Histone H2B-GFP Research Articles

Clonogenic Assays to Detect Cell Fate in Mitotic Catastrophe.

Mitotic catastrophe (MC) is a cell death modality induced by DNA damage that involves the activation of cell cycle checkpoints such as the "DNA structure checkpoint" and "spindle assembly checkpoint" (SAC) leading to aberrant mitosis. Depending on the signal, MC can drive the cell to death or to senescence. The suppression of MC favors aneuploidy. Several cancer therapies, included microtubular poisons and radiations, trigger MC. The clonogenic assay has been used to study the capacity of single cells to proliferate and to generate macroscopic colonies and to evaluate the efficacy of anticancer drugs. Nevertheless, this method cannot analyze MC events. Here, we report an improved technique based on the use of human colon cancer HCT116 stable expressing histone H2B-GFP and DsRed-centrin proteins, allowing to determine the capacity of cells to proliferate, and to determine changes in the nucleus and centrosomes.

Dynamical Signatures of Local DNA Damage in Live Cells

The dynamic organization of chromatin inside the eukaryotic cell nucleus is essential to the regulation of the genome such as maintaining its integrity, gene expression and replication. However, the physical picture of chromatin structure and dynamics remains unclear and requires identifying the dynamical and structural signatures of specific biological processes. In this work, we study the chromatin dynamics in interphase, time between two cell divisions, in response to DNA damage. As shown previously, chromatin dynamics across the whole nucleus change dramatically after DNA damage; chromatin's coherent motion is eliminated, while its local mobility increases and its compaction decreases [1]. Here, we explore the local chromatin dynamics and compaction in response to local DNA damage in human cells. To do so, we induce double stranded DNA breaks (DSBs), which we visualize using 53BP1-mCherry, and observe the spatiotemporal evolution of their dynamics using single particle tracking. Simultaneously, we use histone H2B-GFP as a reporter on the chromatin compaction, to compare DSB dynamics inside the heterochromatin and euchromatin. To elucidate the dynamic signature of the local DNA damage response, we vary the number of DSBs as well as explore the ATP-dependence of these processes. Our preliminary results show the local subdiffusive dynamics of the DSBs changes with an increasing DSB count inside the cell nucleus. We also explore the relationship between the protein aggregation, diffusion coefficient and chromatin compaction at a DSB. [1] Zidovska A, Weitz DA & Mitchison TJ, Proc. Natl. Acad. Sci., 110(39), 15555-60, 2013.

Interphase Chromatin Dynamics in Response to Double Stranded DNA Breaks

The dynamic organization of chromatin inside the eukaryotic cell nucleus is essential to the regulation of the genome such as maintaining its integrity, gene expression and replication. However, the physical picture of chromatin structure and dynamics remains unclear and requires identifying the dynamical and structural signatures of specific biological processes. In this work, we study the chromatin dynamics in interphase, time between two cell divisions, in response to DNA damage. As shown previously, chromatin dynamics across the whole nucleus change dramatically after DNA damage; chromatin's coherent motion is eliminated, while its local mobility increases and its compaction decreases [1]. Here, we explore the local chromatin dynamics and compaction in response to local DNA damage in human cells. To do so, we induce double stranded DNA breaks (DSBs), which we visualize using 53BP1-mCherry, and observe the spatiotemporal evolution of their dynamics using single particle tracking. Simultaneously, we use histone H2B-GFP as a reporter on the chromatin compaction, to compare DSB dynamics inside the heterochromatin and euchromatin. To elucidate the dynamic signature of the local DNA damage response, we vary the number of DSBs as well as explore the ATP-dependence of these processes. Our preliminary results show that with an increasing DSB count the local subdiffusive dynamics of the DSBs remains unchanged. We also explore the relationship between the protein aggregation, diffusion coefficient and chromatin compaction at a DSB. [1] Zidovska A, Weitz DA, Mitchison TJ, Proc. Natl. Acad. Sci. USA, 110(39), 1555560, 2013.

Identifying Quiescent Stem Cells in Hair Follicles.

