Transcriptomic Changes in Internode Explants of Stinging Nettle during Callogenesis

Callogenesis, the process during which explants derived from differentiated plant tissues are subjected to a trans-differentiation step characterized by the proliferation of a mass of cells, is fundamental to indirect organogenesis and the establishment of cell suspension cultures. Therefore, understanding how callogenesis takes place is helpful to plant tissue culture, as well as to plant biotechnology and bioprocess engineering. The common herbaceous plant stinging nettle (Urtica dioica L.) is a species producing cellulosic fibres (the bast fibres) and a whole array of phytochemicals for pharmacological, nutraceutical and cosmeceutical use. Thus, it is of interest as a potential multi-purpose plant. In this study, callogenesis in internode explants of a nettle fibre clone (clone 13) was studied using RNA-Seq to understand which gene ontologies predominate at different time points. Callogenesis was induced with the plant growth regulators α-napthaleneacetic acid (NAA) and 6-benzyl aminopurine (BAP) after having determined their optimal concentrations. The process was studied over a period of 34 days, a time point at which a well-visible callus mass developed on the explants. The bioinformatic analysis of the transcriptomic dataset revealed specific gene ontologies characterizing each of the four time points investigated (0, 1, 10 and 34 days). The results show that, while the advanced stage of callogenesis is characterized by the iron deficiency response triggered by the high levels of reactive oxygen species accumulated by the proliferating cell mass, the intermediate and early phases are dominated by ontologies related to the immune response and cell wall loosening, respectively.

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Effects of Different Fixatives on β-Galactosidase Activity

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540205001015 ◽

2002 ◽

Vol 50 (10) ◽

pp. 1421-1424 ◽

Cited By ~ 26

Author(s):

Wenbin Ma ◽

Keith Rogers ◽

Berton Zbar ◽

Laura Schmidt

Keyword(s):

Gene Expression ◽

Alkaline Phosphatase ◽

Specific Gene ◽

Galactosidase Activity ◽

Time Points ◽

Specific Gene Expression ◽

Carnoy’S Solution ◽

Reporter Mice ◽

Different Temperatures

β-Galactosidase (β-Gal) staining is widely used to demonstrate specific gene expression during evaluation of gene targets in vivo. This technique is extremely sensitive to fixation. Optimal fixation conditions are necessary to obtain the maximal β-Gal activity. In this experiment, Carnoy's and three different aldehyde fixatives were used at different temperatures and over different time points. Kidneys from LacZ-stop-human alkaline phosphatase (ZA/P) double reporter mice were used to generate positive material for the experiment. The results show that glutaraldehyde combinative solution (LacZ) produced the most consistent and reliable results. Paraformaldehyde and formaldehyde were effective as fixatives only at 4C for a period of less than 4 hr, and Carnoy's solution destroyed β-Gal activity.

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Non-Random Integration and Clone Selection by Lentiviral SIN-LTR Vectors.

Blood ◽

10.1182/blood.v108.11.3257.3257 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3257-3257

Author(s):

Cynthia C. Bartholomae ◽

Annette Deichmann ◽

Manfred Schmidt ◽

Rafael J. Yanez-Munoz ◽

Steven J. Howe ◽

...

Keyword(s):

