Licensing regulators Geminin and Cdt1 identify progenitor cells of the mouse CNS in a specific phase of the cell cycle

doi:10.1016/j.neuroscience.2007.03.050

Neuroscience

Volume 147, Issue 2, 29 June 2007, Pages 373-387

https://doi.org/10.1016/j.neuroscience.2007.03.050 Get rights and content

Abstract

Nervous system formation integrates control of cellular proliferation and differentiation and is mediated by multipotent neural progenitor cells that become progressively restricted in their developmental potential before they give rise to differentiated neurons and glial cells. Evidence from different experimental systems suggests that Geminin is a candidate molecule linking proliferation and differentiation during nervous system development. We show here that Geminin and its binding partner Cdt1 are expressed abundantly by neural progenitor cells during early mouse neurogenesis. Their expression levels decline at late developmental stages and become undetectable upon differentiation. Geminin and Cdt1 expressing cells also express Sox2 while no overlap is detected with cells expressing markers of a differentiated neuronal phenotype. A fraction of radial glial cells expressing RC2 and Pax6 are also immunoreactive for Geminin and Cdt1. The majority of the Geminin and Cdt1 expressing cell populations appears to be distinct from fate-restricted precursor cells expressing Mash1 or Neurogenin2. Bromo-deoxy-uridine (BrdU) incorporation experiments reveal a cell cycle specific expression in neural progenitor cells, with Geminin being present from S to M phase, while Cdt1 expression characterizes progenitor cells in G1 phase. Furthermore, in vitro differentiation of adult neurosphere cultures shows downregulation of Geminin/Cdt1 in the differentiated state, in line with our data showing that Geminin is present in neural progenitor cells of the CNS during mouse embryogenesis and adulthood and becomes downregulated upon cell fate specification and differentiation. This suggests a role for Geminin in the formation and maintenance of the neural progenitor cells.

Section snippets

Plasmids

We cloned by PCR the full-length open reading frames of mouse Geminin and Cdt1 into EcoRI/BamHI and BamHI/HindIII sites of pBluescript KS, respectively, using the following set of primers: Geminin, sense 5′-CGGAATTCTGAAAAATGAATCTCAGTATG-3′; anti-sense 5′-GCGGATCCATAGCCCCAGGATTCAAACCTG-3′; Cdt1, sense 5′-GTGGATCCACTACCTTTTTGGCGCCATG-3′; anti-sense 5′-GGTAAGCTTCTCTGTGACAGGAGG-3′ (Xouri et al., 2004).

In situ hybridization

Non-radioactive in situ hybridization was performed using 12 μm fresh-frozen sections of embryonic

Distribution of Geminin and Cdt1 mRNA in the developing CNS

In order to gain an insight into the role of Geminin and Cdt1 in mammalian neurogenesis, we initially characterized their spatial and temporal expression patterns in the CNS during mouse embryonic development, by in situ hybridization (ISH) on E11.5 (data not shown), E12.5 and E15.5 mouse embryos (Fig. 1).

At E12.5, Geminin mRNA can be detected relatively uniformly in the VZ of the cerebral cortex and the ganglionic eminences of the forebrain (Fig. 1A). More posteriorly, it is expressed in the

Discussion

Geminin was first described as a bifunctional protein, regulating both DNA replication and neural cell fate acquisition (Kroll et al 1998, McGarry and Kirschner 1998). Multiple roles have been attributed to Geminin during neurogenesis using different experimental systems (Quinn et al 2001, Del Bene et al 2004, Luo et al 2004, Seo et al 2005a). We undertook an analysis of Geminin and Cdt1 expression during mouse development and in adult brain using different markers to characterize the various

Conclusion

In summary, we have thoroughly defined the expression profiles of Geminin and Cdt1 during mouse neurogenesis. We have demonstrated that both proteins characterize the proliferating neurogenic regions of the developing CNS and that they delineate a specific population of neural precursor cells not yet committed toward a neuronal subtype, providing a potential new tool to further discriminate proliferating and immature cells as they progress from neural stem cells to transit amplifying progenitor

Acknowledgments

We would like to thank Dr. Jordane Malaterre and Mr. Sebastian Dworkin for help and advice and Dr. P. Liodis for critical reading of the manuscript. We are also thankful to Dr. V. Pachnis and Dr D. Kioussis for their support and critical reading of the manuscript. This work was supported by the Karatheodori program of the University of Patras and the Association for International Cancer Research, UK to S. Taraviras.

