Optimizing CRISPR/Cas9 approaches in the polymorphic tunicate Ciona intestinalis.

CRISPR/Cas9 became a powerful tool for genetic engineering and in vivo knockout also in the invertebrate chordate Ciona intestinalis. Ciona (ascidians, tunicates) is an important model organism because it shares developmental features with the vertebrates, considered the sister group of tunicates, and offers outstanding experimental advantages: a compact genome and an invariant developmental cell lineage that, combined with electroporation mediated transgenesis allows for precise and cell type specific targeting in vivo. A high polymorphism and the mosaic expression of electroporated constructs, however, often hamper the efficient CRISPR knockout, and an optimization in Ciona is desirable. Furthermore, seasonality and artificial maintenance settings can profit from in vitro approaches that would save on animals. Here we present improvements for the CRISPR/Cas9 protocol in silico, in vitro and in vivo. Firstly, in designing sgRNAs, prior sequencing of target genomic regions from experimental animals and alignment with reference genomes of C. robusta and C. intestinalis render a correction possible of subspecies polymorphisms. Ideally, the screening for efficient and non-polymorphic sgRNAs will generate a database compatible for worldwide Ciona populations. Secondly, we challenged in vitro assays for sgRNA validation towards reduced in vivo experimentation and report their suitability but also overefficiency concerning mismatch tolerance. Thirdly, when comparing Cas9 with Cas9:Geminin, thought to synchronize editing and homology-direct repair, we could indeed increase the in vivo efficiency and notably the access to an early expressed gene. Finally, for in vivo CRISPR, genotyping by next generation sequencing (NGS) ex vivo streamlined the definition of efficient single guides. Double CRISPR then generates large deletions and reliable phenotypic excision effects. Overall, while these improvements render CRISPR more efficient in Ciona, they are useful when newly establishing the technique and very transferable to CRISPR in other organisms.

Cdt1 overexpression drives colorectal carcinogenesis through origin overlicensing and DNA damage.

Chromatin licensing and DNA replication factor 1 (CDT1), a protein of the pre-replicative complex, is essential for loading the minichromosome maintenance complex (MCM) helicases onto the origins of DNA replication. While several studies have shown that dysregulation of CDT1 expression causes re-replication and DNA damage in cell lines, and CDT1 is highly expressed in several human cancers, whether CDT1 deregulation is sufficient to enhance tumorigenesis in vivo is currently unclear. To delineate its role in vivo, we overexpressed Cdt1 in the mouse colon and induced carcinogenesis using azoxymethane/dextran sodium sulfate (AOM/DSS). Here, we show that mice overexpressing Cdt1 develop a significantly higher number of tumors with increased tumor size, and more severe dysplastic changes (high-grade dysplasia), compared with control mice under the same treatment. These tumors exhibited an increased growth rate, while cells overexpressing Cdt1 loaded greater amounts of Mcm2 onto chromatin, demonstrating origin overlicensing. Adenomas overexpressing Cdt1 showed activation of the DNA damage response (DDR), apoptosis, formation of micronuclei, and chromosome segregation errors, indicating that aberrant expression of Cdt1 results in increased genomic and chromosomal instability in vivo, favoring cancer development. In line with these results, high-level expression of CDT1 in human colorectal cancer tissue specimens and colorectal cancer cell lines correlated significantly with increased origin licensing, activation of the DDR, and microsatellite instability (MSI). © 2022 The Pathological Society of Great Britain and Ireland.

Geminin is essential for DNA re-replication in the silk gland cells of silkworms.

Endoreplication, known as endocycles or endoreduplication, is a cell cycle variant in which the genomic DNA is re-replicated without mitosis leading to polyploidy. Endoreplication is essential for the development and functioning of the different organs in animals and plants. Deletion of Geminin, a DNA replication licensing inhibitor, causes DNA re-replication or damage. However, the role of Geminin in endoreplication is still unclear. Here, we studied the role of Geminin in the endoreplication of the silk gland cells of silkworms by constructing two transgenic silkworm strains, including BmGeminin1-overexpression and BmGeminin1-RNA interference. Interference of BmGeminin1 led to body weight gain, increased silk gland volume, increased DNA content, and enhanced DNA re-replication activity relative to wild-type Dazao. Meanwhile, overexpression of BmGeminin1 showed an opposite phenotype compared to the BmGem1-RNAi strain. Furthermore, RNA-sequencing of the transgenic strains was carried out to explore how BmGeminin1 regulates DNA re-replication. Our data demonstrated a vital role of Geminin in the regulation of endoreplication in the silk gland of silkworms.

