A cell-autonomous requirement for Cip/Kip cyclin-kinase inhibitors in regulating neuronal cell cycle exit but not differentiation in the developing spinal cord

Abstract

Control over cell cycle exit is fundamental to the normal generation of the wide array of distinct cell types that comprise the mature vertebrate CNS. Here, we demonstrate a critical role for Cip/Kip class cyclin-kinase inhibitory (CKI) proteins in regulating this process during neurogenesis in the embryonic spinal cord. Using immunohistochemistry, we show that all three identified Cip/Kip CKI proteins are expressed in both distinct and overlapping populations of nascent and post-mitotic neurons during early neurogenesis, with p27(Kip1) having the broadest expression, and both p57(Kip2) and p21(Cip1) showing transient expression in restricted populations. Loss- and gain-of-function approaches were used to establish the unique and redundant functions of these proteins in spinal cord neurogenesis. Using genetic lineage tracing, we provide evidence that, in the absence of p57, nascent neurons re-enter the cell cycle inappropriately but later exit to begin differentiation. Analysis of p57(Kip2);p27(Kip1) double mutants, where p21 expression is confined to only a small population of interneurons, demonstrates that Cip/Kip CKI-independent factors initiate progenitor cell cycle exit for the majority of interneurons generated in the developing spinal cord. Our studies indicate that p57 plays a critical cell-autonomous role in timing cell cycle exit at G1/S by opposing the activity of Cyclin D1, which promotes cell cycle progression. These studies support a multi-step model for neuronal progenitor cell cycle withdrawal that involves p57(Kip2) in a central role opposing latent Cyclin D1 and other residual cell cycle promoting activities in progenitors targeted for differentiation.

p57Kip2 is a repressor of Mash1 activity and neuronal differentiation in neural stem cells

“…19 Although these experiments clearly indicate that higher number of neurons observed after loss of p57Kip2 is due to aberrant regulation of progenitor cellcycle exit and timing, additional mechanisms should also be considered. In our in vitro assays, we found no association of the increase in nuclear p57Kip2 staining with cell-cycle exit or CDK-interacting function.…”

“…14 The role for p57Kip2 in brain development is less clear, despite the fact that p57Kip2 is the only CKI of the Cip/Kip family that is absolutely required for survival. 18 p57Kip2 has been implicated in the control of neural precursor proliferation at several levels of the developing nervous system, 11,19 and recent in vivo studies have established that the correct expression levels of p57Kip2 is required for proper development of the developing spinal cord. 19 In addition, it was recently shown that p57Kip2 in cooperation with p27Kip2 regulates cell migration in the developing neocortex.…”

“…18 p57Kip2 has been implicated in the control of neural precursor proliferation at several levels of the developing nervous system, 11,19 and recent in vivo studies have established that the correct expression levels of p57Kip2 is required for proper development of the developing spinal cord. 19 In addition, it was recently shown that p57Kip2 in cooperation with p27Kip2 regulates cell migration in the developing neocortex. 17 p57Kip2 has also been shown to play a direct role in regulating the activity of transcription factors implicated in neuronal differentiation.…”

Smad6 promotes neuronal differentiation in the intermediate zone of the dorsal neural tube by inhibition of the Wnt/β-catenin pathway

“…During neurogenesis, the developing spinal cord is organized into a VZ and an MZ composed of proliferative NPCs and postmitotic and differentiating neurons, respectively (19,20). Cells moving out of the VZ and on their way to the MZ express NeuroM (13,31).…”

“…2C). Cells in the IZ are differentiating nascent neurons that undergo cell-cycle exit (19,20). Because Smad6 was expressed mainly in the IZ of dorsal neural tubes and was required for the neuronal differentiation, knockdown of Smad6 in this region may inhibit the differentiation of nascent neurons and result in the reduction in number of neurons that are migrating from the IZ to the MZ.…”