mucin 1 (Muc1), limb bud and heart development homolog (Lbh), desmocollin 3 (Dsc3), POU class 2 associating factor 1 (Pou2af), and fas apoptotic inhibitory molecule 3 (Faim3) (Fig. 3 A). We also validated the absence of Akt1 protein in the adult mammary glands by western blot, as well as the presence of Akt isoforms Akt2 and Akt3 (Fig. 4). Ingenuity pathway analysis indicated several altered biological functions as a result of Akt1 ablation, including cell-to-cell signaling, intercellular interaction, tissue morphology, organismal development, and cancer within the top ten altered functions (Fig. 3 B). Pathway analysis further illustrated that the pattern of differential gene expression was highly associated with cardiovas- cular system development and function, organismal development, and carcinogenesis pathways (Fig. 3 C). Alveolar cell death incidence and alveolar bud diameter are dependent on Akt1 Our previous results indicated that Akt1 is necessary to maintain proper alveolar bud population in adulthood. Given the pro- survival role of Akt1 during pregnancy and lactation [14,19] and the ability of Akt1 to block several pro-apoptotic processes, we examined the effect of Akt12/2 loss on localization of fibrin and collagen in the mammary gland, alveolar bud size, and mammary epithelial cell death. Collagen and fibrin deposition were not altered by the ablation of Akt1, indicating normal stromal architecture in Akt1+/+ and Akt12/2 mammary glands (Fig. 5 A, C). However, there was a strong trend for decreased alveolar bud diameter in Akt12/2 mammary glands, concomitant with increased epithelial cell apoptosis (Fig. 5 B, D–F). Considering that our gene array results denoted cell death and tissue morphology to be two of the top ten altered biological processes altered by Akt1 deficiency, these data further suggest that apoptosis and alveolar bud formation in the mammary gland are dependent, in part, on Akt1. Akt1 affects mammary gland lobuloalveolar development and Btn1a1 expression during psuedopregnancy Prior studies have indicated that Akt1 is necessary for proper lobuloalveologenesis and lactation during pregnancy and nursing. We chose to study the role of Akt1 in alveolar differentiation during pseudopregnancy based on our findings of its effects on mammary gland development during puberty and on subfertility in Akt12/2 females. Similar to previous work using naturally pregnant mice, Akt1 affects the frequency of formation of lobuloalveolar structures in pseudopregnant mice (Fig. 6 A–D). While Akt1 deficiency Figure 4. Western blot analysis of Akt isoform expression in PND 70 Akt1+/+ and Akt1/2 mammary glands. Western blots of Akt1 (A), Akt2 (B), Akt3 (C), and GAPDH (D) in PND 70 Akt1+/+ and Akt12/2 mammary glands are representative of 5–8 animals per genotype. doi:10.1371/journal.pone.0024432.g004 Akt1 in Mammary Development reduces the number of differentiated alveolar buds, it is not required for alveolar differentiation, as indicated by the presence of alveoli (Fig. 6 D). This is in contrast to previous work that concluded that ablation of Akt1 inhibits the formation of lobuloalveolar structures, but this is likely due to the fact that our study induced pseudopregnancy, whereas prior work utilized naturally pregnant animals [14]. Considering the decreased number of alveolar buds and ducts in adult virgin Akt12/2 mammary glands, our results indicate that Akt1 is necessary for the initial formation of alveolar buds during development and relative number of lobuloalveolar structures formed during pregnancy, but is not necessary for alveologenesis. Our gene array and real-time RT-PCR results indicated that the loss of Akt1 in the mammary gland significantly decreased expression of Btn1a1, a member of the butyrophilin Ig superfamily that is basally expressed in the brain, heart, lymph node, ovary and uterus, thymus, lung, spleen, and mammary gland [24]. Bnt1a1 is highly expressed in the secretory epithelium of lactating mammary glands, and is essential for milk lipid secretion [21]. Btn1a1 and Akt1 were co-expressed in pseudopregnant mammary glands (Fig. 6 E, G, I), and expression of Btn1a1 was decreased in Akt12/2 glands, signifying that Akt1 is necessary for proper protein expression of Btn1a1 in the mammary gland (Fig. 6 H). Additionally, while Akt1+/+ mammary glands responded to pseudopregnancy by significantly increasing the levels of Btn1a1 as compared to virgin glands, Akt12/2 mammary glands failed to do so (Fig. 6 K). Btn1a1 expression in Akt12/2 glands during pseudopregnancy was also significantly decreased as compared to pseuodpregnant Akt1+/+ glands. A two-way ANOVA analysis revealed that both Akt1 genotype status and exposure (pseudo- pregnancy) were significant factors in determining Btn1a1 expression. Akt1 promotes cell proliferation and Btn1a1 and ß-casein gene expression in MCF7 human breast epithelial cells In order to directly address whether Btn1a1 or ß-casein are downstream of Akt1, we evaluated the effect of shRNA-mediated knockdown of Akt1 or Btn1a1 in MCF7 human breast epithelial cells on ß-casein, a gene that encodes for a milk protein expressed during early lactational mammary epithelial differentiation [11]. MCF7 cells expressing shRNA targeting Akt1 (shAkt1) exhibited significant decreases in cell proliferation after 4–6 days of growth in culture (Fig. 7 A). Knockdown of Akt1 also resulted in significant decreases in both Btn1a1 and ß-casein expression (Fig. 7 B), while knockdown of Btn1a1 (shBTN1A1) decreased expression of Btn1a1 and ß-casein, but not Akt1 (Fig. 7 B, Fig. S2.). Discussion The mammary gland is a critical reproductive organ, and the abilities of the adult mammary gland to undergo normal development and proper lobuloalveolar remodeling during pregnancy are critical for reproductive health. The mammary gland is distinct from other organs because it undergoes most of its branching postnatally with the onset of puberty rather than in utero. Proper branching morphogenesis is fundamental for functional development and differentiation to occur in adulthood. Using an Akt1+/+ and Akt12/2 mouse model to examine postnatal development in vivo, we found that Akt1 is essential for proper mammary gland organogenesis and appropriate adult mammary homeostasis. Mammary gland development can be separated into three distinct stages: embryonic, adolescent, and adult. We found that the initial branching and formation of the rudimentary duct occurred normally in Akt12/2 mammary glands, indicating that PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e24432