Varying outcomes of triple-negative breast cancer in different age groups-prognostic value of clinical features and proliferation.

PURPOSE: Triple-negative breast cancer (TNBC) is an aggressive disease lacking specific biomarkers to guide treatment decisions. We evaluated the combined prognostic impact of clinical features and novel biomarkers of cell cycle-progression in age-dependent subgroups of TNBC patients. METHODS: One hundred forty seven TNBC patients with complete clinical data and up to 18 year follow-up were collected from Turku University Hospital, Finland. Eight biomarkers for cell division were immunohistochemically detected to evaluate their clinical applicability in relation to patient and tumor characteristics. RESULTS: Age at diagnosis was the decisive factor predicting disease-specific mortality in TNBC (p = 0.002). The established prognostic features, nodal status and Ki-67, predicted survival only when combined with age. The outcome and prognostic features differed significantly between age groups, middle-aged patients showing the most favorable outcome. Among young patients, only lack of basal differentiation predicted disease outcome, indicating 4.5-fold mortality risk (p = 0.03). Among patients aged > 57, the established prognostic features predicted disease outcome with up to 3.0-fold mortality risk for tumor size ≥ 2 cm (p = 0.001). Concerning cell proliferation, Ki-67 alone was a significant prognosticator among patients aged > 57 years (p = 0.009). Among the studied cell cycle-specific biomarkers, only geminin predicted disease outcome, indicating up to 6.2-fold increased risk of mortality for tumor size < 2 cm (p = 0.03). CONCLUSION: Traditional clinical features do not provide optimal prognostic characterization for all TNBC patients. Young age should be considered as an additional adverse prognostic feature in therapeutic considerations. Increased proliferation, as evaluated using Ki-67 or geminin immunohistochemistry, showed potential in detecting survival differences in subgroups of TNBC.

Geminin is required for Hox gene regulation to pattern the developing limb.

Development of the complex structure of the vertebrate limb requires carefully orchestrated interactions between multiple regulatory pathways and proteins. Among these, precise regulation of 5' Hox transcription factor expression is essential for proper limb bud patterning and elaboration of distinct limb skeletal elements. Here, we identified Geminin (Gmnn) as a novel regulator of this process. A conditional model of Gmnn deficiency resulted in loss or severe reduction of forelimb skeletal elements, while both the forelimb autopod and hindlimb were unaffected. 5' Hox gene expression expanded into more proximal and anterior regions of the embryonic forelimb buds in this Gmnn-deficient model. A second conditional model of Gmnn deficiency instead caused a similar but less severe reduction of hindlimb skeletal elements and hindlimb polydactyly, while not affecting the forelimb. An ectopic posterior SHH signaling center was evident in the anterior hindlimb bud of Gmnn-deficient embryos in this model. This center ectopically expressed Hoxd13, the HOXD13 target Shh, and the SHH target Ptch1, while these mutant hindlimb buds also had reduced levels of the cleaved, repressor form of GLI3, a SHH pathway antagonist. Together, this work delineates a new role for Gmnn in modulating Hox expression to pattern the vertebrate limb.

Controlling centriole numbers: Geminin family members as master regulators of centriole amplification and multiciliogenesis.

