CART expression the pituitary hormones.

<p>Co-localization of CART with specific pituitary hormones was determined with a CART-specific antibody (green) and hormone specific antibodies (red) in sections from pituitary glands collected at P8. The hormone specific antibodies were TSHb (A), GH (B), ACTH (C), FSHb (D), LHb (E), and PRL (F). Co-localization is indicated with yellow arrows (A, B). In one CART immunostaining experiment all hormone antibodies were applied together and detected with the same fluorophore (G, H). A few CART positive cells do not express a detectable level of hormone (green arrow) in the <i>Pou1f1</i>^<i>+/+</i> pituitary (G), but almost all CART positive cells do not co-localize with any hormones in the somatotrope deficient <i>Pou1f1</i>^dw/dw anterior pituitary (H). The CART protein (red) does not co-localize with PROP1 (green) in the developing pituitary gland at e14.5 (I). Images A-I were taken at 400X and scale bar is 100 μm.</p

CART is expressed in the proliferating cells of the pituitary gland.

<p>Immunohistochemical staining was used to detect CART (red) and the proliferative markers Ki67 (green) or BrdU (green) in sections of pituitaries collect from <i>Pou1f1</i>^<i>+/+</i> and <i>Pou1f1</i>^dw/dw mice at P8. Co-localization is indicated by white arrows, and CART immunofluorescence in Ki67 or BrdU negative cells is indicated with arrow heads. There are many CART cells in both pituitary genotypes that do not co-localize with any proliferating cells (arrow heads). All images were taken at 400X, and scale bar is 100 μm.</p

CART is co-expressed with several cell specific transcription factors.

<p>Immunostaining with antibodies specific for CART (red, cytoplasmic) and lineage specific transcription factors (green, nuclear) was carried out on paraffin sections of pituitaries collected at P8 from <i>Pou1f1</i>^<i>+/+</i> mice (A, C, E) and <i>Pou1f1</i>^dw/dw mutants (B, D, F). The lineage specific transcription factors were POU1F1 (A, B), TPIT (C, D), and NR5A1 (E, F). All images were taken at 400X and scale bar is 100 μm.</p

CART is expressed throughout normal pituitary development.

<p>CART protein is not detected in e12.5 pituitary gland at e12.5 (A). PROP1 protein is detected in Rathke’s pouch of the e12.5 pituitary gland (B). CART protein was detected in paraffin sections of pituitary glands with a specific antibody (green fluorophor) in <i>Prop1</i>^<i>+/+</i> (C, E, G) and <i>Prop1</i>^<i>df/df</i> (D, F, H) anterior pituitary lobes at e14.5 (C, D), e16.5 (E, F), and P1 (G, H). At P8 CART is expressed abundantly in the anterior pituitary lobe (I), but it is absent in the <i>Prop1</i>^<i>df/df</i> anterior pituitary lobe (J). CART protein is present in the <i>Pou1f1</i>^<i>+/+</i> (normal) (K) and Pou1f1^<i>dw/dw</i> anterior lobe of the pituitary gland (L). Images A-F were taken at 200X and scale bar is 200 μm. Images G-L were taken at 100X and scale bar is 200 μm.</p

Cocaine-and Amphetamine Regulated Transcript (CART) Peptide Is Expressed in Precursor Cells and Somatotropes of the Mouse Pituitary Gland

<div><p>Cocaine-and Amphetamine Regulated Transcript (CART) peptide is expressed in the brain, endocrine and neuroendocrine systems and secreted into the serum. It is thought to play a role in regulation of hypothalamic pituitary functions. Here we report a spatial and temporal analysis of <i>Cart</i> expression in the pituitaries of adult and developing normal and mutant mice with hypopituitarism. We found that <i>Prop1</i> is not necessary for initiation of <i>Cart</i> expression in the fetal pituitary at e14.5, but it is required indirectly for maintenance of <i>Cart</i> expression in the postnatal anterior pituitary gland. <i>Pou1f1</i> deficiency has no effect on <i>Cart</i> expression before or after birth. There is no 1:1 correspondence between CART and any particular cell type. In neonates, CART is detected primarily in non-proliferating, POU1F1-positive cells. CART is also found in some cells that express TSH and GH suggesting a correspondence with committed progenitors of the POU1F1 lineage. In summary, we have characterized the normal temporal and cell specific expression of CART in mouse development and demonstrate that postnatal CART expression in the pituitary gland requires PROP1.</p></div

CART co-localizes with thyrotropes in the adult pituitary.