Hair follicle stem cells (HFSCs) are noted for their relative quiescence and therefore can be distinguished from other cells by their differential history of cell division. Replicating cells can be labeled by pulsing the animals repeatedly with 5-bromo-2'-deoxyuridine (BrdU) or tritiated thymidine ([3H]TdR), thymidine analogs that get incorporated into DNA during DNA synthesis. Because dividing cells dilute the label after each cell division, frequently dividing cells will lose the label over time while slow cycling cells will retain the label and thus are termed label retaining cells (LRCs). [3H]TdR can be visualized by autoradiography and BrdU can be detected by immunofluorescence with anti-BrdU antibodies. Alternatively, a well-established tet-regulatable transgenic mouse model can be used to express histone H2B-GFP in epithelial proliferative cells and their dilution and retention of the GFP signal can be followed. In this chapter, we detail the steps to perform BrdU pulse-chase and H2B-GFP pulse-chase experiments to identify quiescent cells in the hair follicle.

Ap2a2 Is Crucial for LT-HSC Quiescence

Despite the relative rarity of haematopoietic stem cells (HSCs) within the blood system, functional heterogeneity is paramount to their ability to sustain lifelong blood production. The quiescent HSC sits at the functional apex possessed with self-renewal properties and the greatest repopulation output. We previously identified the gene, Ap2a2 as an enhancer of HSC function and its protein as a potential cell fate determinant in HSC asymmetric cell divisions (Ting SB et al., Blood 2012). Mechanistically, we hypothesise Ap2a2 induces a state of HSC quiescence. Using the Tet-On histone H2B-GFP mouse model (Foudi et al., Nat Biotech 2009), we have shown Ap2a2 to be highly and differentially expressed in the predominantly, G0 dormant CD150+48-LSK GFPhigh as opposed to the more cycling GFPlow HSC subpopulation. Competitive transplantation of Ap2a2- versus empty vector-transduced H2B-GFP HSCs results in a three-fold increase of the CD150+48-LSK GFPhigh HSC subpopulation. To further confirm the importance of Ap2a2 in haematopoiesis, we have constructed Ap2a2-LacZ reporter and constitutive Ap2a2 knockout (KO) mouse lines. The Ap2a2 LacZ reporter with b-galactosidase flow cytometry staining of bone marrow subpopulations confirmed high endogenous Ap2a2 expression in the CD150+48-LSK long-term (LT-) versus CD150-48-LSK short-term (ST-) repopulating HSCs. Interim analyses of the constitutive Ap2a2 KO mice have revealed two obvious phenotypes: 14% of Ap2a2-null mice termed "non-survivors" are smaller, paler with failure of fetal liver (FL) development and die between E18.5 and weaning, whilst the remaining 11% are adult viable "survivors". However, at E14.5, Ap2a2-null compared to Ap2a2-wild type fetal livers showed less absolute total FL cells but increased CD150+48-LSM FL HSCs. This was quantitatively correlated via limiting dilution assay assessed at 16 weeks post-transplant with a two-fold increase in Ap2a2-null HSC numbers (1 in 78,917 versus 1 in 150,891, p=0.027). This suggests Ap2a2 has a role in FL HSC differentiation and/or fate with potential impairment of symmetrical versus asymmetrical HSC divisions currently being studied. When E14.5 FL cells were competitively transplanted, the Ap2a2-null HSC had impaired donor reconstitution function measured at 16 weeks post-transplant (19.8% versus 48.6%, p=0.015). Ap2a2-null versus wild-type E14.5 FL cells showed equivalent numbers of primary in vitro methylcellulose colony assays but loss of secondary colonies upon re-plating indicative of loss of in-vitro HSC self-renewal. Importantly, although the Ap2a2 adult "survivors" exhibited normal quantities of bone marrow HSC subpopulations, when functionally assessed, Ap2a2-null adult "survivor" HSCs showed loss of in-vivo HSC self-renewal in secondary transplantation assays. To investigate potential cellular mechanisms, we studied the cell cycle state of Ap2a2-null and wild-type E14.5 FL cells and identified that Ap2a2-null "non-survivors" had a relative loss of quiescent G0, specifically in the LT-HSC (and not seen in the ST-HSC) subpopulation throughout all of (E14.5 to E18.5) FL development. In contrast, the LT-HSC subpopulation in FLs of Ap2a2-null "survivors" had an initial loss of G0 at E14.5 but a compensatory increase in LT-HSC G0 by E18.5. Our preliminary data suggests Ap2a2 is a crucial factor for the quiescent LT-HSC subpopulation, and we propose that both during the highly proliferative fetal liver stage of haematopoiesis and adult HSCs under stress that Ap2a2 maintains a critical balance of dormant ("deep-sleeper") HSCs to ensure global HSC function. DisclosuresNo relevant conflicts of interest to declare.