To ensure that the genetic material is accurately passed down to daughter cells during mitosis, dividing cells must duplicate their chromosomes and centrosomes once and only once per cell cycle. The same key steps-licensing, duplication, and segregation-control both the chromosome and the centrosome cycle, which must occur in concert to safeguard genome integrity. Aberrations in genome content or centrosome numbers lead to genomic instability and are linked to tumorigenesis. Such aberrations, however, can also be part of the normal life cycle of specific cell types. Multiciliated cells best exemplify the deviation from a normal centrosome cycle. They are post-mitotic cells which massively amplify their centrioles, bypassing the rule for once-per-cell-cycle centriole duplication. Hundreds of centrioles dock to the apical cell surface and generate motile cilia, whose concerted movement ensures fluid flow across epithelia. The early steps that control the generation of multiciliated cells have lately started to be elucidated. Geminin and the vertebrate-specific GemC1 and McIdas are distantly related coiled-coil proteins, initially identified as cell cycle regulators associated with the chromosome cycle. Geminin is required to ensure once-per-cell-cycle genome replication, while McIdas and GemC1 bind to Geminin and are implicated in DNA replication control. Recent findings highlight Geminin family members as early regulators of multiciliogenesis. GemC1 and McIdas specify the multiciliate cell fate by forming complexes with the E2F4/5 transcription factors to switch on a gene expression program leading to centriole amplification and cilia formation. Positive and negative interactions among Geminin family members may link cell cycle control to centriole amplification and multiciliogenesis, acting close to the point of transition from proliferation to differentiation. We review key steps of centrosome duplication and amplification, present the role of Geminin family members in the centrosome and chromosome cycle, and discuss links with disease.

Geminin ablation in vivo enhances tumorigenesis through increased genomic instability.

Geminin, a DNA replication licensing inhibitor, ensures faithful DNA replication in vertebrates. Several studies have shown that geminin depletion in vitro results in rereplication and DNA damage, whereas increased expression of geminin has been observed in human cancers. However, conditional inactivation of geminin during embryogenesis has not revealed any detectable DNA replication defects. In order to examine its role in vivo, we conditionally inactivated geminin in the murine colon and lung, and assessed chemically induced carcinogenesis. We show here that mice lacking geminin develop a significantly higher number of tumors and bear a larger tumor burden than sham-treated controls in urethane-induced lung and azoxymethane/dextran sodium sulfate-induced colon carcinogenesis. Survival is also significantly reduced in mice lacking geminin during lung carcinogenesis. A significant increase in the total number and grade of lesions (hyperplasias, adenomas, and carcinomas) was also confirmed by hematoxylin and eosin staining. Moreover, increased genomic aberrations, identified by increased ATR and γH2AX expression, was detected with immunohistochemistry analysis. In addition, we analyzed geminin expression in human colon cancer, and found increased expression, as well as a positive correlation with ATM/ATR levels and a non-monotonic association with γH2AX. Taken together, our data demonstrate that geminin acts as a tumor suppressor by safeguarding genome stability, whereas its overexpression is also associated with genomic instability. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Immunohistochemical assessment of chromatin licensing and DNA replication factor 1, geminin, and γ-H2A.X in oral epithelial precursor lesions and squamous cell carcinoma.

BACKGROUND: Carcinogenesis occurs when the cell cycle is compromised. Chromatin licensing and DNA replication factor 1, geminin, and γ-H2A histone family member X are expressed in cells in G1 phase, S/G2 /M phases, and apoptosis, respectively, and these three markers may be useful for histological evaluation of malignant lesions. Here, we aimed to identify cell cycle phases and apoptosis using immunohistochemistry in oral epithelial precursor lesions and oral squamous cell carcinoma. METHODS: Chromatin licensing and DNA replication factor 1, geminin, and γ-H2A histone family member X expression levels were immunohistochemically examined in tissue specimens from 55 patients with oral epithelial precursor lesions and 50 patients with oral squamous cell carcinoma. Associations of clinicopathological variables with marker expression were assessed. RESULTS: Chromatin licensing and DNA replication factor 1 was expressed in the prickle cell layer of oral epithelial precursor lesions and many carcinoma cells of oral squamous cell carcinoma. Geminin reactivity was widely distributed in high-grade dysplasia and oral squamous cell carcinoma rather than low-grade or no dysplastic cases. γ-H2A histone family member X was expressed in the superficial layer of oral epithelial precursor lesions and scattered carcinoma cells of oral squamous cell carcinoma. In oral squamous cell carcinoma, lower geminin expression was observed in recurrent cases. Geminin and γ-H2A histone family member X were associated with the degree of differentiation and mode of invasion. CONCLUSION: Chromatin licensing and DNA replication factor 1, geminin, and γ-H2A histone family member X expression levels were correlated with oral carcinogenesis; these markers were associated with clinicopathological behaviors in oral squamous cell carcinoma.

