62 BOVINE OCT4 (POU5F1) KNOCKOUT EMBRYOS FAIL DURING THE SECOND LINEAGE DIFFERENTIATION DUE TO LOSS OF NANOG

2017 ◽  
Vol 29 (1) ◽  
pp. 138 ◽  
Author(s):  
K. Simmet ◽  
N. Klymiuk ◽  
V. Zakhartchenko ◽  
T. Güngör ◽  
M. Reichenbach ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Mass ◽  
Total Cell ◽  
Laser Scanning Microscopy ◽  
Immunofluorescent Staining ◽  
Confocal Laser ◽  
Specific Marker ◽  
Lineage Differentiation ◽  
Oct4 Protein

We generated a CRISPR/Cas9 mediated knockout (KO) of the OCT4 gene in adult fibroblasts, where biallelic deletion of a single nucleotide leads to a frameshift mutation. Through reconstruction of embryos by somatic cell NT, we were able to study the role of OCT4 during pre-implantation development until Day 7 in vitro. The presence of OCT4 protein was evaluated after immunofluorescent staining by confocal laser scanning microscopy of Day 5 morulae and Day 7 blastocysts; somatic cell NT embryos reconstructed from nontransfected cells of the same source served as control. Whereas control morulae expressed OCT4 in all cells, OCT4 KO morulae showed expression in only 67.8 ± 11.1% (mean ± SD, n = 6) of cells and overall intensity was decreased. By Day 7, no expression of OCT4 was detected in OCT4 KO blastocysts (n = 24), suggesting that maternal stores of the OCT4 protein had decayed. In contrast, control blastocysts (n = 20) showed OCT4 expression ubiquitously in both inner cell mass (ICM) and trophectoderm (TE). Simultaneously to the OCT4 staining, we differentially stained ICM and TE with the TE specific marker CDX2 and counterstained cell nuclei with 4′,6-diamidino-2-phenylindole. No significant differences between OCT4 KO Day 7 blastocysts and controls were detected in total cell numbers (89.6 ± 27.5 v. 96.3 ± 38) and percentage of CDX2 positive cells (50.7 ± 16.8% v. 59.0 ± 20.8%) (P > 0.05, mean ± SD, unpaired, two-tailed t-test). To analyse the role of OCT4 during the second lineage differentiation, we stained Day 5 morulae and Day 7 blastocysts for the epiblast and hypoblast specific markers NANOG and GATA6, respectively. In morulae, both markers were present and co-expressed in OCT4 KO and control embryos. By Day 7, control blastocysts (n = 6) already showed the typical salt and pepper distribution of NANOG and GATA6 positive cells, but expression was not mutually exclusive in all cells and also not restricted to ICM. OCT4 KO embryos lost all NANOG expression at Day 7 blastocyst stage (n = 8) and only stained positive for GATA6 in both TE and ICM. We conclude that OCT4 is not required for the quantitative allocation of cells to either the ICM or the TE during the first lineage differentiation, as total cell number and percentage of CDX2 positive cells was unchanged. Additionally, expression of NANOG seems to be OCT4 dependent and OCT4 KO embryos fail to establish the epiblast lineage—unlike mouse Oct4 KO embryos, where developmental failure was connected to loss of GATA6 expression during second lineage differentiation. This work was funded by the Bavarian Research Foundation (AZ-1031–12).

scholarly journals Two Enteropathogenic Escherichia coli Type III Secreted Proteins, EspA and EspB, Are Virulence Factors

1998 ◽  
Vol 188 (10) ◽  
pp. 1907-1916 ◽  
Author(s):  
Akio Abe ◽  
Ursula Heczko ◽  
Richard G. Hegele ◽  
B. Brett Finlay
Keyword(s):  
Escherichia Coli ◽  
Virulence Factors ◽  
Enterohemorrhagic Escherichia Coli ◽  
Laser Scanning Microscopy ◽  
Secreted Proteins ◽  
Confocal Laser ◽  
Infection Model ◽  
Enteropathogenic Escherichia Coli ◽  
Type Iii

