The mechanisms through which cell-cycle control and cell-fate decisions are coordinated in proliferating stem cell populations are largely unknown. Here, we show that E2f3 isoforms, which control cell-cycle progression in cooperation with the retinoblastoma protein (pRb), have critical effects during developmental and adult neurogenesis. Loss of either E2f3 isoform disrupts Sox2 gene regulation and the balance between precursor maintenance and differentiation in the developing cortex. Both isoforms target the Sox2 locus to maintain baseline levels of Sox2 expression but antagonistically regulate Sox2 levels to instruct fate choices. E2f3-mediated regulation of Sox2 and precursor cell fate extends to the adult brain, where E2f3a loss results in defects in hippocampal neurogenesis and memory formation. Our results demonstrate a mechanism by which E2f3a and E2f3b differentially regulate Sox2 dosage in neural precursors, a finding that may have broad implications for the regulation of diverse stem cell populations.

The Rb/E2F pathway has long been appreciated for its role in regulating cell cycle progression. Emerging evidence indicates that it also influences physiological events beyond regulation of the cell cycle. We have previously described a requirement for Rb/E2F mediating neuronal migration; however, the molecular mechanisms remain unknown, making this an ideal system to identify Rb/E2F-mediated atypical gene regulation in vivo. Here, we report that Rb regulates the expression of neogenin, a gene encoding a receptor involved in cell migration and axon guidance. Rb is capable of repressing E2F-mediated neogenin expression while E2F3 occupies a region containing E2F consensus sites on the neogenin promoter in native chromatin. Absence of Rb results in aberrant neuronal migration and adhesion in response to netrin-1, a known ligand for neogenin. Increased expression of neogenin through ex vivo electroporation results in impaired neuronal migration similar to that detected in forebrain-specific Rb deficiency. These findings show direct regulation of neogenin by the Rb/E2F pathway and demonstrate that regulation of neogenin expression is required for neural precursor migration. These studies identify a novel mechanism through which Rb regulates transcription of a gene beyond the classical E2F targets to regulate events distinct from cell cycle progression.The Rb pathway is best characterized for its role in regulating cell cycle progression through E2F-mediated transcriptional regulation of classical cell cycle machinery target genes. Recently, however, accumulating in vivo and in vitro evidence is emerging to suggest that Rb and E2F are capable of regulating expression of atypical target genes with functions other than cell cycle regulation in cell-type-specific manners (reviewed in reference 35). In vivo, several studies have emerged that implicate Rb and E2F interaction in novel processes beyond well-characterized roles in cell cycle regulation (10; for a review, see reference 6). In the nervous system, in particular, we have recently shown that an Rb-E2F3 interaction mediates migration of a subpopulation of GABAergic interneurons (34). In the same study, we also observed deregulation of a number of genes with known roles in neuronal migration in cell populations lacking Rb, suggesting a role for E2F3 in regulating transcription of novel targets (34). A second cell cycle-independent role for E2F3a in regulating Rb-mediated interneuron differentiation was also reported in the retina (9). Thus far, in vivo studies have failed to identify the mechanism through which these cell cycle-independent processes occur.In parallel, in vitro several microarray studies examining changes in gene expression in response to various models of deregulated E2F expression have each identified groups of overlapping novel target genes with well-characterized roles in differentiation, development, and migration (5,15,25,31,39,41,60). More recently, chromatin immunoprecipitation (ChIP)-on-chip studies have identified putative E2F binding site...

The retinoblastoma protein (Rb) family members are essential regulators of cell cycle progression, principally through regulation of the E2f transcription factors. Growing evidence indicates that abnormal cell cycle signals can participate in neuronal death. In this regard, the role of Rb (p105) itself has been controversial. Germline Rb deletion leads to massive neuronal loss, but initial reports argue that death is non-cell autonomous. To more definitively resolve this question, we generated acute murine knock-out models of Rb in terminally differentiated neurons in vitro and in vivo. Surprisingly, we report that acute inactivation of Rb in postmitotic neurons results in ectopic cell cycle protein expression and neuronal loss without concurrent induction of classical E2f-mediated apoptotic genes, such as Apaf1 or Puma. These results suggest that terminally differentiated neurons require Rb for continuous cell cycle repression and survival.

SUMMARY The PIWI-interacting RNA (piRNA) pathway has long been thought to function solely in the germline, but evidence for its functions in somatic cells is emerging. Here we report an unexpected role for the piRNA pathway in Caenorhabditis elegans sensory axon regeneration after injury. Loss of function in a subset of components of the piRNA pathway results in enhanced axon regrowth. Two essential piRNA factors, PRDE-1 and PRG-1/PIWI, inhibit axon regeneration in a gonad-independent and cell-autonomous manner. By smFISH analysis we find that prde-1 transcripts are present in neurons, as well as germ cells. The piRNA pathway inhibits axon regrowth independent of nuclear transcriptional silencing but dependent on the slicer domain of PRG-1/PIWI, suggesting post-transcriptional gene silencing is involved. Our results reveal the neuronal piRNA pathway as a novel intrinsic repressor of axon regeneration.

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