Distinct developmental roles of cell cycle inhibitors p57Kip2 and p27Kip1 distinguish pituitary progenitor cell cycle exit from cell cycle reentry of differentiated cells

Abstract

Patterning and differentiation signals are often believed to drive the developmental program, including cell cycle exit of proliferating progenitors. Taking advantage of the spatial and temporal separation of proliferating and differentiated cells within the developing anterior pituitary gland, we investigated the control of cell proliferation during organogenesis. Thus, we identified a population of noncycling precursors that are uniquely marked by expression of the cell cycle inhibitor p57(Kip2) and by cyclin E. In p57(Kip2-/-) mice, the developing pituitary is hyperplastic due to accumulation of proliferating progenitors, whereas overexpression of p57(Kip2) leads to hypoplasia. p57(Kip2)-dependent cell cycle exit is not required for differentiation, and conversely, blockade of cell differentiation, as achieved in Tpit(-/-) pituitaries, does not prevent cell cycle exit but rather leads to accumulation of p57(Kip2)-positive precursors. Upon differentiation, p57(Kip2) is replaced by p27(Kip1). Accordingly, proliferating differentiated cells are readily detected in p27(Kip1-/-) pituitaries but not in wild-type or p57(Kip2-/-) pituitaries. Strikingly, all cells of p57(Kip2-/-);p27(Kip1-/-) pituitaries are proliferative. Thus, during normal development, progenitor cell cycle exit is controlled by p57(Kip2) followed by p27(Kip1) in differentiated cells; these sequential actions, taken together with different pituitary outcomes of their loss of function, suggest hierarchical controls of the cell cycle that are independent of differentiation.

FIG. 1.

Subpopulation of noncycling undifferentiated pituitary cells. (A, B, and G) Colabeling with the S phase marker BrdU (green) and the M phase marker pHH3 (red) identifies proliferating cells at different stages of pituitary development. Nuclei were stained blue with Hoechst 33258 dye. (C) The first corticotroph cells present at e13.5 are revealed by Tpit staining (red) in the AL. Colocalization with BrdU (green) reveals mutually exclusive expression patterns. (D to F) Proliferative cells identified by Ki67 expression (D) are clearly distinct from differentiated POMC-positive cells (E and F). (H) Schematic representation of the spatial organization of the different pituitary cell populations at e11.5 and e13.5. The proliferating progenitors (green) are found mostly around the lumen, while the differentiated cells (red) appear on the ventral side of the developing AL of the gland. A group of noncycling undifferentiated cells (blue) are present between proliferating and differentiated cells.

FIG. 2.

p57^Kip2 and cyclin E mark noncycling undifferentiated cells in the anterior pituitary. Colocalization experiments were performed with different proliferation and differentiation markers at e13.5 and e14.5, a developmental period when noncycling undifferentiated cells are most abundant. (A) BrdU-positive cells are not colabeled with cyclin E, a cyclin thought to be involved in G₁-S transition. (B) Similarly, cyclin E does not colabel any cells with Ki67, a marker of proliferative cells. However, cyclin D1 (C) and cyclin D2 (D) colocalize partially with BrdU or Ki67 (E), suggesting that cyclin D expression is maintained until the beginning of S phase. p57^Kip2-positive cells are BrdU negative (G) and mostly cyclin D2 (H) and Ki67 (I) negative. (J to L) Colocalization of p57^Kip2 and cyclin E expression in cells of the developing anterior pituitary. (M to R) In order to clearly assess the differentiation status of cyclin E- and p57^Kip2-positive cells, we performed colocalization of p57^Kip2 (M) and cyclin E (P) with a mix of proliferation and differentiation markers (N and Q). Proliferating cells are identified by nuclear staining of BrdU and pHH3, while differentiated cells are marked by cytoplasmic labeling of POMC and αGSU (O and R). p57^Kip2- and cyclin E-positive cells do not colabel with any of these markers, indicating that these noncycling cells are undifferentiated. (F) Schematic representation of the different cell populations at e13.5/e14.5 showing noncycling undifferentiated precursors physically located between proliferating progenitors and differentiated cells. WT, wild type.

