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. 2009 Jun 18;150(9):4386–4394. doi: 10.1210/en.2009-0206

The Notch Target Gene Hes1 Regulates Cell Cycle Inhibitor Expression in the Developing Pituitary

Pamela Monahan 1, Sabina Rybak 1, Lori T Raetzman 1
PMCID: PMC2736073  PMID: 19541765

Abstract

The pituitary is an endocrine gland responsible for the release of hormones, which regulate growth, metabolism, and reproduction. Diseases such as hypopituitarism or pituitary adenomas are able to disrupt pituitary function leading to suboptimal function of the entire endocrine system. Growth of the pituitary during development and adulthood is a tightly regulated process. Hairy and enhancer of split (HES1), a transcription factor whose expression is initiated by the Notch signaling pathway, is a repressor of cell cycle inhibitors. We hypothesize that with the loss of Hes1, pituitary progenitors are no longer maintained in a proliferative state, choosing instead to exit the cell cycle. To test this hypothesis, we examined the expression of cell cycle regulators in wild-type and Hes1-deficient pituitaries. Our studies indicate that in early pituitary development [embryonic day (e) 10.5], cells contained in the Rathke’s pouch of Hes1 mutants have decreased proliferation, indicated by changes in phosphohistone H3 expression. Furthermore, pituitaries lacking Hes1 have increased cell cycle exit, shown by significant increases in the cyclin-dependent kinase inhibitors, p27 and p57, from e10.5 to e14.5. Additionally, Hes1 mutant pituitaries have ectopic expression of p21 in Rathke’s pouch progenitors, an area coincident with increased cell death. These observations taken together indicate a role for HES1 in the control of cell cycle exit and in mediating the balance between proliferation and differentiation, allowing for the properly timed emergence of hormone secreting cell types.

HES1, a target of Notch signaling, controls pituitary cell division and inhibits expression of the cell cycle inhibitors p27, p57, and p21 during embryonic development.

Properly timed and tightly regulated development of the pituitary gland is critical to the function of the adult endocrine system. Situated in the head and in intimate contact with the hypothalamus, the pituitary’s origin lies both in neural and oral ectoderm. Beginning at embryonic day 9.5 (e9.5), the pituitary begins as an invagination in the oral ectoderm. As the embryo develops, the rudimentary pituitary pinches off from the underlying ectoderm to form the developmental structure called Rathke’s pouch (RP). Within this structure reside the pituitary progenitor cells, which are a highly proliferative cell population. As progenitors exit the cell cycle, they differentiate into the hormone secreting cells in the ventral aspect of the developing pituitary (1,2,3).

Pituitary organogenesis involves the interplay of various signaling pathways. Early in development, Sonic Hedgehog proteins emanating from the oral ectoderm signal to the surrounding tissue leading to pituitary induction (4). Fibroblast growth factor (FGF)-8 and FGF10 from the infundibulum have been found to promote cell proliferation in the early pituitary and contribute to cell specification. Bone morphogenetic protein (BMP)-2 and BMP7 signaling originating from the ventral juxtapituitary mesenchyme, contribute to early cell fate selection between thyrotrope and corticotrope lineages (5,6). The integration of FGF and BMP actions culminate in dorsal-ventral delineation of the developing gland. Additionally, the Wnt signaling molecules Wnt4 and Wnt5a are expressed in the ventral diencephalon and within RP. Wnt4 mutants display a slightly hypomorphic pituitary, indicating its role in proliferation of the early pituitary (7,8,9). Yet the question remains, how do progenitors present in early development integrate these extrinsic signals to control proliferation and differentiation to form a gland containing the proper number of cells?

Recent studies postulated that Notch signaling in the pituitary maintains a proliferative zone of cells lining RP (10,11). Notch signaling begins with the binding of the transmembrane Notch receptor with its ligand Delta-like/Jagged. After binding, the Notch receptor’s intracellular domain is cleaved, allowing the Notch receptor’s intracellular domain to translocate to the nucleus, which, along with a coactivator complex drives transcription of factors that determine cell fate (12,13). Notch 2 and 3 receptors, Delta-like 1 ligands, and the Hes and Hey genes are present in the pituitary beginning around e9.5, but expression begins to wane around day e13.5–14.5 (14). As the pituitary matures, cells exit the progenitor state and migrate ventrally before fully differentiating. These maturing cells no longer express Notch receptors and factors necessary for lineage specification such as pituitary transcription factor-1 (Pit1) and steroidogenic factor 1 (SF1) are up-regulated (15,16).

