Cell and nuclear packing during tissue growth

Abstract

Highly proliferative tissues require an optimal cellular organisation to use the available space most effectively. One such organisation is pseudostratification, where cells are tightly packed within a monolayer and their nuclei stack on top of each other. This cellular packing enables, for example, the production of numerous neurons in the brain. Although high proliferation is desired, growth needs to terminate eventually. The mechanisms of growth termination are however complex and largely unknown. The distal part of the Drosophila wing imaginal disc, which gives rise to the adult wing blade, is a model for growth of pseudostratified epithelia. In this tissue, mechanical compression of the apical domain was proposed to cause growth termination. Crucially, nuclei have to undergo apical migration for mitosis. Thus, the packing of nuclei might mechanically influence growth as well. My aim was therefore to quantify nuclei packing (density, volume and position) on a tissue wide scale at different growth stages. An imaging pipeline and a complementary user-guided segmentation program were developed to quantify nuclear packing. The thick and highly folded shape of the wing imaginal disc was preserved and imaged using agarose embedding and a tissue clearing solution, respectively. The nuclear envelope served as a marker to extract 2D shapes which were merged as 3D objects and manually corrected. Local averaging of tiled segmented images was used to compare nuclear parameters of mid and late developmental stages. The analysis indicated that nuclei moved closer together and packed more volume in the available space. My hypothesis is that this close packing of nuclei could contribute to growth termination. While the developed imaging and segmentation pipeline was optimised to extract nuclear parameters, it could also be utilised for other tightly packed tissues, such as tumour spheroids, to extract cells in 3D.