p27kip1 independently promotes neuronal differentiation and migration in the cerebral cortex

Abstract

The generation of neurons by progenitor cells involves the tight coordination of multiple cellular activities, including cell cycle exit, initiation of neuronal differentiation, and cell migration. The mechanisms that integrate these different events into a coherent developmental program are not well understood. Here we show that the cyclin-dependent kinase inhibitor p27(Kip1) plays an important role in neurogenesis in the mouse cerebral cortex by promoting the differentiation and radial migration of cortical projection neurons. Importantly, these two functions of p27(Kip1) involve distinct activities, which are independent of its role in cell cycle regulation. p27(Kip1) promotes neuronal differentiation by stabilizing Neurogenin2 protein, an activity carried by the N-terminal half of the protein. p27(Kip1) promotes neuronal migration by blocking RhoA signaling, an activity that resides in its C-terminal half. Thus, p27(Kip1) plays a key role in cortical development, acting as a modular protein that independently regulates and couples multiple cellular pathways contributing to neurogenesis.

Figure 1.

p27^Kip1 is expressed in cortical progenitors and neurons. (A–C) In situ RNA hybridization with p21^Cip1, p27^Kip1, and p57^Kip2 probes on coronal sections through E14.5 dorso-lateral cortex. The three genes are expressed at low (p21^Cip1) or high levels (p27^Kip1, p57^Kip2) in the cortical plate (CP), while only p27^Kip1 transcripts are significantly present in the ventricular zone (VZ), subventricular zone (SVZ), and intermediate zone (IZ). (D–F) Immunofluorescent staining at the same stage reveals a pattern of Cip/Kip protein expression comparable to that of their transcripts, with p27^Kip1 highly expressed from the SVZ to the CP and at lower level in the VZ, while p21^Cip1 and p57^Kip2 are only expressed at low levels in the CP. (G–I) Note the predominantly cytoplasmic localization of p27^Kip1 in VZ and IZ cells and its nuclear localization in CP cells.

Figure 2.

p27^Kip1-null mutant cortices present defects in neuronal differentiation and radial migration. Pregnant females carrying p27^Kip1 mutant embryos were injected with a single dose of BrdU at E14.5 of gestation to mark neurons born at that date, and embryos were harvested at E17.5 and analyzed with antibodies to BrdU and HuC/D. (A) Loss of p27^Kip1 reduces radial migration of BrdU⁺ cortical neurons, as shown by the accumulation of labeled cells in the VZ/SVZ and IZ and the loss of labeled cells in the CP of p27^−/− mutant embryos compared to wild-type littermates. Embryos homozygous for the cell cycle mutant allele p27^ck− do not present defects in distribution of BrdU⁺ neurons. The histogram shows the distribution of BrdU⁺ cells in the different compartments of the cortex (VZ/SVZ, IZ, and CP) as a way to quantify radial migration in the different genotypes. Asterisks indicate significant differences in percentages of BrdU⁺ cells in a given cortical zone in mutant and wild-type cortices (n = 2–3, *p < 0.05, **p < 0.01). Panels on the right illustrate the distribution of BrdU⁺ cortical cells in the different genotypes. (B) Loss of p27^Kip1 also reduces the differentiation of BrdU⁺ cortical neurons, as marked by expression of HuC/D, while p27^CK− embryos do not present this defect. The histogram shows the percentage of BrdU⁺ cells expressing HuC/D in the different genotypes (n = 2–3 brains; **p < 0.01). Panels illustrate double immunostaining for BrdU (green) and HuC/D (red). Arrows in insets indicate double-labeled cells.

Figure 3.

p27^Kip1 overexpression promotes differentiation and radial migration of cortical neurons. (A) Coronal section through an E17.5 mouse brain electroporated in utero with a GFP construct at E14.5. The inset shows GFP fluorescence in the dorso-lateral cortex in the same brain. Three days after electroporation, the progeny of GFP⁺ electroporated cells (green) were distributed in all zones of the developing cortex (marked by TOTO-3 in red) indicating that electroporated cells have undergone radial migration. (B) Distribution of GFP⁺ cells in the different zones of the cortex at E17.5, following in utero electroporation at E14.5 of various bicistronic constructs expressing a Cip/Kip protein and GFP. p27^Kip1 is the only Cip/Kip gene that promotes radial cell migration when overexpressed, and this effect is independent of cell cycle regulation as the cell cycle mutant form p27^ck− is as efficient as the wild-type version p27^wt. Wild-type p21^Cip1 and p57^Kip2 (p21^wt and p57^wt) and mutant versions that lack cell cycle regulation activity (p21^ck− and p57^ck−) lack radial migration activity (n = 3–5 brains; *p < 0.05; **p < 0.01). (C–E) Immunostaining for GFP illustrating the position of cells electroporated with GFP, p27^wt, or p27^ck. (F–H) Differentiation of cortical cells electroporated in utero at E14.5 and analyzed at E17.5 for expression of the neuronal differentiation markers HuC/D (F,H) and βIII-tubulin (G). Overexpression of p27^Kip1 promotes expression of neuronal markers independently of its role in cell cycle regulation, as its action is mimicked by p27^ck−, while overexpression of wild-type or cell cycle mutant forms of p21^Cip1 or p57^Kip2 does not significantly promote neuronal differentiation. The histograms show the percentage of GFP⁺ Hu⁺ (F) and GFP⁺ βIII-tubulin⁺ (G) double-labeled cells over the total population of GFP⁺ electroporated cells (n = 3–5 brains; **p < 0.01). (H) Double immunostaining for GFP (green) and HuC/D (red) illustrating the induction of neuronal differentiation by p27^wt and p27^ck−. Arrows in insets indicate double-labeled cells. Bars: C–E, 50 μm.