Enteropathogenic Escherichia coli (EPEC) belongs to a family of related bacterial pathogens, including enterohemorrhagic Escherichia coli (EHEC) O157:H7 and other human and animal diarrheagenic pathogens that form attaching and effacing (A/E) lesions on host epithelial surfaces. Bacterial secreted Esp proteins and a type III secretion system are conserved among these pathogens and trigger host cell signal transduction pathways and cytoskeletal rearrangements, and mediate intimate bacterial adherence to epithelial cell surfaces in vitro. However, their role in pathogenesis is still unclear. To investigate the role of Esp proteins in disease, mutations in espA and espB were constructed in rabbit EPEC serotype O103 and infection characteristics were compared to that of the wild-type strain using histology, scanning and transmission electron microscopy, and confocal laser scanning microscopy in a weaned rabbit infection model. The virulence of EspA and EspB mutant strains was severely attenuated. Additionally, neither mutant strain formed A/E lesions, nor did either one cause cytoskeletal actin rearrangements beneath the attached bacteria in the rabbit intestine. Collectively, this study shows for the first time that the type III secreted proteins EspA and EspB are needed to form A/E lesions in vivo and are indeed virulence factors. It also confirms the role of A/E lesions in disease processes.

scholarly journals Potential role of nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase in apoptosis and oxidative stress

2001 ◽  
Vol 114 (9) ◽  
pp. 1643-1653 ◽  
Author(s):  
Z. Dastoor ◽  
J.L. Dreyer
Keyword(s):  
Oxidative Stress ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Apoptotic Cells ◽  
Transfected Cells ◽  
And Oxidative Stress

Recent studies indicating a role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in apoptosis or oxidative stress has been reported. Using confocal laser-scanning microscopy, we have investigated the cellular distribution of GAPDH in central nervous system (CNS)-derived cells (neuroblastoma mNB41A3), in non-CNS derived cells (R6 fibroblast) and in an apoptosis-resistant Bcl2 overexpressing cell line (R6-Bcl2). Induction of apoptosis by staurosporine or MG132 and oxidative stress by H(2)O(2) or FeCN enhanced the nuclear translocation of endogenous GAPDH in all cell types, as detected by immunocytochemistry. In apoptotic cells, GAPDH expression is three times higher than in non-apoptotic cells. Consistent with a role for GAPDH in apoptosis, overexpression of a GAPDH-green fluorescent protein (GAPDH-GFP) hybrid increased nuclear import of GAPDH-GFP into transfected cells and the number of apoptotic cells, and made them more sensitive to agents that induce apoptosis. Bcl2 overexpression prevents nuclear translocation of GAPDH and apoptosis in untransfected cells, but not in transfected cells that overexpress GAPDH-GFP. Our observations indicate that nuclear translocation of GAPDH may play a role in apoptosis and oxidative stress, probably related to the activity of GAPDH as a DNA repair enzyme or as a nuclear carrier for pro-apoptotic molecules.

Behaviour of nucleolar proteins in nuclei lacking ribosomal genes. A study by confocal laser scanning microscopy

1991 ◽  
Vol 98 (1) ◽  
pp. 99-105
Author(s):  
D. Hernandez-Verdun ◽  
M. Robert-Nicoud ◽  
G. Geraud ◽  
C. Masson
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Specific Marker ◽  
Nucleolar Proteins ◽  
Fibrillar Component

The behaviour of nucleolar proteins in cycling PtK1 cells and in micronuclei with or without NORs was investigated by immunofluorescence using antibodies from autoimmune sera and confocal laser scanning microscopy. These antibodies were shown by electron microscopy to recognize antigens confined to only one of the three basic nucleolar components: fibrillar centres (FC), dense fibrillar component (DFC) and granular component (GC). Serial optical sections allowed us to determine the three-dimensional organization of these components in the nucleolus of cycling cells. Furthermore, clear differences were found in the distribution of the various antigens in micronucleated cells. Three patterns could be observed: (1) the FC antigens were found mainly in the nucleoli, but also in varying amounts in the dots; (2) surprisingly, the DFC antigens were found to accumulate preferentially in the dots; (3) the GC-specific marker stained intensively the nucleoli as well the dots. The results are interpreted with regard to possible mechanisms for targeting nucleolar proteins to the site of nucleolar formation.

