Jacqueline L. Vanderluit

Telencephalon-specific Rb knockouts reveal enhanced neurogenesis, survival and abnormal cortical development.

Correct cell cycle regulation and terminal mitosis are critical for nervous system development. The retinoblastoma (Rb) protein is a key regulator of these processes, as Rb−/− embryos die by E15.5, exhibiting gross hematopoietic and neurological defects. The extensive apoptosis in Rb−/− embryos has been attributed to aberrant S phase entry resulting in conflicting growth control signals in differentiating cells. To assess the role of Rb in cortical development in the absence of other embryonic defects, we examined mice with telencephalon‐specific Rb deletions. Animals carrying a floxed Rb allele were interbred with mice in which cre was knocked into the Foxg1 locus. Unlike germline knockouts, mice specifically deleted for Rb in the developing telencephalon survived until birth. In these mutants, Rb−/− progenitor cells divided ectopically, but were able to survive and differentiate. Mutant brains exhibited enhanced cellularity due to increased proliferation of neuroblasts. These studies demonstrate that: (i) cell cycle deregulation during differentiation does not necessitate apoptosis; (ii) Rb‐deficient mutants exhibit enhanced neuroblast proliferation; and (iii) terminal mitosis may not be required to initiate differentiation.

MCL‐1 is a stress sensor that regulates autophagy in a developmentally regulated manner

Apoptosis has an important role during development to regulate cell number. In differentiated cells, however, activation of autophagy has a critical role by enabling cells to remain functional following stress. In this study, we show that the antiapoptotic BCL‐2 homologue MCL‐1 has a key role in controlling both processes in a developmentally regulated manner. Specifically, MCL‐1 degradation is an early event not only following induction of apoptosis, but also under nutrient deprivation conditions where MCL‐1 levels regulate activation of autophagy. Furthermore, deletion of MCL‐1 in cortical neurons of transgenic mice activates a robust autophagic response. This autophagic response can, however, be converted to apoptosis by either reducing the levels of the autophagy regulator Beclin‐1, or by a concomitant activation of BAX. Our results define a pathway whereby MCL‐1 has a key role in determining cell fate, by coordinately regulating apoptosis and autophagy.

p107 regulates neural precursor cells in the mammalian brain

Here we show a novel function for Retinoblastoma family member, p107 in controlling stem cell expansion in the mammalian brain. Adult p107-null mice had elevated numbers of proliferating progenitor cells in their lateral ventricles. In vitro neurosphere assays revealed striking increases in the number of neurosphere forming cells from p107−/− brains that exhibited enhanced capacity for self-renewal. An expanded stem cell population in p107-deficient mice was shown in vivo by (a) increased numbers of slowly cycling cells in the lateral ventricles; and (b) accelerated rates of neural precursor repopulation after progenitor ablation. Notch1 was up-regulated in p107−/− neurospheres in vitro and brains in vivo. Chromatin immunoprecipitation and p107 overexpression suggest that p107 may modulate the Notch1 pathway. These results demonstrate a novel function for p107 that is distinct from Rb, which is to negatively regulate the number of neural stem cells in the developing and adult brain.

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.

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.

HES1 regulates 5-HT1A receptor gene transcription at a functional polymorphism: essential role in developmental expression.

Mammalian HES1 and HES5 are abundant in developing CNS and inhibit neurogenesis, while HES6 promotes neurogenesis. An early serotonergic differentiation marker, the 5-HT1A receptor, is repressed by HES5 and DEAF1 which recognize the C(-1019), but not G(-1019) allele of a human 5-HT1A promoter polymorphism associated with mood disorders. We tested whether HES1 and HES6 regulate transcriptional activity at this element. HES1 strongly repressed 5-HT1A transcription in neuronal and non-neuronal cells, while HES6 reversed HES1- and HES5-mediated repression. Mutation of a putative HES consensus site blocked HES1 and HES5, but, unlike HES5, HES1 repressed at the G(-1019) allele. To address its role in vivo, the temporal expression of 5-HT1A receptor RNA and protein was examined in HES1-/- mice, and elevated levels in E12.5 hindbrain and midbrain were observed. Thus, HES1 and HES6 oppositely regulate 5-HT1A receptor transcription and HES1 is required for its correct developmental expression.

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.

The p107/E2F Pathway Regulates Fibroblast Growth Factor 2 Responsiveness in Neural Precursor Cells

ABSTRACT We have previously shown that p107, a member of the retinoblastoma (Rb) cell cycle regulatory family, has a unique function in regulating the pool of neural precursor cells. As the pool of progenitors is regulated by a limiting supply of trophic factors, we asked if the Rb/E2F pathway may control the size of the progenitor population by regulating the levels of growth factors or their receptors. Here, we demonstrate that fibroblast growth factor 2 (FGF2) is aberrantly upregulated in the brains of animals lacking Rb family proteins and that the gene encoding the FGF2 ligand is directly regulated by p107 and E2F3. Chromatin immunoprecipitation assays demonstrated that E2F3 and p107 occupy E2F consensus sites on the FGF2 promoter in the context of native chromatin. To evaluate the physiological consequence of FGF2 deregulation in both p107 and E2F3 mutants, we measured neural progenitor responsiveness to growth factors. Our results demonstrate that E2F3 and p107 are each mediators of FGF2 growth factor responsiveness in neural progenitor cells. These results support a model whereby p107 regulates the pool of FGF-responsive progenitors by directly regulating FGF2 gene expression in vivo. By identifying novel roles for p107/E2F in regulating genes outside of the classical cell cycle machinery targets, we uncover a new mechanism whereby Rb/E2F mediates proliferation through regulating growth factor responsiveness.

Emerging Roles for the Retinoblastoma Gene Family

Research on the retinoblastoma protein has grown from studying its role as a tumour suppressor in cancer to identifying it as a key regulator of the cell cycle G1/S check point and today to exploring its function in numerous cellular processes. The recent development of conditional knockout mice has shed new light on the roles of Rb in embryonic development and has aided in the identification of the cell-of-origin in Retinoblastoma cancer. In this review, we will discuss the role of Rb as a tumour suppressor as well as its role in cell division, differentiation, apoptosis and cancer.