Cortical localization of a stage-specific surface glycoprotein and its polarization during early mouse development

1983 ◽  
Vol 41 ◽  
pp. 508-509
Author(s):  
S. Polak-Charcon ◽  
L. Johnson ◽  
P. Calarco-Gillam
Keyword(s):  
Specific Surface ◽  
Rabbit Antiserum ◽  
Antigen Expression ◽  
Surface Glycoprotein ◽  
Rabbit Serum ◽  
Normal Rabbit Serum ◽  
Mouse Development ◽  
Cell Stage ◽  
Cortical Localization ◽  
Early Mouse

Stage-specific surface antigens synthesized and expressed during mouse preimplantation development have been detected by a rabbit antiserum prepared against mouse blastocysts (A-BL2). Serological and biochemical work showed the antigens (BL) were a glycoprotein doublet of 65-70,000 daltons expressed on the cell surface at the 4-cell stage, reaching a maximum on the 8-16 cell stage, and declining on the blastocyst. Synthesis and expression of BL occur although little classical RER or Golgi material are detectable. The present study utilized immunoelectron microscopy (IEM) and the peroxidase-antiperoxidase technique to determine the intracellular distribution of BL antigens and, potentially, their site of synthesis, and to follow BL antigen expression on the plasma membrane during early mouse development.Mouse embryos from unfertilized eggs to blastocysts were obtained as described. Some embryos were treated with pronase to remove the zona pellucida. Embryos were fixed 10 min in 0.05% gluteraldehyde in phosphate buffer (PB). Some embryos were permeabilized with saponin buffer for 30 min; some were not. All were incubated with either A-BL2 IgG or normal rabbit serum IgG (NRS), washed and incubated with HRP-conjugated goat anti-rabbit IgG.

scholarly journals THE EFFECT OF SALICYLATES ON THE PRECIPITATION OF ANTIGEN WITH ANTIBODY

1943 ◽  
Vol 77 (2) ◽  
pp. 173-183 ◽  
Author(s):  
Alvin F. Coburn ◽  
Eleanor M. Kapp
Keyword(s):  
Immune System ◽  
Sodium Salicylate ◽  
Sodium Tungstate ◽  
Horse Serum ◽  
Rabbit Antiserum ◽  
Rabbit Serum ◽  
Normal Rabbit Serum ◽  
Egg Albumin ◽  
Immune Precipitation ◽  
Lyotropic Series

1. Sodium salicylate modifies the precipitation of normal rabbit serum protein by sodium tungstate, and partially inhibits the precipitation of horse serum euglobulin by rabbit antiserum. Sodium salicylate added to a system containing crystalline egg albumin and its antibody partly prevents the formation of precipitate, the degree of inhibition being related to the concentration of salicylate. 2. Precipitation in the equivalence zone is more readily prevented by salicylate than precipitation in the region of antibody excess, the immune system becoming progressively less sensitive to the action of salicylate as the excess of antibody becomes larger. 3. Formed precipitates were partly dissolved following resuspension in the presence of salicylate. 4. The salicylate effect on immune precipitation is reversible, and appears to be due to inactivation of antibody. 5. Salicylate was more effective in preventing specific precipitation than other anions of a lyotropic series tested.

Cell division and cell allocation in early mouse development

Development ◽  
1978 ◽  
Vol 48 (1) ◽  
pp. 37-51
Author(s):  
S. J. Kelly ◽  
J. G. Mulnard ◽  
C. F. Graham
Keyword(s):  
Cell Division ◽  
Tritiated Thymidine ◽  
Mouse Embryos ◽  
Mouse Development ◽  
Stages Of Development ◽  
Cell Stage ◽  
Cell Cycles ◽  
Short Cell ◽  
Early Mouse

Cell division was observed in intact and dissociated mouse embryos between the 2-cell stage and the blastocyst in embryos developing in culture. Division to the 4-cell stage was usually asynchronous. The first cell to divide to the 4-cell stage produced descendants which tended to divide ahead of those cells produced by its slow partner at all subsequent stages of development up to the blastocyte stage. The descendants of the first cell to divide to the 4-cell stage did not subsequently have short cell cycles. The first cell or last cell to divide from the 4-cell stage was labelled with tritiated thymidine. The embryo was reassembled, and it was found that the first pair of cells to reach the 8-cell stage contributed disproportionately more descendants to the ICM when compared with the last cell to divide to the 8-cell stage.

