scholarly journals A Genome-Wide RNAi Screen for Enhancers of a Germline Tumor Phenotype Caused by Elevated GLP-1/Notch Signaling in Caenorhabditis elegans

2020 ◽  
Vol 10 (12) ◽  
pp. 4323-4334
Author(s):  
Diana Dalfó ◽  
Yanhui Ding ◽  
Qifei Liang ◽  
Alex Fong ◽  
Patricia Giselle Cipriani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Caenorhabditis Elegans ◽  
Germ Cells ◽  
Germ Line ◽  
Tumor Formation ◽  
Permissive Temperature ◽  
Germline Development ◽  
Link Type ◽  
Somatic Gonad

Stem cells are tightly controlled in vivo. Both the balance between self-renewal and differentiation and the rate of proliferation are often regulated by multiple factors. The Caenorhabditis elegans hermaphrodite germ line provides a simple and accessible system for studying stem cells in vivo. In this system, GLP-1/Notch activity prevents the differentiation of distal germ cells in response to ligand production from the nearby distal tip cell, thereby supporting a stem cell pool. However, a delay in germline development relative to somatic gonad development can cause a pool of undifferentiated germ cells to persist in response to alternate Notch ligands expressed in the proximal somatic gonad. This pool of undifferentiated germ cells forms a proximal tumor that, in adulthood, blocks the oviduct. This type of “latent niche”-driven proximal tumor is highly penetrant in worms bearing the temperature-sensitive weak gain-of-function mutation glp-1(ar202) at the restrictive temperature. At the permissive temperature, few worms develop tumors. Nevertheless, several interventions elevate the penetrance of proximal tumor formation at the permissive temperature, including reduced insulin signaling or the ablation of distal-most sheath cells. To systematically identify genetic perturbations that enhance proximal tumor formation, we sought genes that, upon RNAi depletion, elevate the percentage of worms bearing proximal germline tumors in glp-1(ar202) at the permissive temperature. We identified 43 genes representing a variety of functional classes, the most enriched of which is “translation”. Some of these genes also influence the distal germ line, and some are conserved genes for which genetic interactions with Notch were not previously known in this system.

scholarly journals Cellular Analyses of the Mitotic Region in the Caenorhabditis elegans Adult Germ Line

2006 ◽  
Vol 17 (7) ◽  
pp. 3051-3061 ◽  
Author(s):  
Sarah L. Crittenden ◽  
Kimberly A. Leonhard ◽  
Dana T. Byrd ◽  
Judith Kimble
Keyword(s):  
Stem Cells ◽  
Caenorhabditis Elegans ◽  
Germ Cells ◽  
Germ Line ◽  
Germline Stem Cells ◽  
C Elegans ◽  
Brdu Label ◽  
Asymmetric Divisions ◽  
Brdu Labeling ◽  
Cell Niche

The Caenorhabditis elegans germ line provides a model for understanding how signaling from a stem cell niche promotes continued mitotic divisions at the expense of differentiation. Here we report cellular analyses designed to identify germline stem cells within the germline mitotic region of adult hermaphrodites. Our results support several conclusions. First, all germ cells within the mitotic region are actively cycling, as visualized by bromodeoxyuridine (BrdU) labeling. No quiescent cells were found. Second, germ cells in the mitotic region lose BrdU label uniformly, either by movement of labeled cells into the meiotic region or by dilution, probably due to replication. No label-retaining cells were found in the mitotic region. Third, the distal tip cell niche extends processes that nearly encircle adjacent germ cells, a phenomenon that is likely to anchor the distal-most germ cells within the niche. Fourth, germline mitoses are not oriented reproducibly, even within the immediate confines of the niche. We propose that germ cells in the distal-most rows of the mitotic region serve as stem cells and more proximal germ cells embark on the path to differentiation. We also propose that C. elegans adult germline stem cells are maintained by proximity to the niche rather than by programmed asymmetric divisions.

scholarly journals Murine pluripotent stem cells that escape differentiation inside teratomas maintain pluripotency

