|The primary aim of this thesis was to classify the nature of the clonal expansion of cells that gives rise to bovine and murine mammary epithelia. It is generally assumed that the mammary gland is derived from the outgrowth of mammary-determined stem cell progeny, and that this expansion produces a mixture of cellular clones throughout the gland. Molecular techniques that enable the 'visualization' of the size and distribution of these clones will lead to a better appreciation of mammary gland development and architecture.
In female mammals, the simplest 'mark' of clonality is provided by the inactivation of one or the other of the two X chromosomes. This event occurs early in embryogenesis and gives rise to stem cells that propagate this mark in progeny cells throughout the life of the animal. Two approaches, that utilized X-inactivation as a mark of clonality, were investigated, 1) naturally occurring X-linked gene polymorphisms in cattle, and 2) artificially introduced X-linked transgenes in mice.
The first part of this thesis deals with the isolation and utilization of polymorphic markers on the bovine X-chromosome as markers of clonality. Such expressed genetic markers are essential if one is to distinguish which of the two X-chromosomes is active in a given cell. Two X-linked bovine genes, glucose-6-phosphate dehydrogenase (g6pd) and the X-inactive specific transcript (Xist) were selected for sequencing and SNP detection. As no specific sequence data was available for these genes, a bovine lambda genomic DNA library was screened. The G6PD clone selected from a lambda library was shown to be a pseudogene and therefore not useful for the present study. Another clone, that hybridized with an Xist gene probe, was shown to actually represent a bovine Huntingtin Associated Protein 1 like (Hap1-like) pseudogene, making it of no utility in this study. However, screening of regions of exon 1 of the Xist gene by direct PCR and sequencing, led to the discovery of an informative G↔T single nucleotide polymorphism (SNP). The variation, which was found in frequencies of 0.8 and 0.2 for 'G' and 'T' variants respectively (32% of females are heterozygous), was used in a number of different ways in order to assess the clonal profile of small 'blocks' of the gland (≤30 mm3). In these preliminary experiments, RT-PCR analysis of Xist expression patterns within each of the small blocks (each comprising around 3 X 107 cells) showed general heterogeneity but the experiments were not conclusive. Limitations of the experimental methods used are discussed and suggestions of alternative approaches, that will offer higher resolution, are presented.
A second experimental approach, involving mice with a reporter transgene construct introduced into the X-chromosome (H253), was also used. The transgene consisted of 14 tandem repeats of a lacZ nuclear-localized reporter gene coupled with a mouse 3-hydroxyl-3-methylglutaryl coenzyme A reductase (hmgcr) promoter (Tam and Tan, 1992). A number of researchers had already utilized this transgenic model, and had confirmed that the cellular patterns of lacZ expression reflected actual X-inactivation patterns in a number of tissues. While carrying out initial investigations to ascertain the utility of this reporter system in investigations of the mammary gland it was discovered that the usual description of hmgcr as a 'house-keeping' gene is somewhat of a misnomer, as some tissues in the mouse show very strong patterns of lacZ expression while others gave minimal, or no, detectable expression. Furthermore, transgene expression was observed to increase during pregnancy and it was concluded that this effect was almost certainly a response to increased estrogen levels at this time.
Whole mount analysis of five mouse mammary glands from transgenic animals revealed heterogeneous patterns of lacZ staining, indicating that mammary epithelia is generally polyclonal. In total, 42% of terminal buds and alveoli displayed a single lacZ staining pattern. Analysis of these staining patterns induct termini indicated that the majority of individual alveoli are derived from two or three division competent (stem) cells.