105 THE Oct4/Cdx2 EXPRESSION AND CELL FATE OF INDIVIDUAL TWO-CELL BLASTOMERES IN TWO MOUSE STRAINS

2008 ◽  
Vol 20 (1) ◽  
pp. 133
Author(s):  
M. Katayama ◽  
R. M. Roberts
Keyword(s):  
Cell Mass ◽  
Strain Differences ◽  
Lineage Tracing ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Mouse Strains ◽  
Mouse Development ◽  
Cell Stage ◽  
Developmental Potential ◽  
Nih Swiss

Fertile adults and occasionally twins have been derived from murine blastomeres at the 2-cell stage, indicating that such blastomeres may be equivalently totipotent, but there are conflicting reports that individual blastomeres from 2-cell stage murine conceptuses make different contributions to the embryonic and abembryonic regions of the blastocyst, implying that they differ in developmental potential. Here, we have re-examined this subject using 2 mouse strains, CF1 and NIH Swiss (SW), and 2 experimental approaches, random blastomere destruction at the 2-cell stage by repeated insertion of a needle into its nucleus and lineage tracing with the dye, DiI-CM. The manipulated conceptuses and untreated controls were cultured in KSOM-AA to morula and blastocyst stages (84 or 108 h pc, respectively), fixed, and immunostained for Oct4 and Cdx2. Antigen distribution, number of nuclei (stained by 42,6-diamidino-2-phenylindole), and cell progeny labeled with DiI-CM were examined by confocal laser scanning microscopy. Cell numbers are means � SD and were analyzed by a Student t-test. Cells positive for Cdx2 were assumed to represent trophectoderm or trophectoderm precursors, ones positive for Oct4 but negative for Cdx2 (Oct+Cdx–) inner cell mass. Ablation of a blastomere failed to prevent developmental progression in either strain, but the total number of cells at both morula (SW 11.4 � 3.3 v. 19.2 � 7.1; CF1 10.1 � 2.5 v. 22.1 � 6.4) and blastocyst (SW 48.6 � 7.4 v. 69.4 � 9.9; CF1 24.8 � 6.2 v. 53.8 � 13.5) was significantly reduced. In SW, the average fraction of Oct+Cdx– cells after blastomere ablation was significantly lower (P < 0.05) than in controls in morulae (0.47 � 0.2 v. 0.65 � 0.1) but not in blastocysts (0.33 � 0.1 and 0.34 � 0.1). In CF1, the fraction of Oct+Cdx– cells was lower (P < 0.05) than controls in both morulae and blastocysts (0.31 � 0.2 v. 0.58 � 0.2 and 0.18 � 0.1 v. 0.27 � 0.04, respectively). The CF1 morulae fell mainly into 2 groups, one low fraction (≤0.3, 54%) of Oct+Cdx– cells and the other with a more normal fraction (0.3 to 0.8, 43%) relative to controls. A majority of NIH Swiss morulae had an Oct+Cdx– cell fraction >0.4 and in this respect resembled controls. We then examined these strain differences by lineage tracing. The majority of SW blastocysts (65%, n = 34) demonstrated a random localization of DiI-labeled cell progeny (i.e., there was no preferential distribution of labeled cells to either the embryonic or abembryonic poles). By contrast, in CF1 (n = 38), 32% of blastocysts had labeled cells confined to their embryonic end and 42% with DiI-labeled, Cdx2-positive cells clustered at the abembryonic locale. A random localization was observed in 26% of blastocysts. In conclusion, these data confirm that there is plasticity in early mouse development but also suggest that in CF1, but not in SW conceptuses, blastomeres at the 2-cell stage differ in their abilities to contribute to the embryonic pole. Similar strain differences may explain the disagreements among studies on lineage tracing in early cleavage stage conceptuses.

Detection and enumeration of microbial cells within highly porous calcareous reef sands

2006 ◽  
Vol 57 (4) ◽  
pp. 415 ◽  
Author(s):  
Christian Wild ◽  
Christian Laforsch ◽  
Markus Huettel
Keyword(s):  
Laser Scanning ◽  
Complex Surface ◽  
Porous Matrix ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Size Spectrum ◽  
Order Of Magnitude ◽  
Grain Surface ◽  
Highly Porous