Geminin deletion increases the number of fetal hematopoietic stem cells by affecting the expression of key transcription factors.

Balancing stem cell self-renewal and initiation of lineage specification programs is essential for the development and homeostasis of the hematopoietic system. We have specifically ablated geminin in the developing murine hematopoietic system and observed profound defects in the generation of mature blood cells, leading to embryonic lethality. Hematopoietic stem cells (HSCs) accumulated in the fetal liver following geminin ablation, while committed progenitors were reduced. Genome-wide transcriptome analysis identified key HSC transcription factors as being upregulated upon geminin deletion, revealing a gene network linked with geminin that controls fetal hematopoiesis. In order to obtain mechanistic insight into the ability of geminin to regulate transcription, we examined Hoxa9 as an example of a key gene in definitive hematopoiesis. We demonstrate that in human K562 cells geminin is associated with HOXA9 regulatory elements and its absence increases HOXA9 transcription similarly to that observed in vivo. Moreover, silencing geminin reduced recruitment of the PRC2 component SUZ12 to the HOXA9 locus and resulted in an increase in RNA polymerase II recruitment and H3K4 trimethylation (H3K4me3), whereas the repressive marks H3K9me3 and H3K27me3 were reduced. The chromatin landscape was also modified at the regulatory regions of HOXA10 and GATA1. K562 cells showed a reduced ability to differentiate to erythrocytes and megakaryocytes upon geminin silencing. Our data suggest that geminin is indispensable for fetal hematopoiesis and regulates the generation of a physiological pool of stem and progenitor cells in the fetal hematopoietic system.

Mcidas and GemC1 are key regulators for the generation of multiciliated ependymal cells in the adult neurogenic niche.

Multiciliated cells are abundant in the epithelial surface of different tissues, including cells lining the walls of the lateral ventricles in the brain and the airway epithelium. Their main role is to control fluid flow and defects in their differentiation are implicated in many human disorders, such as hydrocephalus, accompanied by defects in adult neurogenesis and mucociliary disorder in the airway system. Here we show that Mcidas, which is mutated in human mucociliary clearance disorder, and GemC1 (Gmnc or Lynkeas), previously implicated in cell cycle progression, are key regulators of multiciliated ependymal cell generation in the mouse brain. Overexpression and knockdown experiments show that Mcidas and GemC1 are sufficient and necessary for cell fate commitment and differentiation of radial glial cells to multiciliated ependymal cells. Furthermore, we show that GemC1 and Mcidas operate in hierarchical order, upstream of Foxj1 and c-Myb transcription factors, which are known regulators of ependymal cell generation, and that Notch signaling inhibits GemC1 and Mcidas function. Our results suggest that Mcidas and GemC1 are key players in the generation of multiciliated ependymal cells of the adult neurogenic niche.

Concise Review: Geminin-A Tale of Two Tails: DNA Replication and Transcriptional/Epigenetic Regulation in Stem Cells.

Molecular mechanisms governing maintenance, commitment, and differentiation of stem cells are largely unexploited. Molecules involved in the regulation of multiple cellular processes are of particular importance for stem cell physiology, as they integrate different signals and coordinate cellular decisions related with self-renewal and fate determination. Geminin has emerged as a critical factor in DNA replication and stem cell differentiation in different stem cell populations. Its inhibitory interaction with Cdt1, a member of the prereplicative complex, ensures the controlled timing of DNA replication and, consequently, genomic stability in actively proliferating cells. In embryonic as well as somatic stem cells, Geminin has been shown to interact with transcription factors and epigenetic regulators to drive gene expression programs and ultimately guide cell fate decisions. An ever-growing number of studies suggests that these interactions of Geminin and proteins regulating transcription are conserved among metazoans. Interactions between Geminin and proteins modifying the epigenome, such as members of the repressive Polycomb group and the SWI/SNF proteins of the permissive Trithorax, have long been established. The complexity of these interactions, however, is only just beginning to unravel, revealing key roles on maintaining stem cell self-renewal and fate specification. In this review, we summarize current knowledge and give new perspectives for the role of Geminin on transcriptional and epigenetic regulation, alongside with its regulatory activity in DNA replication and their implication in the regulation of stem and progenitor cell biology. Stem Cells 2017;35:299-310.

