Clonal Cell Lineages Research Articles

Single-cell analysis reveals different age-related somatic mutation profiles between stem and differentiated cells in human liver.

Accumulating somatic mutations have been implicated in age-related cellular degeneration and death. Because of their random nature and low abundance, somatic mutations are difficult to detect except in single cells or clonal cell lineages. Here, we show that in single hepatocytes from human liver, an organ exposed to high levels of genotoxic stress, somatic mutation frequencies are high and increase substantially with age. Considerably lower mutation frequencies were observed in liver stem cells (LSCs) and organoids derived from them. Mutational spectra in hepatocytes showed signatures of oxidative stress that were different in old age and in LSCs. A considerable number of mutations were found in functional parts of the liver genome, suggesting that somatic mutagenesis could causally contribute to the age-related functional decline and increased incidence of disease of human liver. These results underscore the importance of stem cells in maintaining genome sequence integrity in aging somatic tissues.

MaGIC: a machine learning tool set and web application for monoallelic gene inference from chromatin

BackgroundA large fraction of human and mouse autosomal genes are subject to random monoallelic expression (MAE), an epigenetic mechanism characterized by allele-specific gene expression that varies between clonal cell lineages. MAE is highly cell-type specific and mapping it in a large number of cell and tissue types can provide insight into its biological function. Its detection, however, remains challenging.ResultsWe previously reported that a sequence-independent chromatin signature identifies, with high sensitivity and specificity, genes subject to MAE in multiple tissue types using readily available ChIP-seq data. Here we present an implementation of this method as a user-friendly, open-source software pipeline for monoallelic gene inference from chromatin (MaGIC). The source code for the MaGIC pipeline and the Shiny app is available at https://github.com/gimelbrantlab/magic.ConclusionThe pipeline can be used by researchers to map monoallelic expression in a variety of cell types using existing models and to train new models with additional sets of chromatin marks.

Abstract 2009: Flow cytometric analysis, sorting and molecular analysis of dissociated cells from human solid tumors derived from PDX mouse models

Abstract Functional and genetic heterogeneity in tumor tissue has been a well described phenomenon for many decades but only recently emerged as a potentially crucial contributor to cancer development and progression. The correlation between cellular heterogeneity and aggressiveness, metastatic potential and drug susceptibility of a cancerous lesion have led to models in which the existence of multiple clonal cell lineages is a central feature enabling a neoplastic lesion to overcome selective pressures caused by the surrounding tissues’ defensive capabilities as well as therapeutic interventions. In addition, the role of the tumor microenvironment as an integral part of tumorigenesis was recognized and infiltrating leukocytes or tumor associated fibroblasts are no longer viewed as mere contaminants of a solid tumor biopsy. The emerging picture is compared to macroscopic ecosystems and a detailed understanding of the interactions between numerous cell subgroups seems necessary for the complete understanding of cancer pathogenesis. Scarcity of appropriate tools and model systems are an obstacle to the investigation of this heterogeneity at a molecular level but advances over the last few years have led to a significant acceleration in this field. More sensitive and far cheaper methods for collection of genomic and transcriptomic data have revealed a complex picture of the evolution of individual solid tumors. To turn this deeper understanding of tumorigenesis into improved clinical outcomes, routine methods are required to separate complex tumors into subpopulations. This stratification will provide a more comprehensive characterization of the tumor and enable more detailed prediction of disease progression and resistance development. We have developed an integrated workflow for dissociation and flow cytometric analysis and sorting for multiple downstream analysis modalities. Using patient derived xenograft (PDX) mouse models derived from primary human breast cancer biopsies we have demonstrated the ability to identify distinct immunophenotypes for each model and use this analysis to isolate distinct subpopulations. Our successful optimization of a variety of well characterized surface markers (e.g. CD 24, 44, 133, 184, 326 (EpCAM), and CD45) provides a basis for effective fingerprinting of cancer cells from a variety of sources. In an effort to demonstrate the potential of FACS sorting of solid tumor derived cell populations we have interrogated sorted fractions by NGS as well as RT-PCR array analysis and show distinct genotypic as well as gene expression signatures for each subgroup. The evidence provided by our data suggests that the single cell focused approach flow cytometry has traditionally enabled in hematological cancers is accessible for solid tumors as well and may unlock valuable biological insights. Citation Format: Rainer Blaesius, Friedrich Hahn, Eileen Snowden, Warren Porter, Mitchell Ferguson, Frances Tong, Stewart Jurgensen, Chang Chen, Daphne Clancy, Jamal Sirriyah, John Alianti, Perry Haaland, Shannon Dillmore, Jeff Baker, Aaron Middlebrook, Joyce Ruitenberg, Maria Suni, Smita Ghanekar. Flow cytometric analysis, sorting and molecular analysis of dissociated cells from human solid tumors derived from PDX mouse models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2009. doi:10.1158/1538-7445.AM2015-2009

Camouflage Patterning in Maize Leaves Results from a Defect in Porphobilinogen Deaminase

Maize leaves are produced from polarized cell divisions that result in clonal cell lineages arrayed along the long axis of the leaf. We utilized this stereotypical division pattern to identify a collection of mutants that form chloroplast pigmentation sectors that violate the clonal cell lineages. Here, we describe the camouflage1 (cf1) mutant, which develops nonclonal, yellow-green sectors in its leaves. We cloned the cf1 gene by transposon tagging and determined that it encodes porphobilinogen deaminase (PBGD), an enzyme that functions early in chlorophyll and heme biosynthesis. While PBGD has been characterized biochemically, no viable mutations in this gene have been reported in plants. To investigate the in vivo function of PBGD, we characterized the cf1 mutant. Histological analyses revealed that cf1 yellow sectors display the novel phenotype of bundle sheath cell-specific death. Light-shift experiments determined that constant light suppressed cf1 sector formation, a dark/light transition is required to induce yellow sectors, and that sectors form only during a limited time of leaf development. Biochemical experiments determined that cf1 mutant leaves have decreased PBGD activity and increased levels of the enzyme substrate in both green and yellow regions. Furthermore, the cf1 yellow regions displayed a reduction in catalase activity. A threshold model is hypothesized to explain the cf1 variegation and incorporates photosynthetic cell differentiation, reactive oxygen species scavenging, and PBGD function.

