Akt1 in Mammary Development Figure 5. Akt1 influences alveolar bud diameter and apoptosis incidence in adult mammary glands. Martius yellow, soluble blue, brilliant crystal (MSB) stain of Akt1+/+ (A) and Akt12/2 (C) PND 70 mammary glands. Blue staining indicates collagen and purple staining indicates fibrin. TUNEL staining in Akt1+/+ (B) and Akt12/2 (D) PND 70 mammary glands. Measurement of alveolar bud diameter in Akt1+/+ and Akt12/2 PND 70 mammary glands (E). Measurement of TUNEL positivity in Akt1+/+ and Akt12/2 PND 70 mammary glands (F). Arrow indicates apoptotic cells in the Akt12/2 gland. Scale bars indicate 100 mm. Data represented as mean plus standard error. Statistical analyses were conducted using a two-tailed student’s t test. #p = 0.08, *p,0.05. n = 4 per genotype for (E), n = 3 per genotype for (F). doi:10.1371/journal.pone.0024432.g005 embryonic mammary gland development is Akt1-independent. These results are not surprising considering embryonic mammary development is also hormone-independent, and PI3K/AKT activates ERa [25]. We did find that postnatal mammary gland development is Akt1-dependent. Unlike embryonic growth, postnatal development requires estrogen and progesterone, which are secreted by the ovaries at approximately three weeks of age in the mouse. Ductal outgrowth of inguinal mammary glands was significantly reduced in Akt12/2 glands during early and late pubertal time points. However, by adulthood these glands occupied the same amount of space in the fat pad as their wild-type counterparts. Our previous work indicated that Akt12/2 female mice had higher levels of estradiol and lower levels of progesterone than Akt1+/+ females during late puberty, but that these levels were similar in both genotypes by adulthood irrespective of Akt1 status [20]. Consid- ering that postnatal ductal growth and alveolar expansion are dependent on estradiol and progesterone, the higher levels of progesterone most likely have a compensatory effect on ductal outgrowth, allowing for the Akt12/2 mammary glands to reach full size. Conversely, the levels of estradiol may be a contributing factor to the altered terminal end bud and alveolar expansion seen in the Akt12/2 glands. Mammary gland postnatal ductal development is driven by the rapid growth of the terminal end bud, a unique structure that contains dichotomously dividing stem cells during puberty. The terminal end bud contains a ‘tube within a tube’ structure, with an outer layer of undifferentiated cap cells and an inner layer of luminal epithelial cells. Both layers have high rates of mitosis, and normally have rates of forward growth through the fat pad of about 0.5 mm per day [26]. Akt1 deficiency resulted in a significant decrease in the terminal end bud population during puberty, and a significant decrease in total alveolar buds and a strong trend for decreased alveolar bud size in adulthood. We also demonstrated that suppression of Akt1 in human breast epithelial cells inhibited cell proliferation, further illustrating the importance of Akt1 in mammary epithelial cell growth. The growth of the terminal end bud relies both on estradiol and growth factors elicited by the stroma, such as insulin- like growth factor-1 (IGF-1). Notably, the AKT pathway has been implicated in both estrogen and insulin signaling [9,25]. Moreover, Akt has been shown to be phosphorylated downstream of IGF-1 [27]. In our model, Akt1 could be a potential mediator of IGF-1 and estrogen signaling to the terminal end bud, and its deficiency results in a lack of bifurcation. The permanent structural changes in Akt12/2 mammary glands led us to examine alterations in gene expression in adult glands. We chose to validate a set of genes based on their high rates of fold change and relative importance in growth and differentiation processes. For example, Akt12/2 glands exhibited increased expression of Lbh, a gene normally expressed during development in branched limbs and the heart, which could be a compensatory mechanism for the observed deficiency in postnatal growth and bud development. Over-expression and deregulation of Lbh results in suppression of epithelial cell differentiation and a potential involvement in Wnt-induced breast tumorigenesis [28]. Our findings further suggest that the lack of Akt1 in the mammary gland may also disrupt its functional role in lactation. Akt1 has previously been found to be involved in the proper formation of lobuloalveolar units during pregnancy, as well as in proper milk production during nursing. The phenotype of Akt12/2 mamma- ry glands has been described, including the inhibition of lobuloalveolar units and survival in mammary epithelia during pregnancy [14]. We chose to investigate the molecular basis of abnormal mammary gland function based on our prior results indicating decreased expression of Btn1a1 in Akt12/2 mammary glands. Btn1a1 has recently been found to be a crucial gene for proper milk lipid secretion during lactation. We induced pseuodopregnacy to PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24432