The Notch target gene, Hes1, encodes a basic helix-loop-helix transcriptional repressor that is necessary to maintain progenitor cell populations in various endocrine organ systems such as the pancreas and intestines. Compared with wild type (WT), Hes1 mutant pituitaries are hypomorphic, with reductions in all hormone cell types. Additionally, Hes1 mutants lack the αMSH-producing cells that are found in the late stages of pituitary development (11,17,18). These phenotypes together indicate the necessity for Hes1 in controlling cell number and cell specification in the pituitary. Yet what mechanisms does Notch signaling use to control these vital developmental events?

Regulation of the cell cycle is a key component in cell fate determination and organ size. Evidence suggests that Notch signaling may control progenitor differentiation by this mechanism. Several studies have shown that Hairy and enhancer of split (HES1) can bind promoter regions of the cyclin-dependent kinase inhibitors (CDKIs) of the Cip/Kip family (p21, p27, and p57) and repress their expression (19,20,21). CDKIs modulate cell cycle progression by binding to the cyclin/cyclin-dependent kinase complex, preventing cells from transitioning into the DNA synthesis phase. This action directs cells to a quiescent state in which differentiation can potentially occur. Levels of CDKIs are tightly regulated and pituitary cell numbers are sensitive to alterations in CDKI expression and activity. Mutation of p27 predisposes rodents and humans to develop endocrine tumors. Mice lacking p27 are prone to pituitary tumors of the intermediate lobe, an area that is the vestige of RP (22,23,24). In humans, the loss of p27 results in multiple endocrine neoplasia whose symptoms include a predisposition to form pituitary adenomas (25). These data indicate that tight control of p27 during pituitary formation and in adulthood is necessary to prevent pituitary overgrowth. Unlike p27, p21 mutant mice do not form pituitary tumors or have noticeable developmental abnormalities. Yet when eliminated in conjunction with other tumor suppressor genes, such as p18 or Rb, double-mutant mice display decreased pituitary tumor latency and larger intermediate lobe cell tumors (26,27).

Mutation studies of each of the Cip/Kip family members have shown the necessity for the proper regulation of CDKIs for repression of cellular overgrowth. We predict that in the developing pituitary, Notch signaling through HES1 regulates cell number by controlling the decision between progenitor cell maintenance and differentiation through regulation of CDKIs. By analyzing key regulators of proliferation and cell cycle exit throughout development, we show that in Hes1-deficient mice, the phenotype of a hypomorphic pituitary is the result of decreased proliferation at early ages as well as an increase in cell cycle exit.

Materials and Methods

Mice and embryo collection

Hes1 mutant mice were previously generated by replacing the first three exons with a neomycin-resistance cassette (28). A breeding colony was generated at the University of Illinois at Urbana-Champaign and maintained on a mixed genetic background of C57BL/6J and CD1. The University of Illinois Institutional Animal Care and Use Committee approved all procedures involving mice. Heterozygous males and females were mated to generate mixed genotype litters. Mice were genotyped as previously described (29). Embryos were collected at e10.5 through e16.5 and fixed in 3.8% formaldehyde solution (Fisher, Pittsburg, PA) in PBS. Embryos were dehydrated through a graded series of ethanol and placed in paraffin for sectioning. Sections measuring 6 μm thick were then affixed to positively charged slides. For bromodeoxyuridine (BrdU) experiments, animals were treated as previously published (18).

Immunohistochemistry

Embryo sections affixed to slides were deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol, and washed in PBS solution. Slides were subjected to antigen retrieval using 0.01 m citrate buffer (pH 6.0) for 10 min for samples treated with anti-p57, anti-Ki67, anti-BrdU, and antiphosphohistone H3 (PH3). All slides were blocked for 10 min using 5% normal donkey serum (Jackson ImmunoResearch, West Grove, PA) in an immunohistochemistry blocking solution containing 5% BSA, 0.1% Triton X-100, and PBS. Primary antibodies were diluted in IHC blocking solution at various dilutions: rabbit anti-cyclin D2 (M-20: sc-593; Santa Cruz Biotechnology, Santa Cruz, CA), 1:250; rabbit anti-p27 (C-19: sc-528; Santa Cruz Biotechnology), 1:250; mouse anti-p57 [Ab-3 (clone KP39) Neomarkers, Fremont, CA], 1:750; rabbit anti-PH3 (Ser 10, no. 06-570; Upstate Cell Signaling Solutions, Lake Placid, NY), 1:300; rat anti-Ki67 (Dako, Carpinteria, CA), 1:100; mouse anti-BrdU (no. 555627; BD PharMingen, San Diego, CA), 1:50; mouse anti-p21 (no. 556431; BD PharMingen), 1:200; rabbit anti-glycoprotein hormone α-subunit (αGSU; National Hormone and Peptide Program, Torrance, CA), 1:1500; and rabbit anti-ACTH (Dako), 1:1500. Donkey-derived mouse and rabbit secondary antibodies conjugated to biotin (Jackson ImmunoResearch) were diluted to 1:200 and incubated with sections for 1 h. Slides were then incubated with tertiary antibodies, streptavidin conjugated to either cy2 or cy3 fluorophore (Jackson ImmunoResearch) for 1 h. Ki67 was detected with a secondary rat antibody conjugated to the fluorophore tetramethyl rhodamine isothiocyanate (TRITC). Cell death was assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) as previously described (19). All sides were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma, St. Louis, MO; 28718-90-3) at 1:1000 (stock 1 mg/ml) and mounted using aqueous fluorescence mounting media. Samples were then visualized at ×200 magnification using a DM 2560 microscope (Leica, Wetzlar, Germany) and images were obtained using Q Capture Pro software (QImaging, Surrey, British Columbia, Canada) and processed using Photoshop software (Adobe, San Jose, CA).