Characterizing the DNA Damage Response by Cell Tracking Algorithms and Cell Features Classification Using High-Content Time-Lapse Analysis.

Traditionally, the kinetics of DNA repair have been estimated using immunocytochemistry by labeling proteins involved in the DNA damage response (DDR) with fluorescent markers in a fixed cell assay. However, detailed knowledge of DDR dynamics across multiple cell generations cannot be obtained using a limited number of fixed cell time-points. Here we report on the dynamics of 53BP1 radiation induced foci (RIF) across multiple cell generations using live cell imaging of non-malignant human mammary epithelial cells (MCF10A) expressing histone H2B-GFP and the DNA repair protein 53BP1-mCherry. Using automatic extraction of RIF imaging features and linear programming techniques, we were able to characterize detailed RIF kinetics for 24 hours before and 24 hours after exposure to low and high doses of ionizing radiation. High-content-analysis at the single cell level over hundreds of cells allows us to quantify precisely the dose dependence of 53BP1 protein production, RIF nuclear localization and RIF movement after exposure to X-ray. Using elastic registration techniques based on the nuclear pattern of individual cells, we could describe the motion of individual RIF precisely within the nucleus. We show that DNA repair occurs in a limited number of large domains, within which multiple small RIFs form, merge and/or resolve with random motion following normal diffusion law. Large foci formation is shown to be mainly happening through the merging of smaller RIF rather than through growth of an individual focus. We estimate repair domain sizes of 7.5 to 11 µm2 with a maximum number of ~15 domains per MCF10A cell. This work also highlights DDR which are specific to doses larger than 1 Gy such as rapid 53BP1 protein increase in the nucleus and foci diffusion rates that are significantly faster than for spontaneous foci movement. We hypothesize that RIF merging reflects a "stressed" DNA repair process that has been taken outside physiological conditions when too many DSB occur at once. High doses of ionizing radiation lead to RIF merging into repair domains which in turn increases DSB proximity and misrepair. Such finding may therefore be critical to explain the supralinear dose dependence for chromosomal rearrangement and cell death measured after exposure to ionizing radiation.

Loading of nuclear autoantigens prototypically recognized by systemic lupus erythematosus sera into late apoptotic vesicles requires intact microtubules and myosin light chain kinase activity

Most cases of systemic lupus erythematosus (SLE) are characterized by an impaired clearance of apoptotic cells in various tissues. Non-cleared apoptotic waste is considered an immunogen driving the autoimmune response in patients with SLE. During the execution of apoptosis, membrane blebs are formed and filled with cellular components. Here, we evaluate the cytoskeletal pathway(s) responsible for the loading of SLE prototypic nuclear autoantigens into the apoptotic cell-derived membranous vesicles (ACMV) generated during late phases of apoptosis. HeLa cells expressing a fusion protein of histone H2B with green fluorescent protein (GFP) were irradiated with ultraviolet (UV)-B to induce apoptosis. The appearance and trafficking of chromatin-derived material was monitored by fluorescence microscopy. Specific inhibitors of cytoskeletal pathways were employed to identify the motile elements involved in translocation and trafficking of the nuclear components. We observed that immediately after their appearance the ACMV did not contain histone H2B(GFP) ; in this phase the fluorescence was contained in the nuclear remnants and the cytoplasm. Within consecutive minutes the ACMV were loaded with chromatin-derived material, whereas the loading of simultaneously created ACMV with histone H2B(GFP) was not uniform. Some ACMV were preferentially filled and, consequently, showed a remarkably higher histone H2B(GFP) accumulation. Inhibitors of the cytoskeleton revealed that functional microtubules and myosin light chain kinase are required for nuclear shrinkage and loading of nuclear material into the ACMV, respectively.

Live embryo imaging to follow cell cycle and chromosomes stability after nuclear transfer.

Nuclear transfer (NT) into mouse oocytes yields a transcriptionally and functionally heterogeneous population of cloned embryos. Most studies of NT embryos consider only embryos at predefined key stages (e.g., morula or blastocyst), that is, after the bulk of reprogramming has taken place. These retrospective approaches are of limited use to elucidate mechanisms of reprogramming and to predict developmental success. Observing cloned embryo development using live embryo cinematography has the potential to reveal otherwise undetectable embryo features. However, light exposure necessary for live cell cinematography is highly toxic to cloned embryos. Here we describe a protocol for combined bright-field and fluorescence live-cell imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This protocol, which can be adapted to observe other reporters such as Oct4-GFP or Nanog-GFP, allowed us to quantitatively analyze cleavage kinetics of cloned embryos.

