The notch target gene HES1 regulates cell cycle inhibitor expression in the developing pituitary

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 We hypothesize that with the loss of Hes1, pituitary progenitors are no longer maintained in a proliferative state, choosing instead to exit the cell cycle.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.

Figure 1

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.

Figure 2

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.

Figure 3

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).

Figure 4

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.

Figure 5

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.

Figure 6

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).