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To define the consequences of defective replication in cortical development, we investigated the response of early progenitors (herein referred to as NECs) and mid-neurogenesis progenitors (referred to as NPs) to impaired replication licensing by depleting Geminin (GMNN). GMNN is a known inhibitor of DNA replication licensing that is expressed in the proliferating neural stem and progenitor cells and is downregulated upon cell-cycle exit and differentiation (Spella et al., 2007). Previous studies have shown that suppression of GMNN in cultured cells induces aberrant licensing and incomplete genome replication (Huang et al., 2015; Klotz-Noack et al., 2012; Melixetian et al., 2004).
Impaired replication has been previously linked to growth retardation and microcephaly; however, why the brain is critically affected compared with other organs remains elusive. Here, we report the differential response between early neural progenitors (neuroepithelial cells [NECs]) and fate-committed neural progenitors (NPs) to replication licensing defects. Our results show that, while NPs can tolerate altered expression of licensing factors, NECs undergo excessive replication stress, identified by impaired replication, increased DNA damage, and defective cell-cycle progression, leading eventually to NEC attrition and microcephaly. NECs that possess a short G1 phase license and activate more origins than NPs, by acquiring higher levels of DNA-bound MCMs. In vivo G1 shortening in NPs induces DNA damage upon impaired licensing, suggesting that G1 length correlates with replication stress hypersensitivity. Our findings propose that NECs possess distinct cell-cycle characteristics to ensure fast proliferation, although these inherent features render them susceptible to genotoxic stress.
Adult Neural Stem Cells and Multiciliated Ependymal Cells Share a Common Lineage Regulated by the Geminin Family Members
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Then the DNA fragment corresponding to the GemC1 cDNA size was isolated from an agarose gel and ligated to the pBlueScriptKS backbone, upstream of the T3 promoter sequence. The pBlueScriptKS-Geminin plasmid was a gift from the laboratory of S. Taraviras (Spella et al., 2007). Briefly, in this study, the open reading frame of Geminin was cloned between the EcoRI/BamHI sites of the pBlueScriptKS plasmid, upstream of the T7 promoter sequence.
Adult neural stem cells and multiciliated ependymal cells are glial cells essential for neurological functions. Together, they make up the adult neurogenic niche. Using both high-throughput clonal analysis and single-cell resolution of progenitor division patterns and fate, we show that these two components of the neurogenic niche are lineally related: adult neural stem cells are sister cells to ependymal cells, whereas most ependymal cells arise from the terminal symmetric divisions of the lineage. Unexpectedly, we found that the antagonist regulators of DNA replication, GemC1 and Geminin, can tune the proportion of neural stem cells and ependymal cells. Our findings reveal the controlled dynamic of the neurogenic niche ontogeny and identify the Geminin family members as key regulators of the initial pool of adult neural stem cells.
Inactivation of geminin in neural crest cells affects the generation and maintenance of enteric progenitor cells, leading to enteric aganglionosis
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NCCs delaminate from the dorsal part of the NT by undergoing an epithelial to mesenchymal transition (EMT). Geminin expression starts as cells enter into S phase, a stage that has been shown to be essential for the efficient formation of the NCCs (Burstyn-Cohen and Kalcheim, 2002; Spella et al., 2007; Xouri et al., 2004). Moreover, a role in EMT and regulation of E-cadherin expression has been attributed to Geminin during early gastrulation (Emmett and O'Shea, 2012), which suggests a potential mechanism for the defects in the generation of early NCCs observed in mice that lack Geminin expression
Neural crest cells comprise a multipotent, migratory cell population that generates a diverse array of cell and tissue types, during vertebrate development. Enteric Nervous System controls the function of the gastrointestinal tract and is mainly derived from the vagal and sacral neural crest cells. Deregulation on self-renewal and differentiation of the enteric neural crest cells is evident in enteric nervous system disorders, such as Hirschsprung disease, characterized by the absence of ganglia in a variable length of the distal bowel. Here we show that Geminin is essential for Enteric Nervous System generation as mice that lacked Geminin expression specifically in neural crest cells revealed decreased generation of vagal neural crest cells, and enteric neural crest cells (ENCCs). Geminin-deficient ENCCs showed increased apoptosis and decreased cell proliferation during the early stages of gut colonization. Furthermore, decreased number of committed ENCCs in vivo and the decreased self-renewal capacity of enteric progenitor cells in vitro, resulted in almost total aganglionosis resembling a severe case of Hirschsprung disease. Our results suggest that Geminin is an important regulator of self-renewal and survival of enteric nervous system progenitor cells.
Lice;nsing of DNA replication, cancer, pluripotency and differentiation: An interlinked world?
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Recent findings provide evidence for a functional interplay between DNA replication and the seemingly distinct areas of cancer, development and pluripotency. Protein complexes participating in DNA replication origin licensing are now known to have roles in development, while their deregulation can lead to cancer. Moreover, transcription factors implicated in the maintenance of or reversal to the pluripotent state have links to the pre-replicative machinery. Several studies have shown that overexpression of these factors is associated to cancer.
The Geminin and Idas coiled coils preferentially form a heterodimer that Inhibits Geminin function in DNA replication licensing
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High levels of Idas have been previously reported in the choroid plexus and the cortical hem of the mouse telencephalon (24). In this specific part of the brain Geminin is predominantly expressed by neural progenitors (48), whereas Idas levels become higher in specific differentiated cell types (Pefani et al. (24)). High levels of Idas have also been reported in other developing multiciliated cells (Stubbs et al. (25)).
Geminin is an important regulator of proliferation and differentiation in metazoans, which predominantly inhibits the DNA replication licensing factor Cdt1, preventing genome over-replication. We show that Geminin preferentially forms stable coiled-coil heterodimers with its homologue, Idas. In contrast to Idas-Geminin heterodimers, Idas homodimers are thermodynamically unstable and are unlikely to exist as a stable macromolecule under physiological conditions. The crystal structure of the homology regions of Idas in complex with Geminin showed a tight head-to-head heterodimeric coiled-coil. This Idas-Geminin heterodimer binds Cdt1 less strongly than Geminin-Geminin, still with high affinity (∼30 nm), but with notably different thermodynamic properties. Consistently, in Xenopus egg extracts, Idas-Geminin is less active in licensing inhibition compared with a Geminin-Geminin homodimer. In human cultured cells, ectopic expression of Idas leads to limited over-replication, which is counteracted by Geminin co-expression. The properties of the Idas-Geminin complex suggest it as the functional form of Idas and provide a possible mechanism to modulate Geminin activity.
Background: Idas is a homologue of the proliferation and differentiation regulator, Geminin.
Results: Idas and Geminin form preferentially a stable heterodimeric coiled-coil that interacts with Cdt1 in vitro and in cells.
Conclusion: The Idas-Geminin complex inhibits Geminin function in DNA replication licensing.
Significance: The Idas-Geminin complex, possibly the only functional form of Idas, likely regulates the diverse functions of Geminin.
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Cellular senescence is a permanent out-of-cycle state regulated by molecular circuits acting during the G1 phase of the cell cycle. Cdt1 is a central regulator of DNA replication licensing acting during the G1 phase and it is negatively controlled by Geminin. Here, we characterize the cell cycle expression pattern of Cdt1 and Geminin during successive passages of primary fibroblasts and compare it to tumour-derived cell lines. Cdt1 and Geminin are strictly expressed in distinct subpopulations of young fibroblasts, similarly to cancer cells, with Geminin accumulating shortly after the onset of S phase. Cdt1 and Geminin are down-regulated when primary human and mouse fibroblasts undergo replicative or stress-induced senescence. RNAi-mediated Geminin knock-down in human cells enhances the appearance of phenotypic and molecular features of senescence. Mouse embryonic fibroblasts heterozygous for Geminin exhibit accelerated senescence compared to control fibroblasts. In contrast, ectopic expression of Geminin in mouse embryonic fibroblasts delays the appearance of the senescent phenotype. Taken together, our data suggest that changes in Geminin expression levels affect the establishment of senescence pathways.

