Lack of the cell-cycle inhibitor p27Kip1 results in selective increase of transit-amplifying cells for adult neurogenesis

Abstract

The subventricular zone (SVZ) is the largest germinal layer in the adult mammalian brain and comprises stem cells, transit-amplifying progenitors, and committed neuroblasts. Although the SVZ contains the highest concentration of dividing cells in the adult brain, the intracellular mechanisms controlling their proliferation have not been elucidated. We show here that loss of the cyclin-dependent kinase inhibitor p27Kip1 has very specific effects on a population of CNS progenitors responsible for adult neurogenesis. Using bromodeoxyuridine and [(3)H]thymidine incorporation to label cells in S phase and cell-specific markers and electron microscopy to identify distinct cell types, we compared the SVZ structure and proliferation characteristics of wild-type and p27Kip1-null mice. Loss of p27Kip1 had no effect on the number of stem cells but selectively increased the number of the transit-amplifying progenitors concomitantly with a reduction in the number of neuroblasts. We conclude that cell-cycle regulation of SVZ adult progenitors is remarkably cell-type specific, with p27Kip1 being a key regulator of the cell division of the transit-amplifying progenitors.

Fig. 1.

Increased BrdU incorporation in the SVZ of adult p27Kip1-null mice. A computer-generated map of BrdU-labeled cells in the SVZ of adult wild-type (A) and knock-out (B) mice is shown. Nine-week-old mice received a single pulse of BrdU and were killed after 1 hr. The number of proliferating cells was determined by staining SVZ whole mounts with anti-BrdU antibodies. Each dot represents the nucleus of a BrdU-positive cell. The black lines indicate arbitrary anatomical subdivisions of the SVZ, as described in Materials and Methods. Note that the increased number of BrdU-positive cells in the p27Kip1-null mice is localized predominantly to the anterior horn (ah) rather than the intermediate bridge (ib) or the posterior horn (ph). The topographic location of the BrdU-positive cells parallels the distribution of type A and C cells in the adult SVZ.

Fig. 2.

Loss of p27Kip1 function results in increased C-cell number and disorganization of the SVZ. A, Photomicrograph of the typical structure of the adult mouse SVZ as observed in wild-type animals. The migrating neuroblasts (A cells), identified by scant dark cytoplasm and dense heterochromatin, are surrounded by type B cells (B), characterized by light cytoplasm with several inclusions and irregular contours. Type C cells (C) are interspersed in clusters, whereas ependymal cells (E) line the ventricular wall (4812× magnification). B, Photomicrograph of the SVZ structure in p27Kip1-null littermates. There are significantly more type C cells, whereas type B cells and ependymal cells are constant and type A cells are reduced in number (4156× magnification).

Fig. 3.

Displaced A cells in the p27Kip1-null mice. Electron micrograph of the anterior horn of the SVZ of mutant mice showing the ependymal cells (E) and the transit amplifying progenitors (C) close to the ventricular lumen (left). Note the unusual location of the migratory A cells (arrow) away from the lumen of the lateral ventricle.

Fig. 4.

Identification of proliferating type C cells in p27Kip1-null mice. Triple immunofluorescence of frozen coronal sections obtained from the SVZ of wild-type (+/+, A, C, E) and p27Kip1-null (−/−, B, D, F) mice and stained for BrdU (green nuclei), PSA-NCAM (blue immunofluorescence), and Dll (redimmunofluorescence). The arrowhead indicates a cell that is BrdU+/PSA-NCAM− (B, F) but Dll+ (D, F) and therefore identified as a proliferating type C cell. Note that in the mutant, the accumulation of proliferating C cells close to the ventricular lumen (on theleft of each panel) displaces the PSA-NCAM+ type A cells laterally. Scale bar, 100 μm.

Fig. 5.

Increased apoptosis in whole mounts of SVZ from p27Kip1-null mice. A computer-generated map of the SVZ of adult wild-type (A) and knock-out (B) mice whole mounts stained with TUNEL. Eachdot represents the nucleus of a TUNEL-positive cell, and the black lines indicate arbitrary subdivisions of the SVZ based on anatomical landmarks, as defined in Materials and Methods. Note that the increased number of TUNEL-positive cells in the p27Kip1-null mice is localized predominantly to the anterior horn (ah) of the SVZ rather than the intermediate bridge (ib) or the posterior horn (ph), which parallels the distribution of type A and C cells in the adult SVZ.

Fig. 6.

Microglial cells in the p27Kip1-null mouse SVZ.A, Photomicrograph of the adult mouse SVZ, as observed in wild-type animals. Occasional microglial cells (M,arrows) can be observed (4812× magnification).B, A typical section from p27Kip1-null mice reveals an increased number of microglial cells (M,arrows) (6453× magnification). A, A cells; B, B cells; E, ependymal cells.

Fig. 7.

Identification of apoptotic cells in the SVZ of wild-type and p27Kip1-null mice using type A- and type C-specific cell markers. Triple immunofluorescence of whole mounts from wild-type mice (+/+, left column) and p27Kip1-null mice (−/−,right column) stained for TUNEL (green nuclei), PSA-NCAM (blueimmunofluorescence), and Dll (red immunofluorescence). The arrow indicates the apoptotic nucleus (B) of a cell that is Dll-positive (F) and PSA-NCAM-negative (D) and therefore identified as a type C apoptotic cell in the mutant mice. Scale bar, 50 μm.

Fig. 8.

Identification of apoptotic cells in the SVZ of wild-type and p27Kip1-null mice using type B-specific (gfap) and microglial-specific (mac-1) cell markers. Double immunofluorescence of whole mounts from wild-type mice (+/+) and p27Kip1-null mice (−/−) stained for TUNEL (green nuclei), GFAP (redimmunofluorescence; A–F), or Mac-1 (red immunofluorescence; G–L). Thearrow indicates an apoptotic nucleus of a microglial cell in wild-type mice. The arrowheads indicate apoptotic cells that do not stain with Mac-1 in the mutant mice. Scale bar, 150 μm.

Fig. 9.

Neurospheres isolated from p27Kip1-null mice maintain their ability to differentiate in vitro. Pluripotent cells were isolated from the SVZ of p27Kip1-null mice and grown in vitro to generate neurospheres. After dissociation and plating on laminin-coated dishes, these cells differentiate into glial cells (A) or into neurons (B). A, p27Kip1-null GFAP-positive astrocyte (greenimmunofluorescence). B, p27Kip1-null A cell stained with the neural marker TuJ-1 (red immunofluorescence). Individual nuclei are labeled by DAPI (blueimmunofluorescence). Scale bar, 25 μm.

Fig. 10.

Schematic model of population dynamics in the anterior horn of the SVZ. A model of SVZ lineage progression is shown. Type B cells are the GFAP-positive primary precursors that give rise to type C transit-amplifying progenitors, which can further differentiate into type A migratory neuroblasts. The number of cells in each stage of the lineage is determined by self-renewal, differentiation, or death.