Molecular and biophysical investigation of epithelial cell sheet invagination

Early morphogenetic movements transform the new embryo from an amorphous ball of cells into a complex compartimentalised structure in which the main organs are laid out. Examples of such movements are gastrulation which creates the primitive gut and neurulation which creates the spinal chord. Invagination is a morphogenetic movement during which a cell sheet buckles (along a line in the case of neurulation and along a circle in the case of gastrulation). Abnormalities in invagination lead to conditions such as spina bifida that affect approximately 1 in 1000 live births. Cell sheets also undergo invagination during the creation of branched organs (lungs, kidneys, blood vessels) or during cancer when the tumour causes new blood vessels to grow to provide it with nutrients. During invagination, one group of cells has to express different genes than the surrounding cells. Invagination has been extensively studied in embryos and several theories have been proposed to explain the mechanism through which a cell sheet invaginates. However, since all cells in embryos undergo a defined sequence of gene expression, it is difficult to know what is responsible for invagination and what is just a side effect. All of the proposed theories for invagination require that one subset of the cell population express a different set of genes than the surrounding cells. Modern molecular biology techniques enable us to force cells to express a gene of choice. Microprinting techniques and microfluidic techniques enable us to selectively treat cells with micrometer precision. By combining both sets of techniques, I will devise an experimental system that will allow me to force a subset of cells within a monolayer to express a gene of my choice. In addition, the cells will be cultured on a soft substrate that they can deform. This system will enable me to test each of the different theories proposed for invagination directly. For each proposed theory, I will replicate the proposed movement by forcing a stripe of cells within the monolayer to undergo the type of movement that the theory hypothesizes is the cause of invagination. This will be done either through forced gene expression or through chemical treatment. Then, I will simply observe the cell sheet over a period of 24 hours and see if it invaginates. Once I have found which treatments give rise to invaginations, I will examine the mechanical forces at play. This project will enable us to better understand the mechanisms of invagination and identify proteins that can be targeted to inhibit it during cancer progression.

Epithelial cell sheet invagination is a ubiquitous phenomenon in embryogenesis and organogenesis. During embryonic development, cells of the neural plate invaginate to form the neural tube and spinal chord. Many models of invagination have been proposed and these include contraction of an apical band of actin, cell proliferation, cell migration, or differential adhesion. Though invagination is of great importance, to date there exist no in vitro culture systems to study it. I propose to devise such a system and utilize it to study the molecular biology and biophysics of epithelial cell sheet invagination. All of the proposed models for invagination involve localized gene expression or cell behaviour. I will devise a culture system to locally transfect a stripe of cells within a sheet with the relevant gene products under the control of inducible promoters using either microfluidic channels or micropatterning. The gene product will be expressed only in the central region or only in the peripheral regions. This will enable me to replicate each of the proposed models of invagination in vitro. To test the role of apical contraction in invagination, I will perfuse the cells with contraction inducing drugs or retroviral constructs containing constitutively active forms of the small GTPase Rho or the neural crest protein shroom. To test the role of differential adhesion, cells in the central zone will be transfected with different types of cadherins. To test the role of cell proliferation, we will perfuse the cells in the central regions with promitogenic drugs. The role of cell migration will be tested by transfecting the cells in the peripheral regions with dominant forms of the small GTPase Rac. To directly assess the appearance of an invagination and investigate the forces involved, I will culture the cells on deformable acrylamide substrates and directly observe whether invaginations result from a specific pattern of gene expression.