Akt1 in Mammary Development adulthood, the growth past the lymph node was comparable in Akt12/2 and Akt1+/+ glands (Fig. 1 J). Overall, these results suggest that Akt1 is not necessary for the initial formation of the rudimentary ductal tree during the neonatal period, but that it does mediate ductal growth during puberty until adulthood when Akt12/2 glands reach full outgrowth similar to their wild-type counterparts. Akt1 regulates the cell population in developing and adult mammary glands Considering the effects that Akt1 deficiency elicited on ductal growth in the mammary gland, we next investigated the effects of Akt1 loss on ductal branching and alveolar expansion in the developing gland. Initial results indicated the Akt1 deficiency had little effect on initial side branching at either PND 11 or PND 25 (Fig. S1). We evaluated terminal end bud bifurcation by quantifying the number of terminal end buds, alveolar buds, and terminal ducts in the Zone C area of the mammary gland throughout development (Fig. 2, A–H) [22]. Akt1 deficiency decreased the number of terminal end buds during the juvenile time period (Fig. 2 I). The terminal end bud is characterized by having an inner layer of body cells surrounded by an outer layer of cap cells, which give rise to luminal cells and basal cells, respectively [3]. This outer layer of cap cells is evident in the Akt1+/+ mammary glands during puberty, but is near absent in the Akt12/2 glands (Fig. 2 B, F). These terminal end buds are normally present during puberty, and are permanently replaced with alveolar buds and ducts when the mammary gland reaches the end of the fat pad [7]. Therefore, calculating the percentage of terminal end buds is useful for gauging the relative stage of development of the mammary gland. Although Akt12/2 glands contained a lower percentage of TEBs at PND25, there was a trend for a higher percentage of TEBs during puberty (PND 50), suggesting a more immature phenotype for the Akt12/2 glands at puberty (Fig. 2 K). Figure 2. Akt1 mediates the terminal end bud, terminal duct and alveolar bud population. Representative H&E images of Akt1+/+ (A–D) and Akt12/2 (E–H) mammary glands at PND 11 (A,E), PND 25 (B,F), PND 50 (C,G), and PND 70 (D,H). Arrow indicates terminal end buds (B) Quantification of terminal end buds in Akt1+/+ and Akt12/2 mammary glands at PND 11, 25, 50 and 70 (I). Quantification of total buds and terminal ducts in Akt1+/+ and Akt12/2 mammary glands at PND 11, 25, 50 and 70 (J). Quantification of the terminal end buds relative to the total number of buds of Akt1+/+ and Akt12/2 mammary glands at PND 11, 25, 50 and 70 (K). Scale bars indicate 100 mm. Data represented in line plots as mean +/2 standard error. Statistical analyses were conducted using a two-tailed student’s t test. **p,0.01, *p,0.05, #p,0.1. n = 3–8 per genotype, per age group. doi:10.1371/journal.pone.0024432.g002 PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24432