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Emergence of tissue mechanical properties from molecular and cellular activity during morphogenesis

Final Report Summary - MORPHOMECHANICS (Emergence of tissue mechanical properties from molecular and cellular activity during morphogenesis.)

Building organs and organisms is a question of generating different cell types and also of organizing them in correct 3D architectures. The control of organ morphology and size is a fundamental problem that is ill understood and its study can impact biomedical fields such as regenerative biology, tissue engineering and cancer biology. Tissue morphogenesis requires that groups of cells are folded, stretched and compressed in a coordinated manner to generate precise organ shape. One of the most salient discoveries of the last decade is that the principles driving morphogenesis are not only genetic and biochemical but also mechanical [1]. How cells integrate biochemical and mechanical information at different scales to drive coherent morphogenesis remains largely unknown.

My scientific project aims at investigating how the stereotyped patterns of cell shape changes in a tissue emerge through the interplay between cytoskeletal activity, the dynamics of cell-cell adhesion and mechanics. For this, I use a multi-disciplinary approach to interrogate the process of Dorsal Closure of the Drosophila embryo. This is a reference morphogenetic process for the study of collective cell movements that is being a source of knowledge and inspiration for the understanding of other processes with biomedical interest such as wound healing, neural tube closure and palate fusion [2, 3]. Dorsal closure (DC) takes place during the final phases of Drosophila development and is the process by which an epidermal gap on the dorsal side of the embryo, bridged by an extra-embryonic epithelium called the amnioserosa (AS), is closed to generate epidermal continuity [2, 4]. During DC the lateral epidermis of the embryo converges towards the dorsal midline, while the AS reduces its global area until it disappears inside the embryo. The process relies on the coordinated cell shape changes of these two tissues: epidermal cells elongate in the dorso-ventral direction and AS cells undergo apical constriction. My work focus on the amnioserosa, one of the main force-generating tissues during DC, whose constituent cells undergo pulsated apical contraction driven by the transient activity of the actomyosin cytoskeleton [5-7]. The main goal of this project is to understand the molecular basis of oscillatory actomyosin activity, how it translates into effective cell shape changes and how this activity is coordinated across the whole tissue to give rise to the contractile activity and the mechanical properties of the tissue.

To tackle these questions, we use a combination of live imaging, quantitative image analysis, mechanical and genetic perturbation and modelling. To explore possible mechanisms of the emergence of cell area and cytoskeletal oscillations, we have developed a theoretical model where such oscillations arise through the coupling of myosin-driven active forces, actomyosin turnover and cell deformation, and are modulated by neighbour-coupling [8]. In wild type embryos, we have previoulsy found that the amplitude and frequency of cytoskeletal and cell oscillations evolve with time [5], and this temporal pattern can be reproduced by the model providing that fundamental parameters governing actin turnover and Myosin force change over the course of DC [8]. Interestingly, we have found that the evolution of Myosin and cell oscillations is not completely coupled. The frequency of Myosin and cell oscillations both increase as DC progresses but the amplitude of fluctuations show distinct features. While the amplitude of cell oscillations decrease over DC, the amplitude of Myosin oscillations remains constant. A viscoelastic framework accounting for these observations predicts that the mechanical properties of AS cells evolve during the process and cells become stiffer and more solid-like as tension increases [9]. These are phenomenological parameters that suggest changes in the dynamics of the cytoskeleton and cell-cell adhesion. Our work on the mechanical properties of amnioserosa cells measured by laser ablation, shows that these are determined by the level of Myosin phosphorylation and the architecture of the actin network [10]. Interestingly, tension propapgation is facilitated in a tissue where ECadherin is stabilised at the level of cell-cell junctions [10], and we have observed a satbilization of both ECad and α-catenin, a central component of adherens junctions linking the membrane to the actin cytoskeleton, as DC progresses (Jurado, de Navascués and Gorfinkiel, in preparation). We have also investigated the molecular basis of the oscillatory and contractile behaviour of AS cells. Our results reveal a key role for α-catenin in modulating the frequency of cytoskeletal and cell area oscillations. Furthermore, we uncovered the importance of adhesion dynamics in maintaining the integrity of the tissue as tension develops (Duque and Gorfinkiel, in preparation).