Evidence from the stop‐EGFP mouse supports a niche‐sharing model of epidermal proliferative units

The classical model of epidermal proliferative units (EPUs) postulates that each EPU is composed of a single column of corneocytes plus epidermal cells directly below the column and is maintained by a single stem cell within the unit. Using the stop-enhanced green fluorescent protein (stop-EGFP) transgenic mouse system, we previously showed epidermal stem cell clonal lineages could produce multiple adjacent corneocytes (i.e. epidermal cells belonging to multiple adjacent EPUs), contradicting the classical EPU model. One possible problem with our earlier study was that N-ethyl-N-nitrosourea (ENU) was used to generate mutations for clonal analysis. This could alter the normal environment of the epidermal tissue and might lead to an artificial expansion of stem cell clonal lineages. In this study, we replicate our earlier findings using untreated stop-EGFP mice and relying on spontaneous mutations to generate clonal cell lineages. We propose an alternative to the classical EPU model to explain the dynamic nature of epidermal proliferation. Our niche-sharing model of EPUs allows epidermal cells to horizontally migrate among EPUs, so that multiple stem cells cooperatively maintain a larger proliferative compartment.

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.

PHENOTYPES MEDIATED BY THE IOJAP GENOTYPE IN MAIZE.

The phenotype of maize plants that are homozygous recessive for the nuclear gene iojap is commonly an "anemic," grainy green appearance accompanied by white leaf stripes, white leaf margins, and reduced growth. In plants in which the iojap effect is occurring (homozygous ij/ij), we find affected tissue varies in extent, from wide, discrete striping to layered striping to short striations; and in distribution, from white leaves to boldly white-patterned to white-margined. In homozygous iojap plants the patterns are predictable and are consistent within a particular genetic background. Leaf striping patterns in iojap seedlings and plants are expressed primarily in the leaf margins, in positional patterns that are inconsistent with cellular proliferation. Reversion sectors on iojap plants (green stripes resulting from nuclear reversion of the ij gene), however, follow clonal cell lineages and indicate either that the iojap defects do not arise until after a leaf differentiates, or that they remain correctable in leaf tissue. On transmission through the egg, iojap-affected plastids become altered to a stable, nongreening state and can be transmitted maternally to subsequent generations. Defects in eggs borne on iojap ears, as revealed by ear maps of seedling progeny, have positional rather than clonal distributions on the ear. The expression of striping nonetheless is clonal in the occasional sectored plants that occur among such maternal progeny. The contrast of maternal sectorials with iojap plants is profound, and shows the period of meiosis and gametogenesis to be a critical passage for plastids in the egg as well as in the pollen. In some genetic backgrounds aborted embryos occur at variable stages in embryo development in ij ij zygotes and in the maternal progeny from ij ij plants. These effects presumably reflect a participation of the organelles in embryogenesis that is subject to the influence of nuclear genes. We conclude that the functional basis of the effects of the iojap gene must be 1) position-dependent (i.e., dependent on the location of a cell in the meristem or organ at a particular time in development of an organ) and 2) differentiation-dependent (i.e., dependent on the attainment of a certain state, specific to the genetic background). The iojap phenotype is thus a reflection of developmental progression of the organ rather than cell-specific, clonal changes. The rate of cell proliferation and growth of one portion of an organ vs. another is related to position-specific and background-specific expressions, and the range of iojap expression is similarly related. Maternally inherited sectoring, on the other hand, shows phenotypes that are derived from clonal changes specific to each cell lineage, presumably attributable to sorting of mixed organelles in the apical meristem cells.

Clonal analysis and cell lineages of the vertebrate central nervous system.

Very little progress in tracing cell lineages in the CNS of vertebrates had been made before the introduction of horseradish peroxidase (HRP) as an intra­ cellular lineage tracer in the frog embryo (Jacobson & Hirose 1978, 1981, Hirose & Jacobson 1979). Conceptual weaknesses and lack of adequate tech­ niques reinforced one another to ensure slow progress (Jacobson 1978, p. 98). Thus, a distinction has traditionally been made between embryos that have development, in which lineage determines cell fate, and those with regulative development, in which cell interactions determine cell fate, but, in fact, most if not all embryos show a combination of both (Harrison 1933, Weiss 1939, Davidson 1976). I shall propose that there are several lineage modes with different degrees of autonomy. In addition to the determinate lineage mode. which is completely autonomous and in which the relationship between a given progenitor and the phenotype(s) of its descendants is all-or­ none, there are others called indeterminant lineage modes in which the rela­ tionship between ancestral cells and the phenotypes of their descendants is contingent on external conditions to some degree. It is now clear that cell interactions can result in changes in of cellular fates in Uroand Cephalo­ chordates (Tung et a11958, 1960, Nakauchi & Takashita 1983), in which there is rigorous proof that materials localized in the egg and differentially distrib­ uted into different lineages determine the ultimate fates of the differentiated cells (Whittaker 1973, 1979. Jeffery 1983, Jeffery et al 1983). Such unim­ peachable evidence of extensive regulation in mosaic embryos should help to open the minds of those who have been unwilling to admit that determination

Clonal Analysis and Cell Lineages of the Vertebrate Central Nervous System

Clonal Analysis and Cell Lineages of the Vertebrate Central Nervous System