Nicolas PLACHTA 
                       
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  Nicolas PLACHTA  
  Lab Location: #5-01B

email:
plachtan[at]imcb.a-star.edu.sg
tel: 65869515
 
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  Key Publications  
 


Zenker J, White M, Templin R, Parton R, Thorn-Seshold, Bissiere S, Plachta N.
A microtubule organizing center directing intracellular transport in the early mouse embryo.
Science
(2017)

Zhao Z, White M, Alvarez Y, Zenker J, Bissiere S, Plachta N.
Quantifying transcription factor–DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy.
Nature Protocols (2017)

White M, Angiolini J, Alvarez Y, Kaur G, Zhao Z, Moksos E, Bruno L, Bissiere S, Levi, Plachta N. Long-lived binding of Sox2 to DNA predicts cell fate in the four-cell mouse embryo.
Cell  (2016) 
[See Preview by Wu & Ispizua-Belmonte] [Cover image]

Samarage R, White M, Alvarez Y, Fierro-Gonzalez J, Jesudason E, Hanon Y, Fouras A, Plachta N.
Cortical tension positions the first inner cells of the mammalian embryo.
Developmental Cell (2015)
[See Dev Cell Preview by Goldstein & Kiehart, 2015]

Bouveret R et al.
NKX2-5 mutations causative for congenital heart disease retain functionality and are directed to hundreds of targets.
eLife (2015)

Angiolini J, Plachta N, Mocskos E, Levi V.
Exploring the Dynamics of Cell Processes through Simulations of Fluorescence Microscopy Experiments.
Biophysical Journal (2015)

Fierro-Gonzalez J, White M, Silva J, Plachta N.
Cadherin-dependent filopodia control preimplantation embryo compaction.
Nature Cell Biology (2013)
[2 reviews by Faculty 1000]

Kaur G, Nefzger C, Costa M, Silva J, Fierro-Gonzalez J, Polo J, Bell T, Plachta N.
Probing transcription factor diffusion in the developing mammalian embryo with photoactivatable fluorescence correlation spectroscopy.
Nature Communications (2013)

Plachta N, Bollenbach T, Pease S, Fraser SE, Pantazis P.
Oct4 kinetics predict cell lineage patterning in the early mammalian embryo.
Nature Cell Biology (2011)
[Journal Cover. See NCB News & Views by Zernicka-Goetz, 2011. 3 reviews by Faculty 1000.]

Bissiere S, Plachta N, McAllister K, Hoyer D, Olpe HR, Grace A, Cryan JF.
The anterior cingulate cortex modulates the efficiency of amygdala dependent fear learning.
Biological Psychiatry (2008) (Journal Cover)

Nikoletopoulou V, Plachta N, Allen ND, Haubst N, Götz M, Barde Y-A.
Neurotrophin receptor-mediated death of misspecified neurons generated from embryonic stem cells lacking Pax6.
Cell Stem Cell (2007)

Plachta N, Annaheim C, Bissiere S, Hoving S, Voshol V, Bibel M, Barde Y-A.
Identification of a lectin causing the degeneration of neuronal processes using engineered embryonic stem cells.
Nature Neuroscience (2007)

Plachta N, Bibel M, Tucker KL, Barde Y-A.
Developmental potential of defined neural progenitors derived from mouse embryonic stem cells.
Development (2004)

Plachta N, Traister A, Weil M.
Nitric oxide is involved in establishing the balance between cell cycle progression and cell death in the developing neural tube.
Experimental Cell Research (2003)

Traister A, Abashidze S, Gold V, Michael E, Plachta N, Patel K, Fainsod A, Weil M.
BMP controls nitric oxide-mediated regulation of cell numbers in the neural tube.
Cell Death and Differentiation (2004)

Traister A, Abashidze S, Gold V, Plachta N, Karchovsky E, Patel K, Weil M.
Evidence that nitric oxide regulates cell cycle progression in the developing chick neuroepithelium.
Developmental Dynamics (2002)

REVIEWS

Plachta N.
New embryo users.
Cell  (2017)