This project has also contributed to develop new approaches to the quantification of tissue deformation during in vivo morphogenesis. Although frameworks to quantitatively analyze cell shape changes are being increasingly applied to the understanding of morphogenesis, it is starting to be acknowledged that important properties and patterns emerge at the subcellular level and at the level of tissue domains whose size and shape are not straightforward to determine [11]. This notion reveals the limitations of cell-based quantitative analysis tools and calls for new methods to measure subcellular and supracellular deformations. In collaboration with the Biomedical Image Technologies group lead by Andrés Santos at the Universidad Complutense de Madrid, we are developing a new framework based on B-splines registration and continuum mechanics to quantify continuous tissue deformation and provide subcellular and supracellular descriptions of tissue dynamics. This framework maps the deformation between consecutive frames and extracts kinematics descriptors either showing the rate of change (eulerian kinematics) or the accumulated deformation (lagrangian kineamtics) at any spatial point of the tissue, giving rise to continuous measurements rather than cell-averaged quantities (Pastor-Escuredo, Butler, Jurado, Gorfinkiel, Ledesma and Santos, in preparation). This kind of approach is revealing unexpected patterns of organization both at the subcellular and supracellular level.

This project lies at the interphase between developmental biology and tissue mechanics, and thus required an interdisciplinary approach. We have undertaken such approach involving a combination of genetics, live imaging, quantitative image analysis and biophysical modelling, and this project has contributed to the increasing shift developmental biology is experiencing towards a more quantitative and predictive field of knowledge. In the context of this project, I have supervised the projects of three students from the Master in Biophysics of the Universidad Autónoma de Madrid. These students obtained their degree in Biology or in Physics and have been trained in my lab in genetics, confocal microscopy and quantitative analysis of cell behaviours. Two of them are now doing their PhD in my lab and the third one is doing his PhD at the University of St. Andrews (Scotland). Thus, this project has contributed to the development of a multi-disciplinary approach to understand biological systems. The results from this project have been presented at different international conferences, some of the work has been published in peer-reviewed journals, and the rest of the work will be sent for publication during this year.


References

1. Mammoto, T. and D.E. Ingber, Mechanical control of tissue and organ development. Development, 2010. 137(9): p. 1407-20.
2. Jacinto, A., S. Woolner, and P. Martin, Dynamic analysis of dorsal closure in Drosophila: from genetics to cell biology. Dev Cell, 2002. 3(1): p. 9-19.
3. Wood, W., et al., Wound healing recapitulates morphogenesis in Drosophila embryos. Nat Cell Biol, 2002. 4(11): p. 907-12.
4. Gorfinkiel, N., S. Schamberg, and G.B. Blanchard, Integrative approaches to morphogenesis: lessons from dorsal closure. Genesis, 2011. 49(7): p. 522-33.
5. Blanchard, G.B. et al., Cytoskeletal dynamics and supracellular organisation of cell shape fluctuations during dorsal closure. Development, 2010. 137(16): p. 2743-52.
6. David, D.J. A. Tishkina, and T.J. Harris, The PAR complex regulates pulsed actomyosin contractions during amnioserosa apical constriction in Drosophila. Development, 2010. 137(10): p. 1645-55.
7. Solon, J., et al., Pulsed forces timed by a ratchet-like mechanism drive directed tissue movement during dorsal closure. Cell, 2009. 137(7): p. 1331-42.
8. Machado, P.F. Blanchard, G.B. Duque, J., and Gorfinkiel, N. , Cytoskeletal turnover and Myosin contractility drive cell autonomous oscillations in a model of Drosophila Dorsal Closure. EUROPEAN PHYSICAL JOURNAL-SPECIAL TOPICS, 2014. 223(7): p. 11.
9. Machado, P.F. Duque, J., Etienne, J., Martinez Arias, A., Blanchard, G.B. and Gorfinkiel, N., Active rheology and emergent material properties of developing epithelial tissues. Submitted to PNAS.
10. Fischer, S.C. et al., Contractile and mechanical properties of epithelia with perturbed actomyosin dynamics. PLoS One, 2014. 9(4): p. e95695.
11. Blanchard, G.B. and R.J. Adams, Measuring the multi-scale integration of mechanical forces during morphogenesis. Curr Opin Genet Dev, 2011. 21(5): p. 653-63.