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4177
Author(s):  
Yangli Pei ◽  
Liang Yue ◽  
Wei Zhang ◽  
Jinzhu Xiang ◽  
Zhu Ma ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Fluorescent Protein ◽  
Embryonic Stem ◽  
Tumor Formation ◽  
Immunofluorescent Staining ◽  
Pluripotent Cells ◽  
Teratoma Formation

Background Pluripotent stem cells (PSCs) offer immense potential as a source for regenerative therapies. The teratoma assay is widely used in the field of stem cells and regenerative medicine, but the cell composition of teratoma is still elusive. Methods We utilized PSCs expressing enhanced green fluorescent protein (EGFP) under the control of the Pou5f1 promoter to study the persistence of potential pluripotent cells during teratoma formation in vivo. OCT4-MES (mouse embryonic stem cells) were isolated from the blastocysts of 3.5-day OCT4-EGFP mice (transgenic mice express EGFP cDNA under the control of the Pou5f1 promoter) embryos, and TG iPS 1-7 (induced pluripotent stem cells) were generated from mouse embryonic fibroblasts (MEFs) from 13.5-day OCT4-EGFP mice embryos by infecting them with a virus carrying OCT4, SOX2, KLF4 and c-MYC. These pluripotent cells were characterized according to their morphology and expression of pluripotency markers. Their differentiation ability was studied with in vivo teratoma formation assays. Further differences between pluripotent cells were examined by real-time quantitative PCR (qPCR). Results The results showed that several OCT4-expressing PSCs escaped differentiation inside of teratomas, and these escaped cells (MES-FT, GFP-positive cells separated from OCT4-MES-derived teratomas; and iPS-FT, GFP-positive cells obtained from teratomas formed by TG iPS 1-7) retained their pluripotency. Interestingly, a small number of GFP-positive cells in teratomas formed by MES-FT and iPS-FT (MES-ST, GFP-positive cells isolated from MES-FT-derived teratomas; iPS-ST, GFP-positive cells obtained from teratomas formed by iPS-FT) were still pluripotent, as shown by alkaline phosphatase (AP) staining, immunofluorescent staining and PCR. MES-FT, iPS-FT, MES-ST and iPS-ST cells also expressed several markers associated with germ cell formation, such as Dazl, Stella and Stra8. Conclusions In summary, a small number of PSCs escaped differentiation inside of teratomas, and these cells maintained pluripotency and partially developed towards germ cells. Both escaped PSCs and germ cells present a risk of tumor formation. Therefore, medical workers must be careful in preventing tumor formation when stem cells are used to treat specific diseases.

Germ-line tumor formation caused by activation of glp-1, a Caenorhabditis elegans member of the Notch family of receptors

Development ◽  
1997 ◽  
Vol 124 (4) ◽  
pp. 925-936 ◽  
Author(s):  
L.W. Berry ◽  
B. Westlund ◽  
T. Schedl
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Fate ◽  
Germ Cells ◽  
Transmembrane Domain ◽  
Germ Line ◽  
Late Onset ◽  
Family Members ◽  
Mitotic Cell ◽  
Tumor Formation ◽  
Mutant Phenotypes

Caenorhabditis elegans germ-line proliferation is controlled by an inductive interaction between the somatic distal tip cell and the germ line. GLP-1, a member of the Notch family of transmembrane receptors, is required continuously in the germ line to transduce the proliferative signal. In the absence of GLP-1, all proliferative germ cells exit the mitotic cell cycle and enter meiotic prophase. We have characterized an activating mutation in glp-1, oz112gf, that has the opposite phenotype. Homozygous glp-1(oz112gf) hermaphrodites and males have a completely tumorous germ line in which germ cells never leave the mitotic cycle. In glp-1(oz112gf) heterozygotes, germ-line polarity is established correctly, but as adults age, the distal proliferative population expands leading to a late-onset tumorous phenotype. The mutant receptor is constitutively active, promoting proliferation in the absence of ligand. The normal distal-proximal spatial restriction of GLP-1 expression is lost in tumorous and late-onset tumorous animals; ectopically proliferating germ cells contain membrane-associated GLP-1. The correlation between proliferation and expression, both in wild-type where glp-1 signalling is limited by localized ligand and in glp-1(oz112gf) where signalling is ligand-independent, suggests that glp-1 signalling positively regulates GLP-1 expression. In addition to germ-line defects, glp-1(oz112gf) causes inappropriate vulval cell fate specification. A missense mutation in a conserved extracellular residue, Ser642, adjacent to the transmembrane domain, is sufficient to confer the glp-1(oz112gf) mutant phenotypes. Two mammalian Notch family members, TAN-1 and int-3, are proto-oncogenes. Thus, activating mutations in both invertebrate and vertebrate Notch family members can lead to tumor formation.

