The mammary gland is a dynamic organ that undergoes many cycles of proliferation and death throughout both oestrus cycling and the gestation/lactation/involution cycle. Moreover, it is a unique tissue in that the majority of its development occurs postnatally coincident with the production of ovarian hormones in puberty. This capacity of the mammary gland for rapid growth and regeneration has been attributed to mammary stem cells (MaSCs). However, despite extensive efforts over the past 60 years, definitive characterisation of the stem and progenitor cells of the mammary gland has yet to be achieved. A number of recent conflicting, lineage tracing studies have served only to fuel the fires of controversy, with previous characterisation of the mammary gland largely carried out using two-dimensional tissue sections. However, in order to fully appreciate the capacity of MaSCs and to maintain spatial information of the complex topological structure, the mammary gland must be investigated in its intact form. Accordingly, there is still much disagreement regarding the potency, capacity and location of MaSCs.

Consequently, in order to unequivocally elucidate the MaSC hierarchy, a variety of novel techniques have been combined in this thesis. The first involves development and optimisation of optical tissue clearing techniques to allow the visualisation of the native mammary gland, in situ, and in three dimensions. To do so, a number of different optical tissue clearing methods have been assessed and combined with a variety of microscopy techniques to allow the multiple focal planes of the ductal network to be examined. These imaging techniques were then combined with two neutral lineage tracing models; the first, the Rosa26[CA]30 model, utilises stochastic continuous clonal labelling to allow for the fate tracking of the progeny from single functional stem and progenitor cells. The second unbiased lineage tracing approach, the Rosa26-Confetti model, allows for the mammary stem and progenitor progeny to be traced with precise timing, with the additional benefit of a multicolour reporter. Next, proliferation was examined in wholemount tissues to investigate the functional requirements for MaSCs, and their potential locations. Finally, these techniques have been combined with an ex vivo 3D organoid culture system to investigate the use of culture methods in examining mammary epithelial cell dynamics.

By combination of these techniques, clonally marked regions can be investigated throughout the development of the mammary gland, from the formation of the embryonic mammary iii rudiment, to expansion of the ductal tree in puberty, and ultimately their fate in lactation and involution, where the mammary gland fulfils its evolutionary purpose. The mammary gland provides a unique opportunity to investigate epithelial development extra-embryonically that is not available in other tissues. Moreover, study of maintenance and turnover of this organ has important implications for other epithelial systems. Finally, elucidation of the normal MaSC hierarchy also has important implications for understanding the complex heterogeneity of breast cancer and the cell(s) of origin of breast cancer. Given the proposed longevity and suggested ability of MaSCs to survive multiple waves of cell death in involution, they represent a logical candidate for a potential cell of origin of breast cancer.

The work presented in this thesis provides novel insights into MaSCs and progenitors and their potential to contribute to mammary gland development.