Localization of trophoblast-defined surface antigens during early mouse embryogenesis

Development ◽  
1978 ◽  
Vol 43 (1) ◽  
pp. 147-156
Author(s):  
R. F. Searle ◽  
E. J. Jenkinson
Keyword(s):  
Rabbit Antiserum ◽  
Cell Populations ◽  
Electron Microscope Level ◽  
Cell Stage ◽  
Component Cell ◽  
Low Levels ◽  
Early Mouse ◽  
Post Implantation ◽  
Embryonic Ectoderm ◽  
Ectoplacental Cone

The binding pattern of a rabbit antiserum raised against mouse ectoplacental-cone trophoblast on component cell populations in the pre-implantation and early post-implantation mouse embryo has been examined at the electron-microscope level using an immunoperoxidase-labelling technique. Binding was not detectable on the 1-cell stage, appeared at low levels at the 8-cell stage ana was heavy on the trophectoderm and its trophoblast giant cell and extra-embryonic ectoderm descendants in the post-implantation embryo. In contrast, immunosurgically isolated 3½-day inner cell masses (ICM) showed only slight labelling, whilst ICM derivatives in the 7½-day embryo were unlabelled. The results indicate that the antiserum may be identifying a trophoblast-specific surface determinant(s), which appears with the differentiation of the trophectoderm and is maintained on some of the cell populations derived from this tissue at least until the early postimplantation stages.

Localisation of tight junction protein cingulin is temporally and spatially regulated during early mouse development

Development ◽  
1993 ◽  
Vol 117 (3) ◽  
pp. 1135-1144 ◽  
Author(s):  
T.P. Fleming ◽  
M. Hay ◽  
Q. Javed ◽  
S. Citi
Keyword(s):  
Tight Junction ◽  
Cell Types ◽  
Early Embryo ◽  
Mouse Development ◽  
Cell Stage ◽  
Tight Junction Formation ◽  
Junction Protein ◽  
Junction Site ◽  
Mouse Early Embryo ◽  
Early Mouse

The molecular maturation of the tight junction in the mouse early embryo has been investigated by monitoring the distribution of cingulin, a 140 × 10(3) M(r) peripheral (cytoplasmic) membrane constituent of the junction, at different stages of development and in different experimental situations. Although tight junction formation does not begin until compaction at the 8-cell stage, cingulin is detectable in oocytes and all stages of cleavage, a factor consistent with our biochemical analysis of cingulin expression (Javed et al., 1992, Development 117, 1145–1151). Using synchronised egg and embryo stages and isolated cell clusters, we have identified three sites where cingulin is localised, the cytocortex, punctate cytoplasmic foci and tight junctions themselves. Cytocortical cingulin is present at the cumulus-oocyte contact site (both cell types), in unfertilised and fertilised eggs and in cleavage stages up to 16-cell morulae, particularly at microvillous domains on the embryo outer surface (eg. apical poles at compaction). Embryo manipulation experiments indicate that cortical cingulin is labile and dependent upon cell interactions and therefore is not merely an inheritance from the egg. Cingulin cytoplasmic foci are evident only in outer cells (prospective trophectoderm) from the 32-cell stage, just prior to cavitation, and decline from approx. 8 hours after cavitation has initiated. The appearance of these foci is insensitive to cycloheximide treatment and they colocalise with apically derived endocytic vesicles visualised by FITC-dextran, indicating that the foci represent the degradation of cytocortical cingulin by endocytic turnover. Cingulin is detectable at the tight junction site between blastomeres usually from the 16-cell stage, although earlier assembly occurs in a minority (up to 20%) of specimens. Cingulin assembly at the tight junction is sensitive to cycloheximide and is identifiable approx. 10 hours after cell adhesion is initiated and ZO-1 protein assembles. Collectively, our results indicate that (i) cingulin from nonjunctional sites does not contribute to tight junction assembly and (ii) the molecular maturation of the junction appears to occur progressively over at least two cell cycles.

scholarly journals THE EFFECT OF ANTISERUM, ALONE AND WITH HYDROCORTISONE, ON FOETAL MOUSE BONES IN CULTURE

1965 ◽  
Vol 121 (4) ◽  
pp. 551-560 ◽  
Author(s):  
Honor B. Fell ◽  
L. Weiss
Keyword(s):  
Lysosomal Enzymes ◽  
Protective Action ◽  
Rabbit Antiserum ◽  
Normal Serum ◽  
Rabbit Serum ◽  
Normal Rabbit Serum ◽  
Limb Bones ◽  
Mouse Tissues ◽  
Antigen Antibody ◽  
Indirect Action

1. The effects of normal rabbit serum and of rabbit antiserum to whole foetal mouse tissues, on the isolated limb bones of late foetal mice were studied in organ culture, and the influence of hydrocortisone on these effects was investigated. 2. Unheated normal serum caused slight loss of metachromatic material from the cartilage matrix, and some resorption of both cartilage and bone. 3. In unheated antiserum to foetal mouse tissues, the terminal cartilage was smaller and less metachromatic than in paired controls in normal serum, while osteoclasis was so intense that in many explants the bone had almost disappeared. The amount of necrosis varied with different batches of antiserum. 4. The changes produced by normal serum and antiserum could be largely prevented by heating the sera to 57°C for 45 minutes. 5. The effects could also be inhibited by the addition of hydrocortisone to the unheated sera; as little as 0.1 µg hydrocortisone per ml of medium had a well marked protective action. 6. It is suggested that (a) unheated antiserum causes a release of lysosomal enzymes with consequent breakdown of intercellular material, (b) this release is due to an indirect action on the lysosome via an increased permeability of the cell membrane, (c) hydrocortisone does not affect the antigen-antibody reaction, but inhibits the autolytic changes that normally follow this reaction, possibly by stabilising both the lysosomal and cell membranes.

