Cooperation between the Cdk inhibitors p27(KIP1) and p57(KIP2) in the control of tissue growth and development

Abstract

Cell cycle exit is required for terminal differentiation of many cell types. The retinoblastoma protein Rb has been implicated both in cell cycle exit and differentiation in several tissues. Rb is negatively regulated by cyclin-dependent kinases (Cdks). The main effectors that down-regulate Cdk activity to activate Rb are not known in the lens or other tissues. In this study, using multiple mutant mice, we show that the Cdk inhibitors p27(KIP1) and p57(KIP2) function redundantly to control cell cycle exit and differentiation of lens fiber cells and placental trophoblasts. These studies demonstrate that p27(KIP1) and p57(KIP2) are critical terminal effectors of signal transduction pathways that control cell differentiation.

Figure 1

Expression of cell cycle regulatory genes during lens development. In situ hybridization was performed on transverse sections through the eye region of an E15.5 embryo using antisense probes as indicated. The arrow in A points to the pigmented epithelium (PE), which falsely stains positive for all probes because of the presence of pigmented granules in these cells. Arrows in D indicate cells in the equatorial zone of the lens that express high levels of p57. p27 is expressed in the equatorial zone and in the retina (r). Scale bar, 200 μm.

Figure 2

p57 and p27 are required for lens development. (A–C) Hematoxylin and eosin-stained sections of E15.5 lenses from the indicated genotypes. Nuclei at the posterior edge of p57^+/−m (E) and p27^−/−p57^+/−m (F) lenses are indicated by the arrow. (G–I) Nuclei in lens sections derived from E14.5 mice revealed by DAPI staining. Scale bar, 200 μm.

Figure 3

Overproliferation of lens fiber cells in p27/p57 double mutants leads to compromised differentiation and increased apoptosis. (A–C) Nuclei of lens sections derived from E15.5 embryos revealed by DAPI staining. (D–F) BrdU incorporation assays demonstrate defects in cell cycle exit in p27^−/− p57^+/−m lenses. (G–L) Immunofluorescence staining of β- and γ-crystallins demonstrates reduced expression of these two differentiation markers in the lens of p57^+/−m mice (H,K) and the absence of expression in the lens of p27^−/−p57^+/−m mice (I, L). (M–O) TUNEL assays detect apoptotic cells (arrows) in both p57^+/−m and p27^−/−p57^+/−m lenses. (P) Quantitation of BrdU incorporation assays at E13.5 and E15.5. BrdU-positive nuclei from a total of six sections for each genotype were determined, and the average is shown in the histogram along with the s.d.. Scale bars in C, F, I, and L, 200 μm; O, 50 μm.

Figure 4

p57 and p27 are required for the proper development of the mouse hemochorial placenta. (A) Hematoxylin and eosin-stained E12.5 placenta sections from p27^+/− (a), p57^+/−m (b), and p27^+/−p57^+/−m (c) mice. Arrows in c indicate hyaline membranes. (B) Expression of p57 (a) and p27 (c) in E12.5 placenta as detected by in situ hybridization. The specificity of the p57 probe is demonstrated through the absence of signal in a placental section from a p57^+/−m mouse (b). (C) BrdU incorporation assays reveal overproliferation in the labyrinth zone of p27^−/− p57^+/−m placentas, as compared with p27^+/−, p27^−/−, or p27^+/− p57^+/−m placentas. Placentas were harvested at E18.5. Quantitation of BrdU assays on embryos collected from a single litter are presented in c. Error bars represent the s.d. (lz) Labyrinth zone; stz, spongiotrophoblasts zone. Scale bars, 200 μm.