RESEARCH ARTICLE Stromal Hoxa5 Function Controls the Growth and Differentiation of Mammary Alveolar Epithelium E ´ lisabeth Garin, 1 Margot Lemieux, 1 Yan Coulombe, 1 Gertraud W. Robinson, 2 and Lucie Jeannotte 1 * Recent progress has enlightened the involvement of Hox genes in organogenesis. Several Hox genes are expressed in normal and neoplastic mammary glands. Using Hoxa5 mutant mice, we showed that Hoxa5 -/- females present nursing defects. Characterization of the Hoxa5 -/- mammary gland phenotype reveals changes in proliferation and differentiation of the epithelium of nulliparous and pregnant Hoxa5 -/- females that precede the abnormal secretory activity at parturition. These defects likely underlie the incapacity of Hoxa5 -/- dams to properly feed their pups. Hoxa5 expression is restricted to the mammary stroma at specific stages of mammary gland development. The loss of Hoxa5 function causes accelerated lobuloalveolar epithelium development, a phenotype that can be rescued upon grafting of mutant mammary epithelium into wild-type fat pads. Conversely, reciprocal grafting experiments demonstrate that Hoxa5 -/- stroma cannot support normal proliferation of wild-type mammary epithelium. These data establish the essential contribution of Hoxa5 to mammary epithelium instruction by means of mesenchymal– epithelial crosstalk. Developmental Dynamics 235:1858 –1871, 2006. © 2006 Wiley-Liss, Inc. Key words: Hox genes; Hoxa5; mammary gland development; mouse; lactation; proliferation; differentiation Accepted 14 March 2006 INTRODUCTION Mammary gland formation is initi- ated during embryogenesis, but its complete development predominantly occurs after birth in two distinct growth stages, puberty and preg- nancy, that are contingent on ovarian and pituitary hormones (Hen- nighausen and Robinson, 1998). From birth to puberty, the mammary epi- thelium growth is limited and results in a small ductal tree surrounded by the mesenchyme. At puberty, the on- set of ovarian hormone secretion to- gether with locally acting growth fac- tors induce duct elongation and branching through the formation and the extensive mitosis of terminal end buds (TEBs) at the tip of the growing ducts. A precise balance between pro- liferation and apoptosis controls the formation of the ducts (Humphreys et al., 1996). TEBs regress once the en- tire fat pad is enlaced with mammary ducts. In adult nulliparous females, the growth of mammary glands re- mains relatively quiescent. The final differentiation occurs during preg- nancy when increased proliferation and subsequent differentiation of the ductal and alveolar epithelial cells produce extensive ductal lateral branching and lobuloalveolar struc- tures with secretory functions. Al- though secretory differentiation ini- tiates at mid-pregnancy, functional lactogenesis and active secretion is at- tained at parturition (McManaman and Neville, 2003). Upon weaning and milk stasis, the gland involutes due to epithelial cell apoptosis and remodels to a virgin-like state. A complex interplay between sys- temic hormones, local growth factors, transcription factors, and mesen- 1 Centre de recherche en cance ´rologie de l’Universite ´ Laval, Centre Hospitalier Universitaire de Que ´bec, L’Ho ˆtel-Dieu de Que ´bec, Que ´bec, Canada 2 Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland Grant sponsor: Cancer Research Society; Grant sponsor: Fonds de la Recherche en Sante ´ du Que ´bec. *Correspondence to: Lucie Jeannotte, Centre de recherche de L’Ho ˆ tel-Dieu de Que ´ bec, 9, rue McMahon, Que ´ bec, QC, Canada, G1R 2J6. E-mail: lucie.jeannotte@crhdq.ulaval.ca DOI 10.1002/dvdy.20822 Published online 10 April 2006 in Wiley InterScience (www.interscience.wiley.com). DEVELOPMENTAL DYNAMICS 235:1858 –1871, 2006 © 2006 Wiley-Liss, Inc.