scholarly journals G9a regulates temporal preimplantation developmental program and lineage segregation in blastocyst

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Jan J Zylicz ◽  
Maud Borensztein ◽  
Frederick CK Wong ◽  
Yun Huang ◽  
Caroline Lee ◽  
...  
Keyword(s):  
Cell Fate ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Vital Role ◽  
Mouse Development ◽  
Cell Stage ◽  
Developmental Progression ◽  
Early Mouse ◽  
The Impact

Early mouse development is regulated and accompanied by dynamic changes in chromatin modifications, including G9a-mediated histone H3 lysine 9 dimethylation (H3K9me2). Previously, we provided insights into its role in post-implantation development (Zylicz et al., 2015). Here we explore the impact of depleting the maternally inherited G9a in oocytes on development shortly after fertilisation. We show that G9a accumulates typically at 4 to 8 cell stage to promote timely repression of a subset of 4 cell stage-specific genes. Loss of maternal inheritance of G9a disrupts the gene regulatory network resulting in developmental delay and destabilisation of inner cell mass lineages by the late blastocyst stage. Our results indicate a vital role of this maternally inherited epigenetic regulator in creating conducive conditions for developmental progression and on cell fate choices.

scholarly journals Maternal LSD1/KDM1A is an essential regulator of chromatin and transcription landscapes during zygotic genome activation

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Katia Ancelin ◽  
Laurène Syx ◽  
Maud Borensztein ◽  
Noémie Ranisavljevic ◽  
Ivaylo Vassilev ◽  
...  
Keyword(s):  
Transcriptional Level ◽  
Mouse Development ◽  
Cell Stage ◽  
Genome Expression ◽  
Maternal To Zygotic Transition ◽  
Histone H3k4 ◽  
Histone Modifying Enzymes ◽  
Early Mouse ◽  
Methylation Patterns ◽  
Genome Activation

Upon fertilization, the highly specialised sperm and oocyte genomes are remodelled to confer totipotency. The mechanisms of the dramatic reprogramming events that occur have remained unknown, and presumed roles of histone modifying enzymes are just starting to be elucidated. Here, we explore the function of the oocyte-inherited pool of a histone H3K4 and K9 demethylase, LSD1/KDM1A during early mouse development. KDM1A deficiency results in developmental arrest by the two-cell stage, accompanied by dramatic and stepwise alterations in H3K9 and H3K4 methylation patterns. At the transcriptional level, the switch of the maternal-to-zygotic transition fails to be induced properly and LINE-1 retrotransposons are not properly silenced. We propose that KDM1A plays critical roles in establishing the correct epigenetic landscape of the zygote upon fertilization, in preserving genome integrity and in initiating new patterns of genome expression that drive early mouse development.

scholarly journals A framework for TRIM21-mediated protein depletion in early mouse embryos: recapitulation of Tead4 null phenotype over three days

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Steffen Israel ◽  
Ellen Casser ◽  
Hannes C.A. Drexler ◽  
Georg Fuellen ◽  
Michele Boiani
Keyword(s):  
Effective Means ◽  
Absolute Quantification ◽  
Mammalian Development ◽  
Mouse Development ◽  
Cell Stage ◽  
Knock Out ◽  
Protein Depletion ◽  
Dna And Rna ◽  
Gene And Protein Expression ◽  
Early Mouse