Cell count quantification: PH3, p21, and TUNEL

Slides containing midsagittal sections from e10.5 and e11.5 WT and Hes1 mutant embryos were stained and imaged as previously described. Images were taken at ×200 magnification. Cells that were solid in nature were counted. A DAPI counterstaining was used to obtain an overall cell count for the whole 6-μm-thick pituitary section. For each embryo at e10.5, 100–200 cells were examined and at e11.5, 200–300 cells for at least three embryos for each genotype. The proportion of immunoreactive cells was compared with the total number of DAPI-positive cells contained within RP. A percentage of positive cells per pituitary section was determined and then tested for statistical significance using a Student’s t test (SAS 9.1 software; SAS Institute, Cary, NC).

Immunostaining intensity quantification

Slides containing e14.5 embryo sections were stained for the presence of p27 or p57 proteins and visualized using previously mentioned methods. At least three separate embryos and two sections per embryo were processed and analyzed in parallel. Images of the anterior lobe of the pituitary at ×400 magnification were converted to gray scale and contrasted to 90% using Adobe Photoshop. These images were then processed through Image J software (National Institutes of Health, Bethesda, MD) to provide for a gradation of pixel intensity (red, dark orange, light orange, and yellow, denoting positive signal for either p27 or p57 protein) by pseudocolor appointment to intensity within the image. Pseudocolored images were then again processed in Adobe Photoshop to provide pixel counts for positive signal. Positive pixel counts were then divided by total pixel amounts for the image (120,000), resulting in a percentage of positive signal per section of anterior lobe of pituitary denoted by relative absorbance units similar to previously published protocols (30). Percentages were then tested for statistical significance using a Student t test (SAS 9.1 software).

Results

RP progenitors have decreased proliferation in Hes1 mutants

Hes1 mutant pituitaries are smaller than WT pituitaries at e18.5. We hypothesized that Hes1 is necessary to maintain progenitor proliferation during early embryonic development to generate sufficient quantities of cells to populate the e18.5 pituitary. At e10.5, the majority of RP cells are highly proliferative progenitor cells. Pituitaries were first analyzed with markers of the cell cycle, such as Ki67, which marks cells in all active phases of the cell cycle. In e10.5 pituitaries, Ki67 is present throughout RP of both WT (Fig. 1A) and Hes1 mutants (Fig. 1F). Cyclin D2 is a protein required for the G1 to S transition. Without cyclin proteins, cells are unable to enter the synthesis phase and will exit the cell cycle. The majority of cells in the pituitary express cyclin D2 in a similar pattern in both WT (Fig. 1B) and Hes1 mutant embryos (Fig. 1G). BrdU incorporation indicates active DNA synthesis during the S phase of the cell cycle. BrdU labeling in WT pituitaries shows active DNA synthesis throughout RP (Fig. 1C). Hes1 mutants have a decrease in BrdU-positive cells in the caudal region of RP (Fig. 1H), similar to previously published data (11,17,18). To clarify whether cells are progressing, although the cell cycle and entering into the mitosis and G2 phase, we used PH3 labeling. WT embryos have a mean percentage of 6.50 ± 0.964% (n = 5) of mitosing progenitors in RP (Fig. 1D), whereas Hes1 mutant pituitaries (Fig. 1I) have significantly fewer mitosing cells (mean percentage 4.77 ± 0.890%, n = 5, P ≤ 0.0092). Although cells in the pituitaries of both genotypes are actively in the G1 stage, there is a lack of progression to the next stages of the cell cycle in Hes1 mutants.

