FOXO1 is present in the nuclei of pituitary cells at an increasing frequency as development progresses.

<p>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 <i>Foxo1^+/LacZ</i> mice identifies cells in which the endogenous <i>Foxo1</i> promoter is active. (H) An overlay of immunohistochemical staining for FOXO1 (green) and β-galactosidase staining of pituitary from <i>Foxo1^+/LacZ</i> 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).</p

The number of FOXO1-positive pituitary cells is increased in the absence of <i>p27^Kip1</i>.

<p>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 <i>Hes1</i> null embryos and wild type littermates at e16.5. (D–F) The number of FOXO1-positive cells is significantly reduced in <i>Prop1^df/df</i> 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 <i>p27^Kip1</i> 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 <i>p27^Kip1</i>. 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.</p

FOXO1 is present in quiescent pituitary cells during development.

<p>(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.</p

Approximately half of somatotrope cells contain nuclear FOXO1.

<p>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.</p

The number of cells containing GH, LHB, and CGA are reduced in <i>Foxm1</i>^<i>-/-</i> pituitary glands.

<p>Immunohistochemistry for (A-B) GH (red), (D-E) CGA (red), (G-H) ACTH (red), (J-K) LHB (red), (M-N) TSHB (red) was performed on e17.5/18.5 coronal sections. DAPI (blue) labels all cell nuclei. Scale bar represents 100 μm. The total number of cells containing (C) GH, (F) CGA, (I) ACTH, (L) LHB, (O) TSHB and total DAPI in each pituitary were counted for <i>Foxm1</i>^<i>-/-</i> embryos (KO) and wild type (WT) littermates. The KO ratio (hormone/DAPI) was then normalized to the WT ratio. Data are expressed as mean ± SEM of 4–6 littermate pairs for each age. The data were analyzed by Student <i>t</i>-test to determine significant difference between WT and KO (*<i>P</i> < 0.05).</p

No significant difference in M phase or G2 phase is apparent in <i>Foxm1</i>^<i>-/-</i> pituitary glands.

<p>Immunohistochemistry was performed on midsagittal pituitaries from <i>Foxm1</i>^<i>-/-</i> embryos (KO) and wild type littermates (WT) at embryonic day 10.5 (e10.5), e12.5, and e14.5 embryos to identify cellular proliferation. (A-F) Staining of phosphohistone H3 (pHH3, green) allows for differentiation between cells in M phase (brightly stained, white arrow) and G2 phase (dimly stained; red arrow). Cells in M phase, G2 phase and total cells per pituitary section were counted for each age and genotype. Graphs represent the ratio of cells in (G-I) M phase or (J-L) G2 phase to total DAPI (blue) counts per pituitary section. Data are expressed as mean ± SEM of four or five littermate pairs for each age. The data were analyzed by Student <i>t</i>-test to determine significant difference between WT and KO.</p

No significant difference in S phase is apparent in <i>Foxm1</i>^<i>-/-</i> pituitary glands.

<p>(A-F) BrdU (red) was used as a marker for cells in S phase and DAPI (blue) labels all cell nuclei. Scale bar represents 100 μm. (G-I) A ratio of BrdU-positive cells to total DAPI count per pituitary section was calculated for <i>Foxm1</i>^<i>-/-</i> embryos (KO) and wild type littermates (WT) at each age. Data are expressed as mean ± SEM of four or five littermate pairs for each age. The data were analyzed by Student <i>t</i>-test to determine significant difference between WT and KO. (J) The total number of cells per pituitary section was counted for embryonic day 10.5 (e10.5), e12.5, e14.5, e18.5 in <i>Foxm1</i>^<i>-/-</i> (KO) and wild type littermates (WT). Data are expressed as mean ± SEM of six littermate pairs for each age. The data were analyzed by Student <i>t</i>-test to determine significant difference between WT and KO. (K) Area of approximately three pituitary sections per individual was measured. Values are shown relative to WT littermate controls. Data are expressed as mean ± SEM of four littermate pairs for e12.5 and eight littermate pairs for e14.5 and e18.5. The data were analyzed by Student <i>t</i>-test to determine significant difference between WT and KO (*<i>P</i><0.05).</p

Expression of <i>Gh1</i> is reduced in <i>Foxm1</i>^<i>-/-</i> pituitary glands.

<p>Real time RT-PCR for the genes encoding growth hormone (<i>Gh1</i>), luteinizing hormone <b>β</b> (<i>Lhb</i>), the common α subunit (<i>Cga</i>), the precursor for ACTH (<i>Pomc</i>) and thyroid stimulating hormone (<i>Tshb</i>) was performed on RNA isolated from pituitary of e17.5 and e18.5 <i>Foxm1</i>^<i>-/-</i> embryos (KO) and wild type littermates (WT). Values were calculated using the ΔΔC_T method and normalized to WT. Data are expressed as mean ± SEM of 7 littermate pairs for each age and were analyzed by Student <i>t</i>-test to determine significant difference between WT and KO (*<i>P</i> < 0.05).</p

No significant difference in M phase or G2 phase is apparent in <i>Foxm1</i>^<i>-/-</i> pituitary glands.

<p>Immunohistochemistry was performed on midsagittal pituitaries from <i>Foxm1</i>^<i>-/-</i> embryos (KO) and wild type littermates (WT) at embryonic day 10.5 (e10.5), e12.5, and e14.5 embryos to identify cellular proliferation. (A-F) Staining of phosphohistone H3 (pHH3, green) allows for differentiation between cells in M phase (brightly stained, white arrow) and G2 phase (dimly stained; red arrow). Cells in M phase, G2 phase and total cells per pituitary section were counted for each age and genotype. Graphs represent the ratio of cells in (G-I) M phase or (J-L) G2 phase to total DAPI (blue) counts per pituitary section. Data are expressed as mean ± SEM of four or five littermate pairs for each age. The data were analyzed by Student <i>t</i>-test to determine significant difference between WT and KO.</p

FOXM1 is present in actively proliferating cells.

<p>FOXM1 (green) and a marker for cells in S phase, BrdU (red), were labeled in midsagittal pituitary sections from wild type e16.5 mouse embryos (A-C). FOXM1 (green) and a marker for non-cycling precursors, p57 (red), were labeled in midsagittal pituitary sections from wild type e14.5 mouse embryos (D-F). Scale bar represents 100 μm.</p