Abstract
The terminal end bud (TEB) is the growing part of the ductal mammary epithelium during puberty, enabling the formation of a primary epithelial network. These highly proliferative bulbous end structures that drive the ductal expansion into the mammary fat pad comprise an outer cap cell layer, containing the progenitor cells of the ductal myoepithelium, and the body cells, which form the luminal epithelium. As TEB make up only a very small part of the whole mammary tissue, TEB-associated factors can be easily missed when whole-tissue sections are being analyzed. Here we describe a method to enzymatically separate TEB and ducts, respectively, from the surrounding stroma of pubertal mice in order to perform transcriptomic or proteomic analysis on the isolated structures and identify potential novel regulators of epithelial outgrowth, or to allow further cell culturing. This approach has previously allowed us to identify novel TEB-associated proteins, including several axonal guidance proteins. We further include protocols for the culturing of isolated TEB, processing of mammary tissue into paraffin and immunohistochemical/fluorescent staining for verification, and localization of protein expression in the mammary tissue at different developmental time points.
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References
Silberstein GB (2001) Postnatal mammary gland morphogenesis. Microsc Res Tech 52(2):155–162. doi:10.1002/1097-0029(20010115)52:2<155::AID-JEMT1001>3.0.CO;2-P
Propper AY, Howard BA, Veltmaat JM (2013) Prenatal morphogenesis of mammary glands in mouse and rabbit. J Mammary Gland Biol Neoplasia 18(2):93–104. doi:10.1007/s10911-013-9298-0
Silberstein GB, Daniel CW (1982) Glycosaminoglycans in the basal lamina and extracellular matrix of the developing mouse mammary duct. Dev Biol 90(1):215–222, doi:0012-1606(82)90228-7 [pii]
Master SR, Hartman JL, D'Cruz CM, Moody SE, Keiper EA, Ha SI, Cox JD, Belka GK, Chodosh LA (2002) Functional microarray analysis of mammary organogenesis reveals a developmental role in adaptive thermogenesis. Mol Endocrinol 16(6):1185–1203. doi:10.1210/mend.16.6.0865
McBryan J, Howlin J, Kenny PA, Shioda T, Martin F (2007) ERalpha-CITED1 co-regulated genes expressed during pubertal mammary gland development: implications for breast cancer prognosis. Oncogene 26(44):6406–6419. doi:10.1038/sj.onc.1210468
Kouros-Mehr H, Werb Z (2006) Candidate regulators of mammary branching morphogenesis identified by genome-wide transcript analysis. Dev Dyn 235(12):3404–3412. doi:10.1002/dvdy.20978
Richards J, Guzman R, Konrad M, Yang J, Nandi S (1982) Growth of mouse mammary gland end buds cultured in a collagen gel matrix. Exp Cell Res 141(2):433–443
Davies CR, Morris JS, Griffiths MR, Page MJ, Pitt A, Stein T, Gusterson BA (2006) Proteomic analysis of the mouse mammary gland is a powerful tool to identify novel proteins that are differentially expressed during mammary development. Proteomics 6(21):5694–5704. doi:10.1002/pmic.200600202
Morris JS, Stein T, Pringle MA, Davies CR, Weber-Hall S, Ferrier RK, Bell AK, Heath VJ, Gusterson BA (2006) Involvement of axonal guidance proteins and their signaling partners in the developing mouse mammary gland. J Cell Physiol 206(1):16–24
Emig D, Salomonis N, Baumbach J, Lengauer T, Conklin BR, Albrecht M (2010) AltAnalyze and DomainGraph: analyzing and visualizing exon expression data. Nucleic acids research 38 (Web Server issue):W755-762. doi:10.1093/nar/gkq405
Olijnyk D, Ibrahim AM, Ferrier RK, Tsuda T, Chu ML, Gusterson BA, Stein T, Morris JS (2014) Fibulin-2 is involved in early extracellular matrix development of the outgrowing mouse mammary epithelium. Cell Mol Life Sci 71(19):3811–3828. doi:10.1007/s00018-014-1577-4
Acknowledgements
We would like to thank Dr. Daria Olijnyk for the images of the whole mounts, Rod Ferrier for critical reading of the IHC protocol, Stephanie Stengel for culturing and IF staining of TEBs, and Ayman Ibrahim for photographs of the isolation procedure.
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Morris, J.S., Stein, T. (2017). Pubertal Ductal Morphogenesis: Isolation and Transcriptome Analysis of the Terminal End Bud. In: Martin, F., Stein, T., Howlin, J. (eds) Mammary Gland Development. Methods in Molecular Biology, vol 1501. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6475-8_5
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DOI: https://doi.org/10.1007/978-1-4939-6475-8_5
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-6475-8
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