Figure 1.

Figure 1

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Hes1 is necessary for appropriate cell cycle progression. Midsagittal sections at e10.5 were immunostained with Ki67 (A), which marks actively proliferating cells in RP (bracket) progenitor cells throughout WT and Hes1 mutant pituitaries (F). Cyclin D2 expression, marking G1 phase of the cell cycle, is found throughout WT (B) and Hes1 mutant RPs (G). As RP progenitors progress to S phase, as visualized by BrdU incorporation and immunostaining, cells are not readily present in the caudal section of the Hes1 mutant pituitary (H), unlike WT RP (C). PH3 marks the cells progressing into the G2 and M phase. Hes1 mutant pituitaries (I, arrows) have significantly fewer cells entering into these later stages than their WT littermates (D). WT pituitaries have few cells expressing p57 that are present in the dorsal region of RP (E), whereas Hes1 mutant pituitary cells express p57 in the rostral region (J). Scale bar, 50 μm. c, Caudal; d, dorsal; r, rostral; v, ventral for all images.

Instead of progressing through the cell cycle, cells may enter a quiescent phase, a critical decision marked by the increase in CDKIs. HES1 has been shown to transcriptionally repress the CIP/KIP family of inhibitors, specifically p27 and p57 (20,21). Consequently, increased expression of CDKIs may have led to the decrease in mitosis that was observed in Hes1 mutants. p57 expression in WT pituitaries is restricted to the dorsal aspect of the pituitary, with few cells extending ventrally down RP (Fig. 1E). In Hes1 mutant pituitaries, the presence of p57-expressing cells can be found throughout RP (Fig. 1J) with similar findings in p27 expression (data not shown). Taken together, these data show that Hes1 mutants at e10.5 have decreased progression through the cell cycle that could be attributed to cells being signaled to exit the cell cycle.

Pituitary progenitor proliferation is altered after pituitary induction in Hes1 mutants

By e11.5, the pituitary is distinct from the underlying oral ectoderm. At this age, cells contained within RP still exhibit characteristics of progenitors: highly proliferative with no evidence of differentiation into hormone-producing cells. Cells in G1 phase are apparent in the majority of RP, although a cohort of cells toward the ventral region are not actively expressing cyclin D2 in WT pituitaries (Fig. 2A). This observation is also consistent in the Hes1 mutant pituitaries (Fig. 2E). Cells entering into the mitosis phase of the cell cycle remain restricted to the inner lumen of RP in both WT (Fig. 2B) and Hes1 mutant pituitaries (Fig. 2F). By e11.5 there are no apparent changes in proliferation based on numbers of cycling cells, like that of e10.5, but relative numbers may be changed due to prior pituitary progenitor loss through cell death or reduced proliferation at e10.5.

Figure 2.

Figure 2

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Increased cell cycle exit is evident in Hes1 mutant pituitaries. Sagittal sections at e11.5 were stained with cell cycle markers. Cyclin D2 expression is found in RP progenitors in both WT (A) and Hes1 mutant (E) pituitaries, whereas a small population in the ventral aspect lack cyclin D2 in both genotypes (arrows). PH3 expression is found in cells lining the lumen of RP in both the WT (B) and Hes1 mutant (F) RP. p27, a cell cycle inhibitor, is found throughout RP in WT (C) and Hes1 (G) pituitaries. p57 expression is found in few cells throughout RP in WT pituitaries (D, arrows), whereas Hes1 mutants (H, arrows) have an increase in cells expressing p57 in the ventral region of RP, the area of the future anterior lobe.

Although we observed no overall change in the proportion of cells undergoing proliferation, there is still evidence for cell cycle alteration, as reflected in the expression patterns of the CDKIs. p27 expression in both WT (Fig. 2C) and Hes1 mutant (Fig. 2G) pituitaries show a dispersed pattern of expression in midsagittal sections of RP, with an increased concentration of cells expressing CDKIs in the ventral region of RP when the pouch is in close contact with the oral ectoderm. p57 expression reiterates the observation that cells of the Hes1 mutant are readily exiting the cell cycle and progressing toward differentiation. In WT pituitaries, few cells express p57, with positive cells found in the periphery of RP (Fig. 2D). Hes1 mutant pituitaries, however, display an increase in p57-positive cells, localized ventrally (Fig. 2H).

These data at e10.5 and e11.5 paint a picture of early pituitary progenitor exit from the cell cycle, possibly providing a mechanism for development of the hypomorphic pituitary seen in Hes1 mutants.