<p>Immunohistochemical staining was used to detect co-localization of CART (green) and hormones (red) in sections prepared from adult pituitary glands. The hormone specific antibodies were GH (A), PRL (B), TSHβ (C), LHβ (D), ACTH (E), and FSHβ (F). Representative cells (Inset panel C) exhibited co-localization of CART with TSHβ (yellow arrow), or lacked TSH expression (green arrow), and some TSHβ positive cells lacked CART staining (red arrow). All images were taken at 200X and scale bar is 100 μm. Inset of image C was taken at 400X and scale bar is 100 μm.</p

Cocaine-and Amphetamine Regulated Transcript (CART) Peptide Is Expressed in Precursor Cells and Somatotropes of the Mouse Pituitary Gland - Table 1

<p>Cocaine-and Amphetamine Regulated Transcript (CART) Peptide Is Expressed in Precursor Cells and Somatotropes of the Mouse Pituitary Gland</p> - Table

Elevated basal corticosterone levels in young adult <i>Prop1</i> deficient mice become higher in response to restraint stress.

<p>RIA analysis of circulating corticosterone was carried out on serum from 8 to 10 week males (A) and females (B) of segregating the <i>Prop1</i> null allele at N4 B6 prior to (white bars) and following restraint stress (black bars). Male <i>Prop1</i>^-/- (n = 6) had significantly elevated basal and post-stress levels of corticosterone compared to <i>Prop1^+/−</i> (n = 7) and <i>Prop1^+/+</i> (n = 3). Values represent the mean corticosterone (ng/mL of blood) ± SE. *, <i>P</i><0.0001. Female <i>Prop1^-/-</i> (n = 3) mice had both elevated basal and post-stress levels of corticosterone compared to <i>Prop1^+/−</i> (n = 5) and <i>Prop1^+/+</i> (n = 6). Values represent the mean corticosterone (ng/mL of blood) ± SE. *, <i>P</i><0.005.</p

<i>Prop1</i>-defiency results in low blood glucose levels.

<p>Blood glucose levels were measured in normal and <i>Prop1</i> mutant mice at four ages. (A) Basal glucose levels in 3.5 to 5 week <i>Prop1^-/-</i> mice (n = 6) were lower than <i>Prop1^+/+</i> (n = 10) and <i>Prop1^+/−</i> (n = 10) mice from mixed genetic backgrounds, but the difference was not statistically significant at this age. (B) On mixed genetic backgrounds the blood-glucose measurements from 5 to 6.5 wk old <i>Prop1^+/+</i> (n = 6) and <i>Prop1</i>^df/df (n = 6) were normal, but <i>Prop1</i>^df/- (n = 3), <i>Prop1^-/-</i> healthy (n = 12) and <i>Prop1^-/-</i> wasting (n = 7) mice had significantly decreased blood-glucose levels. Values represent the mean blood glucose levels (mg glucose/dL blood) ± SE. *, <i>P</i><0.01; **, <i>P</i><0.005. (C) The low glucose levels in mutants shown in panel B are associated with elevated corticosterone levels (ng corticosterone/ml blood +/− SE.) (D) Blood-glucose levels were measured in 8 to 10 week old mice of the N4 B6 background prior to (white bars) and following restraint stress (black bars). <i>Prop1^-/-</i> (n = 8) mice had decreased basal and post-stress blood-glucose levels compared to <i>Prop1^+/+</i> (n = 9) and <i>Prop1^+/−</i> (n = 11). Values represent the mean blood glucose levels (mg glucose/dL blood) ± SE. *, <i>P</i><0.0001; **, <i>P</i><0.0005. (E) Blood-glucose levels in 34 to 52 wk old mice on mixed genetic background were decreased in all genotypes of <i>Prop1</i> mutants, <i>Prop1^df/df</i> (n = 4), <i>Prop1^df/-</i> (n = 11), <i>Prop1^-/-</i> (n = 7), compared to normals, <i>Prop1^+/+</i> (n = 4). Values represent the mean blood glucose levels (mg glucose/dL blood) ± SE. *, <i>P</i><0.005; **, <i>P</i><0.0005; ***, <i>P</i><0.0001.</p

Adrenal glands of <i>Prop1</i> deficient mice are not hypotrophic.

<p>Adrenal glands were dissected from 5 and 8 week old female N4 B6 <i>Prop1^+/+</i> and <i>Prop1^-/-</i> mice, fixed, embedded, sectioned, and stained with hemotoxylin and eosin (Panels A, C, E, G) and immunostained for 20α-hydroxysteroid dehydrogenase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028355#pone.0028355-Hershkovitz1" target="_blank">[59]</a> and developed with diaminobenzidine (brown, Panels B, D, F, H) to visualize the X-zone (brackets). The ratio of adrenal weight to body weight (Panel I) was increased in <i>Prop1</i>^-/- (n = 5) compared to <i>Prop1^+/−</i> (n = 6) or <i>Prop1^+/+</i> (n = 3) N4 B6 male mice at 8 to 10 wks. Values represent the mean adrenal weight (mg) per body weight (g) ± SE. *, <i>P</i><0.0001; **, <i>P</i><0.0005.</p