI foci and with a maternally-compensated gene expression (Romero, Wyder, Köhler, Baroux, unpublished). Whether those also relate to parental chromosome associations in the triploid endosperm (Baroux et al 2016) remains to be determined.

Figure7

 

 

 

9. Functional characterization of atypical H1-related variants

Kinga Rutowicz, Marek Whitehead, Célia Baroux
Collaborations: Lukasz Knizewski, Krzysztof Ginalski, Prof. Andrzej Jermanowski (IBB, University of Warsaw, Poland); Anja Schmidt, Prof. Ueli Grossniklaus (IPMB, University of Zurich)
Funding: SNF project 31003A_149974 (2014-2017)

The reproductive lineage is largely devoid of the canonical H1s (with the exception of the transient, meiotic chromosomes). This raises the possibility that plants possess germline-specific H1 variants that may enable a relaxed chromatin structure favorable to reprogramming in the germline. By analyzing protein variants sharing homology with the globular domain of H1’s (GH1), we identified three atypical variants (AH1L.1-.3, Knizewski, Ginalski Jermanowski, unpublished) that show transcript enrichment in the SMCs, gametes and embryo according to published cell-specific transcriptome profiles (Schmid et al, PLoS Genetics 2011). We are characterizing their spatial and temporal expression profile using reporter lines, molecular and developmental function using a combination of genetic, cell and molecular approaches.

Figure2

 

 

Videos and Animations

 

  • An Efficient Method for Quantitative, Single-Cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-Mount Ovules in Arabidopsis (video tutorial)



 

 

  • Movie: Arabidopis embryo development (morph animation)


 

  • Movie: Arabidopsis embryo sac (animated 3D reconstruction, powered by Imaris)

     


 

  • Movie: 3D segmentation and cell identity labeling for morphodynamic studies of Arabidopsis ovule primordia.
    Animation powered by Imaris, Bitplane AG


 

  • Movie: Lightsheet microscopy imaging and Multiview angle reconstruction of an Arabidopsis carpel stained with the mPS-PI method (Truernit et al, Plant Cell 2008)
    Animation powered by Imaris, Bitplane AG


 

  • Movie: A 3D travel through the plant cells - animation for the Scientifica public exhibition 2015