Cell cycle inhibitor expression is affected by loss of Hes1

As the embryo ages, hormone producing cell types begin to emerge in the spatially restricted anterior lobe. Proliferation at e14.5, as seen by cyclin D2-positive cells, is restricted to the dorsal aspect of RP and few cells detectable in the expanding anterior lobe (Fig. 3A). Hes1 mutants similarly show the restriction of cyclinD2 expression to the dorsal part of RP (Fig. 3D). With this decrease in proliferation, there is an increase in cells expressing CDKIs, mainly p27 and p57. Both p27 and p57 protein expression in WT tissues are concentrated away from the proliferative zone and extend into the anterior lobe in which hormone-producing cells have begun to emerge (Fig. 3, B and C). Hes1 mutant pituitaries have a larger population of cells that express these inhibitors within the developing anterior lobe (Fig. 3, E and F). Further supporting this theory, NIH Image J quantification revealed a significant increase of the intensity of p57 expression in Hes1 mutants anterior pituitaries compared with WT (Fig. 3G). Furthermore, p27 expression intensity in Hes1 mutant pituitaries was also higher than WT tissues (Fig. 3H). These data indicate that pituitary progenitors, in the absence of Hes1, are signaled to exit the cell cycle before a proper pituitary size is achieved.

Figure 3.

Figure 3

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Hes1 mutants have significant increases in cell cycle inhibitor expression in the developing anterior lobe at e14.5. In e14.5 pituitaries, many cells are actively in the G1 phase of the cell cycle, as marked by cyclin D2 expression. These cells can be found in RP but rarely in the developing anterior lobe (AL) of WT (A) and Hes1 mutant pituitaries (D). p27 expression in WT pituitaries (B) is found in few cells throughout both RP and anterior lobe cells. Hes1 mutant pituitaries (E) have significantly more cells in the anterior lobe expressing p27, compared with WT pituitaries. p57 expression in WT pituitaries (C) is mainly concentrated in RP cells with few found in the developing anterior lobe. Hes1 mutants (F), on the other hand, have a significant increase in p57 expressing cells in the AL. G, Image J signal quantification of p57 reveals a significant (P ≤ 0.001) increase in mean signal strength between WT (2.335, n = 3) and Hes1 mutants (5.087, n = 3) (denoted by asterisk). H, Significant (P ≤ 0.001) p27 increase between WT (12.665, n = 3) and Hes1 mutant (18.280, n = 3) (denoted by asterisk).

Few hormone-producing cell types at e14.5 show colocalization with cell cycle inhibitors

At e14.5, fully differentiated hormone-producing cell types are present in the anterior lobe. We have already shown that p27 and p57 expression is increased in Hes1 mutant pituitaries at this age. Yet we questioned whether cells expressing CDKIs have begun to express hormones. Cells expressing the CDKI, p27 (solid arrow, pink), colocalize with few cells also expressing the hormone ACTH (open arrow, hormone in green) in the anterior lobe of both WT (Fig. 4, A and A′) and Hes1 mutant pituitaries (Fig. 4E). Although there appears to be more p27 and αGSU colocalization in WT (Fig. 4, B and B′) and Hes1 mutant pituitaries (Fig. 4F), the majority of the overlap is localized in the rostral tip thyrotrope lineage (denoted by bracket), a lineage of undetermined origin that does not secrete hormone. ACTH colocalization with p57 also reveals no overlap in both WT (Fig. 4, C and C′) and Hes1 mutant pituitaries (Fig. 4G). Again, colocalization of p57 and αGSU shows that the vast majority of overlapping expression patterns are found in cells of the rostral tip thyrotrope lineage in WT (Fig. 4, D and D′) and Hes1 mutant pituitaries (Fig. 4H). Taken together, this may indicate that although Hes1 mutant pituitaries have a significant increase in the amount of cells expressing cell cycle inhibitors, there does not seem to be an acceleration of these exiting cells to form fully differentiated cells.

Figure 4.

