Regulation of cell behaviour and identity in a branching epithelium

Abstract

Branching morphogenesis is critical for the development of many organs including the lung, pancreas, kidney, breast and prostate. In the developing kidney, the branching epithelium is called the ureteric bud; it is divided into tip regions, at its ends, and the stalk regions everywhere else. The tip regions are capable of inducing nephron formation, unlike the stalk regions. Tip regions are also the regions where most branching occurs. The cells of the tip regions of the ureteric bud have the ability to proliferate and differentiate into cells of the stalk region. Although differentiation and morphogenesis of the ureteric bud have been studied for many years, the mechanisms that control their overall pattern remain unknown. In this thesis, I have tested a specific set of hypotheses in which both differentiation and morphogenesis are controlled by a self-organization based on inhibitory interactions between tip and stalk cells.Using micro dissection and organ culture I show that;• The ureteric bud is composed of at least two distinct populations of cells, those that bind Dolichos biflorus agglutinin (DBA) and those that do not. These correspond to the stalk and tip regions respectively. DBA is a marker of regions of the ureteric bud in which branching morphogenesis is inactivated. When branching morphogenesis is inactivated a change in cell behaviour of the tip cells of the ureteric bud takes place. Tip regions change to a stalk-like behaviour as they loose the expression of tip-specific markers with a parallel increase in stalk markers.• Stalk cells are capable of giving rise to tip cells. Using DBA as a marker of stalk cells, I investigate the mechanisms controlling branching of the ureteric bud. Firstly, I tested the hypothesis that branches rarely arise from the stalks of the ureteric bud because they have lost the ability to branch; it seems that the stalk cells retain their ability to become tips when provided with an appropriate environment. Differentiation of ureteric bud cells is therefore surprisingly plastic.• I also tested the hypothesis that tips of the ureteric bud space out by sensing and responding to other tips in the vicinity. There are two components to this hypothesis;(i) that tips are separated within the epithelium by a lateral inhibition mechanism that prevents new tips forming close to existing ones, and(ii) that tips of extending epithelia are repelled by the presence of nearby tips, so that they spread out to fill space optimally.I have gained evidence against the first hypothesis and show that tips can form from stalk regions. I have investigated the second hypothesis by manipulating cultures so that the tips of separate ureteric bud are placed on a collision course. These clustered ureteric buds prematurely ceased branching and appeared to be compressed in the direction of the collision with the neighbouring clustered kidney. This supports the idea that the tips of the ureteric bud interact with each other to avoid colliding. I did not find convincing evidence to suggest that the tips at the periphery of the colliding ureteric buds were influencing the position of their nearest neighbour. In addition to this, I attempted to compare the closest distance between tips when kidneys are placed on a collision course or are cultured in isolation.The data presented in this thesis provide evidence both to partially support, and also to limit, the specific self-organization hypothesis tested.