Kelly A. McClellan

Dissociating the dual roles of apoptosis‐inducing factor in maintaining mitochondrial structure and apoptosis

The mitochondrial protein apoptosis‐inducing factor (AIF) translocates to the nucleus and induces apoptosis. Recent studies, however, have indicated the importance of AIF for survival in mitochondria. In the absence of a means to dissociate these two functions, the precise roles of AIF remain unclear. Here, we dissociate these dual roles using mitochondrially anchored AIF that cannot be released during apoptosis. Forebrain‐specific AIF null (tel. AifΔ) mice have defective cortical development and reduced neuronal survival due to defects in mitochondrial respiration. Mitochondria in AIF deficient neurons are fragmented with aberrant cristae, indicating a novel role of AIF in controlling mitochondrial structure. While tel. AifΔ Apaf1−/− neurons remain sensitive to DNA damage, mitochondrially anchored AIF expression in these cells significantly enhanced survival. AIF mutants that cannot translocate into nucleus failed to induce cell death. These results indicate that the proapoptotic role of AIF can be uncoupled from its physiological function. Cell death induced by AIF is through its proapoptotic activity once it is translocated to the nucleus, not due to the loss of AIF from the mitochondria.

Specific In Vivo Roles for E2Fs in Differentiation and Development

E2Fs have been historically considered as key interacting factors for the retinoblastoma (Rb) family of pocket proteins, acting as universal regulators of cell cycle progression. Often exhibiting overlapping function, deregulated E2F activity is thought to cancer or cell death. While early reports hypothesized that E2Fs may be capable of regulating distinct functions beyond proliferation, several recent reports have characterized increasingly diverse, context dependent functions for different E2Fs in vivo, often in what appears to a manner beyond traditional cell cycle regulation. Ironically, many of these new functions are still mediated through the classical cell cycle regulatory Rb family of interacting factors. Here we review the recent advances, focusing on differentiation and development, to emphasize that E2F function is likely more complex than the simple model suggests, capable of exhibiting both specificity of function, and roles beyond cell cycle progression in vivo.

Unique Requirement for Rb/E2F3 in Neuronal Migration: Evidence for Cell Cycle-Independent Functions

ABSTRACT The cell cycle regulatory retinoblastoma (Rb) protein is a key regulator of neural precursor proliferation; however, its role has been expanded to include a novel cell-autonomous role in mediating neuronal migration. We sought to determine the Rb-interacting factors that mediate both the cell cycle and migration defects. E2F1 and E2F3 are likely Rb-interacting candidates that we have shown to be deregulated in the absence of Rb. Using mice with compound null mutations of Rb and E2F1 or E2F3, we asked to what extent either E2F1 or E2F3 interacts with Rb in neurogenesis. Here, we report that E2F1 and E2F3 are both functionally relevant targets in neural precursor proliferation, cell cycle exit, and laminar patterning. Each also partially mediates the Rb requirement for neuronal survival. Neuronal migration, however, is specifically mediated through E2F3, beyond its role in cell cycle regulation. This study not only outlines overlapping and distinct functions for E2Fs in neurogenesis but also is the first to establish a physiologically relevant role for the Rb/E2F pathway beyond cell cycle regulation in vivo.

A cell‐autonomous requirement for the cell cycle regulatory protein, Rb, in neuronal migration

Precise cell cycle regulation is critical for nervous system development. To assess the role of the cell cycle regulator, retinoblastoma (Rb) protein, in forebrain development, we studied mice with telencephalon‐specific Rb deletions. We examined the role of Rb in neuronal specification and migration of diverse neuronal populations. Although layer specification occurred at the appropriate time in Rb mutants, migration of early‐born cortical neurons was perturbed. Consistent with defects in radial migration, neuronal cell death in Rb mutants specifically affected Cajal–Retzius neurons. In the ventral telencephalon, although calbindin‐ and Lhx6‐expressing cortical neurons were generated at embryonic day 12.5, their tangential migration into the neocortex was dramatically and specifically reduced in the mutant marginal zone. Cell transplantation assays revealed that defects in tangential migration arose owing to a cell‐autonomous loss of Rb in migrating interneurons and not because of a defective cortical environment. These results revealed a cell‐autonomous role for Rb in regulating the tangential migration of cortical interneurons. Taken together, we reveal a novel requirement for the cell cycle protein, Rb, in the regulation of neuronal migration.

