Polymorphism and evolution of vulval precursor cell lineages within two nematode genera, Caenorhabditis and Oscheius

BACKGROUND: The cell lineage of nematodes is mostly invariant for a given species, but varies between species. One can thus wonder how a cell lineage varies during evolution. We have started a microevolutionary approach within two genera by observing lineage variations of vulval precursor cells in different natural nematode populations of the same and closely related species. RESULTS: In Caenorhabditis elegans, the P3.p cell lineage is variable within a genetically homogeneous population and polymorphic between wild strains. Irrespective of its division pattern, P3.p is competent to form vulval tissue in different C. elegans strains, whereas it is not competent in C. briggsae. In Oscheius sp. 1, P4.p and P8.p lineages are strongly polymorphic. Within each genus, these intraspecies polymorphisms in cell lineages are amplified between closely related species. In Oscheius sp. 1, the large polymorphisms in P4.p and P8.p lineages allowed us to undertake a genetic analysis of the variation between two pairs of strains. Multiple loci are involved in cell lineage differences, and variation at one locus appears to have a relatively strong effect. In addition to these large lineage variations in cells that do not normally contribute to the vulva, we find minor variations (errors) in vulval lineages, which represent the precision level of the vulval-patterning process and point to a selection pressure for maintenance of a large vulval equivalence group. CONCLUSIONS: Polymorphisms in vulval cell lineage are found within a given nematode species, and could be instrumental in explaining evolutionary variations between closely related species.     

Lineage analysis of the hemangioblast as defined by FLK1 and SCL expression

Accumulating studies support the idea that a common progenitor, termed the hemangioblast, generates both hematopoietic and endothelial cell lineages. To better define the relationship between these cell lineages, we have generated knock-in embryonic stem (ES) cells carrying a non-functional human CD4 at the Scl locus. By using in vitro differentiated Scl(+/CD4) ES cells, we demonstrate that FLK1 and SCL are molecular determinants of the hemangioblast. Furthermore, our studies demonstrate that hematopoietic and endothelial cells develop via distinct, sequential generation of FLK1 and SCL-expressing cells. FLK1(+)CD4(-) cells first arise in developing embryoid bodies. The Scl gene is turned on within FLK1(+)CD4(-) cells to give rise to FLK1(+)CD4(+) cells. Alternatively, a subpopulation of the initial FLK1(+)CD4(-) cells remains as SCL negative. Within the FLK1(+)CD4(+) cells, FLK1 is down regulated to generate FILK1(-)CD4(+) cells. Replating studies demonstrate that hematopoietic progenitors are enriched within FLK1(+)CD4(+) and FLK1(-)CD4(+) cells, while endothelial cells develop from FLK1(+)CD4(+) and FLK1(+)CD4(-) cell populations.     

Extra-embryonic endoderm cells derived from ES cells induced by GATA Factors acquire the character of XEN cells

Background  

Three types of cell lines have been established from mouse blastocysts: embryonic stem (ES) cells, trophoblast stem (TS) cells, and extra-embryonic endoderm (XEN) cells, which have the potential to differentiate into their respective cognate lineages. ES cells can differentiate in vitro not only into somatic cell lineages but into extra-embryonic lineages, including trophectoderm and extra-embryonic endoderm (ExEn) as well. TS cells can be established from ES cells by the artificial repression of Oct3/4 or the upregulation of Cdx2 in the presence of FGF4 on feeder cells. The relationship between these embryo-derived XEN cells and ES cell-derived ExEn cell lines remains unclear, although we have previously reported that overexpression of Gata4 or Gata6 induces differentiation of mouse ES cells into extra-embryonic endoderm in vitro.     