Gene Therapy ◽

Gene Ontology ◽

Clonal Selection ◽

Severe Combined Immunodeficiency ◽

Specific Gene ◽

Gene Ontology Analysis ◽

Chromosomal Distribution ◽

Time Points ◽

Vector Systems ◽

Integration Sites

Abstract The need for safer gene therapy vectors was highlighted by the occurrence of three cases of retroviral vector-induced leukemia in children after the cure of severe combined immunodeficiency by gene therapy. These severe adverse events enhanced the development of new gene therapy vector systems, which aim to reduce influence of insertions on integrity and expression of genomic host DNA. Lentiviral vectors with self-inactivating (SIN) long terminal repeats (LTRs) have no LTR enhancer activity and are not expected to produce insertional gene activation. In our study we analyzed the integrations sites of three different lentiviral SIN-HIV-based- vectors. These vectors differed in their internal elements (Promotor, Transgene, WPRE) which allowed us to investigate whether internal elements are influencing target site selection and clone survival. A total of 1422 integration sites were analyzed at three different time points (1 day, 30 days, 60 days) after transduction of identical HeLa cells by LAM- PCR. Our results showed similar gene involvement and chromosomal distribution of integration sites for all vector types analyzed, independent of vector composition. 62–63% of the integrations were detected in Refseq genes, 82–86% were found within Refseq genes and their surrounding 10kb. Surprisingly, 271 of 1422 integration sites were clustered as common integration sites (CIS). Computer simulations allowed us to show that this high number of CIS was significantly different from a modeled random distribution of integration sites. Gene ontology analysis showed no difference in significantly overrepresented gene categories between the distinct vector types. Interestingly, we detected a time dependent increase or decrease in significance. Specific gene categories like phosphorylation, protein kinase or ATP binding activity increased in significance from freshly transduced cells (1 day) to 30 days. This observation was even more pronounced at the latest time point analyzed (60 days). Other gene categories showed the exactly opposite effect. Gene ontology analysis of freshly transduced cells showed a significant overrepresentation of genes involved in cell cycle regulation, whereas the analysis of the later time points did not show overrepresentation of this gene category. These results suggest that lentiviral SIN-HIV-based vectors may induce clonal selection in vitro independently of internal vector elements. The character of such effects as well as any putative relevance of such genotoxicity for the in vivo situation will have to be investigated in detail for the role of individual vector/cell type configurations.

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Oct4 regulates embryonic pluripotency via metabolic mechanisms and Stat3 signalling

10.1101/2020.01.28.922856 ◽

2020 ◽

Author(s):

Giuliano Giuseppe Stirparo ◽

Agata Kurowski ◽

Stanley Eugene Strawbridge ◽

Hannah Stuart ◽

Thorsten Edwin Boroviak ◽

...

Keyword(s):

Transcription Factors ◽

Single Cell ◽

Inner Cell Mass ◽

Cell Mass ◽

Ectopic Expression ◽

Specific Gene ◽

List Type ◽

Early Mouse

AbstractOCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 protects the pluripotent lineage from differentiation into trophoblast, we used single cell transcriptomics and quantitative immunofluorescence on blastocysts and established differentially expressed genes and pathways between control and OCT4 null cells. Activation of most pluripotency-associated transcription factors in the early mouse inner cell mass appears independent of OCT4, whereas JAK/STAT signalling requires OCT4, via activation of IL6ST. Single cell deconvolution, diffusion component and trajectory inference dissected the process of differentiation of OCT4 null cells by activating specific gene-network and transcription factors. Downregulation of glycolytic and oxidative metabolism was observed. CHIPseq analysis suggests OCT4 directly targets rate-limiting glycolytic enzymes. Concomitant with significant disruption of the STAT3 pathway, oxidative respiration is significantly diminished in OCT4 null cells. Upregulation of the lysosomal pathway detected in OCT4 null embryos is likely attributable to aberrant metabolism.Highlights and noveltyMajor pluripotency-associated transcription factors are activated in OCT4-deficient early mouse ICM cells, coincident with ectopic expression of trophectoderm markersJAK/STAT signalling is defective in OCT4 null embryosOCT4 promotes expression of KATS enzymes by means of glycolytic production of Acetyl CoA to secure chromatin accessibility for acquisition of epiblast identityOCT4 regulates the metabolic and biophysical processes required for establishment of embryonic pluripotency

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Interactome comparison of human embryonic stem cell lines with the inner cell mass and trophectoderm

10.1101/411439 ◽

2018 ◽

Author(s):

Adam Stevens ◽

Helen Smith ◽

Terence Garner ◽

Ben Minogue ◽

Sharon Sneddon ◽

...

Keyword(s):

Human Embryonic Stem Cell ◽

Transcriptional Activity ◽

Inner Cell Mass ◽

Cell Mass ◽

Embryonic Stem ◽

Specific Gene ◽

Transcriptomic Data ◽

Network Properties ◽

Stem Cell Lines ◽

Hesc Lines

AbstractHuman embryonic stem cells (hESCs) derived from the pluripotent Inner cell mass (ICM) of the blastocyst are fundamental tools for understanding human development, yet are not identical to their tissue of origin. To investigate this divergence we compared the transcriptomes of genetically paired ICM and trophectoderm (TE) samples with three hESC lines: MAN1, HUES3 and HUES7 at similar passage. We generated inferred interactome networks using transcriptomic data unique to the ICM or TE, and defined a hierarchy of modules (highly connected regions with shared function). We compared network properties and the modular hierarchy and show that the three hESCs had limited overlap with ICM specific transcriptome (6%-12%). However this overlap was enriched for network properties related to transcriptional activity in ICM (p=0.016); greatest in MAN1 compared to HUES3 (p=0.048) or HUES7 (p=0.012). The hierarchy of modules in the ICM interactome contained a greater proportion of MAN1 specific gene expression (46%) compared to HUES3 (28%) and HUES7 (25%) (p=9.0×10−4).These findings show that traditional methods based on transcriptome overlap are not sufficient to identify divergence of hESCs from ICM. Our approach also provides a valuable approach to the quantification of differences between hESC lines.And Manchester Academic Health Sciences Centre