White M, Zenker J, Bissiere S, Plachta N.
How cells chose their shape and position in the early mouse embryo.
Current Opinion in Cell Biology  (2016)

Zhao Z, White MD, Bissiere S, Levi V, Plachta N.
Quantitative imaging of mammalian nuclear dynamics: From single cells to whole embryos.
BMC Biology  (2016)

White M, Bissiere S, Plachta N.
Mouse embryo compaction.
Current Topics Developmental Biology  (2016)

White M, Plachta N.
How adhesion forms the early mammalian embryo.
Current Topics Developmental Biology (Review, 2005)

White M, Plachta N.
The first cell fate decision during mammalian development
Stem Cells, Tissue Engineering & Regenerative Medicine
Imperial College Press (Book Chapter, 2005)

 

 

 
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  Nicolas PLACHTA

Nicolas studied biology at the University of Buenos Aires (Argentina) and University of Tel-Aviv (Israel). He did his PhD with Yves-Alain Barde at the Biozentrum (University of Basel, Switzerland) and postdoc in biological imaging with Scott Fraser at Caltech (US), supported by fellowships from Swiss National Foundation, EMBO and CIRM.

He was appointed Group Leader at EMBL Australia in 2011 and joined A*STAR in 2015 as Senior PI. He received the A*STAR Investigatorship, Viertel Medical Fellowship, Australian NHMRC and ARC Fellowships. In 2015 he become the third Singapore-based scientist to join the EMBO Young Investigator Program. In 2016 he was awarded the Gibco–Emerging Leader Prize of the American Society for Cell Biology (ASCB). In 2017 he became an HHMI International Scholar.

 

     
  Seeing How Mammalian Life Starts:
Imaging how cells decide their fate, shape and position in the living mammalian embryo
 


During development, each cell in our body must resolve its fate, shape and position. Revealing how these decisions are made is critical to understand how embryos form, and what problems compromise fertility, yet their real time control in mammals is unknown. Because fixed specimens cannot capture in vivo cell dynamics, we apply advanced imaging technologies to study cells directly in live mouse embryos.

We recently provided the first biophysical explanation of how transcription factors (TFs) search and bind on-and-off to DNA to control cell fates in vivo. Combining fluorescence correlation spectroscopy with photo-activation (paFCS), we showed how the dynamics of Sox2 and Oct4 control pluripotency at the single-cell level in the embryo (White et al 2016 Cell; Kaur et al 2013 Nat. Comms.; Zhao et al 2017 Nat. Prot.).

Using live imaging, we also discovered that cells use a new class of long filopodia to form the first tissue-like structures of the embryo. These protrusions enable cells to draw their neighbours closer and achieve embryo compaction, providing a new picture of how mammalian cells form epithelial structures in vivo (Fierro-Gonzalez et al 2013, Nat. Cell Biol.).

We also established computer segmentation and laser ablation techniques to demonstrate that anisotropies in cortical tension (a force generated by contractility of the cell cortex) drive the formation of the pluripotent inner mass of the embryo. Our findings challenged models based on spatially orientated divisions and established a role for mechanical forces in preimplantation development (Samarage et al 2015, Dev. Cell).

Recently, we explored the dynamics of microtubules in vivo. The microtubules of most animal cells are organized by an organelle called the centrosome. Using live imaging in the early mouse embryo, which lacks centrosomes, we revealed that the cells are connected by a stable microtubule bridge. This bridge functions as a non-centrosomal  organizing center, directing the growth of microtubules within the cell. Moreover, the microtubules emanating from this bridge transport key proteins to the cell membrane, including E-cadherin, to control cell polarization during development (Zenker et al 2017, Science).

Our discoveries reveal how multiple mechanisms regulating cell fate, shape and position are integrated in vivo to direct the earliest stages of mammalian life. We now extend these studies to more complex morphogenetic processes and other cell types like neurons and stem cells.

We use early mouse embryos to reveal how mammalian cells choose their fate and interactions with other cells in real time and in vivo. These live embryos can be microinjected with RNA to label key regulatory proteins inside cells. We then follow how proteins and cells function in real time with time-lapse laser scanning microscopy.