Inactivation of Geminin in neural crest cells affects the generation and maintenance of enteric progenitor cells, leading to enteric aganglionosis.

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.

Different effects of neuropeptide Y on proliferation of vascular smooth muscle cells via regulation of Geminin.

The proliferation-promoting effect of neuropeptide Y (NPY) always functions in low-serum-cultured vascular smooth muscle cells (VSMCs), and the phenotypic switch of VSMCs is regulated by concentrations of serum. Whether the property of the NPY proliferative effect in VSMCs relies on phenotype of VSMCs is unclear. We aimed to explore the role of NPY on proliferation of different VSMC phenotypes in the pathogenesis of atherosclerosis. By stimulating A10 cells with 200 nM NPY in 0.5 or 10% serum, 3H-thymidine and 5-ethynyl-2'-deoxyuridine (EdU) and CCK8 measurements were used to detect VSMC proliferation. RT-PCR and Flow cytometry were performed to detect the factors involved in different properties of the NPY proliferative effect in VSMCs. Instead of facilitating proliferation, NPY had no significant effect on the growth of VSMCs when cultured in 10% serum (VSMCs stayed at synthetic states). The underlying mechanism may be involved in down-regulation of Y1 receptor (P < 0.05 vs. Vehicle) and up-regulation of Geminin (P < 0.05 vs. Vehicle) in 10% serum-cultured VSMCs co-incubated with 200 nM NPY. Besides, modulation of Geminin was effectively blocked by the Y1 receptor antagonist. The stimulation of NPY on proliferation of VSMCs could be a double-edged sword in the development of atherosclerosis and thus provides new knowledge for therapy of atherosclerosis.

Geminin deletion in pre-meiotic DNA replication stage causes spermatogenesis defect and infertility.

Geminin plays a critical role in cell cycle regulation by regulating DNA replication and serves as a transcriptional molecular switch that directs cell fate decisions. Spermatogonia lacking Geminin disappear during the initial wave of mitotic proliferation, while geminin is not required for meiotic progression of spermatocytes. It is unclear whether geminin plays a role in pre-meiotic DNA replication in later-stage spermatogonia and their subsequent differentiation. Here, we selectively disrupted Geminin in the male germline using the Stra8-Cre/loxP conditional knockout system. Geminin-deficient mice showed atrophic testes and infertility, concomitant with impaired spermatogenesis and reduced sperm motility. The number of undifferentiated spermatogonia and spermatocytes was significantly reduced; the pachytene stage was impaired most severely. Expression of cell proliferation-associated genes was reduced in Gmnnfl/Δ; Stra8-Cre testes compared to in controls. Increased DNA damage, decreased Cdt1, and increased phosphorylation of Chk1/Chk2 were observed in Geminin-deficient germ cells. These results suggest that geminin plays important roles in pre-meiotic DNA replication and subsequent spermatogenesis.

Geminin prevents DNA damage in vagal neural crest cells to ensure normal enteric neurogenesis.