Rb/E2F Regulates Expression of Neogenin during Neuronal Migration

ABSTRACT 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 Retinoblastoma family member p107 regulates the rate of progenitor commitment to a neuronal fate

The Retinoblastoma protein p107 regulates the neural precursor pool in both the developing and adult brain. As p107-deficient mice exhibit enhanced levels of Hes1, we questioned whether p107 regulates neural precursor self-renewal through the repression of Hes1. p107 represses transcription at the Hes1 promoter. Despite an expanded neural precursor population, p107-null mice exhibit a striking reduction in the number of cortical neurons. Hes1 deficiency rescues neurosphere numbers in p107-null embryos. We find that the loss of a single Hes1 allele in vivo restores the number of neural precursor cells at the ventricular zone. Neuronal birthdating analysis reveals a dramatic reduction in the rate of neurogenesis, demonstrating impairment in p107−/− progenitors to commit to a neuronal fate. The loss of a single Hes1 allele restores the number of newly generated neurons in p107-deficient brains. Together, we identify a novel function for p107 in promoting neural progenitor commitment to a neuronal fate.

Opposing Regulation of Sox2 by Cell-Cycle Effectors E2f3a and E2f3b in Neural Stem Cells

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.

Novel Functions for Cell Cycle Genes in Nervous System Development

Many cell cycle genes are known to play important roles in regulating proliferation in the nervous system, however, a growing body of research has proposed that these genes have diverse functions beyond cell cycle regulation. Through the study of new genetic models, cell cycle regulatory genes have been shown to impact on a number of processes during nervous system development including apoptosis, differentiation, and, most recently, neuronal migration. Here we emphasize that the proposed roles for cell cycle genes in neuronal differentiation and migration are not the consequence of deregulated cell cycle, but represent truly novel functions for cell cycle genes.

The Rb/E2F Pathway Modulates Neurogenesis through Direct Regulation of the Dlx1/Dlx2 Bigene Cluster

During brain morphogenesis, the mechanisms through which the cell cycle machinery integrates with differentiation signals remain elusive. Here we show that the Rb/E2F pathway regulates key aspects of differentiation and migration through direct control of the Dlx1 and Dlx2 homeodomain proteins, required for interneuron specification. Rb deficiency results in a dramatic reduction of Dlx1 and Dlx2 gene expression manifested by loss of interneuron subtypes and severe migration defects in the mouse brain. The Rb/E2F pathway modulates Dlx1/Dlx2 regulation through direct interaction with a Dlx forebrain-specific enhancer, I12b, and the Dlx1/Dlx2 proximal promoter regions, through repressor E2F sites both in vitro and in vivo. In the absence of Rb, we demonstrate that repressor E2Fs inhibit Dlx transcription at the Dlx1/Dlx2 promoters and Dlx1/2-I12b enhancer to suppress differentiation. Our findings support a model whereby the cell cycle machinery not only controls cell division but also modulates neuronal differentiation and migration through direct regulation of the Dlx1/Dlx2 bigene cluster during embryonic development.

Cell Cycle Regulator E2F4 Is Essential for the Development of the Ventral Telencephalon

Early forebrain development is characterized by extensive proliferation of neural precursors coupled with complex structural transformations; however, little is known regarding the mechanisms by which these processes are integrated. Here, we show that deficiency of the cell cycle regulatory protein, E2F4, results in the loss of ventral telencephalic structures and impaired self-renewal of neural precursor cells. The mechanism underlying aberrant ventral patterning lies in a dramatic loss of Sonic hedgehog (Shh) expression specifically in this region. The E2F4-deficient phenotype can be recapitulated by interbreeding mice heterozygous for E2F4 with those lacking one allele of Shh, suggesting a genetic interaction between these pathways. Treatment of E2F4-deficient cells with a Hh agonist rescues stem cell self-renewal and cells expressing the homeodomain proteins that specify the ventral telencephalic structures. Finally, we show that E2F4 deficiency results in impaired activity of Shh forebrain-specific enhancers. In conclusion, these studies establish a novel requirement for the cell cycle regulatory protein, E2F4, in the development of the ventral telencephalon.