scholarly journals Murine pluripotent stem cells that escape differentiation inside teratomas maintain pluripotency

2017 ◽  
Author(s):  
Yangli Pei ◽  
Liang Yue ◽  
Wei Zhang ◽  
Jinzhu Xiang ◽  
Zhu Ma ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Fluorescent Protein ◽  
Embryonic Stem ◽  
Tumor Formation ◽  
Immunofluorescent Staining ◽  
Pluripotent Cells ◽  
Teratoma Formation

Background. Pluripotent stem cells (PSCs) offer immense potential as a source for regenerative therapies. The teratoma assay is widely used in the field of stem cells and regenerative medicine, but the cell composition of teratoma is still elusive. Methods. We utilized PSCs expressing enhanced green fluorescent protein (EGFP) under the control of the Pou5f1 promoter to study the persistence of potential pluripotent cells during teratoma formation in vivo. OCT4-MES (mouse embryonic stem cells) were isolated from the blastocysts of 3.5-day OCT4-EGFP mice (transgenic mice express EGFP cDNA under the control of the Pou5f1 promoter) embryos, and TG iPS 1-7 (induced pluripotent stem cells) were generated from mouse embryonic fibroblasts (MEFs) from 13.5-day OCT4-EGFP mice embryos by infecting them with a virus carrying OCT4, SOX2, KLF4 and c-MYC. These pluripotent cells were characterized according to their morphology and expression of pluripotency markers. Their differentiation ability was studied with in vivo teratoma formation assays. Further differences between pluripotent cells were examined by real-time quantitative PCR (qPCR). Results. The results showed that s everal OCT4-expressing PSCs escaped differentiation inside of teratomas, and these escaped cells (MES-FT, GFP-positive cells separated from OCT4-MES-derived teratomas; and iPS-FT, GFP-positive cells obtained from teratomas formed by TG iPS 1-7) retained their pluripotency. Interestingly, a small number of GFP-positive cells in teratomas formed by MES-FT and iPS-FT ( MES-ST, GFP-positive cells isolated from MES-FT-derived teratomas; iPS-ST, GFP-positive cells obtained from teratomas formed by iPS-FT ) were still pluripotent, as shown by alkaline phosphatase (AP) staining, immunofluorescent staining and PCR. MES-FT, iPS-FT, MES-ST and iPS-ST cells also expressed several markers associated with germ cell formation, such as Dazl, Stella and Stra8. Conclusions. In summary, a small number of PSCs escaped differentiation inside of teratomas , and these cells maintained pluripotency and partially developed towards germ cells. Both escaped PSCs and germ cells present a risk of tumor formation. Therefore, medical workers must be careful in preventing tumor formation when stem cells are used to treat specific diseases.

scholarly journals Murine pluripotent stem cells that escape differentiation inside teratomas maintain pluripotency

2017 ◽  
Author(s):  
Yangli Pei ◽  
Liang Yue ◽  
Wei Zhang ◽  
Jinzhu Xiang ◽  
Zhu Ma ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Fluorescent Protein ◽  
Embryonic Stem ◽  
Tumor Formation ◽  
Immunofluorescent Staining ◽  
Pluripotent Cells ◽  
Teratoma Formation