Adult Thymic Epithelium Contains Nonsenescent Label-Retaining Cells

Progress in our understanding of thymic epithelial cell (TEC) renewal and homeostasis is hindered by the lack of markers for TEC progenitors. Stem and progenitor cell populations display remarkable diversity in their proliferative behavior. In some but not all tissues, stemness is associated with quiescence. The primary goal of our study was to discover whether quiescent cells were present in neonatal and adult TECs. To this end, we used a transgenic label-retaining cell (LRC) assay in which a histone H2B-GFP fusion protein is expressed under the control of the reverse tetracycline-controlled transactivator and the tetracycline operator minimal promoter. In adult mice, we found that both cortical and medullary TECs (cTECs and mTECs) proliferated more actively in females than males. Moreover, we observed three main differences between neonatal and adult TECs: 1) neonatal TECs proliferated more actively than adult TECs; 2) whereas cTECs and mTECs had similar turnover rates in young mice, the turnover of mTECs was more rapid than that of cTECs in adults; and 3) although no LRCs could be detected in young mice, LRCs were detectable after a 16-wk chase in adults. In female mice, LRCs were found almost exclusively among cTECs and expressed relatively low levels of p16INK4a, p19ARF, and Serpine1, and high levels of Bmi1, Foxn1, Trp63, and Wnt4. We conclude that LRCs in adult TECs are not senescent postmitotic cells and may represent the elusive progenitors responsible for TEC maintenance in the adult thymus.

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1. The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones, forming foci within 5-15 minutes. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis. In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

Nuclear Reprogramming: Kinetics of Cell Cycle and Metabolic Progression as Determinants of Success

Establishment of totipotency after somatic cell nuclear transfer (NT) requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI). Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development.

Orderly Inactivation of the Key Checkpoint Protein Mitotic Arrest Deficient 2 (MAD2) during Mitotic Progression

Anaphase is promoted by the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) only when all the chromosomes have achieved bipolar attachment to the mitotic spindles. Unattached kinetochores or the absence of tension between the paired kinetochores activates a surveillance mechanism termed the spindle-assembly checkpoint. A fundamental principle of the checkpoint is the activation of mitotic arrest deficient 2 (MAD2). MAD2 then forms a diffusible complex called mitotic checkpoint complex (designated as MAD2(MCC)) before it is recruited to APC/C (designated as MAD2(APC/C)). Large gaps in our knowledge remain on how MAD2 is inactivated after the checkpoint is satisfied. In this study, we have investigated the regulation of MAD2-containing complexes during mitotic progression. Using selective immunoprecipitation of checkpoint components and gel filtration chromatography, we found that MAD2(MCC) and MAD2(APC/C) were regulated very differently during mitotic exit. Temporally, MAD2(MCC) was broken down ahead of MAD2(APC/C). The inactivation of the two complexes also displayed different requirements of proteolysis; although APC/C and proteasome activities were dispensable for MAD2(MCC) inactivation, they are required for MAD2(APC/C) inactivation. In fact, the degradation of CDC20 is inextricably linked to the breakdown of MAD2(APC/C). These data extended our understanding of the checkpoint complexes during checkpoint silencing.

The Dynactin Complex Maintains the Integrity of Metaphasic Centrosomes to Ensure Transition to Anaphase

The dynactin complex is required for activation of the dynein motor complex, which plays a critical role in various cell functions including mitosis. During metaphase, the dynein-dynactin complex removes spindle checkpoint proteins from kinetochores to facilitate the transition to anaphase. Three components (p150(Glued), dynamitin, and p24) compose a key portion of the dynactin complex, termed the projecting arm. To investigate the roles of the dynactin complex in mitosis, we used RNA interference to down-regulate p24 and p150(Glued) in human cells. In response to p24 down-regulation, we observed cells with delayed metaphase in which chromosomes frequently align abnormally to resemble a "figure eight," resulting in cell death. We attribute the figure eight chromosome alignment to impaired metaphasic centrosomes that lack spindle tension. Like p24, RNA interference of p150(Glued) also induces prometaphase and metaphase delays; however, most of these cells eventually enter anaphase and complete mitosis. Our findings suggest that although both p24 and p150(Glued) components of the dynactin complex contribute to mitotic progression, p24 also appears to play a role in metaphase centrosome integrity, helping to ensure the transition to anaphase.