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MON-719 The Cellular and Molecular Landscape of Hypothalamic Patterning and Differentiation

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.1237 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Thomas Kim

Keyword(s):

Cell Fate ◽

Regulatory Networks ◽

Large Scale ◽

Molecular Mechanisms ◽

Brain Atlas ◽

Specific Gene ◽

Cell Fate Specification ◽

Human Hypothalamus ◽

Fate Specification ◽

Time Points

Abstract The hypothalamus is a central regulator of physiological homeostasis. During development, multiple transcription factors coordinate the patterning and specification of hypothalamic nuclei. However, the molecular mechanisms controlling hypothalamic patterning and cell fate specification are poorly understood. To identify genes that control these processes, we have used single-cell RNA sequencing (scRNA-Seq) to profile mouse hypothalamic gene expression across multiple developmental time points. We have further utilised scRNA-Seq to phenotype mutations in genes that play major roles in early hypothalamic patterning. To first understand hypothalamic development, hypothalami were collected at both embryonic (E10-E16, E18) and postnatal (PN4, PN8, PN14, PN45) time points. At early stages, when the bulk of hypothalamic patterning occurs (E11-E13), we observe a clear separation between mitotic progenitors and postmitotic neural precursor cells. We likewise observed clean segregation among cells expressing regional hypothalamic markers identified in previous large-scale analysis of hypothalamic development. This analysis reveals new region-specific markers and identifies candidate genes for selectively regulating patterning and cell fate specification in individual hypothalamic regions. With our rich dataset of developing mouse hypothalamus, we integrated our dataset with the Allen Brain Atlas in situ data, publicly available adult hypothalamic scRNA-Seq dataset to understand hierarchy of hypothalamic cell differentiation, as well as re-defining cell types of the hypothalamus. We next used scRNA-Seq to phenotype multiple mutant lines, including a line that has been extensively characterised as a proof of concept (Ctnnb1 overexpression), and lines that have not been characterised (Nkx2.1, Nkx2.2, Dlx1/2 deletion). We show that this approach can rapidly and comprehensively characterize mutants that have altered hypothalamic patterning, and in doing so, have identified multiple genes that simultaneously repress posterior hypothalamic identity while promoting prethalamic identity. This result supports a modified columnar model of organization for the diencephalon, where prethalamus and hypothalamus are situated in adjacent dorsal and ventral domains of the anterior diencephalon. These data serve as a resource for further studies of hypothalamic development and dysfunction, and able to delineate transcriptional regulatory networks of hypothalamic formation. Lastly, using our mouse hypothalamus as a guideline, we are comparing dataset of developing chicken, zebrafish and human hypothalamus, to identify evolutionarily conserved and divergent region-specific gene regulatory networks. We aim to use this knowledge and information of key molecular pathways of human hypothalamic development and produce human hypothalamus organoids.

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In vitro morphogenesis of Syngonanthus mucugensis Giul: subsp. mucugensis

Ciência e Agrotecnologia ◽

10.1590/s1413-70542011000300010 ◽

2011 ◽

Vol 35 (3) ◽

pp. 502-510 ◽

Cited By ~ 8

Author(s):

Alone Lima-Brito ◽

Sheila Vitória Resende ◽

Carolina Oliveira de Cerqueira Lima ◽

Bruno Matos Alvim ◽

Claudia Elena Carneiro ◽

...