BACKGROUND: In vertebrate organisms, the neural crest (NC) gives rise to multipotential and highly migratory progenitors which are distributed throughout the embryo and generate, among other structures, the peripheral nervous system, including the intrinsic neuroglial networks of the gut, i.e. the enteric nervous system (ENS). The majority of enteric neurons and glia originate from vagal NC-derived progenitors which invade the foregut mesenchyme and migrate rostro-caudally to colonise the entire length of the gut. Although the migratory behaviour of NC cells has been studied extensively, it remains unclear how their properties and response to microenvironment change as they navigate through complex cellular terrains to reach their target embryonic sites. RESULTS: Using conditional gene inactivation in mice we demonstrate here that the cell cycle-dependent protein Geminin (Gem) is critical for the survival of ENS progenitors in a stage-dependent manner. Gem deletion in early ENS progenitors (prior to foregut invasion) resulted in cell-autonomous activation of DNA damage response and p53-dependent apoptosis, leading to severe intestinal aganglionosis. In contrast, ablation of Gem shortly after ENS progenitors had invaded the embryonic gut did not result in discernible survival or migratory deficits. In contrast to other developmental systems, we obtained no evidence for a role of Gem in commitment or differentiation of ENS lineages. The stage-dependent resistance of ENS progenitors to mutation-induced genotoxic stress was further supported by the enhanced survival of post gut invasion ENS lineages to γ-irradiation relative to their predecessors. CONCLUSIONS: Our experiments demonstrate that, in mammals, NC-derived ENS lineages are sensitive to genotoxic stress in a stage-specific manner. Following gut invasion, ENS progenitors are distinctly resistant to Gem ablation and irradiation in comparison to their pre-enteric counterparts. These studies suggest that the microenvironment of the embryonic gut protects ENS progenitors and their progeny from genotoxic stress.

Neural transcription factors bias cleavage stage blastomeres to give rise to neural ectoderm.

The decision by embryonic ectoderm to give rise to epidermal versus neural derivatives is the result of signaling events during blastula and gastrula stages. However, there also is evidence in Xenopus that cleavage stage blastomeres contain maternally derived molecules that bias them toward a neural fate. We used a blastomere explant culture assay to test whether maternally deposited transcription factors bias 16-cell blastomere precursors of epidermal or neural ectoderm to express early zygotic neural genes in the absence of gastrulation interactions or exogenously supplied signaling factors. We found that Foxd4l1, Zic2, Gmnn, and Sox11 each induced explants made from ventral, epidermis-producing blastomeres to express early neural genes, and that at least some of the Foxd4l1 and Zic2 activities are required at cleavage stages. Similarly, providing extra Foxd4l1 or Zic2 to explants made from dorsal, neural plate-producing blastomeres significantly increased the expression of early neural genes, whereas knocking down either significantly reduced them. These results show that maternally delivered transcription factors bias cleavage stage blastomeres to a neural fate. We demonstrate that mouse and human homologs of Foxd4l1 have similar functional domains compared to the frog protein, as well as conserved transcriptional activities when expressed in Xenopus embryos and blastomere explants. genesis 54:334-349, 2016. © 2016 Wiley Periodicals, Inc.

Geminin is Essential to Prevent DNA Re-Replication-Dependent Apoptosis in Pluripotent Cells, but not in Differentiated Cells.

Geminin is a dual-function protein unique to multicellular animals with roles in modulating gene expression and preventing DNA re-replication. Here, we show that geminin is essential at the beginning of mammalian development to prevent DNA re-replication in pluripotent cells, exemplified by embryonic stem cells, as they undergo self-renewal and differentiation. Embryonic stem cells, embryonic fibroblasts, and immortalized fibroblasts were characterized before and after geminin was depleted either by gene ablation or siRNA. Depletion of geminin under conditions that promote either self-renewal or differentiation rapidly induced DNA re-replication, followed by DNA damage, then a DNA damage response, and finally apoptosis. Once differentiation had occurred, geminin was no longer essential for viability, although it continued to contribute to preventing DNA re-replication induced DNA damage. No relationship was detected between expression of geminin and genes associated with either pluripotency or differentiation. Thus, the primary role of geminin at the beginning of mammalian development is to prevent DNA re-replication-dependent apoptosis, a role previously believed essential only in cancer cells. These results suggest that regulation of gene expression by geminin occurs only after pluripotent cells differentiate into cells in which geminin is not essential for viability.

Early neural ectodermal genes are activated by Siamois and Twin during blastula stages.