In order to assess and to compare the abundances of prokaryotes in coral sands from three different areas in the Indo-Pacific, a technique was developed and evaluated for enumeration of prokaryotes living on and within calcareous grains. Propidium iodide labelling of prokaryotes and consecutive confocal laser scanning microscopy showed microbial colonisation within pores and small fissures of the coral sands. This embedded microbial colonisation required at least four extractions with weak acetic acid to dissolve the grain surface layer in order to detach 97% of the prokaryotic cells. Microbial enumeration based on this technique revealed that the abundance of prokaryotes in the carbonate sands were not significantly different among the three sites, but were about one order of magnitude higher than reported for silicate sands of a similar grain size spectrum. A possible reason for this high abundance of prokaryotes is the complex surface structure of the biogenic calcareous grains, their correspondingly highly porous matrix and the associated ability of prokaryotes to penetrate into carbonate grains. Our results highlight the role of calcareous reef sands as a substratum with a large specific surface area for prokaryotic colonisation and emphasise the contribution of calcium carbonate reef sands for element cycles in subtropical and tropical ecosystems.

scholarly journals Antibody response to the 45 kDa Candida albicans antigen in an animal model and potential role of the antigen in adherence

2008 ◽  
Vol 57 (12) ◽  
pp. 1466-1472 ◽  
Author(s):  
Helena Bujdáková ◽  
Ema Paulovičová ◽  
Silvia Borecká-Melkusová ◽  
Juraj Gašperík ◽  
Soňa Kucharíková ◽  
...  
Keyword(s):  
Animal Model ◽  
Candida Albicans ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Vaccine Development ◽  
Laser Scanning Microscopy ◽  
Model Systems ◽  
Confocal Laser

The Candida antigen CR3-RP (complement receptor 3-related protein) is supposed to be a ‘mimicry’ protein because of its ability to bind antibody directed against the α subunit of the mammalian CR3 (CD11b/CD18). This study aimed to (i) investigate the specific humoral isotypic response to immunization with CR3-RP in vivo in a rabbit animal model, and (ii) determine the role of CR3-RP in the adherence of Candida albicans in vitro using the model systems of buccal epithelial cells (BECs) and biofilm formation. The synthetic C. albicans peptide DINGGGATLPQ corresponding to 11 amino-acids of the CR3-RP sequence DINGGGATLPQALXQITGVIT, determined by N-terminal sequencing, was used for immunization of rabbits to obtain polyclonal anti-CR3-PR serum and for subsequent characterization of the humoral isotypic response of rabbits. A significant increase of IgG, IgA and IgM anti-CR3-RP specific antibodies was observed after the third (P<0.01) and the fourth (P<0.001) immunization doses. The elevation of IgA levels suggested peptide immunomodulation of the IgA1 subclass, presumably in coincidence with Candida epithelial adherence. Blocking CR3-RP with polyclonal anti-CR3-RP serum reduced the ability of Candida to adhere to BECs, in comparison with the control, by up to 35 % (P<0.001), and reduced biofilm formation by 28 % (P<0.001), including changes in biofilm thickness and integrity detected by confocal laser scanning microscopy. These properties of CR3-RP suggest that it has potential for future vaccine development.

scholarly journals Hypoblast Formation in Bovine Embryos Does Not Depend on NANOG

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2232 ◽  
Author(s):  
Claudia Springer ◽  
Valeri Zakhartchenko ◽  
Eckhard Wolf ◽  
Kilian Simmet
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Model Organism ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Lineage Differentiation ◽  
Species Specific ◽  
And Control

The role of the pluripotency factor NANOG during the second embryonic lineage differentiation has been studied extensively in mouse, although species-specific differences exist. To elucidate the role of NANOG in an alternative model organism, we knocked out NANOG in fibroblast cells and produced bovine NANOG-knockout (KO) embryos via somatic cell nuclear transfer (SCNT). At day 8, NANOG-KO blastocysts showed a decreased total cell number when compared to controls from SCNT (NT Ctrl). The pluripotency factors OCT4 and SOX2 as well as the hypoblast (HB) marker GATA6 were co-expressed in all cells of the inner cell mass (ICM) and, in contrast to mouse Nanog-KO, expression of the late HB marker SOX17 was still present. We blocked the MEK-pathway with a MEK 1/2 inhibitor, and control embryos showed an increase in NANOG positive cells, but SOX17 expressing HB precursor cells were still present. NANOG-KO together with MEK-inhibition was lethal before blastocyst stage, similarly to findings in mouse. Supplementation of exogenous FGF4 to NANOG-KO embryos did not change SOX17 expression in the ICM, unlike mouse Nanog-KO embryos, where missing SOX17 expression was completely rescued by FGF4. We conclude that NANOG mediated FGF/MEK signaling is not required for HB formation in the bovine embryo and that another—so far unknown—pathway regulates HB differentiation.

