Elucidating the mechanical role of internal tissues in morphogenesis – MechInMorph

The overarching aim of this proposal is to elucidate the mechanical role of internal tissues in morphogenesis. Over the past 20 years, numerous studies have demonstrated that morphogenetic gradients control tissue growth; in this framework, morphogens and/or signalling molecules control the developmental patterns of organs and tissues by coordinating the expression of cell identity genes. It has long been overlooked that cells are also physical objects that experience internal and external forces and thus obey the laws of physics. Recent work has established that cell shape changes depend on the generation of intrinsic forces such as through actomyosin activity in animals or osmotic pressure in plants. However, we are far from understanding how extrinsic physical interactions, and the resultant cell shape changes, bring about tissue morphogenesis. In particular, most studies have focussed on epidermal shape changes, and it is unknown how internal tissues contribute to morphogenesis.
To go beyond these statements, it is crucial to comprehensively measure the physical properties of cells and tissues, and to unravel the molecular mechanisms allowing shape changes in response to extrinsic physical forces. To do so, we will combine molecular tools, morphometry, and physical measurements, and we will integrate these data in computational models, which we will test using mutants, pharmacological agents and physical manipulations of cells, so as to obtain an integrated view of morphogenesis.
Our work will be validated in both plants and animals, on the ovarian follicle of Drosophila melanogaster and on flower primordia in Arabidopsis thaliana. In both systems, the morphogenesis of the outermost cell layer (the follicular cells and the epidermal cell layer L1, respectively) likely occurs under the tension generated by an internal growing tissue (germline cells and subepidermal L2 and L3 layers, respectively) and is limited by the extracellular matrix (basal membrane and cell wall, respectively). Thus, although these models are evolutionarily distant, they raise similar questions. Crucially, both systems permit similar physical measurements and mechanical manipulations to be carried out. Furthermore, as a great number of molecular and genetic tools are available in both models, experiments can be set up in wildtype and in mutant conditions, allowing us to identify correlations between mechanics, cell identity and cell shape.
Conceptually, this project is divided into four parts:
First, we will obtain a global view of morphogenesis in these systems by combining cell morphometry and measuring the relative stiffness of the different cell types or layers using micromechanical compressions.
Second, we will use an atomic force microscope to measure the turgor (osmotic) pressure of the internal cells using the osmolarity of external media, and the stiffnesses of the extracellular matrix in wildtype.
Third, we will test the accuracy of the WT measurements by using mutant conditions and we will analyse the morphogenetic consequences of manipulating the physical properties of internal or of external cells.
Fourth, all physical and biological data will be integrated into mechanical growth models, which we will test using further manipulations of cell behaviour.
In summary, the final result we will generate is the development of in silico mechanical models that reproduce the morphogenetic processes observed in living organisms. Furthermore, as we will develop innovative protocols and methods to measure internal physical properties, one of the outcomes of this project will be their transmission to the scientific community, especially the developmental biologists. Finally, since the two systems (animals and plants) are evolutionarily distant, this project will help us identify the unifying mechanical concepts of morphogenesis.