Olijnyk, Daria (2011) Pubertal mouse mammary gland development - transcriptome analysis and the investigation of Fbln2 expression and function. PhD thesis. http://theses.gla.ac.uk/2996/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] Pubertal Mouse Mammary Gland Development- Transcriptome Analysis and the Investigation of Fbln2 Expression and Function Daria Olijnyk (B.Sc, MRes) Thesis submitted to the University of Glasgow in fulfilment of the requirements for the degree of Doctor of Philosophy June 2011 Institute of Cancer Sciences College of Medical, Veterinary and Life Sciences University of Glasgow 2 For my parents, Bogusława and Bogusław Olijnyk and grandmother, Anna Bandurak 3 Abstract Mouse mammary gland morphogenesis at puberty is a complex developmental process, regulated by systemic hormones, local growth factors and dependent on the epithelial/epithelial and epithelial/stromal interactions. TEBs which invade the fat pad are important in laying down the epithelial framework of the gland at this time point. The objective of this thesis was to use a combination of „pathway-„ and „candidate gene analysis‟ of the transcriptome of isolated TEBs and ducts and associated stroma, combined with detailed analyses of selected proteins, to further define the key proteins and processes involved at puberty. Using GeneChip® Mouse Exon 1.0 ST Arrays we identified the epithelial-, epithelial-stromal- and stromal transcriptomes of TEBs and ducts and defined the major functional pathways/biological processes in each compartment. By ranking the transcripts according to their expression levels and known functions in other systems, we identified genes of potential importance for pubertal mammary morphogenesis. We focused our study on Upk3a and Fbln2 and their protein products. Upk3a could only be detected at mRNA level and thus further analysis was based on Fbln2. We demonstrated that Fbln2 V1 and Fbln2 protein are predominantly expressed in the epithelium and stroma of TEBs. Using hormone primed mice we demonstrated that Fbln2 expression and localisation in the mouse mammary gland is positively regulated by E2 and P. Furthermore, by a combination of further in silico analysis, in vitro functional assays, IHC and IF we identified Vcan, Lama1, Fbn1, ColVIαIII, ColIVαI, ColXVIIIαI, Eln, Per, Acan, Nid, Itgb3 and Itga5 as potential binding partners of Fbln2 in mammary gland. Finally, we reported lack of an obvious mammary phenotype in Fbln2 KO-/- mice at puberty but demonstrated that this may be attributed to the over- compensation by Fbln1. This thesis demonstrates the benefit of DNA microarray analysis in studying pubertal development of mouse mammary gland. It identifies Fbln2 as a potential pubertal mammary regulator which by interacting with various ECM proteins at different sites of mammary milieu may contribute to an array of structural and migratory functions during mammary morphogenesis. These data substantially add to the understanding of the development of mammary gland at puberty and reveal many potential avenues for further investigations. 4 Table of Contents 1 Introduction ........................................................................... 22 1.1 Overview of mouse mammary gland development ........................ 22 1.1.1 Embryonic development of mouse mammary gland .................. 23 1.1.2 Postnatal development of mouse mammary gland ................... 27 1.2 Mouse mammary gland morphogenesis at puberty ........................ 30 1.2.1 The morphology and characteristics of pubertal mouse mammary gland .............................................................................. 31 1.2.1.1 Epithelium ............................................................ 31 1.2.1.1.1 TEBs ................................................................. 33 1.2.1.1.2 Ducts ................................................................ 35 1.2.1.2 Fat pad ................................................................ 37 1.2.1.2.1 Adipose tissue ..................................................... 38 1.2.1.2.2 Fibroblasts ......................................................... 39 1.2.1.2.3 Migratory cells of the immune system ......................... 39 1.2.1.2.4 ECM ................................................................. 40 1.2.2 Motility in the mouse mammary gland at puberty ................... 42 1.2.2.1 TEBs .................................................................... 42 1.2.2.1.1 Forward movement ............................................... 43 1.2.2.1.2 Bifurcation ......................................................... 44 1.2.2.1.3 Turning ............................................................. 47 1.2.2.2 Ducts ................................................................... 48 1.2.3 Regulation of ductal morphogenesis at puberty ...................... 49 1.2.3.1 Epithelial-epithelial communication .............................. 49 1.2.3.2 Epithelial-stromal interactions .................................... 51 1.2.3.2.1 Hormonal control of pubertal mouse mammary gland morphogenesis .................................................................. 51 1.2.3.2.2 Local growth control of pubertal mouse mammary gland development .................................................................... 57 1.2.3.2.3 Transcriptional control of pubertal mouse mammary gland development .................................................................... 63 1.2.3.2.4 Immune response cells and cytokines in the regulation of pubertal mammary gland development ..................................... 68 1.2.3.2.5 The role of other stromal components in regulation of pubertal mammary morphogenesis .......................................... 74 1.2.3.2.6 Axonal guidance proteins ........................................ 78 1.2.3.2.7 Other regulators of pubertal mouse mammary gland morphogenesis .................................................................. 80 1.2.4 Summary .................................................................... 80 1.3 Microarray analysis of gene expression to study pubertal mouse mammary gland development ........................................................ 81 2 Materials and methods .............................................................. 90 2.1 Materials ......................................................................... 90 2.1.1 Reagents and materials .................................................. 90 2.1.2 Solutions and Buffers ..................................................... 95 2.1.3 Electric equipment ........................................................ 97 2.2 Methodology ..................................................................... 98 2.2.1 Sample Collection ......................................................... 98 2.2.1.1 Mouse husbandry ..................................................... 98 2.2.1.2 Dissection of mouse mammary glands ............................ 99 2.2.1.3 Dissection of other mouse organs and tissues .................. 101 5 2.2.1.4 Collection of mouse mammary gland tissue strips ............. 101 2.2.1.5 Collection of isolated epithelium ................................. 101 2.2.2 Cell culture ................................................................ 103 2.2.2.1 Cell lines and Cell maintenance .................................. 103 2.2.2.2 Cell Counting ........................................................ 104 2.2.2.3 Plasmid information and plasmid DNA purification ............ 104 2.2.2.4 Transfections ........................................................ 106 2.2.2.4.1 Optimisation of transfections .................................. 106 2.2.2.4.2 Fbln2 over-expression in HC11 cells ........................... 108 2.2.2.5 Wound Healing Assay ............................................... 108 2.2.2.6 Adhesion Assays ..................................................... 109 2.2.2.6.1 Adhesion of pRc/CMV-Fbln2 transfected HC11 cells to plastic surface ..................................................................... 109 2.2.2.6.2 Adhesion of pRc/CMV-Fbln2 transfected HC11 cells to ECM proteins ..................................................................... 109 2.2.3 Protein work .............................................................. 110 2.2.3.1 Protein extraction from the cell lines ........................... 110 2.2.3.2 Protein extraction from the frozen tissues ..................... 111 2.2.3.3 Protein Quantification ............................................. 111 2.2.3.4 Concentration of Protein Extracts ................................ 112 2.2.3.5 Electrophoresis and Western blotting............................ 112 2.2.4 RNA and DNA .............................................................. 116 2.2.4.1 Preparation of total RNA ........................................... 116 2.2.4.1.1 Preparation of collected frozen tissue for RNA extraction 116 2.2.4.1.2 Preparation of isolated mammary