We are testing the mechanical trigger of the activation of the developmental pathway activating embryonic mesoderm invagination.
In Drosophila embryos, mesoderm invagination is generated by mesoderm cells apical constrictions ensured by Fog dependent apical coalescence of Myo-II. This movement is preceded by unstable puslations of apex cells, associated to unstavle apical coalescence of Myo-II. We have suggested that the mechanical strain associated to unstable apilca pulsations activate the Fog pathway by mechano-transduction, based on experiments of apical stabilisation of Myo-II and mesoderm invagination rescue, by soft indent of the mesoderm of snail mutants defective in pulsations, apical stabilisation of Myo-II and mesoderm invagination. The MIME project consists in testing definitiuvely such hypothesis by magnetically mimicking the physiological pulsations in vivo in a snail mutant.
We already set up a method of magnetisation of embryonic tissue in vivo to use magnetic fields in order to mimic morphogenetic movements. Today, the goal is to strongly refine the method to allow single cells resolution to control cell shape changes inside a full tissue in vivo.
First we checked the dynamical response of Myo-II apical stabilisation in response to the indent, using Myo-GFP. Then we characterized by Fourrier analysis the amplitude and frequency of the pulsations, checked their inhibition in sna mutants, and demonstrated the possibility of rescuying it by micromagnet applications in sna mutants, in vivo. As a result, we found a rescue of both apical stabilisation of Myo-II and mesoderm invagination.
The same type of methods was used to mimic epiboly in epiboly defective embryos. This allowed to demonstrate the mechanical induction of beta-catenin nuclear translocation and of the notail target mesoderm gene expression by the epiboly morphogenetic, movement (Figure en annexe, Bouclet, Brunet et al., Nature Communications, 2013).
The work realized on drosophila embryos mesoderm invagination is conclusive. We are now testing the underlying mechanotransductive pathway under magnetic mimicking of endogenous morphogenetic movements.
The work realized on zebrafish, published in Nature Communcations in 2013, was coupled to a similar analysis of mesoderm differentiation in the drosophila embryos, allowing to demonstrate the conservation of mesoderm mechanical induction in two species having diverged around 600 millions years ago, at the time of emergence of mesoderm in the common ancestor. This allowed to propose the underlying process, i.e the Y667-beta-catenin phosphorylation mechancial activation in response to the very first morphogenetic movements of embryogenesis, at the possible evolutionary origin of mesoderm ermergence, one of the important opened question of Evo-devo today.
Brunet, T. et al. Evolutionary conservation of early mesoderm specification by mechanotransduction in Bilateria. Nature communications 4, 2821, doi:10.1038/ncomms3821 (2013).
D. Le Roy et al., Magnetic flux micro-sources for bio-applications, poster at Intermag2014, Dresden, Germany, May 2014
V. Zablotskii et al. Life on Magnets: Stem Cell Networking on Micro-Magnet Arrays. PLoS ONE 8(8) (2013) e70416
DEMPSEY et al., Micro-magnetic imprinting
N° Dépôt : FR1254667 (2012), US 61/650,398; Date de dépôt 22/05/2012
Embryogenesis involves biochemical patterning as well as mechanical morphogenetic movements, both controlled by the expression of the master genes of development. The reciprocal interplay of morphogenetic movements with developmental genes expression and biochemical patterning is becoming an increasingly intense subject of investigation. The study of the control of morphogenetic movements by biochemical patterning, initiated two decades ago, has seen important progress. More recently, experimental evidences revealed that mechanical cues generated by morphogenesis control, in turn, the gene expression and biochemical patterning that drive embryonic development, through the activation of mechano-transduction pathways. In one specific case, magnetic tools acting at the multi-cellular scale permitted a full decoupling of genetic inputs from mechanical inputs in embryogenesis, which allowed the thorough demonstration of mechano-transduction processes involved in development. This demonstrated mechanical induction of Twist expression driving future gut cell functional differentiation in the early Drosophila embryo. However, the latter method, developed by the coordinator of the present project, was highly specific to the unique geometry and developmental stage of interest characterizing the early Drosophila embryo just before gastrulation.
Beyond this primary work, the objective of the present project is thus to generically study mechanical effects on mechano-transduction in any tissue and at any stage of the early embryonic development of potentially any species. A generic biocompatible magnetic technology will be developed allowing local (from individual cells to sub-mm assemblies of cells) and time-resolved control of mechanical deformations of embryonic tissues. Magnetic field and field-gradient sources will be developed by patterning hard magnetic films, forming a network of structures of typical thickness ranging from 5 µm to 100 µm, from the sub-mm to the micron-scale. The local magnetic force applied on cells will result from the interaction of such magnetic field sources with magnetic nanoparticles embedded in embryo cells. The effects of the mechanical forces will be analysed in-vivo, based on the observation of the mechano-transduction response of the GFP tagged protein involved, from the sub-cellular to the cell-assemblies scale, using spinning disk fluorescence microscopy.
Initially, one will concentrate on mesoderm invagination, a key morphogenetic movement of Drosophila embryo gastrulation. This movement is driven by an apical accumulation of Myosin-II. It was suggested that this accumulation was induced by a mechano-transduction pathway activated in response to initial active pulsations of mesoderm cell apex size variations depending on the expression of the gene snail. Here we will test such mechanical induction process by using magnetic forces to quantitatively mimick the physiological deformations with single cell length resolution, in the mutant of snail lacking endogenous pulsations. We will primarily check for GFP tagged Myo-II apical accumulations and mesoderm invagination rescue, both lacking in the mutant. The analysis of mechanical induction processes in embryonic development will be generalised by studying the biochemical response to deformation of zebrafish embryo tissue to check the role of mechano-transduction in embryogenesis more generally. It is hoped that this type of study will contribute to reconciling the two main approaches of developmental biology, namely the physico-centered and gene-centered approaches.
The project gathers the group of physico-biologists which has initiated the study of mechano-transduction processes in embryo development and a group of physicists, specialised in hard magnetic films generating micrometric magnetic source. This association of two groups, each one having a unique key expertise, should provide the best chance of success to this in vivo mechano-biology project.
Monsieur Emmanuel FARGE (INSTITUT CURIE) – email@example.com
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
NEEL CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES
Institut Curie INSTITUT CURIE
Help of the ANR 438,838 euros
Beginning and duration of the scientific project: June 2011 - 36 Months