Production of imidazole alkaloids in cell cultures of jaborandi as affected by the medium pH

The effect of pH (from 4.8 to 9.8) on the production of pilosine and pilocarpine and on their partition between cell and medium was studied in two lineages (P and PP) of Pilocarpus microphyllus cell suspension cultures. Highest mass accumulation was observed at high pHs and both lineages produced pilocarpine while only lineage PP produced pilosine. Both alkaloids were released in the medium but higher accumulation occurred in the cells. The highest production of pilocarpine was at pH 8.8–9.8 in both cell lineages. Other imidazole alkaloids were also identified in both lineages. At all pHs tested, the pH in the media cultures tended to stabilize around 6 after 10–15 days of cultivation. NO₃ ⁻ and NH₄ ⁺ variation in the media might partially explain the pH stabilization.     

A CTX family cell adhesion molecule, JAM4, is expressed in stem cell and progenitor cell populations of both male germ cell and hematopoietic cell lineages

Stem cells are maintained in an undifferentiated state by interacting with a microenvironment known as the "niche," which is comprised of various secreted and membrane proteins. Our goal was to identify niche molecules participating in stem cell-stem cell and/or stem cell-supporting cell interactions. Here, we isolated genes encoding secreted and membrane proteins from purified male germ stem cells using a signal sequence trap approach. Among the genes identified, we focused on the junctional adhesion molecule 4 (JAM4), an immunoglobulin type cell adhesion molecule. JAM4 protein was actually localized to the plasma membrane in male germ cells. JAM4 expression was downregulated as cells differentiated in both germ cell and hematopoietic cell lineages. To analyze function in vivo, we generated JAM4-deficient mice. Histological analysis of testes from homozygous nulls did not show obvious abnormalities, nor did liver and kidney tissues, both of which strongly express JAM4. The numbers of hematopoietic stem cells in bone marrow were indistinguishable between wild-type and mutant mice, as was male germ cell development. These results suggest that JAM4 is expressed in stem cells and progenitor cells but that other cell adhesion molecules may substitute for JAM4 function in JAM4-deficient mice both in male germ cell and hematopoietic lineages.     

Generation of megakaryocytes and platelets from preadipocyte cell line 3T3-L1, but not the parent cell line 3T3, in vitro

Platelets are produced from megakaryocytes (MKs), although the pathway leading from stem cells to MK lineages are not yet fully understood. Recently, we reported to obtain abundant MKs and platelets from human subcutaneous adipose tissues. Adipose tissues contain various cell types, most of which are lineage cells from mesenchymal or adipocyte-derived stem cells, distinct from hematopoietic cells. To identify the cells responsible for the differentiation MK lineages in adipose tissues, this study examined whether the preadipocyte cell line 3T3-L1 and fibroblast cell line 3T3 differentiated into MK lineages in vitro. Cells were cultured in megakaryocyte lineage induction medium. By day 4, most of 3T3 cell-derived cells leaded to cell death. In contrast, 3T3-L1-derived cells on days 8 showed to have typical characterizations of MK lineages in analyses for specific marker, DNA ploidy, transmission electro micrograph. 3T3-L1-derived platelet-sized cells on day 12 could be stimulated by ADP and PAR4-activating peptide. This study clearly shows in vitro differentiation from 3T3-L1 cells, not from 3T3 cells, into MK lineages.     

Global mapping of H3K4me1 and H3K4me3 reveals the chromatin state-based cell type-specific gene regulation in human Treg cells

Regulatory T cells (Treg) contribute to the crucial immunological processes of self-tolerance and immune homeostasis. Genomic mechanisms that regulate cell fate decisions leading to Treg or conventional T cells (Tconv) lineages and those underlying Treg function remain to be fully elucidated, especially at the histone modification level. We generated high-resolution genome-wide distribution maps of monomethylated histone H3 lysine 4 (H3K4me1) and trimethylated H3K4 (H3K4me3) in human CD4(+)CD25(+)FOXP3(+) Tregs and CD4(+)CD25(+)FOXP3(-) activated (a)Tconv cells by DNA sequencing-by-synthesis. 2115 H3K4me3 regions corresponded to proximal promoters; in Tregs, the genes associated with these regions included the master regulator FOXP3 and the chemokine (C-C motif) receptor 7 (CCR7). 41024 Treg-specific H3K4me1 regions were identified. The majority of the H3K4me1 regions differing between Treg and aTconv cells were located at promoter-distal sites, and in vitro reporter gene assays were used to evaluate and identify novel enhancer activity. We provide for the first time a comprehensive genome-wide dataset of lineage-specific H3K4me1 and H3K4me3 patterns in Treg and aTconv cells, which may control cell type-specific gene regulation. This basic principle is likely not restricted to the two closely-related T cell populations, but may apply generally to somatic cell lineages in adult organisms.     

