Mechanics and Genetics of Embryonic and Tumoral Development
Group leader : Emmanuel Farge
Read the scientific activity report. (pdf 438Ko, last update 26th, march 2010).
The thematic of the Mechanics and Genetics of Embryonic and Tumour Development team focuses on the role of mechanical strain and deformation of macroscopic biological structures at the cell or multi-cellular level, into the regulation and the generation of active biochemical processes at the microscopic molecular level, including gene expression, in vivo.
Our findings chronologically goes from the mechanical modulation of the endocytosis of signalling proteins as a mechanotransductive underlying molecular mechanism of cell trans-differentiation (early 2000’s), to its role in the involvement of mechanical cues in the trigger of early Drosophila embryos mesoderm invagination (late 2000’s). It additionally goes from our finding of the mechanosenstivity of the beta-catenin pathway as involved in the mechanical induction of early Drosophila embryos endoderm differentiation (from early to late 2000’s), most recently found as at the probable evolutionary origins of mesoderm emergence in the common ancestor of bilaterians, a process anomalously reactivated as a tumorigenic signal in compressed healthy epithelial tissues in response to tumour growth pressure in vivo (2010’s). From latest to earliest research:
Tumourigenesis: mechanical induction of tumourigenesis in compressed healthy cells, in response to the mechanical strains developed by tumorous growing tissues
We found β-catenin dependent mechanical induction of oncogenes expression and tumour initiation in both pre-tumorous and wild type mice colon healthy epithelia, in response to tumour growth pressure in vivo (M-E Fernandez-Sanchez, S. Barbier et al, Nature 2015, in press (http://www.nature.com/nature/journal/vaop/ncurrent/full/nature14329.html), Figure 1).
Figure 1. Mechanical induction of the β-catenin tumorigenic pathway in healthy epithelia in response to tumour growth pressure, in vivo. Magnetic loading of mesenchemial cells conjunctive of epithelial crypt colonic cells (in orange), submitted to a millimetric magnetic field gradient, generates a permanent 1kPa pressure quantitatively mimicking tumour growth pressure on weeks to months, in vivo. Right- Resulting mechanical activation of the phosphorylation of the Y654 site of β-catenin, leading to its release into the cytoplasm and nucleus, and leading to the expression of its tumorigene target gene c-Myc.
To do so, we mimicked the 1kPa tumour growth pressure in vivo by magnetically loading the mesenchemial conjunctive tissue with ultra-magnetic liposomes, which we submitted to a permanent magnetic field gradient due to a millimetric magnet sub-cutaneoulsy localized in front of the colon. Such mechanical strain activated the phosphorylation of both the Y654-beta-catenin leading to the release of a junctional pool into the cytoplasm. It additionally led to the phosphorylation of Ser9-GSK3b allowing the nuclear translocation of the cytoplasmic beta-catenin into the nucleus and the expression of its tumorigenic target genes. The same responses are observed in the non-tumorous crypts compressed by neighbouring Notch-hyperproliferative crypts of a mice model of tumour progression.
Evo-Devo: a mechano-transductive origin of mesoderm emergence in the common ancestor of bilaterian complex animals
We found that the mechanical activation of the beta-catenin pathway, anomalously activated in the process of tumour development, is an ancestral property, having been probably involved in the emergence of first differentiation patterns in ancient organism embryos, such as in the evolutionary emergence of the mesoderm in the last common ancestor of bilaterians. We effectively demonstrated the conservation of mechanical induction as involved in early mesoderm differentiation in both the zebrafish and Drosophila embryo, initiated by the mechanotransductive phosphorylation of the Y654 site of beta-catenin impairing its interaction with E-cadherins, leading to its release from the junctions to the cytoplasm and nuclei, and subsequently to the brackury and twist earliest mesoderm target genes expression, respectively (Figure 2).
Figure 2. Conserved mechanical induction of earliest embryonic mesodermal genes as a possible evolutionary origin of mesoderm emergence in the last common ancestor of Bilaterians. Left- Mechanical induction of earliest mesoderm genes expression brackury (in zebrafish) and gene product Twist (in Drosophila) commonly triggered by the mechanical activation of the phosphorylation of the Y667 conserved site of -catenin (Y654 in mammalians) leading to its release from the junctions to the nucleus, in response to the first morphogenetic movement of gastrulation, in both species. Right- Mechanotransductive evolutionary emergence of the mesoderm proposal, in response to the first morphogenetic movement of embryogenesis in the last common ancestor of the vertebrate zebrafish and the arthropod Drosophila, i.e in the 570 millions years old last common ancestor of bilaterians.
