scholarly journals The zebrafishspiel-ohne-grenzen(spg) gene encodes the POU domain protein Pou2 related to mammalianOct4and is essential for formation of the midbrain and hindbrain, and for pre-gastrula morphogenesis

Development ◽  
2002 ◽  
Vol 129 (4) ◽  
pp. 905-916 ◽  
Author(s):  
Shawn Burgess ◽  
Gerlinde Reim ◽  
Wenbiao Chen ◽  
Nancy Hopkins ◽  
Michael Brand
Keyword(s):  
Insertional Mutagenesis ◽  
Inner Cell Mass ◽  
Insertion Site ◽  
Cell Mass ◽  
Brain Regions ◽  
Chemical Mutagenesis ◽  
Gene Encoding ◽  
Specific Expression ◽  
Pou Domain ◽  
Similar Phenotype

In early embryonic development, the brain is divided into three main regions along the anteroposterior axis: the forebrain, midbrain and hindbrain. Through retroviral insertional mutagenesis and chemical mutagenesis experiments in zebrafish, we have isolated mutations that cause abnormal hindbrain organization and a failure of the midbrain-hindbrain boundary (MHB) to form, a region that acts as an organizer for the adjacent brain regions. The mutations fail to complement the spiel-ohne-grenzen (spg) mutation, which causes a similar phenotype, but for which the affected gene is unknown. We show through genetic mapping, cloning of the proviral insertion site and allele sequencing that spg mutations disrupt pou2, a gene encoding the Pou2 transcription factor. Based on chromosomal synteny, phylogenetic sequence comparison, and expression and functional data, we suggest that pou2 is the zebrafish ortholog of mouse Oct3/Oct4 and human POU5F1. For the mammalian genes, a function in brain development has so far not been described. In the absence of functional pou2, expression of markers for the midbrain, MHB and the hindbrain primordium (pax2.1, wnt1, krox20) are severely reduced, correlating with the neuroectoderm-specific expression phase of pou2. Injection of pou2 mRNA restores these defects in spg mutant embryos, but does not activate these markers ectopically, demonstrating a permissive role for pou2. Injections of pou2-morpholinos phenocopy the spg phenotype at low concentration, further proving that spg encodes pou2. Two observations suggest that pou2 has an additional earlier function: higher pou2-morpholino concentrations specifically cause a pre-gastrula arrest of cell division and morphogenesis, and expression of pou2 mRNA itself is reduced in spg-homozygous embryos at this stage. These experiments suggest two roles for pou2. Initially, Pou2 functions during early proliferation and morphogenesis of the blastomeres, similar to Oct3/4 in mammals during formation of the inner cell mass. During zebrafish brain formation, Pou2 then functions a second time to activate gene expression in the midbrain and hindbrain primordium, which is reflected at later stages in the specific lack in spg embryos of the MHB and associated defects in the mid- and hindbrain.

Specific expression of a retinoic acid-regulated, zinc-finger gene, Rex-1, in preimplantation embryos, trophoblast and spermatocytes

Development ◽  
1991 ◽  
Vol 113 (3) ◽  
pp. 815-824 ◽  
Author(s):  
M.B. Rogers ◽  
B.A. Hosler ◽  
L.J. Gudas
Keyword(s):  
Retinoic Acid ◽  
Germ Cell ◽  
Cell Fate ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Preimplantation Embryos ◽  
Specific Expression

We have previously isolated a cDNA clone for a gene whose expression is reduced by retinoic acid (RA) treatment of F9 embryonal carcinoma cells. The nucleotide sequence indicated that this gene, Rex-1, encodes a zinc-finger protein and thus may be a transcriptional regulator. The Rex-1 message level is high in two lines of embryonic stem cells (CCE and D3) and is reduced when D3 cells are induced to differentiate using four different growth conditions. As expected for a stem-cell-specific message, Rex-1 mRNA is present in the inner cell mass (ICM) of the day 4.5 mouse blastocyst. It is also present in the polar trophoblast of the blastocyst. One and two days later, Rex-1 message is found in the ectoplacental cone and extraembryonic ectoderm of the egg cylinder (trophoblast-derived tissues), but its abundance is much reduced in the embryonic ectoderm which is directly descended from the ICM. Rex-1 is expressed in the day 18 placenta (murine gestation is 18 days), a tissue which is largely derived from trophoblast. The only tested adult tissue that contains detectable amounts of Rex-1 mRNA is the testis. In situ hybridization and northern analyses of RNA from germ-cell-deficient mouse testis and stage-specific germ cell preparations suggest that Rex-1 expression is limited to spermatocytes (germ cells undergoing meiosis). These results suggest that Rex-1 is involved in trophoblast development and spermatogenesis, and is a useful marker for studies of early cell fate determination in the ICM.