The microtubule cytoskeleton of most animal cells is organized by an organelle called the centrosome. We recently used live imaging to study microtubules in the early mouse embryo, which lacks centrosomes. We found that that the cells of the embryo are connected by a stable microtubule bridge (shown in this image). This structure functions as a non-centrosomal  organizing center, directing the growth of microtubules within the cell. The microtubules emanating from the bridge transport key proteins to the cell membrane, including E-cadherin, to control cell polarization during early mammalian development. This study reveals a new form of cytoskeleton organization in vivo (Zenker et al, 2017, Science).

 

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Movie shows microtubules growing outwards from the bridge connecting cells in a live embryo

To understand how gene-regulatory molecules control cell fates in vivo, we developed new methods to study the DNA search mechanisms and DNA binding properties of transcription factors (TFs) in complex systems. (A) Using photo-activatable FCS (paFCS), we can uncage TFs fused to paGFP with multiphoton lasers deep in live embryo. (B) Controlling the number of fluorescent TFs by photo-activation makes FCS feasible in vivo. (C) Comparing normal and mutant TFs reveals the mechanisms controlling diffusion and DNA binding. In this example we studied the dynamics of the TF Oct4, a key regulator of pluripotency and early embryogenesis (see Kaur et al, 2013, Nature Communications).



Cover image (White et al, 2016, Cell)

YouTubeCell PaperFlick:





We recently discovered that heterogeneities in Sox2–DNA binding between the cells of the 4-cell mouse embryo predict cell fate (White et al, 2016, Cell). We performed the first quantifications of TF–DNA binding in single cells of a live embryo. Combining FCS and modelling, we can now measure how stably or transiently TFs bind to DNA in single cells in vivo, and how long TFs remain bound to DNA (residence times) as cells make physiological differentiation decisions. This work demonstrated how TFs repartition between their specific and non-specific DNA targets to control pluripotency during mammalian development.

Example of selective uncaging of the transcription factor Oct4 fused to the photoactivatable GFP within a defined volume in a living mouse embryo. The use of two-photon lasers allows targeting the photoactivation event within small subcellular volumes, and then quantitatively follow how the TF moving over time (see Plachta et al, 2011, Nature Cell Biology).

 

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We also use photoactivation and time-lapse imaging to follow selective pool of TFs moving inside stem cells during division.

Using time-lapse imaging, we discovered a new class of filopodia that draw cells closer to achieve compaction of the mouse embryo. Compaction is one of the first morphogenetic processes of embryogenesis. This discovery offers a new in vivo system to understand how cellular protrusions control morphogenesis in real time and to reveal rules for generating tissue-like structures (see Fierro-Gonzalez et al, 2013, Nature Cell Biology).

 

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Example of filopodia dynamics in a living mouse embryo.



Revealing how cells adopt defined positions is key to understand how our body structures form. We combine non-invasive imaging and computer segmentation to reveal how cells are first segregated to form the pluripotent inner mass in the living the mouse embryo. Unlike previous models based on highly orientated asymmetric divisions, we show the pluripotent mass forms primarily by apical constriction.  Using highly precise laser ablations, we show how anisotropies in cortical tension –a force generated by contractility of the actomyosin cell cortex– drives this key morphogenetic process.



We use computational methods to flatten live embryos and study the distribution of rproteins regulating mechanical forces in vivo. We then apply femtosecond laser ablation of acto-myosin networks to probe the directionalities and magnitudes of tensile forces acting in the living embryo. These experiments enable us to build maps showing the organization of key mechanical forces directing the earliest morphogenetic process during mammalian development.

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Imaging how the pluripotent inner mass forms in real time.

Optimization of ex-utero embryo culture allows us to also image intact developing mouse embryos as they undergo more complex morphogenetic processes, like gastrulation. We follow the formation of key body structures during development and the movement of large groups of cells with non-invasive multiphoton imaging.

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Two-photon non-invasive imaging of a live mouse embryo undergoing gastrulation outside the uterus. Over a ~12 hours period the head and trunk become visible. We use this approach to study cell dynamics of gastrulation and the formation of early embryonic structures like the head, trunk and nervous system.