Deletion of bem46 retards spore germination and may be related to the polar growth of Aspergillus fumigatus

2019 ◽  
Vol 58 (5) ◽  
pp. 690-697
Author(s):  
Yan Ma ◽  
Ying Ji ◽  
Jing Yang ◽  
Wen Li ◽  
Jiajuan Li ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Polarized Growth ◽  
Bud Emergence ◽  
Green Fluorescent

Abstract Bud emergence 46 (BEM46), a member of the α/β hydrolase superfamily, has been reported to be essential for polarized growth in Neurospora crassa. However, the role of BEM46 in aspergillus fumigatus (A. fumigatus) remains unclear. In this study, we constructed an A. fumigatus strain expressing BEM46 fused with enhanced green fluorescent protein, and a Δbem46 mutant, to explore the localization and the role of growth of BEM46 in A. fumigatus, respectively. Confocal laser scanning microscopy revealed that BEM46 was dominantly expressed in the sites where hyphae germinated from conidia in A. fumigatus. When compared with the control strain, the Δbem46 mutant exhibited insignificant morphological changes but delayed germination. No significant changes were found regarding the radial growth of both strains in response to various antifungal agents. These results suggest that BEM46 plays an essential role in timely germination in A. fumigatus. From the observation of fluorescence localization, we infer that that BEM46 might be involved in polarized growth in A. fumigatus.

Neuron model system: Correlative confocal laser scanned microscopy and membrane physiology

1990 ◽  
Vol 48 (3) ◽  
pp. 170-171
Author(s):  
Donald H. Szarowski ◽  
Michael Fejtl ◽  
Paul McCauley ◽  
David O. Carpenter ◽  
James N. Turner
Keyword(s):  
Laser Scanning ◽  
Model System ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Patch Clamping ◽  
Surface Geometry ◽  
Receptor Stimulation ◽  
Membrane Physiology ◽  
Conventional Light Microscopy

Confocal laser scanning microscopy (CLSM) has been used to correlate morphology and membrane physiology in cultured neurons, providing a model system for studying physiologic and pathologic conditions. Ion channels are studied by patch-clamp methods as a function of receptor stimulation and toxic excitatory amino acids, including those implicated in Alzheimer’s dementia. Glial cells are often closely associated with the neurons, and are difficult to detect in living cultures due to the relative sizes of glia and neurons (5-20 μm versus 125 μm), compounded with the fact that they are thick phase objects. Groups of glia can also be confused with neurons. Thus it is difficult to select appropriate cells and/or cell regions for patch-clamping. We are correlating physiology and conventional light microscopy with CLSM to determine the role of glia, and neuron surface geometry on the ability to establish Gigaohm membrane-micropipette seals. Morphology of the system as observed by CLSM is presented here.

Quantitation of the spatial distribution of ‘prespore vacuoles’ in pseudoplasmodia of Dictyostelium discoideum

Development ◽  
1976 ◽  
Vol 35 (3) ◽  
pp. 499-505
Author(s):  
Paul A. Farnsworth ◽  
William F. Loomis
Keyword(s):  
Electron Microscopy ◽  
Spatial Distribution ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Axial Distribution ◽  
Total Cell Number ◽  
Total Complement ◽  
Number Of Cells

The axial distribution of an organelle, the prespore vacuole (PV), previously reported absent from the prestalk region, was determined in pseudoplasmodia of varying sizes, under differing conditions of photostimulation of migration. The distribution of these organelles, determined quantitatively by electron microscopy ofsections from known axial locations, was found to have a spatial pattern which varied with pseudoplasmodial size. The total complement of these organelles appeared constant for any size of pseudoplasmodium under similar conditions of illumination. Increased illumination decreased the total number of the organelles. The spatial distribution of PV varies with total cell number, and the size of the region with no PV bears no relationship to the proportion of the cell mass which would form stalk cells. Similarly, the number of cells containing PV bears no fixed relationship to the number of cells which will form spores. On these grounds, the reported role of PV, that of directing or reflecting spore differentiation, appears unlikely.

Sign in / Sign up

Export Citation Format

Share Document