Figure 4

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The cell cycle inhibitors p27 and p57 localize with few fully differentiated cells at e14.5. By e14.5, the hormone-producing cells (αGSU, ACTH) that reside in the developing anterior lobe are evident. The WT AL (A′) with p27 expression (solid arrows, pink) overlaid with ACTH-producing cell types shows few cells that colocalize (open arrows, green), expression that is comparable with Hes1 mutant ALs (E). This expression patterned is also reflected in αGSU hormone overlays, with few cells expressing both p27 and hormone in WT (B′) and Hes1 mutants (F, brackets mark rostral tip thyrotropes, which do contain CDKI and αGSU). p57 expression (solid arrows, pink) and ACTH do not colocalize (green) at e14.5 in WT (C′) Hes1 mutants (G), whereas few cells in the anterior lobe colocalize both p57 and αGSU in WT (D′) and Hes1 mutants (H, brackets mark rostral tip thyrotropes). A–D indicate magnified images from WT anterior lobes that were taken for A′–D′. Magnified images from Hes1 mutants (E–H) were taken from the same location as WT. Scale bar, 50 μm.

Pituitary progenitor proliferation wanes in e16.5 embryos

By e16.5, the remnant of RP begins to differentiate into melanotropes. Few cells in the WT RP are proliferating, as seen by cyclin D2 and PH3 expression (Fig. 5, A and B). Similar expression patterns are observed in the Hes1 mutant RP (Fig. 5, E and F). The inhibitors, p27 and p57, also show a decrease in expression in WT (Fig. 5, C and D) and Hes1 mutant pituitaries (Fig. 5, G and H). Only a few p27-expressing cells colocalize with the hormone producing cells, αGSU, and ACTH (supplemental Fig. 1). By this age it is clear that without the action of HES1 to repress CDKI activity, a hypomorphic pituitary can develop due to the loss of early pituitary progenitors.

Figure 5.

Figure 5

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The cell cycle does not appear altered in Hes1 mutants at e16.5. Cyclin D2 expression is found in the remnant of RP with few cells in the developing anterior lobe in the WT (A) and Hes1 mutant (E). Few cells are actively undergoing mitosis, as seen by PH3 expression (marked by arrows) in WT (B) and Hes1 mutants (F). p27 expression is relegated to few cells in the remnant of RP and in the developing anterior lobe in WT (C) and Hes1 mutant (G) pituitaries. p57 expression is reduced, with few cells present in the anterior lobe of both the WT (D) and Hes1 mutant pituitaries.

Ectopic p21 expression is seen in RP during early development in Hes1 mutants

HES1 can repress transcription of p21 (19), so we examined p21 expression during pituitary ontogeny. In WT pituitaries at e10.5, a small cohort of p21-expressing cells can be found at the interface between RP and the surrounding oral ectoderm (Fig. 6B, arrow). Interestingly, this expression correlates with the area in which cell death occurs (Fig. 6A, arrow). By e11.5 and through the rest of embryonic development, p21 expression is undetectable in midsagittal sections of the pituitary (Fig. 6D and data not shown), whereas cell death is restricted to the underlying oral ectoderm (Fig. 6C, arrow). Hes1 mutant pituitaries at e10.5 show a significant increase in cells expressing p21 compared with WT, although cells are not restricted to the interface between pituitary and oral ectoderm and instead can be found extending dorsally into RP (Fig. 6F, bracket, and quantification table). Additionally, Hes1 mutant pituitaries have a significant increase in the amount of cell death present in RP at e10.5 compared with WT (Fig. 6E, arrows, and quantification table). At e11.5, ectopic p21 expression is evident with cells concentrated on the caudal side of RP in Hes1 mutants (Fig. 6H) with quantification revealing a significant increase of p21 containing cells in Hes1 mutants compared with WT (Fig. 6, quantification table). Additionally, cell death is present in RP even at e11.5 in Hes1 mutants (Fig. 6G and quantification table). By the next day and throughout the rest of development, however, p21 expression and cell death are no longer detected in either genotype (data not shown). With the increase in p21 expression and an increase in apoptosis, the pituitary progenitor population early on may be significantly reduced and, with additional misregulation of other CDKIs, could lead to the hypomorphic pituitary seen in Hes1 mutant mice at e18.5.

Figure 6.

Figure 6

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Ectopic p21 expression is found in Hes1 mutant pituitaries. At e10.5, WT pituitaries (A, arrow) exhibit cells undergoing death at the junction of oral ectoderm, as assayed by TUNEL staining. These cells also appear to be the population that expresses p21 (B, arrow). In Hes1 mutants (E), there is an increase in cell death within RP (marked with arrows). This correlates with increased expression of p21 extending into RP (F, bracket). At e11.5 (C, arrow) cell death is mostly restricted to the underlying oral ectoderm in the WT pituitary but is still detectable in the Hes1 mutant RP (G, arrows). Increased p21 expression is still evident in the RP of Hes1 mutant pituitaries (Fig. 6H and lower panel), whereas WT pituitaries have little to no p21 expression at e11.5 and through the rest of development (Fig. 6D, data not shown, and quantification table).