A stop-EGFP transgenic mouse to detect clonal cell lineages generated by mutation

The investigation of cell lineages and clonal organization in tissues is facilitated by techniques that allow labelling of clonal cell lineages. Here, we describe a novel transgenic mouse that allows clonal cell lineages to be traced in virtually any tissue. A green fluorescent cell lineage is generated by a random mutation at an enhanced green fluorescent protein gene that carries a premature stop codon, ensuring clonality. The transgenic system allows efficient detection of mutations and stem-cell fate mapping in the epidermis using live mice, as well as in the kidney and liver post-mortem. Cell lineages that descended from single epidermal stem cells were found to be capable of generating three adjacent corneocytes using the system, providing evidence for horizontal migration of epidermal cells between epidermal proliferative units (EPUs), in contrast to the classical EPU model. The transgenic mouse system is expected to provide a novel tool for stem-cell lineage studies.     

Distinct mechanisms triggering glial differentiation in Drosophila thoracic and abdominal neuroblasts 6-4

Neurons and glia are produced in stereotyped patterns after neuroblast cell division during development of the Drosophila central nervous system. The first cell division of thoracic neuroblast 6-4 (NB6-4T) is asymmetric, giving rise to a glial precursor cell and a neuronal precursor cell. In contrast, abdominal NB6-4 (NB6-4A) divides symmetrically to produce two glial cells. To understand the relationship between cell division and glia-neuron cell fate determination, we examined and compared the effects of known cell division mutations on the NB6-4T and NB6-4A lineages. Based on observation of expression of glial fate determination and early glial differentiation genes, the onset of glial differentiation occurred in NB6-4A but not in NB6-4T when both cell cycle progression and cytokinesis were genetically arrested. On the other hand, glial differentiation started in both lineages when cytokinesis was blocked with intact cell cycle progression. These results showed that NB6-4T, but not NB6-4A, requires cell cycle progression for acquisition of glial fate, suggesting that distinct mechanisms trigger glial differentiation in the different lineages.     

Survival of cells in mammalian tissues after low doses of irradiation: a short review

A survey of data in the literature indicates that the radiosensitivity of cells to doses less than 1 Gy varies widely within cell lineages and less so between lineages. This is due in large part to the differentiation status and division capacities of the cells, and possibly also to the grouping of cells into 'viable units'. In addition, the mode of cell death is important, and cells susceptible to natural apoptosis are particularly radiosensitive. There are also quite marked differences in cell sensitivity between species.     

Magnifying Stem Cell Lineages: The Stop-EGFP Mouse

Cell fate mapping techniques which can label clonal cell lineages are of importance because they allow one to investigate the distribution and types of daughter cells arising from single precursor cells. Thus, the potential of precursor cells to generate various types of descendent cells can be studied at the single-cell level. The stop-EGFP transgenic mouse carries a premature stop codon-containing enhanced green fluorescent protein (EGFP) gene as a target gene for mutations. A cell having undergone a mutation at the premature stop codon and its descendant cell lineage will express EGFP, thus a clonal cell lineage can be traced in vivo using a fluorescent microscope. Using the stop-EGFP mouse, stem cell clonal lineages in the mouse dorsal epidermis can be investigated in vivo and repeated analyses of the same cell lineages can be performed over time. In vivo imaging studies possible with the stop-EGFP mouse provide new insights into the structure of epidermal proliferative units (EPUs). The stop-EGFP system provides a novel tool for investigating clonal cell lineages in developmental studies as well as in stem cell biology.     