Keyword(s):

Shoot Regeneration ◽

Callus Formation ◽

Economic Value ◽

Direct Organogenesis ◽

Naphthalene Acetic Acid ◽

Herbaceous Plant ◽

Indirect Organogenesis ◽

In Vitro Morphogenesis ◽

Different Types

Syngonanthus mucugensis Giul. subsp. mucugensis is an herbaceous plant with significant economic value in the ornamental dry flower business. The restricted occurrence of the municipality Mucugê-BA, Brazil, exclusively associated with extractive exploitation, has considered this species as endangered. The objective of this work was to evaluate the organogenic potential of three different types of S. mucugensis subsp. mucugensis explants to promote the development of an alternative method to the propagation of the genetic resources of this important plant. The morphogenetic capacities of the leaf, stem and root this species was tested using Murashige and Skoog culture medium at half salt concentration and different concentrations of growth of regulators benzylaminopurine - BAP (0.00; 2.22 and 4.44 µM), and naphthalene acetic acid - NAA (0.00; 1.34 and 2.68 µM). The morphoanatomic events that lead to formation of shoots were described. Stems proved to be the best source of explants, showing 58.75% regeneration of shoot by direct organogenesis in the absence of growth regulators, and 32.18 and 47.55% of shoot regeneration by indirect organogenesis in the presence of 2.22 and 4.44 µM BAP, respectively. As for leaves, there was callus formation, but without regenerating shoots. Morphogenesis was not observed when roots were used as explants. The histological analyses showed that shoot regeneration in S. mucugensis subsp. mucugensis occurred both indirectly, by unorganized tissue differentiation, and directly through returning to merismatic activity in differentiated mature cells and preexisting bud proliferation.

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1 DIFFERENCES IN RESUMPTION OF OOCYTE MATURATION IN YOUNG AND OLD MARES

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab1 ◽

2008 ◽

Vol 20 (1) ◽

pp. 81 ◽

Cited By ~ 2

Author(s):

L. F. Campos-Chillon ◽

C. M. Clay ◽

J. L. Altermatt ◽

G. J. Bouma ◽

E. M. Carnevale

Keyword(s):

Granulosa Cells ◽

Expression Patterns ◽

Cell Number ◽

Specific Gene ◽

Meiotic Arrest ◽

Temporal Differences ◽

Time Points ◽

Follicular Maturation ◽

Copy Numbers

The decline in fertility of aged mares is linked with declining oocyte quality. Oocyte viability is dependent on the ability of oocytes to remain in meiotic arrest until the initiation of maturation. We hypothesize that aging is associated with quantitative and temporal differences in meiotic arrest and resumption in oocytes, ultimately resulting in a dissociation of oocyte and follicular maturation. The objectives of this study were to determine temporal differences in the mRNA content of amphiregulin and epiregulin in granulosa cells; PDE4 in cumulus and granulosa cells; and PDE3A, GPR3, GDF9, and BMP15 in oocytes during in vivo maturation in young (3–12 years) v. old (>20 years) mares. Oocytes and follicular cells were collected by transvaginal follicular aspiration. Maturation was induced in estrous mares with a follicle >30 mm by injection of 750 �g of recombinant equine LH. Aspirations were attempted at 0, 6, 9, and 12 h after LH administration. Six oocytes and follicular cell samples from each age group and time point were collected and stored immediately after aspiration. Total RNA was isolated from single denuded oocytes and lysed cumulus and granulosa cells. A fraction of the total lysate was used to determine cell numbers from the DNA copy number of the equine CG� subunit gene. DsRED RNA was added to each RNA isolate to serve as an exogenous standard. Quantitative RT-PCR was performed from cDNA with equine primer pairs. Copy numbers were calculated with an intra assay standard curve of plasmid containing the specific gene and corrected with the exogenous DsRED RNA and cell number. For each gene, mean mRNA copy numbers for time points and age groups were compared by ANOVA and Tukey's HSD test. Expression of PDE4D in cumulus cells was similar between young and old mares and time points. However, PDE4D peaked (P < 0.05) at 6 h in granulosa cells from young, but not old mares. Amphiregulin expression in granulosa cells of young mares peaked (P < 0.05) at 9 h and did not increase in the old mares. Epiregulin expression in granulosa cells peaked (P < 0.05) at 9 h and 6 h in young and old mares, respectively. The pattern of expression of PDE3A for oocytes of young and old mares was similar with an increase (P < 0.05) at 9 h. There was an interaction (P < 0.05) in the expression of GPR3 for age � time. Expression peaked at 9 and 6 h in young and old mares, respectively. Pattern of expression of GDF9 was similar between young and old mares except for a decrease (P < 0.05) in expression in old mares at 9 h. There was an interaction (P < 0.05) in the expression of BMP15 for age � time. Expression in young mares peaked at 9 h while that in old mares peaked at 6 h and decreased at 9 h. These results suggest that key gene expression patterns involved in oocyte and follicular maturation cascades are asynchronous for young versus old mares and could explain some aspects of the age-associated decline in fertility.