Abstract Background While DNA and RNA methods are routine to disrupt the expression of specific genes, complete understanding of developmental processes requires also protein methods, because: oocytes and early embryos accumulate proteins and these are not directly affected by DNA and RNA methods. When proteins in the oocyte encounter a specific antibody and the TRIpartite Motiv-containing 21 (TRIM21) ubiquitin-protein ligase, they can be committed to degradation in the proteasome, producing a transient functional knock-out that reveals the role of the protein. However, there are doubts about whether this targeted proteolysis could be successfully used to study mammalian development, because duration of the transient effect is unknown, and also because amounts of reagents delivered must be adequate in relation to the amount of target protein, which is unknown, too. Results We show that the mouse egg contains up to 1E-02 picomoles/protein, as estimated by mass spectrometry using the intensity-based absolute quantification (iBAQ) algorithm. However, the egg can only accommodate ≈1E-04 picomoles of antibody or TRIM21 without incurring toxic effects. Within this framework, we demonstrate that TRIM21-mediated protein depletion efficiently disrupts the embryonic process of trophectoderm formation, which critically depends on the TEA domain family member 4 (Tead4) gene. TEAD4 depletion starting at the 1-cell stage lasts for 3 days prior to a return of gene and protein expression to baseline. This time period is long enough to result in a phenotype entirely consistent with that of the published null mutation and RNA interference studies: significant underexpression of trophectodermal genes Cdx2 and Gata3 and strongly impaired ability of embryos to cavitate and implant in the uterus. Omics data are available via ProteomeXchange (PXD012613) and GEO (GSE124844). Conclusions TRIM21-mediated protein depletion can be an effective means to disrupt gene function in mouse development, provided the target gene is chosen carefully and the method is tuned accurately. The knowledge gathered in this study provides the basic know-how (prerequisites, requirements, limitations) to expedite the protein depletion of other genes besides Tead4.

scholarly journals Analysis of human myelogenous leukemia cells in the fluorescence- activated cell sorter using a tumor-specific antiserum

Blood ◽  
1983 ◽  
Vol 61 (5) ◽  
pp. 858-866 ◽  
Author(s):  
AJ Malcolm ◽  
PM Logan ◽  
RC Shipman ◽  
R Kurth ◽  
JG Levy
Keyword(s):  
Bone Marrow ◽  
Acute Myelogenous Leukemia ◽  
Rabbit Antiserum ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Rabbit Serum ◽  
Normal Rabbit Serum ◽  
Cell Sorter ◽  
Chronic Granulocytic Leukemia ◽  
Acute Myelogenous

Abstract The properties of a rabbit antiserum (anti-AML) raised to a purified protein from membranes of human acute myelogenous leukemia (AML) cells is described. Bone marrow and peripheral blood leukocytes (PBL) from either normal individuals or patients with either myeloproliferative or other disorders were analyzed in a fluorescence-activated cell sorter (FACS IV) after labeling with anti-AML, normal rabbit serum (NRS), or antiserum raised to normal human membrane antigens. Of 40 cell samples from patients with acute myelogenous leukemia, 39 reacted strongly with the anti-AML antiserum. Similarly, all of 19 specimens from patients with chronic granulocytic leukemia reacted with the anti-AML. When 42 bone marrow or PBL samples from patients with a variety of lymphoproliferative disorders were examined, 2 specimens reacted with the antiserum, both from individuals with diagnoses of acute lymphocytic leukemia (ALL). None of the 14 normal bone marrow or PBL donor specimens tested reacted with the antiserum. It was also found that essentially all samples from patients in clinical remission from AML had high numbers of cells reactive with the anti-AML. When cells from such individuals were labeled and sorted on the FACS IV, it was found that cells fluorescing strongly with the anti-AML contained cells of both myeloid and lymphoid origin. The implications of these results are discussed.

scholarly journals Maternal Argonaute 2 Is Essential for Early Mouse Development at the Maternal-Zygotic Transition

2008 ◽  
Vol 19 (10) ◽  
pp. 4383-4392 ◽  
Author(s):  
Karin Lykke-Andersen ◽  
Michael J. Gilchrist ◽  
Joanna B. Grabarek ◽  
Partha Das ◽  
Eric Miska ◽  
...  
Keyword(s):  
Mouse Development ◽  
Rnai Machinery ◽  
Cell Stage ◽  
P Bodies ◽  
Argonaute 2 ◽  
Zygotic Gene ◽  
Specific Mrnas ◽  
Maternal Mrnas ◽  
Early Mouse ◽  
Cell Mouse Embryo

Activation of zygotic gene expression in the two-cell mouse embryo is associated with destruction of maternally inherited transcripts, an important process for embryogenesis about which little is understood. We asked whether the Argonaute (Ago)/RNA-induced silencing complex, providing the mRNA “slicer” activity in gene silencing, might contribute to this process. Here we show that Ago2, 3, and 4 transcripts are contributed to the embryo maternally. By systematic knockdown of maternal Ago2, 3, and 4, individually and in combination, we find that only Ago2 is required for development beyond the two-cell stage. Knockdown of Ago2 stabilizes one set of maternal mRNAs and reduces zygotic transcripts of another set of genes. Ago2 is localized in mRNA-degradation P-bodies analogous to those that function in RNAi-like mechanisms in other systems. Profiling the expression of microRNAs throughout preimplantation development identified several candidates that could potentially work with Ago2 to mediate degradation of specific mRNAs. However, their low abundance raises the possibility that other endogenous siRNAs may also participate. Together, our results demonstrate that maternal expression of Ago2 is essential for the earliest stages of mouse embryogenesis and are compatible with the notion that degradation of a proportion of maternal messages involves the RNAi-machinery.

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