BMP signaling distinguishes between neural and non-neural fates by activating epidermis-specific transcription and repressing neural-specific transcription. The neural ectoderm forms after the Organizer secrets antagonists that prevent these BMP-mediated activities. However, it is not known whether neural genes also are transcriptionally activated. Therefore, we tested the ability of nine Organizer transcription factors to ectopically induce the expression of four neural ectodermal genes in epidermal precursors. We found evidence for two pathways: Foxd4 and Sox11 were only induced by Sia and Twn, whereas Gmnn and Zic2 were induced by Sia, Twn, as well as seven other Organizer transcription factors. The induction of Foxd4, Gmnn and Zic2 by Sia/Twn was both non-cell autonomous (requiring an intermediate protein) and cell autonomous (direct), whereas the induction of Sox11 required Foxd4 activity. Because direct induction by Sia/Twn could occur endogenously in the dorsal-equatorial blastula cells that give rise to both the Organizer mesoderm and the neural ectoderm, we knocked down Sia/Twn in those cells. This prevented the blastula expression of Foxd4 and Sox11, demonstrating that Sia/Twn directly activate some neural genes before the separation of the Organizer mesoderm and neural ectoderm lineages.

The dual roles of geminin during trophoblast proliferation and differentiation.

Geminin is a protein involved in both DNA replication and cell fate acquisition. Although it is essential for mammalian preimplantation development, its role remains unclear. In one study, ablation of the geminin gene (Gmnn) in mouse preimplantation embryos resulted in apoptosis, suggesting that geminin prevents DNA re-replication, whereas in another study it resulted in differentiation of blastomeres into trophoblast giant cells (TGCs), suggesting that geminin regulates trophoblast specification and differentiation. Other studies concluded that trophoblast differentiation into TGCs is regulated by fibroblast growth factor-4 (FGF4), and that geminin is required to maintain endocycles. Here we show that ablation of Gmnn in trophoblast stem cells (TSCs) proliferating in the presence of FGF4 closely mimics the events triggered by FGF4 deprivation: arrest of cell proliferation, formation of giant cells, excessive DNA replication in the absence of DNA damage and apoptosis, and changes in gene expression that include loss of Chk1 with up-regulation of p57 and p21. Moreover, FGF4 deprivation of TSCs reduces geminin to a basal level that is required for maintaining endocycles in TGCs. Thus, geminin acts both like a component of the FGF4 signal transduction pathway that governs trophoblast proliferation and differentiation, and geminin is required to maintain endocycles.

Geminin inhibits a late step in the formation of human pre-replicative complexes.

The initial step in initiation of eukaryotic DNA replication involves the assembly of pre-replicative complexes (pre-RCs) at origins of replication during the G1 phase of the cell cycle. In metazoans initiation is inhibited by the regulatory factor Geminin. We have purified the human pre-RC proteins, studied their interactions in vitro with each other and with origin DNA, and analyzed the effects of HsGeminin on formation of DNA-protein complexes. The formation of an initial complex containing the human origin recognition complex (HsORC), HsCdt1, HsCdc6, and origin DNA is cooperative, involving all possible binary interactions among the components. Maximal association of HsMCM2-7, a component of the replicative helicase, requires HsORC, HsCdc6, HsCdt1, and ATP, and is driven by interactions of HsCdt1 and HsCdc6 with multiple HsMCM2-7 subunits. Formation of stable complexes, resistant to high salt, requires ATP hydrolysis. In the absence of HsMCM proteins, HsGeminin inhibits the association of HsCdt1 with DNA or with HsORC-HsCdc6-DNA complexes. However, HsGeminin does not inhibit recruitment of HsMCM2-7 to DNA to form complexes containing all of the pre-RC proteins. In fact, HsGeminin itself is a component of such complexes, and interacts directly with the HsMcm3 and HsMcm5 subunits of HsMCM2-7, as well as with HsCdt1. Although HsGeminin does not prevent the initial formation of DNA-protein complexes containing the pre-RC proteins, it strongly inhibits the formation of stable pre-RCs that are resistant to high salt. We suggest that bound HsGeminin prevents transition of the pre-RC to a state that is competent for initiation of DNA replication.