Background. Pluripotent stem cells (PSCs) offer immense potential as a source for regenerative therapies. The teratoma assay is widely used in the field of stem cells and regenerative medicine, but the cell composition of teratoma is still elusive. Methods. We utilized PSCs expressing enhanced green fluorescent protein (EGFP) under the control of the Pou5f1 promoter to study the persistence of potential pluripotent cells during teratoma formation in vivo. OCT4-MES (mouse embryonic stem cells) were isolated from the blastocysts of 3.5-day OCT4-EGFP mice (transgenic mice express EGFP cDNA under the control of the Pou5f1 promoter) embryos, and TG iPS 1-7 (induced pluripotent stem cells) were generated from mouse embryonic fibroblasts (MEFs) from 13.5-day OCT4-EGFP mice embryos by infecting them with a virus carrying OCT4, SOX2, KLF4 and c-MYC. These pluripotent cells were characterized according to their morphology and expression of pluripotency markers. Their differentiation ability was studied with in vivo teratoma formation assays. Further differences between pluripotent cells were examined by real-time quantitative PCR (qPCR). Results. The results showed that s everal OCT4-expressing PSCs escaped differentiation inside of teratomas, and these escaped cells (MES-FT, GFP-positive cells separated from OCT4-MES-derived teratomas; and iPS-FT, GFP-positive cells obtained from teratomas formed by TG iPS 1-7) retained their pluripotency. Interestingly, a small number of GFP-positive cells in teratomas formed by MES-FT and iPS-FT ( MES-ST, GFP-positive cells isolated from MES-FT-derived teratomas; iPS-ST, GFP-positive cells obtained from teratomas formed by iPS-FT ) were still pluripotent, as shown by alkaline phosphatase (AP) staining, immunofluorescent staining and PCR. MES-FT, iPS-FT, MES-ST and iPS-ST cells also expressed several markers associated with germ cell formation, such as Dazl, Stella and Stra8. Conclusions. In summary, a small number of PSCs escaped differentiation inside of teratomas , and these cells maintained pluripotency and partially developed towards germ cells. Both escaped PSCs and germ cells present a risk of tumor formation. Therefore, medical workers must be careful in preventing tumor formation when stem cells are used to treat specific diseases.

scholarly journals glp-3 Is Required for Mitosis and Meiosis in the Caenorhabditis elegans Germ Line

Genetics ◽  
1997 ◽  
Vol 145 (1) ◽  
pp. 111-121 ◽  
Author(s):  
Lisa C Kadyk ◽  
Eric J Lambie ◽  
Judith Kimble
Keyword(s):  
Caenorhabditis Elegans ◽  
Germ Cells ◽  
Germ Line ◽  
Stem Cell Population ◽  
Phenotypic Characterization ◽  
Loss Of Function ◽  
Dapi Staining ◽  
Cell Cycles ◽  
C Elegans ◽  
Mitosis And Meiosis

The germ line is the only tissue in Caenorhabditis elegans in which a stem cell population continues to divide mitotically throughout life; hence the cell cycles of the germ line and the soma are regulated differently. Here we report the genetic and phenotypic characterization of the glp-3 gene. In animals homozygous for each of five recessive loss-of-function alleles, germ cells in both hermaphrodites and males fail to progress through mitosis and meiosis, but somatic cells appear to divide normally. Germ cells in animals grown at 15° appear by DAPI staining to be uniformly arrested at the G2/M transition with <20 germ cells per gonad on average, suggesting a checkpoint-mediated arrest. In contrast, germ cells in mutant animals grown at 25° frequently proliferate slowly during adulthood, eventually forming small germ lines with several hundred germ cells. Nevertheless, cells in these small germ lines never undergo meiosis. Double mutant analysis with mutations in other genes affecting germ cell proliferation supports the idea that glp-3 may encode a gene product that is required for the mitotic and meiotic cell cycles in the C. elegans germ line.

scholarly journals Mesenchymal stem cells accelerated growth and metastasis of neuroblastoma and preferentially homed towards both primary and metastatic loci in orthotopic neuroblastoma model

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jiao-Le Yu ◽  
Shing Chan ◽  
Marcus Kwong-Lam Fung ◽  
Godfrey Chi-Fung Chan
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Primary Tumor ◽  
Imaging System ◽  
Ex Vivo ◽  
Tumor Formation ◽  
Human Neuroblastoma Cell ◽  
Fluorescence Signal ◽  
Human Neuroblastoma