FIG. 3.

p27^Kip1 replaces p57^Kip2 during pituitary differentiation. The expression of another member of the Cip/Kip family, p27^Kip1 (B and E), is complementary to the expression of p57^Kip2 (A) and cyclin E (D) at e13.5. There is a small overlap between the two populations (C and F). The expression of p27^Kip1 is not correlated with the proliferation marker cyclin D2 (G) or Ki67 (H). The ventral/rostral expression pattern of p27^Kip1 suggests that it is expressed in nonproliferating differentiated cells. (I) Indeed, all Tpit-positive corticotroph cells coexpress p27^Kip1. (J to L) Quantification of colabeling for different markers in the population of noncycling cells. These data are presented relative to p57^Kip2 (J)-, cyclin E (K)-, and Tpit (L)-positive cells. WT, wild type.

FIG. 4.

p57^Kip2 controls progenitor cell cycle exit but not differentiation. (A to J) Knockout of p57^Kip2 affects pituitary development, as revealed by hematoxylin-eosin staining between e12.5 and e18.5. The absence of p57^Kip2 leads to early pituitary hyperplasia between e12.5 and e17.5 (compare panels F to I to panels A to D) and later (e18.5) to tissue loss (compare panel J to panel E). (Z1) The pituitary hyperplasia was quantitated at e14.5 by surface area measurement for two to four wild-type and p57^Kip2−/− pituitaries. Data represent the means ± standard errors of the means for measurements performed on six to eight sections for each of two to four pituitaries. Similar quantitations were performed for the control and transgenic pituitaries described in the legend to Fig. 5. (K to T) Proliferation and apoptosis in p57^Kip2−/− pituitaries. p57^Kip2 gene disruption leads to an increased number of proliferating cells in the AL, as revealed by BrdU incorporation (compare panel P to panel K) and by the proliferation markers Ki67 and pHH3 (compare panel Q to panel L). An increased number of cyclin E-positive cells is also observed in p57^Kip2−/− pituitaries, but these cells appear to have exited the cell cycle, as they do not label for Ki67 (compare panel R to panel M). Expression of p27^Kip1 is not altered in p57^Kip2−/− pituitaries (compare panel S to panel N), but apoptotic cells are detected by cleaved caspase 3 immunofluorescence in knockout pituitaries (quantification is shown in panel Z2) and never in normal pituitaries (compare panel T to panel O), thus explaining the tissue loss observed in e18.5 pituitaries (J). (U to Z) Unaltered differentiation in p57^Kip2−/− pituitaries. Expression of differentiation markers in wild-type (U to W) and p57^Kip2 knockout (X to Z) pituitaries is similar for Tpit (corticotrophs) (U and X), αGSU (gonadotrophs) (V and Y), and Pit1 (somatotrophs, lactotrophs, and thyrotrophs) (W and Z). It is noteworthy that p57^Kip2-dependent hyperplasia is accounted for by progenitor proliferation but does not involve proliferation of differentiated cells, as indicated by the absence of overlap between BrdU and differentiation markers.

FIG. 5.

Early p57^Kip2 expression leads to pituitary hypoplasia but does not affect differentiation. A transgene driving p57^Kip2 expression under the control of the Pitx1 promoter (A) will lead to early expression throughout the developing pituitary (B), including BrdU-positive cells (C and D). The ectopic expression of p57^Kip2 leads to pituitary hypoplasia (compare panel F to panel E; see quantification in Fig. 4Z1). Two independent transgenic pituitaries are shown for p57^Kip2 expression and BrdU incorporation (compare panels I and J to panel H) as well as for Tpit- and αGSU-positive cells (compare panels L and M to panel K). Differentiation is not impaired, as revealed by Tpit- and αGSU-positive cells (L and M). (G) The proportions of p57^Kip2-positive and BrdU-positive cells in wild-type (WT) and transgenic pituitaries were quantitated for six to eight sections from three different embryos, and the differences are statistically significant (P ≤ 0.02).

FIG. 6.

