Drosophila germ-band extension as a model for understanding the integration of cell intrinsic and extrinsic forces during animal morphogenesis

When fertilized eggs start their development they are faced with an enormous challenge. The egg must divide many times to produce huge numbers of cells. In turn, these cells must be directed to become differentiated from one another and simultaneously rearrange in stereotypical ways to shape the tissues and organs of the body. This programme of movements is called morphogenesis. One of the most important early transformations that takes place in all animal embryos is the elongation of the embryonic head-tail axis from a short, wide tissue to an elongated narrow body. If this process of convergence and extension should fail, it is highly detrimental or more usually lethal to embryos. In humans, neural tube defects such as spina bifida and anencephaly are examples of the consequences of abnormal convergence and extension.

Two types of information are vital in trying to understand how morphogenesis happens. Firstly, identifying which genes are turned on to orchestrate it and, secondly, working out how the relevant genes control the generation of forces that drive embryonic reshaping. The last 30 years or so of developmental biology have seen a focus primarily on genetic explanations. This has begun to change with the recent revolution in our ability to image live embryonic development and even more recent progress in the automation of the tracking of cells and cell shapes within developing embryos. Our laboratories have been at the forefront d of these recent developments, and we are in an excellent position to be able to investigate the nature and changing balance of forces during development, and how these are orchestrated by the genes.

The aim of this project is to use fruit-fly embryos as a model with which to develop methods to understand the forces that drive embryonic development. The fruit-fly embryo has a relatively simple single-layered 'epithelium' of cells on the outside of the embryo that converges and extends, and its genetics is very well understood. Forces in embryos are generated by contractions of cells in ways analogous to how muscles contract. During convergence and extension movements, cell contractions drive changes in cell shape and cause cell to rearrange. Importantly, forces generated by active cell behaviour will exert effects on neighbouring cells, that can transfer force onto their neighbours in turn, or respond by dissipating the force through changing shape or arrangement. Thus cells can experience a variety of extrinsic forces from cells near and far. Disentangling active cell forces from forces imposed extrinsically (from elsewhere) is one of the goals of this project.

Convergence and extension does not happen in a homogenous tissue. Gene expression varies across the tissue, and is correlated with patterns of the strength of active cell rearrangement behaviour. We will investigate the patterns of behaviour variation in detail, and correlate these with the patterns of gene expression and of the contraction of the cell. We will apply new methods to distinguish intrinsic cell rearrangement from passive rearrangement induced by extrinsic forces.

We will extend our current automated methods for tracking cells to track the full three-dimensional shapes of cells during convergence and extension. With such data we will be able to ask whether there are differences in the shapes and orientations of cells, whether they are tilted or wedge-shaped in ways that indicate the presence of local or distant forces. We will test hypotheses generated above about the nature of tissue forces using focused laser ablation, making punctures or cut lines to test if the tissue pulls apart in ways predicted by our hypotheses.

The combination of developing new generic methods to disentangle embryonic forces in this simple model will be a major step in being able to tackle more complicated vertebrate models, such as the zebrafish and mouse and other models relevant to human birth defects and disease states.

Tissue morphogenesis results from the integration of intrinsic and extrinsic forces. Intrinsic forces are generated within a cell, while extrinsic forces are produced outside of the cell but both will determine cell shape and movement. Here, we aim at understanding how intrinsic and extrinsic forces integrate during the morphogenetic movement of convergence and extension, using an optically and genetically tractable model, the Drosophila embryo.

During gastrulation in Drosophila, the germ-band tissue undergoes extension in the anterior/posterior (AP) axis and convergence in the dorsal/ventral (DV) axis. Polarized cell intercalation is the main cell behaviour contributing to this tissue shape change. By quantifying for the first time the strain rates of tissue deformation, we have found evidence that an extrinsic axial force deforms germ-band cells and contributes to tissue extension in parallel to polarized cell intercalation. We have also found that rather than being homogenous, the rate of polarized intercalation is patterned in the germ-band, along both AP and DV axes. These behavioural patterns might be explained by either intrinsic patterns controlled by gene expression, or by extrinsic factors, such as the axial force deforming the germ-band, or both.

To distinguish between these, we will investigate i) which populations of cells are intercalating most actively, ii) whether gene expression patterns correlate with these patterns, and iii) if fluctuating cortical actomyosin can account for the differences in intercalation strength. We will use automated cell tracking of cell shape in 2D and in 3D, combined with quantification of actomyosin concentrations. We will develop novel methods to quantify the dynamics of cell-cell interfaces and cell packing during cell neighbour exchange and tissue shear. This work will identify signatures of intrinsic and extrinsic forces in the germ-band, which can then be utilised in more complex tissues.

In addition to the academic beneficiaries, the research from this proposal will benefit the public by increasing the stock of useful knowledge, by contributing to the creation of new scientific methodologies, by stimulating the development of scientific networks and thus increasing social interaction, by increasing the supply of skilled graduates and researchers, and by enhancing the problem-solving capacity through undergraduate, graduate and postdoctoral teaching.

Increasing the stock of knowledge on morphogenesis is very important as this will impact many other fields and will support progress in medicine. Gaining a deeper understanding of morphogenetic mechanisms is essential to improve human health in three areas. First, to better understand birth defects, which are now the leading cause of infant mortality in developing countries, with neural tube closure malformations alone affecting 0.5-2/1000 pregnancies. Second, to understand cancer metastasis: there is evidence that many cancers invade healthy tissues through collective cell movements that are very reminiscent of embryonic morphogenetic movements. Finally, to develop regenerative medicine: tissue and organ engineering will require in-depth knowledge of morphogenetic mechanisms to be able to build three-dimensional structures following stem cell manipulation. Creation of new scientific methodologies will complement the increase in knowledge about morphogenesis and support progress in the above areas as well. In addition, software tools and protocols developed through the proposed work will increase the stock of scientific methodologies that can be applied to a variety of other problems.

In conducting the proposed research, reducing a complex biological problem to a more simple numerical description will involve the development of numerical skills that are directly applicable to a wide range of problems in science and society. We are actively training students at undergraduate, graduate and postdoctoral level, who will benefit from gaining numerical and problem-solving skills. The knowledge and methodologies gained from the proposed research will also be disseminated through the creation of specific forums such as a wiki site, the Cambridge Advanced Imaging Centre and the QuanTissue Research Network Programme. This will develop scientific networks at national and international levels and thus increasing social interaction within the UK and beyond.

An indirect impact of the proposed project is that Dr G. Blanchard's (named Researcher co-Investigator) growing expertise as a scientist in the field of animal development will continue to support his essential role in the medical voluntary sector as a Research Trustee of The Neuroblastoma Society (Reg. Char. 326385, http://www.nsoc.co.uk/). Neuroblastoma is a neuro-developmental solid childhood tumour with very poor survival rates. The impact is two-fold, allowing him to know how to galvanise the UK scientific and clinical neuroblastoma community and providing the knowledge base to help inform the lay parent community of recent developments. He oversaw the Society's biennial grant round (2009/10) in which £0.5M was allocated in research grants. He also conceived and organised the first UK Neuroblastoma Research Symposium, held on 3/12/10 at the C.R.I. in Cambridge (programme http://www.nsoc.co.uk/symposium2010.html). This Symposium featured 16 talks from international and UK world leaders in neuroblastoma research, 20 posters and 150 delegates. He co-authored a lay meeting report of the Research Symposium, 1000 copies of which have been disseminated to parents via charities.