Recruitment of healthy mitochondria fuels transmissible cancers

The replication of mitochondrial DNA (mtDNA) is not under strict control of the nucleus. Therefore, within-cell selection can favour mtDNA variants with a replication or survival advantage even if deleterious for the cell. Here, we consider how the balance between selection within and among cells is shifted in cancer cell lineages, and how this affects the somatic evolution of mitochondria. Cancer cell lineages are known to be prone to mitochondrial genetic erosion. Nevertheless, some cancer lineages are long lived and a few exceptional lineages even can survive their host because of horizontal transmission to other individuals. Recent work now shows that such transmissible cancer cell lineages occasionally secondarily recruit the mitochondrial genome of their host, which we propose as a means to replace genetically eroded mitochondrial genomes. Studying the dynamics of the horizontal exchange of mtDNA between somatic cells may provide important insight into the evolution of mitochondria during somatic growth and in mitochondrial diseases.     

Stochastic Dynamics of Interacting Haematopoietic Stem Cell Niche Lineages

Since we still know very little about stem cells in their natural environment, it is useful to explore their dynamics through modelling and simulation, as well as experimentally. Most models of stem cell systems are based on deterministic differential equations that ignore the natural heterogeneity of stem cell populations. This is not appropriate at the level of individual cells and niches, when randomness is more likely to affect dynamics. In this paper, we introduce a fast stochastic method for simulating a metapopulation of stem cell niche lineages, that is, many sub-populations that together form a heterogeneous metapopulation, over time. By selecting the common limiting timestep, our method ensures that the entire metapopulation is simulated synchronously. This is important, as it allows us to introduce interactions between separate niche lineages, which would otherwise be impossible. We expand our method to enable the coupling of many lineages into niche groups, where differentiated cells are pooled within each niche group. Using this method, we explore the dynamics of the haematopoietic system from a demand control system perspective. We find that coupling together niche lineages allows the organism to regulate blood cell numbers as closely as possible to the homeostatic optimum. Furthermore, coupled lineages respond better than uncoupled ones to random perturbations, here the loss of some myeloid cells. This could imply that it is advantageous for an organism to connect together its niche lineages into groups. Our results suggest that a potential fruitful empirical direction will be to understand how stem cell descendants communicate with the niche and how cancer may arise as a result of a failure of such communication.     

Characterization of Trophoblast and Extraembryonic Endoderm Cell Lineages Derived from Rat Preimplantation Embryos

Background

Previous attempts to isolate pluripotent cell lines from rat preimplantation embryo in mouse embryonic stem (ES) cell culture conditions (serum and LIF) were unsuccessful, however the resulting cells exhibited the expression of such traditional pluripotency markers as SSEA-1 and alkaline phosphatase. We addressed the question, which kind of cell lineages are produced from rat preimplantation embryo under “classical” mouse ES conditions.

Results

We characterized two cell lines (C5 and B10) which were obtained from rat blastocysts in medium with serum and LIF. In the B10 cell line we found the expression of genes known to be expressed in trophoblast, Cdx-2, cytokeratin-7, and Hand-1. Also, B10 cells invaded the trophectodermal layer upon injection into rat blastocysts. In contrast to mouse Trophoblast Stem (TS) cells proliferation of B10 cells occurred independently of FGF4. Cells of the C5 line expressed traditional markers of extraembryonic-endoderm (XEN) cells, in particular, GATA-4, but also the pluripotency markers SSEA-1 and Oct-4. C5 cell proliferation exhibited dependence on LIF, which is not known to be required by mouse XEN cells.

Conclusions

Our results confirm and extend previous findings about differences between blastocyst-derived cell lines of rat and mice. Our data show, that the B10 cell line represents a population of FGF4-independent rat TS-like cells. C5 cells show features that have recently become known as characteristic of rat XEN cells. Early passages of C5 and B10 cells contained both, TS and XEN cells. We speculate, that mechanisms maintaining self-renewal of cell lineages in rat preimplantation embryo and their in vitro counterparts, including ES, TS and XEN cells are different than in respective mouse lineages.     