Double p27^Kip1 and p57^Kip2 knockout prevents pituitary cell cycle exit but not differentiation. In order to test whether p27^Kip1 and p57^Kip2 are sufficient for cell cycle exit during pituitary organogenesis, we crossed mice carrying null alleles for both genes. Data are shown for e14.5 pituitaries of p27^Kip1+/−; p57^Kip2+/+ (A, E, I, and M) mice, which are exactly like their wild-type sibs (not shown); for p27^Kip1+/+; p57^Kip2−/− mice (B, F, J, and N); for mice that are p27^Kip1−/−; p57^Kip2+/−, with the mutant allele being on the nonexpressed paternal chromosome (C, G, K, and O); and for double mutant mice (D, H, L, and P). For each genotype, hematoxylin-eosin stains are shown (A to D) together with immunofluorescence for p27^Kip1 and p57^Kip2 (E to H), Ki67 and POMC (I to L), and Tpit and BrdU (M to P). For double labeling, insets show colabeling or the absence thereof. Detailed analysis of Ki67-positive cells in double knockout pituitaries (L) revealed that the vast majority of the cells are Ki67 positive, including those that are POMC positive (P). Thus, the absence of cell cycle arrest in double knockout mice does not prevent differentiation into the corticotroph lineage.

FIG. 7.

In the absence of Tpit, undifferentiated cells are blocked in a p57^Kip2-positive noncycling precursor state. Tpit loss of function (Tpit^−/−) prevents melanotroph and corticotroph differentiation. The normal adult pituitary IL (between dotted lines) does not express cytoplasmic αGSU (A), but all of its melanotroph cells express nuclear Tpit (B). This tissue does not have p57^Kip2-positive cells (C), but most cells are p27^Kip1 positive (D). As previously described (32), a small number of gonadotroph cells appear in the IL in the absence of Tpit, as revealed by αGSU staining together with a few POMC-expressing cells (E). The Tpit^−/− IL has a large number of p57^Kip2-positive cells (F), in contrast to the normal IL (C). There is no overlap between differentiated and p57^Kip2-positive cells (G). The Tpit targeting vector included a β-Gal gene fused in frame with the Tpit gene's first exon, leading to production of a Tpit-β-Gal fusion protein; expression of Tpit-β-Gal (I) is not codetected with αGSU (H) in gonadotrophs with a changed fate (J). The p57^Kip2-positive cells of the mutant IL (L) are not identical to their counterparts in normal development, since they are also p27^Kip1 positive (K to M). (N) Quantification of marker expression in the IL of normal and Tpit^−/− pituitaries. (O and P) Tpit knockout does not affect proliferation of cells in either the adult IL or AL, as revealed by Ki67 labeling. (Q and R) Colabeling of Tpit^−/− (R) and Tpit^+/− (Q) pituitaries for Tpit-β-Gal (blue) and p57^Kip2 (brown) reveals a significant persistence of p57^Kip2 expression in both the AL and IL of the mutant pituitary. Both normal and mutant AL contain weak p57^Kip2-positive nuclei close to the AL-IL border (arrows), but the Tpit^−/− AL also has stronger-staining p57^Kip2-positive cells that either coexpress Tpit-β-Gal (arrowhead) or do not (double arrow).

FIG. 8.

Uncoupling of proliferation and differentiation during pituitary development. The present work defined a transient population of noncycling undifferentiated precursors (blue) during normal pituitary development that are marked by the expression of p57^Kip2 and cyclin E. (A) Following their expansion, proliferative pituitary progenitors (green) exit the cell cycle under the control of p57^Kip2 to yield noncycling precursors (blue). (B) These transient cells then switch off p57^Kip2 and switch on p27^Kip1 in parallel with differentiation into various hormone-producing lineages. This sequence was characterized in detail for the Tpit-dependent corticotroph (POMC) lineage (red), which is the earliest to reach terminal differentiation in the anterior pituitary. (C) In the absence of p57^Kip2, the cdki that appears to drive normal cell cycle exit, pituitary progenitors initially overgrow and then undergo extensive apoptosis late in fetal life. However, the absence of p57^Kip2 does not prevent the later expression of p27^Kip1 or differentiation into various lineages. The p57^Kip2-dependent cell cycle exit of progenitors therefore appears to be controlled independently of cell differentiation. (D) Expression of p27^Kip1 occurs with differentiation and protects differentiated cells from reentering the cell cycle. (E) In agreement with this, the blockade of cell differentiation observed in Tpit^−/− mice leads to the accumulation of noncycling undifferentiated precursors (orange) that are p57^Kip2 and p27^Kip1 positive. However, a small fraction of cells in the pituitary IL of Tpit^−/− mutant mice do differentiate, either through cell fate change into gonadotrophs (yellow) (αGSU and SF1 positive) or, more rarely, into POMC-positive cells. The cells that do differentiate switch off p57^Kip2 expression, suggesting that the differentiation process itself is responsible for p57^Kip2 extinction.