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Expression of an activated rasD gene changes cell fate decisions during Dictyostelium development.

Molecular Biology of the Cell ◽

10.1091/mbc.8.2.303 ◽

1997 ◽

Vol 8 (2) ◽

pp. 303-312 ◽

Cited By ~ 7

Author(s):

S A Louis ◽

G B Spiegelman ◽

G Weeks

Keyword(s):

Gene Expression ◽

Cell Fate ◽

Cell Mass ◽

Cell Formation ◽

Specific Gene ◽

Wild Type ◽

Multicellular Development ◽

Cell Fate Decisions ◽

Prespore Cell

It has been previously demonstrated that the expression of an activated rasD gene in wild-type Dictyostelium cells results in formation of aggregates with multitips, instead of the normal single tips, and a block in further development. In an attempt to better understand the role of activated RasD development, we examined cell-type-specific gene expression in a strain stably expressing high levels of RasD[G12T]. We found that the expression of prestalk cell-specific genes ecmA and tagB was markedly enhanced, whereas the expression of the prespore cell-specific gene cotC was reduced to very low levels. When the fate of cells in the multitipped aggregate was monitored with an ecmA/lacZ fusion, it appeared that most of the cells eventually adopted prestalk gene expression characteristics. When mixtures of the [G12T]rasD cells and Ax3 cells were induced to differentiate, chimeric pseudoplasmodia were not formed. Thus, although the [G12T]rasD transformant had a marked propensity to form prestalk cells, it could not supply the prestalk cell population when mixed with wild-type cells. Both stalk and spore cell formation occurred in low cell density monolayers of the [G12T]rasD strain, suggesting that at least part of the inhibition of stalk and spore formation during multicellular development involved inhibitory cell interactions within the cell mass. Models for the possible role of rasD in development are discussed.

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PDTM-11. GAINING INSIGHTS INTO THE INFLAMMATORY MICROENVIRONMENT OF PEDIATRIC HIGH-GRADE GLIOMAS USING GEMMs AND PATIENT SAMPLES

Neuro-Oncology ◽

10.1093/neuonc/noz175.787 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi189-vi189

Author(s):

James Ross ◽

Zhihong Chen ◽

Cameron Herting ◽

Frank Szulzewsky ◽

Oren Becher ◽

...

Keyword(s):

Immune Cell ◽

Cell Mass ◽

Driver Mutations ◽

Specific Gene ◽

High Grade ◽

Human Patient ◽

Inflammatory Microenvironment ◽

Inflammatory Monocytes ◽

High Grade Gliomas

Abstract Pediatric high-grade gliomas (pHGG) account for the most cancer-related deaths in children, as there are no effective therapies available. It is known tumor associated macrophages (TAM) can make up 30–40% of the total tumor cell mass in adult high-grade gliomas, promoting tumor growth and immune evasion. This raises the question of whether pHGGs possess a distinct constituency of TAMs due to their unique genetic and epigenetic landscapes. To uncover the composition and behavior of TAMs in pHGG we utilize RCAS/tva, a somatic cell-type specific gene transfer system which allows us to recapitulate all major subtypes of pHGG in newborn immunocompetent mice, including histone wild-type and histone-mutant tumors. We combine RCAS-H3.3K27M, RCAS-H3.3G34R/V, or RCAS-H3.3WT along with their driver mutations such as RCAS-shp53 and RCAS-PDGFA or RCAS-PDGFB. These tumors are induced in Nestin-positive cells, each in their respective locations found in the human population. Tumors driven by PDGFB have a significantly lower median survival compared to PDGFA-driven tumors and have increased infiltration of lymphocytes and TAMs, specifically inflammatory monocytes. In vitro bone marrow derived monocyte and microglial cultures demonstrate the BMDM population is most responsible for the production of inflammatory chemokines and angiogenic factors in the tumor microenvironment. We performed histological analyses on over 40 human patient samples to determine the role of the stromal population in TAM infiltration. Matched human samples were also utilized for pan-cancer immune profiling with NanoString to further characterize the innate and adaptive immune microenvironments. These analyses indicate DIPG/K27M tumors have a higher immune cell infiltrate compared to G34R/V and histone wildtype tumors. Further, we observe higher infiltration of T-cell populations in pHGGs compared to adult HGGs, suggesting these tumors may be amenable to immunotherapy despite being considered “immune cold.” These studies provide the critical foundation needed for the development of novel therapeutics targeting these tumors.

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