The evolutionary origin of mesoderm emergence remains a major persisting opened question of Evo-Devo. Our results allow to suggest mechanostransductive Y654 phosphorylation in response to first embryonic morphogenetic movements at the origin of mesoderm emergence in the 570 millions years ago last common ancestor of bilaterians (Bouclet, Brunet et al, Nature Comm. 2013).
Developmental Biology: mechano-genetic and mechano-proteic reciprocal coupling in the regulation of gastrulating embryos development
Embryonic development is a coordination of multi-cellular biochemical patterning and morphogenetic movements. Last decades revealed the close control of Myosin-II dependent biomechanical morphogenesis by patterning gene expression, with constant progress in the understanding of the underlying molecular mechanisms. We recently revealed reversed control of the Twist developmental differentiation patterning gene expression (Figure 3) and of Myosin-II active relocalisation (Figure 4) by the mechanical strains developed by morphogenetic movements at Drosophila gastrulation, through mechanotransduction processes involving the Armadillo/beta-catenin and the down-stream of Fog signalling pathways (due mechanical inhibition of Fog endocytosis in this case, see next paragraph), respectively.
Figure 3. Mechanical induction of Twist by convergence extension in the early anterior endoderm determination. A Ectopic mechanical induction of Twist-lacZ expression in response to uniaxial global deformation of about 10% of the Drosophila embryo dorso-ventral size. B Mechanical rescue of the Twist protein expression by an indent of the anterior endoderm lacking Twist expression associated to its defect of compression in a bcd, nos tsl mutant defective in convergent-extension. C Up- Magnetic loading with super-paramagnetic nano-particles to quantitatively rescue physiological compression, of wild-type photo-ablated embryos lacking endoderm cells compression. Down- Rescue of the strong expression of the Twist protein by the magnetically induced rescue of the anterior endoderm compression in the photo-ablated embryo lacking both compression and the strong expression of Twist. Such high level of Twist expression is vitally required for anterior mid-gut functional differentiation of the larvae (Desprat et al, Dev Cell, 2008).
Figure 4. Mechanical trigger of mesoderm invagination in sna defective mutants. a- Indent of a mutant of snail that does not invaginate (of 5 microns), 5 minutes after the end of cellularisation. b- Rescue of both the apical accumulation of Myo-II and mesoderm invagination wild-type phenotypes, lacking in the mutant of snail, after the indentation of the mutant of snail mesoderm.
We used experimental tools (genetic and biophysical control of morphogenetic movements, Figure 3), and theoretical tools (simulations integrating the accumulated knowledge in the genetics of early embryonic development and morphogenesis) (Figure 5), to uncouple genetic inputs from mechanical inputs in the regulation of Twist meso-endoderm gene expression and Myosin-II active relocalisation. Specifically, we set-up an innovative magnetic tweezers tool to measure and apply physiological strains and forces in vivo, allowing to mimic morphogenetic movements from the inside of the tissue in living embryos (Figure 3). Farge, Curr. Biol., 2003; Desprat et al, Dev Cell, 2008; Pouille et al Phys. Biol. 2008; Ahmadi, Pouille et al, Science Signalling, 2009).
Figure 5. Hydrodynamic simulation of embryonic gastrulation in response to the apical constriction of mesoderm cells. a- Before gastrulation (red arrows delimit the mesoderm domain). b- Gastrulation response to apical constriction into the mesoderm, regulated by membrane-cortical elasticity, and the hydrodynamic flow inside and outside the embryo.
Endocytosis: vesicle budding driving force; mechanical modulation of endocytosis as a mechanotransduction process triggering transdifferentiation
Historically, the first main thematic studied in the team was the motor role of biological membrane soft matter elasticity into the budding driving force of vesiculation initiating plasma membrane endocytosis (Rauch et al, Bioph. J, 2000), as well as the role of mechanical inhibition of morphogene endocytosis in mechanical induction of cell transdifferentiation (Rauch et al, Am. J. Cell Phys, 2002) (Figure 6).
Figure 6. Mechanotranductive cell trans-differentiation by mechanical inhibition of signalling proteins endocytosis due to tension induced membrane flattening. A Membrane tension flatten membranes, leading to the inhibition of endocytosis of secreted signalling proteins. In the case of an involvement of endocytosis in the inhibition of downstream signalling, mechanical blocking of endocytosis leads to an enhancement of signalling. B This is the case for mechanical inhibition of BMP2 (a,b) which leads to the enhancement of C2C12 myoblast-osteoblast transdifferentiation initiated by JunB expression (c,d).