CD4+ CD25+ regulatory T cell repertoire formation in response to varying expression of a neo-self-antigen

We have examined the development of self-peptide-specific CD4+ CD25+ regulatory T cells in lineages of transgenic mice that express the influenza virus PR8 hemagglutinin (HA) under the control of several different promoters (HA transgenic mice). By mating these lineages with TS1-transgenic mice expressing a TCR that recognizes the major I-E(d)-restricted determinant from HA (site 1 (S1)), we show that S1-specific T cells undergo selection to become CD4+ CD25+ regulatory T cells in each of the lineages, although in varying numbers. In some lineages, S1-specific CD4+ CD25+ regulatory T cells are highly abundant; indeed, TS1xHA-transgenic mice can contain as many S1-specific CD4+ T cells as are present in TS1 mice, which do not express the neo-self HA. In another lineage, however, S1-specific thymocytes are subjected to more extensive deletion and far fewer S1-specific CD4+ CD25+ regulatory T cells accumulate in the periphery. We show that radioresistant stromal cells can direct both deletion and CD4+ CD25+ regulatory T cell selection of S1-specific thymocytes. Interestingly, even though their numbers can vary, the S1-specific CD4+ CD25+ regulatory T cells in all cases coexist with clonally related CD4+ CD25- T cells that lack regulatory function. These findings show that the formation of the CD4+ CD25+ regulatory T cell repertoire is sensitive to variations in the expression of self-peptides.     

Target cells for avian myeloblastosis virus in embryonic yolk sac and relationship of cell differentiation to cell transformation

The yolk sac of the 12-day chicken embryo retains the blast stage progenitors to cells of the myeloid lineages with a very low level of contamination by more mature myeloid cells which have begun to express the characteristic myeloid cell markers. Both in vivo and in vitro experiments have supported the hypothesis that target cells for the BAI-A strain of avian myeloblastosis virus are contained within the myeloid lineages. An assay system for avian myeloblastosis virus was developed which utilizes this yolk sac cell system and which appears to be more sensitive than previous published assays. In addition, the kinetics of a liquid culture transformation system is presented in which at least 4% of the yolk sac cell population was transformed in a relatively synchronous fashion at 2 days after infection. The morphological transformation preceded an increased rate of cell proliferation. Cell separation procedures provided a 10- to 20-fold enrichment of target cells and demonstrated that the target cell population copurifies with macrophage colony-forming cells which are the committed progenitors to the macrophage lineage. In combination with earlier work, this work demonstrated that cells committed to the macrophage lineage at all stages of differentiation may serve as target cells for infection by avian myeloblastosis virus.     

Cell death shapes embryonic lineages of the central complex in the grasshopper Schistocerca gregaria

We have investigated cell death in identified lineages of the central complex in the embryonic brain of the grasshopper Schistocerca gregaria. Progeny from these lineages lie in the pars intercerebralis and direct projections to the protocerebral bridge and then the central body via the w, x, y, z tracts. Osmium‐ethyl gallate staining reveals pycnotic cells exclusively in cortical regions, and concentrated specifically within the lineages of the W, X, Y, Z neuroblasts. Minimal cell death occurs in a sporadic, nonpatterned manner, in other protocerebral regions. Immunohistochemistry reveals pycnotic cells express the enzyme cleaved Caspase‐3 in their cytoplasm and are therefore undergoing programmed cell death (apoptosis). The number of pycnotic bodies in lineages of the pars intercerebralis varies with age: small numbers are present in the Y, Z lineages early in embryogenesis (42%), the number peaks at 67–80%, and then declines and disappears late in embryogenesis. Cell death may encompass up to 20% of a lineage at mid‐embryogenesis. Peak cell death occurs shortly after maximum neurogenesis in the Y, Z lineages, and is maintained after neurogenesis has ceased in these lineages. Cell death within a lineage is patterned. Apoptosis is more pronounced among older cells and almost absent among younger cells. This suggests that specific subsets of progeny will be culled from these lineages, and we speculate about the effect of apoptosis on the biochemical profile of such lineages. J. Morphol. 271:949–959, 2010. © 2010 Wiley‐Liss, Inc.