Discussion

The maintenance of pituitary progenitors is essential to the development of a pituitary containing an optimal number of hormone secreting cells that control growth, fertility, and metabolism. Proliferation of RP progenitors generates the population of cells that will comprise the functioning adult anterior lobe.

Alterations in signaling factors, such as Sonic Hedgehog, FGF, and BMP, that control proliferation of pituitary progenitors, can lead to disorders of reduced pituitary function. The Notch signaling pathway has also been shown to be critical in the maintenance of pituitary progenitors through the action of the transcriptional repressor HES1 (11,17,18). We now show that in the absence of Hes1, pituitary progenitors have decreased proliferation and increased expression of the cell cycle inhibitors, p27, p57, and p21, which leads to early cell cycle exit and depletion of the progenitor pool. In addition, Hes1 mutants have increased cell death at e10.5 and e11.5, a pattern not seen in WT pituitaries. These data indicate that Notch signaling control over progenitor proliferation and cell death is a critical regulator of pituitary cell number (model, supplemental Fig. 2).

HES1 is normally found in RP progenitor cells. Additionally, Notch receptors, ligands, and downstream molecules have been found in putative stem cell populations in the adult pituitary (31). As differentiation begins, Hes1 expression wanes and cells migrate ventrally to form the anterior lobe (10,11,18). Pituitaries of mice lacking Hes1 have decreased numbers of proliferative progenitors. Although the RP contains many cells that are actively in the G1 phase of the cell cycle, there are reduced numbers of cells in S phase and undergoing mitosis, which are important for replenishing the progenitor pool. The decrease in proliferation that we see in Hes1 mutant pituitaries indicates that progenitor proliferation is impaired by the G1-S phase check point, instead shuttling cells into cell cycle exit.

As progenitors differentiate, cells must first exit from a proliferative state. This transition is mediated by the actions of a class of CDKIs of the Cip/Kip family: p21, p27, and p57. As these proteins are up-regulated, they inhibit cyclin/cyclin-dependent kinase complexes, causing cells to cease proliferation and enter into a quiescent state. Recent p27/p57 double-mutant studies revealed that in the absence of these inhibitors, there is continued unchecked proliferation of pituitary progenitors (32). HES1 has been shown to transcriptionally repress the inhibitor p27. Hes1-deficient mice have increased expression of p27 in brain, liver, and thymus tissues during development. Additionally, repression of endogenous Hes1 expression in embryonic carcinoma cells results in enhanced p27 expression followed by cell cycle arrest (21). Interestingly, adult p27 mutant animals have a preponderance to form pituitary tumors, indicating that tight control of p27 throughout development is critical to the maintenance of proper pituitary size (22,23,24). In the pituitary, p27 expression indicates that progenitors must exit the cell cycle to fully differentiate. Hes1 mutant mice have increased numbers of cells that express p27 in the developing anterior lobe, indicating that RP progenitors without HES1 exit the cell cycle in increasing numbers.

HES1 has also been shown to directly modulate the transcription of p57 during progenitor maintenance. In Hes1-deficient mice, there is a significant increase in the expression (Pdx1) of p57 in the pancreatic duodenal homeobox-1-expressing progenitor cells in the dorsal bud of the developing pancreas. This indicates that the absence of Hes1 in pancreatic progenitors causes an increase in p57, which shifts cells from the progenitor state to cell cycle arrest (20). Furthermore, studies in intestinal crypt progenitors show that inactivation of the Notch receptor and decreased levels of Hes1 are accompanied by an increase in cells expressing both p57 and p27, resulting in a loss of progenitor maintenance (33). Similarly, in the Hes1-deficient pituitary, the reduced number of proliferative progenitors correlates with a significant increase in p57-expressing cells located ventrally in the developing anterior lobe throughout development. The critical role p57 plays in the maintenance of proper pituitary size is further highlighted in recent studies that show p57 mutant pituitaries are hyperplastic during development, whereas p57 overexpression produces a profound reduction in pituitary size (32).

