Overexpression of p27Kip1, Probability of Cell Cycle Exit, and Laminar Destination of Neocortical Neurons

Abstract

Neocortical projection neurons arise from a pseudostratified ventricular epithelium (PVE) from embryonic day 11 (E11) to E17 in mice. The sequence of neuron origin is systematically related to mechanisms that specify neuronal class properties including laminar fate destination. Thus, the neurons to be assembled into the deeper layers are the earliest generated, while those to be assembled into superficial layers are the later generated neurons. The sequence of neuron origin also correlates with the probability of cell cycle exit (Q) and the duration of G1-phase of the cell cycle (T(G1)) in the PVE. Both Q and T(G1) increase as neuronogenesis proceeds. We test the hypothesis that mechanisms regulating specification of neuronal laminar destination, Q and T(G1) are coordinately regulated. We find that overexpression of p27(Kip1) in the PVE from E12 to E14 increases Q but not T(G1) and that the increased Q is associated with a commensurate increase in the proportion of exiting cells that is directed to superficial layers. We conclude that mechanisms that govern specification of neocortical neuronal laminar destination are coordinately regulated with mechanisms that regulate Q and are independent of mechanisms regulatory to cell cycle duration. Moreover, they operate prior to postproliferative mechanisms necessary to neocortical laminar assembly.

Proliferation and Cell Cycle Dynamics in the Developing Stellate Ganglion

“…S-phase labeling index and total cell cycle length were measured using techniques modified from published methods (Hayes and Nowakowski, 2000;Nowakowski et al, 2002;Tarui et al, 2005). The major changes were to use ethyldeoxyuridine (EdU) as the second S-phase marker instead of tritiated thymidine, to use Ki67 instead of cumulative bromodeoxyuridine (BrdU) labeling to identify cycling cells and to adapt all methods to allow multiple labeling immunofluorescences to identify specific cell types.…”

“…The calculation of cell cycle length requires measurement of three factors: the S-phase length, growth fraction (the proportion of cells in the cell cycle), and labeling index (the number of cells in S-phase at any instance). A full explanation of the derivation of the formulae used below and the experimental data underlying these techniques has been described previously (Hayes and Nowakowski, 2000;Nowakowski et al, 2002;Tarui et al, 2005). Confocal microscope images of transverse sections of embryos through the stellate ganglia and, at E9.5 only, the region between the neural tube and the stellate ganglion, were analyzed.…”

In utero exposure to dioxin causes neocortical dysgenesis through the actions of p27 Kip1

“…GABA immunohistochemistry was performed as described previously using anti-GABA antibody (Chemicon International) (16). Cumulative BrdU labeling analysis (18) and Q fraction analysis (9,15,34) were performed as previously described. Detailed methods are described in SI Materials and Methods.…”

“…We then identified the NPCs in the Q fraction and P fraction (the fraction of daughter cells that remain proliferative; P = 1.0 − Q) on E12 by using two S-phase tracers, iododeoxyuridine (IdU) and BrdU ( Fig. 2F) (15,19). In the dorsomedial cerebral wall of the TCDD-treated mice, the IdU-positive nuclei (blue nuclei) were located in the outer margin of the S-phase zone in both the P + Q and Q experiments (Fig.…”

“…Specifically, overexpression of p27 Kip1 in NPCs in vivo increases the Q fraction (that is, promotes differentiation of the NPCs), with a resultant thinner neocortex (14,15), whereas the lack of p27 Kip1 decreases the Q fraction, resulting in a thicker neocortex (16). It is worthy of note in this context that TCDD has been reported to induce p27…”