Dose-dependent regulation of primitive erythroid maturation and identity by the transcription factor Eklf

AbstractThe primitive erythroid (EryP) lineage is the first to differentiate during mammalian embryogenesis. Eklf/Klf1 is a transcriptional regulator that is essential for definitive erythropoiesis in the fetal liver. Dissection of the role(s) of Eklf within the EryP compartment has been confounded by the simultaneous presence of EryP and fetal liver–derived definitive erythroid (EryD) cells in the blood. To address this problem, we have distinguished EryP from their definitive counterparts by crossing Eklf+/− mutant and ϵ-globin::histone H2B-GFP transgenic mice. Eklf-deficient EryP exhibit membrane ruffling and a failure to acquire the typical discoidal erythroid shape but they can enucleate. Flow cytometric analyses of H2B-GFP+ EryP revealed that Eklf heterozygosity results in the loss of Ter119 surface expression on EryP but not on EryD. Null mutation of Eklf resulted in abnormal expression of a range of surface proteins by EryP. In particular, several megakaryocyte markers were ectopically expressed by maturing Eklf-null EryP. Unexpectedly, the platelet tetraspanin CD9 was detected on nucleated wild-type EryP but not on mature EryD and thus provides a useful marker for purifying circulating EryP. We conclude that Eklf gene dosage is crucial for regulating the surface phenotype and molecular identity of maturing primitive erythroid cells.

A role for PDGF signaling in expansion of the extra-embryonic endoderm lineage of the mouse blastocyst

The inner cell mass (ICM) of the implanting mammalian blastocyst comprises two lineages: the pluripotent epiblast (EPI) and primitive endoderm (PrE). We have identified platelet-derived growth factor receptor alpha (PDGFRα) as an early marker of the PrE lineage and its derivatives in both mouse embryos and ex vivo paradigms of extra-embryonic endoderm (ExEn). By combining live imaging of embryos and embryo-derived stem cells expressing a histone H2B-GFP fusion reporter under the control of Pdgfra regulatory elements with the analysis of lineage-specific markers, we found that Pdgfra expression coincides with that of GATA6, the earliest expressed transcriptional regulator of the PrE lineage. We show that GATA6 is required for the activation of Pdgfra expression. Using pharmacological inhibition and genetic inactivation we addressed the role of the PDGF pathway in the PrE lineage. Our results demonstrate that PDGF signaling is essential for the establishment, and plays a role in the proliferation, of XEN cells, which are isolated from mouse blastocyst stage embryos and represent the PrE lineage. Implanting Pdgfra mutant blastocysts exhibited a reduced number of PrE cells, an effect that was exacerbated by delaying implantation. Surprisingly, we also noted an increase in the number of EPI cells in implantation-delayed Pdgfra-null mutants. Taken together, our data suggest a role for PDGF signaling in the expansion of the ExEn lineage. Our observations also uncover a possible role for the PrE in regulating the size of the pluripotent EPI compartment.

Identification of epithelial label-retaining cells at the transition between the anal canal and the rectum in mice

In certain regions of the body, transition zones exist where stratified squamous epithelia directly abut against other types of epithelia. Certain transition zones are especially prone to tumorigenesis an example being the anorectal junction, although the reason for this is not known. One possibility is that the abrupt transition of the simple columnar epithelium of the colon to the stratified squamous epithelium of the proximal portion of the anal canal may contain a unique stem cell niche. We investigated whether the anorectal region contained cells with stem cell properties relative to the adjacent epithelium. We utilized a tetracycline-regulatable histone H2B-GFP transgenic mice model, previously used to identify hair follicle stem cells, to fluorescently label slow-cycling anal epithelial cells (e.g. prospective stem cells) in combination with a panel of putative stem cell markers. We identified a population of long-term GFP label-retaining cells concentrated at the junction between the anal canal and the rectum. These cells are BrdU-retaining cells and expressed the stem cell marker CD34. Moreover, tracking the fate of the anal label-retaining cells in vivo revealed that the slow-cycling cells only gave rise to progeny of the anal epithelium. In conclusion, we identified a unique population of cells at the anorectal junction which can be separated from the other basal anal epithelial cells based upon the expression of the stem cell marker CD34 and integrin a6, and thus represent a putative anal stem cell population.

