Regulation of neuronal gene expression is critical to central nervous system development. Here, we show that REST regulates the transitions from pluripotent to neural stem/progenitor cell and from progenitor to mature neuron. In the transition to progenitor cell, REST is degraded to levels just sufficient to maintain neuronal gene chromatin in an inactive state that is nonetheless poised for expression. As progenitors differentiate into neurons, REST and its co-repressors dissociate from the RE1 site, triggering activation of neuronal genes. In some genes, the level of expression is adjusted further in neurons by CoREST/MeCP2 repressor complexes that remain bound to a site of methylated DNA distinct from the RE1 site. Expression profiling based on this mechanism indicates that REST defines a gene set subject to plasticity in mature neurons. Thus, a multistage repressor mechanism controls the orderly expression of genes during development while still permitting fine tuning in response to specific stimuli.

Ceramide engagement in apoptotic pathways has been a topic of controversy. To address this controversy, we tested loss-of-function (lf) mutants of conserved genes of sphingolipid metabolism in Caenorhabditis elegans. Although somatic (developmental) apoptosis was unaffected, ionizing radiation-induced apoptosis of germ cells was obliterated upon inactivation of ceramide synthase and restored upon microinjection of long-chain natural ceramide. Radiation-induced increase in the concentration of ceramide localized to mitochondria and was required for BH3-domain protein EGL-1-mediated displacement of CED-4 (an APAF-1-like protein) from the CED-9 (a Bcl-2 family member)/CED-4 complex, an obligate step in activation of the CED-3 caspase. These studies define CEP-1 (the worm homolog of the tumor suppressor p53)-mediated accumulation of EGL-1 and ceramide synthase-mediated generation of ceramide through parallel pathways that integrate at mitochondrial membranes to regulate stress-induced apoptosis.

Neurogenesis requires mechanisms that coordinate early cell-fate decisions, migration, and terminal differentiation. Here, we show that the transcriptional repressor, repressor element 1 silencing transcription factor (REST), regulates radial migration and the timing of neural progenitor differentiation during neocortical development, and that the regulation is contingent upon differential REST levels. Specifically, a sustained presence of REST blocks migration and greatly delays-but does not prevent-neuronal differentiation, resulting in a subcortical band heterotopia-like phenotype, reminiscent of loss of doublecortin. We further show that doublecortin is a direct gene target of REST, and that its overexpression rescues, at least in part, the aberrant phenotype caused by persistent presence of REST. Our studies support the view that the targeted down-regulation of REST to low levels in neural progenitors, and its subsequent disappearance during neurogenesis, is critical for timing the spatiotemporal transition of neural progenitor cells to neurons.in utero electroporation | neuronal differentiation | neuronal cell fate N ervous system development relies on extrinsic and intrinsic signaling to regulate the precise spatial and temporal acquisition of the different neural lineages. Neurons and glia arise from neural stem cells in a temporally defined order, where generation of neurons precedes glia (1-3). Furthermore, the generation and migration of neurons occur in a stereotyped pattern to construct the distinctive structure of the central nervous system. For example, the neocortex, which consists of six layers of neurons, is built through precisely orchestrated waves of newly born neurons that migrate past their precursors (2, 4). This orderly acquisition of the different neural lineages is mediated by specific networks of transcriptional activators and repressors in response to environmental and intrinsic cues (for reviews see refs. 3, 5, and 6). How the precise timing of this signaling cascade is accomplished and whether migration and differentiation are linked obligatorily during development is still obscure.One key factor in this process could be the transcriptional repressor REST (also called NRSF), which regulates a large number of neuronal genes as well as brain-specific microRNA genes (7-11). In nonneuronal cells, REST binds to a conserved 23-bp DNA motif known as RE1 (repressor element 1), located in the regulatory regions of these genes, and blocks their transcription, via the corepressors . We showed previously that REST repression in pluripotent ES cells and multipotent neural stem/progenitor (NS/P) cells creates a chromatin status poised for subsequent activation (12,14). Importantly, REST itself is regulated differentially throughout development, expressed to high levels in ES cells but present in minimal levels in NS/P cells. The down-regulation of REST in NS/P cells is mediated, at least in part, by targeted proteasomal degradation via the E3 ubiquitin ligase β-TRCP (15, 12). As NS/P cells diffe...