One striking finding from these studies is that in both WT and Hes1 mutant pituitaries, p27 and p57 colocalize with few hormone producing cell types (αGSU and ACTH). These data indicate that for differentiation to occur, cells must first exit the cell cycle, as indicated by CDKI expression, and likely must turn off CDKI expression to and progress into the final stages of differentiation. This up-regulation of p27 and p57 in the Hes1 mutant anterior lobe at e14.5 may explain why we do not observe premature differentiation of cells when Hes1 alone is lost (29). Expression of CDKIs in anterior lobe cells may also indicate a mechanism to prevent cell lineage expansion in prenatal development. In support of this theory, p27 mutant mice have ACTH-positive cells that colocalize with Ki-67, indicating prenatal proliferation of differentiated cells, something that is never detected in WT pituitaries (32). As animals enter into postnatal development, a period of lineage specific proliferation occurs (34,35,36,37,38), and disruption of this expansion during later development can perturb proper pituitary cell numbers. Studies show that mice mutant for Prop1 and Pit1 have hypomorphic pituitaries that can be attributed to reduced proliferation of anterior lobe cells (39,40). Taken together these data reiterate the need to control proliferation during precise periods in both prenatal and postnatal development to maintain a proper pituitary cell number.

Another mechanism to restrict the progenitor pool is to limit progenitor expansion through programmed cell death. At e10.5, pituitaries have a small population of cells at the periphery of RP that undergo apoptosis. Interestingly, this incidence of cell death is marked by the presence of p21, a factor that is under direct transcriptional control by Notch signaling (41). In the absence of HES1 repression, p21 expression is detected in a greater proportion of cells in concert with an increase in cell death. These data may indicate that in the pituitary, p21 mediates cell death in response to the loss of HES1. This could be through inducing inappropriate cell cycle arrest, leading to death or through a more direct mechanism. For example, overexpression of p21 in thymocytes leads to a hypersensitivity to p53-dependent cell death in response to radiation (42). Alternatively, like p27 and p57, p21 may also cause progenitor cell cycle exit coupled with differentiation. Studies have shown that by blocking HES1 action, p21 is induced, leading to γ-aminobutyric acid differentiation of neural stem cells (43). Additionally, in the Pttg/securin knockout animals, p21 expression is induced in the adult pituitary, and it serves to reduce proliferation and attenuate tumor formation (44). Our current studies point toward a role for progenitor limitation by cell cycle exit and cell death early in pituitary induction. This indicates that tight control of progenitor proliferation is necessary to control pituitary size.

Although the role of Hes1 in progenitor expansion has been studied extensively, its role in progenitor differentiation remains to be clarified. Studies have shown that constitutive expression of Hes1 can prevent αGSU and TSH expression, indicating that Hes1 is needed to prevent cellular differentiation (18). Additionally, when the transcription factor Prop1 is mutated in Hes1 mutant pituitaries, progenitors are found to prematurely differentiate within RP (29). These data, coupled with studies that have shown that Hes1 is necessary to specify melanotrope cell fate (18), indicate that the actions of HES1 on pituitary development include not only early events that govern progenitor expansion but may also include late events that determine hormone cell fate.

The most significant changes in Hes1 mutant pituitaries seen here is the misregulation of CDKIs. Indeed, it has been shown that the loss of p27 in both rats and humans results in multiple endocrine neoplasia with a high prevalence of pituitary tumors (25). These data suggest that regulation of pituitary proliferation by CDKIs is essential to prevent tumor formation. HES1 expression in the developing pituitary maintains progenitors in a proliferative, undifferentiated state by transcriptionally repressing CDKIs, preventing cells from exiting the cell cycle and preventing progenitor death. It is tempting to speculate that misregulation of Notch signaling may also contribute to pituitary tumor formation. Interestingly, Notch molecules have been found to be up-regulated during pituitary tumor development, reiterating that Notch molecules are needed to maintain cells in a proliferative state (45,46). These studies provide evidence that Notch signaling is a critical regulator of pituitary organogenesis through progenitor maintenance and may modulate organ size in the adult organ.

Supplementary Material

[Supplemental Data]
en.2009-0206_index.html (1.5KB, html)

Acknowledgments

We thank Tyler Moran and Ashley Himes, who provided critical review of the manuscript, and to Ryoichiro Kageyama for supplying the Hes1 mutant mice.

Footnotes

This work was supported by Grant R01 DK076647 from the National Institutes of Health (to L.T.R).

Disclosure Summary: The authors have nothing to disclose.

First Published Online June 18, 2009

Abbreviations: BMP, Bone morphogenetic protein; BrdU, bromodeoxyuridine; CDKI, cyclin-dependent kinase inhibitor; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; e9.5, embryonic day 9.5; FGF, fibroblast growth factor; αGSU, glycoprotein hormone α-subunit; HES1, hairy and enhancer of split; PH3, phosphohistone H3; RP, Rathke’s pouch; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling; WT, wild type.

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Supplementary Materials

[Supplemental Data]
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en.2009-0206_1.pdf (119.5KB, pdf)
en.2009-0206_2.pdf (405.5KB, pdf)

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