Reversible whole-organism cell cycle arrest in a living vertebrate

In vivo cell cycle analysis in higher eukaryotes has been limited by the challenge of preserving the integrity of the living organism while visualizing dividing cells. Here, we propose a new model, which uses the unique combination of features of the Japanese medaka in order to visualize and manipulate the cell cycle progression in a live vertebrate. Our stable transgenic histone H2B-GFP medaka line allows fluorescence-based monitoring of the chromosomes. The system has a high specificity, with a strong GFP signal labeling the chromatin architecture. The subcellular resolution ensures detection of both normal and abnormal divisions in live recordings. This translates into the possibility to quantify temporal and spatial aspects of the cell cycle, such as length or nuclear size, as well as to expose drug toxicity at the earliest stage. We also show that acclimation to cold, a prominent feature of the eurytherm medaka, is a valuable natural way of inducing a reversible cell cycle arrest in the entire living organism. Our results suggest that this manipulation can be performed from the early stages of development, has no toxicity and does not alter the cell-cycle profile of the embryo.

Production of Transgenic Medaka Fish Carrying Fluorescent Nuclei and Chromosomes

As with zebrafish, attention has focused on the teleost medaka Oryzias latipes as an experimental animal representative of non-mammalian vertebrates in various fields of biological science. To enable real-time analyses of the dynamics of nuclei and chromosomes in living medaka cells, we produced a transgenic medaka expressing a fusion protein between histone H2B and green fluorescent protein (GFP) under the control of a cytomegalovirus (CMV) promoter. Since the nuclei and chromosomes of transgenic medaka cells are labeled with GFP, their morphological changes can be instantly monitored throughout the mitotic cell cycle progression under a fluorescent microscope without any fixation and staining of samples. However, GFP-labeling of nuclei and chromosomes is not successful during early embryonic development until zygotic expression begins and during the meiotic cell cycle progression, because the CMV promoter does not work in these stages. In addition, histone H2B-GFP fusion proteins are expressed in an organ-specific manner; strong and ubiquitous expression occurs in cells comprising the gut and fin, whereas the expression is restricted to certain types of cells in the liver and brain. These findings suggest that the CMV-driven expression of the histone H2B-GFP transgene is modified depending on the integration site of the transgene in the genome. Nevertheless, easy and precise monitoring of cytological changes in nuclei and chromosomes in the majority of mitotic cells by using the transgenic medaka will greatly contribute to a better understanding of control mechanisms of nuclear and chromosomal behaviors in vertebrate cells.

Generation of a tightly regulated doxycycline‐inducible model for studying mouse intestinal biology

To develop a sensitive and inducible system to study intestinal biology, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the 12.4 kb murine Villin promoter. The newly generated Villin-rtTA2-M2 mice were then bred with the previously developed tetO-HIST1H2BJ/GFP model to assess inducibility and tissue-specificity. Expression of the histone H2B-GFP fusion protein was observed exclusively upon doxycycline induction and was uniformly distributed throughout the intestinal epithelium. The Villin-rtTA2-M2 was also found to drive transgene expression in the developing mouse intestine. Furthermore, we could detect transgene expression in the proximal tubules of the kidney and in a population of alleged gastric progenitor cells. By administering different concentrations of doxycycline, we show that the Villin-rtTA2-M2 system drives transgene expression in a dosage-dependent fashion. Thus, we have generated a novel doxycycline-inducible mouse model, providing a valuable tool to study the effect of different gene dosages on intestinal physiology and pathology.

Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst

The first two lineages to differentiate from a pluripotent cell population during mammalian development are the extraembryonic trophectoderm (TE) and the primitive endoderm (PrE). Whereas the mechanisms of TE specification have been extensively studied, segregation of PrE and the pluripotent epiblast (EPI) has received comparatively little attention. A current model of PrE specification suggests PrE precursors exhibit an apparently random distribution within the inner cell mass of the early blastocyst and then segregate to their final position lining the cavity by the late blastocyst. We have identified platelet-derived growth factor receptor alpha (Pdgfralpha) as an early-expressed protein that is also a marker of the later PrE lineage. By combining live imaging of embryos expressing a histone H2B-GFP fusion protein reporter under the control of Pdgfra regulatory elements with the analysis of lineage-specific markers, we investigated the events leading to PrE and EPI lineage segregation in the mouse, and correlated our findings using an embryo staging system based on total cell number. Before blastocyst formation, lineage-specific factors are expressed in an overlapping manner. Subsequently, a gradual progression towards a mutually exclusive expression of PrE- and EPI-specific markers occurs. Finally, cell sorting is achieved by a variety of cell behaviours and by selective apoptosis.