Abstract Background Majority of neuroblastoma patients develop metastatic disease at diagnosis and their prognosis is poor with current therapeutic approach. Major challenges are how to tackle the mechanisms responsible for tumorigenesis and metastasis. Human mesenchymal stem cells (hMSCs) may be actively involved in the constitution of cancer microenvironment. Methods An orthotopic neuroblastoma murine model was utilized to mimic the clinical scenario. Human neuroblastoma cell line SK-N-LP was transfected with luciferase gene, which were inoculated with/without hMSCs into the adrenal area of SCID-beige mice. The growth and metastasis of neuroblastoma was observed by using Xenogen IVIS 100 in vivo imaging and evaluating gross tumors ex vivo. The homing of hMSCs towards tumor was analyzed by tracing fluorescence signal tagged on hMSCs using CRI Maestro™ imaging system. Results hMSCs mixed with neuroblastoma cells significantly accelerated tumor growth and apparently enhanced metastasis of neuroblastoma in vivo. hMSCs could be recruited by primary tumor and also become part of the tumor microenvironment in the metastatic lesion. The metastatic potential was consistently reduced in lung and tumor when hMSCs were pre-treated with stromal cell derived factor-1 (SDF-1) blocker, AMD3100, suggesting that the SDF-1/CXCR4 axis was one of the prime movers in the metastatic process. Conclusions hMSCs accelerated and facilitated tumor formation, growth and metastasis. Furthermore, the homing propensity of hMSCs towards both primary tumor and metastatic loci can also provide new therapeutic insights in utilizing bio-engineered hMSCs as vehicles for targeted anti-cancer therapy.

Genetic regulation of entry into meiosis in Caenorhabditis elegans

Development ◽  
1998 ◽  
Vol 125 (10) ◽  
pp. 1803-1813 ◽  
Author(s):  
L.C. Kadyk ◽  
J. Kimble
Keyword(s):  
Signal Transduction ◽  
Caenorhabditis Elegans ◽  
Germ Cells ◽  
Genetic Regulation ◽  
Signal Transduction Pathway ◽  
Mitotic Cell ◽  
Transduction Pathway ◽  
Germline Development ◽  
Large Tumor ◽  
Regulatory Pathways

The Caenorhabditis elegans germline is composed of mitotically dividing cells at the distal end that give rise to meiotic cells more proximally. Specification of the distal region as mitotic relies on induction by the somatic distal tip cell and the glp-1 signal transduction pathway. However, the genetic control over the transition from mitosis to meiosis is not understood. In this paper, we report the identification of a gene, gld-2, that has at least two functions in germline development. First, gld-2 is required for normal progression through meiotic prophase. Second, gld-2 promotes entry into meiosis from the mitotic cell cycle. With respect to this second function, gld-2 appears to be functionally redundant with a previously described gene, gld-1 (Francis, R., Barton, M. K., Kimble, J. and Schedl, T. (1995) Genetics 139, 579–606). Germ cells in gld-1(o) and gld-2 single mutants enter meiosis at the normal time, but germ cells in gld-2 gld-1(o) double mutants do not enter meiosis. Instead, the double mutant germline is mitotic throughout and forms a large tumor. We suggest that gld-1 and gld-2 define two independent regulatory pathways, each of which can be sufficient for entry into meiosis. Epistasis analyses show that gld-1 and gld-2 work downstream of the glp-1 signal transduction pathway. Therefore, we hypothesize that glp-1 promotes proliferation by inhibiting the meiosis-promoting functions of gld-1 and gld-2.

Regulation of Life-Span by Germ-Line Stem Cells in Caenorhabditis elegans

Science ◽  
2002 ◽  
Vol 295 (5554) ◽  
pp. 502-505 ◽  
Author(s):  
N. Arantes-Oliveira
Keyword(s):  
Stem Cells ◽  
Caenorhabditis Elegans ◽  
Life Span ◽  
Germ Line
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