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¹: Present address: Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA (O. Britz); Department of Pharmaceutical Biology, Victorian College of Pharmacy, Monash University, Victoria 3052, Australia (T. Mantamadiotis).

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Cellular neuroscienceLicensing regulators Geminin and Cdt1 identify progenitor cells of the mouse CNS in a specific phase of the cell cycle

Abstract

Section snippets

Plasmids

In situ hybridization

Distribution of Geminin and Cdt1 mRNA in the developing CNS

Discussion

Conclusion

Acknowledgments

Dev Cell

Neuron

Cell

Neuron

Neuron

Neuron

Cell

Curr Opin Genet Dev

Biochem Biophys Res Commun

Cell

Neuron

Dev Biol

Cell

Brain Res Dev Brain Res

Neuron

J Biol Chem

Curr Opin Neurobiol

Trends Neurosci

Mutat Res

Cell

Dev Biol

Dev Biol

Radial glia serve as neuronal progenitors in all regions of the central nervous system

Neuron

Hu protein as an early marker of neuronal phenotypic differentiation by subependymal zone cells of the adult songbird forebrain

J Neurobiol

Proneural genes and the specification of neural cell types

Nat Rev Neurosci

A role for proneural genes in the maturation of cortical progenitor cells

Cereb Cortex

Transient expression of doublecortin during adult neurogenesis

J Comp Neurol

Doublecortin expression levels in adult brain reflect neurogenesis

Eur J Neurosci

Direct interaction of geminin and Six3 in eye development

Nature

Cellular neuroscience
Licensing regulators Geminin and Cdt1 identify progenitor cells of the mouse CNS in a specific phase of the cell cycle