Figure 4.

p27^Kip1 stabilizes Ngn2 protein in cortical progenitors. (A) Double immunostaining for p27^Kip1 (green) and Ngn2 (red) in a E15.5 mouse cortex showing coexpression of the two proteins in a subset of SVZ/VZ progenitors. Insets show enlargements of regions indicated in boxes. (B) Double immunostainings for electroporated cells overexpressing or down-regulating p27^Kip1 as indicated (GFP in green) and for endogenous Ngn2 (red). (C) Percentage of VZ cells expressing Ngn2 protein after electroporation of various forms of p27^Kip1 and siRNA in E15.5 VZ progenitors and 24-h slice culture. Electroporation of p27 siRNA#1 down-regulates Ngn2 protein and overexpression of p27^wt, p27^ck−, or the N-terminal half of p27^ck− (p27^ck−N-term) up-regulate Ngn2 protein, while the C-terminal part (p27 C-term) has no activity (n = 3–5 slices; *p < 0.05, **p < 0.01). (D) Percentage of VZ cells expressing Ngn2 protein in E15.5 cortical progenitors from wild-type, p27^−/−-null mutant p27^ck− embryos (n = 3–6 brains; *p < 0.05). Panels showing immunostainings for Ngn2 (green) and TOTO-3 nuclear staining (red) illustrate the reduction in Ngn2 expression in absence of p27^Kip1 (p27^−/−) and its restoration to wild-type levels by the cell cycle mutant allele p27^CK−. (E) Ngn2 protein degradation analyzed by pulse-chase in rabbit reticulocytes (see Materials and Methods for details). Coexpression of Ngn2 with p27^wt and p27^ck− markedly increased Ngn2 protein stability. In vitro translated p27^wt and p27^ck− proteins were stable during the course of the experiment. Three experiments were performed and one representative example is shown.

Figure 5.

Ngn2 overexpression rescues the neuronal differentiation defect but not the radial migration defect induced by p27^Kip1 knockdown. (A) Loss of Ngn2 function results in neuronal differentiation defects, as shown by reduced HuC/D expression in cortices of Ngn2 homozygous null mutant embryos (Ngn2^null) electroporated with GFP at E14.5 and cultivated as slices for 4 d, and in cortices of embryos homozygous for a floxed allele of Ngn2 (Ngn2^floxed) coelectroporated with Cre and GFP. Ngn2 overexpression conversely promotes neuronal differentiation, as shown by increased HuC/D expression in Ngn2 electroporated cells. The defect in HuC/D expression in cells electroporated with p27 siRNA#1 is fully rescued by coelectroporation of Ngn2 (n = 5–13 slices; *p < 0.05, **p < 0.01). Panels showing double immunostainings for GFP (green) and HuC/D (red) illustrate the rescue by Ngn2 of the neuronal differentiation defect induced by p27^Kip1 knockdown. High magnification pictures show electroporated cortical neurons that have differentiated and remained in the VZ/SVZ compartment. (B) Loss of Ngn2 results in radial migration defects, as shown by electroporation of GFP in E14.5 Ngn2^null cortices or of Cre and GFP in E14.5 Ngn2^floxed cortices. Ngn2 overexpression results in a small but significant increase in the percentage of electroporated cells reaching the CP in wild-type cortex. However, Ngn2 overexpression does not rescue the migration defect resulting from knockdown of p27^Kip1 when coelectroporated with p27 siRNA#1 (n = 3–8 slices; *p < 0.05, **p < 0.01, **p < 0.001). Panels illustrate the migration phenotypes resulting from Ngn2 overexpression (Ngn2) and p27^Kip1 knockdown (p27 siRNA#1) and the lack of rescue when Ngn2 is coelectroporated with p27 siRNA#1. Electroporated cells are labeled for GFP.

Figure 6.

Blocking RhoA-ROCK signaling pathway rescues the radial migration defect induced by p27^Kip1 knockdown. (A) In situ hybridization of a coronal section through E14.5 telencephalon with a RhoA probe showing high levels of RhoA transcripts in VZ and SVZ cells. (B) Blocking RhoA activity by electroporating a dominant-negative form of RhoA (DNRhoA) or blocking ROCK1/2 activity by exposing cortical slices to the specific inhibitor CY27632 fully rescues the radial migration defect resulting from electroporation of p27 siRNA#1. Overexpression of Rac1 does not rescue this phenotype (n = 5–13 slices; *p < 0.05, **p < 0.01). Panels on the right illustrate the rescue of the radial migration defect induced by p27 siRNA#1 when RhoA signaling is inhibited.

Figure 7.

The N- and C-terminal halves of p27^Kip1 harbor different activities. (A) The N-terminal half of p27^ck− (p27^ck−N-term) efficiently promotes expression of the neuronal marker HuC/D, while the C-terminal half (p27 C-term) only has a moderate differentiation activity. (B) p27 C-term promotes cell migration to the CP as efficiently as DNRhoA, while p27^ck−N-term has no migration activity. (C) Summary scheme illustrating the modular nature of the p27^Kip1 molecule and its role in coordinating multiple cellular pathways during neurogenesis. p27^Kip1 N-term harbors the cell cycle regulation function and the neuronal differentiation promoting activity, involving stabilization of Ngn2, while p27^Kip1 C-term harbors the radial migration promoting activity, involving inactivation of RhoA. p27^Kip1 and Ngn2 form a regulatory loop that coordinates cell cycle exit with differentiation and radial migration of cortical neurons. Black arrows represent nontranscriptional interactions, and white arrows represent transcriptional interactions.