Figure 1.
FOXO1 is present in the nuclei of pituitary cells at an increasing frequency as development progresses.
Immunohistochemistry for FOXO1 (green) was performed on midsagittal pituitary sections. (A) FOXO1 is present in the developing pituitary by e10.5. Nuclear FOXO1 is apparent where the invaginating Rathke’s pouch is joined to the oral ectoderm that will form the mouth (see inset). (B) By e12.5 FOXO1 is present almost entirely in the cytoplasm of pituitary cells. (C) A few pituitary cells contain nuclear FOXO1 at e14.5. (D) At e18.5 the developing pituitary contains a mostly nuclear FOXO1. (E) In adults, FOXO1 is present in the anterior and intermediate lobes of the pituitary gland, but not in the posterior lobe (data not shown). In the adult pituitary FOXO1 is primarily nuclear (inset, arrow). Some cytoplasmic FOXO1 (inset, arrow head) is also present. (F) Immunohistochemistry for FOXO1. (G) Beta-galactosidase staining of pituitary from Foxo1+/LacZ mice identifies cells in which the endogenous Foxo1 promoter is active. (H) An overlay of immunohistochemical staining for FOXO1 (green) and β-galactosidase staining of pituitary from Foxo1+/LacZ mice (blue). (F–H) Arrows highlight examples of co-localized cells. Pictures are taken at 200X (A–E) or 630X (F–H). Insets are magnified 600X. Scale bars represent 100 µm. All cell nuclei were marked with DAPI (A–E, blue). Oral ectoderm (OE), infundibulum (INF), ventral diencephalon (VD), Rathke’s pouch (RP), posterior lobe (PL), intermediate lobe (IL), anterior lobe (AL).
Figure 2.
Approximately half of somatotrope cells contain nuclear FOXO1.
Hormone co-localizations were performed on embryonic pituitary tissue at e18.5 (A–E) and in adults (F–K). FOXO1 (green) and LH (A, F), TSH (B, G), ACTH (C, H), GH (D, I), or PRL (J) were labeled by immunohistochemistry. Hormones are shown in red. The total number of hormone-positive cells and the number of hormone cells containing nuclear FOXO1 were counted manually. Pictures were taken at 630X. Scale bars represent 100 µm. (E) In the e18.5 embryonic pituitary, nuclear FOXO1 is present in 8% of gonadotrope cells, 8% of thyrotrope cells, 13% of corticotrope cells, and 42% of somatotrope cells. (K) In the adult pituitary gland, nuclear FOXO1 is present in 7% of gonadotrope cells, 9% of thyrotrope cells, 30% of corticotrope cells, 63% of somatotrope cells, and 15% of lactotrope cells. Graphs represents mean ± SEM.
Figure 3.
FOXO1 is present in quiescent pituitary cells during development.
(A) FOXO1 (green) is not present in actively dividing cells labeled with BrdU (red) at e16.5. Picture was taken at 400X. (B) FOXO1 (green) is present in a subset of p27-positive cells (red) at e14.5. Picture was taken at 200X. (C) FOXO1 (green) is not present in cells exiting the cell cycle that are expressing p57 (red) at e14.5. Picture was taken at 200X. Scale bars represent 100 µm.
Figure 4.
The number of FOXO1-positive pituitary cells is increased in the absence of p27Kip1.
FOXO1 (green) was analyzed in mouse models that lack several different pituitary transcription factors. The number of FOXO1-positive pituitary cells and the total number (DAPI-positive) of pituitary cells were counted manually. Graphs represent mean ± SEM. The left y-axis shows the total number of FOXO1-positive pituitary cells. The right y-axis represents the number of FOXO1-positive pituitary cells as a percentage of the total number of pituitary cells. (A–C) FOXO1 is not different between Hes1 null embryos and wild type littermates at e16.5. (D–F) The number of FOXO1-positive cells is significantly reduced in Prop1df/df mice as compared to wild type littermates at e16.5. However, when the reduction in the total number of pituitary cells is considered the difference is no longer significant. (G–I) The number of cells containing FOXO1 is increased approximately 3-fold in p27Kip1 null mice at e14.5. The percentage of pituitary cells containing FOXO1 is increased by approximately 2.5-fold in these mutants. (J–K) FOXO1 (green) does not co-localize with BrdU (red) suggesting that FOXO1-positive cells remain quiescent in the absence of p27Kip1. Student’s t-test was performed to determine significance (*P<0.05). Sample size is three for these studies. Pictures are taken at 200X. Scale bars represent 100 µm.