054. TROPHOBLAST, PLACENTA AND EARLY EMBRYO: HOW THE MARSUPIAL DEVELOPS

2010 ◽  
Vol 22 (9) ◽  
pp. 15 ◽  
Author(s):  
M. B. Renfree ◽  
S. R. Frankenberg ◽  
C. Freyer
Keyword(s):  
Differential Expression ◽  
Inner Cell Mass ◽  
Cell Metabolism ◽  
Cell Mass ◽  
Early Embryo ◽  
Yolk Sac ◽  
Lineage Specification ◽  
Specific Expression ◽  
Embryonic Tissues ◽  
Pluripotency Genes

In marsupials, the blastocyst forms as a single cell layer of cells. The marsupial blastocyst has no inner cell mass, so the 80–100 cell tammar embryo remains in diapause as a unilaminar blastocyst. All marsupials have a unilaminar stage, but what is unusual is that in the tammar the total cessation of cell division and cell metabolism lasts for 11 months each year. Marsupials are placental mammals. The yolk sac forms the definitive placenta up to birth. Only very few marsupials, such as the bandicoot, have a chorio-allantoic placenta, which supplements the placental functions of the yolk sac. However, the understanding how the unilaminar layer of trophoblast cells of the diapausing blastocyst become specified into placental and embryonic tissues has been an ongoing puzzle. To identify genes that do become differentially expressed in tammar development, we targeted the stage of the earliest appearance of the embryonic disc, at which the remainder of the blastocyst is then defined as trophoblast, as well as early cleavage stages. Intriguingly, we found no evidence for early differential expression of the canonical pluripotency genes POU5F1, SOX2 and NANOG, or of CDX2. By contrast, we found overt differential expression of GATA3, the closely related gene GATA2, and FGF4. This expression profile suggests that in the tammar, mechanisms regulating trophoblast- and pluriblast-specific expression of POU5F1, SOX2, NANOG and CDX2 are temporally secondary to those regulating GATA2 & -3 and FGF4 expression. Together, our results may signify the evolution of alternative mechanisms of early lineage specification in marsupials, or alternatively reveal a general hierarchy of signalling mechanisms that are masked in the relatively rapid and ‘compressed’ development of mice. The results of our ongoing study have important implications for understanding not only marsupial stem cells but the early development of all therian mammals.

scholarly journals Disruption of the Mouse mTOR Gene Leads to Early Postimplantation Lethality and Prohibits Embryonic Stem Cell Development

2004 ◽  
Vol 24 (21) ◽  
pp. 9508-9516 ◽  
Author(s):  
Yann-Gaël Gangloff ◽  
Matthias Mueller ◽  
Stephen G. Dann ◽  
Petr Svoboda ◽  
Melanie Sticker ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Mammalian Target Of Rapamycin ◽  
Embryonic Stem ◽  
Gene Encoding ◽  
Protein Levels ◽  
Maternal Mrna ◽  
Regulate Cell Growth ◽  
A Site

ABSTRACT The mammalian target of rapamycin (mTOR) is a key component of a signaling pathway which integrates inputs from nutrients and growth factors to regulate cell growth. Recent studies demonstrated that mice harboring an ethylnitrosourea-induced mutation in the gene encoding mTOR die at embryonic day 12.5 (E12.5). However, others have shown that the treatment of E4.5 blastocysts with rapamycin blocks trophoblast outgrowth, suggesting that the absence of mTOR should lead to embryonic lethality at an earlier stage. To resolve this discrepancy, we set out to disrupt the mTOR gene and analyze the outcome in both heterozygous and homozygous settings. Heterozygous mTOR (mTOR +/ −) mice do not display any overt phenotype, although mouse embryonic fibroblasts derived from these mice show a 50% reduction in mTOR protein levels and phosphorylation of S6 kinase 1 T389, a site whose phosphorylation is directly mediated by mTOR. However, S6 phosphorylation, raptor levels, cell size, and cell cycle transit times are not diminished in these cells. In contrast to the situation in mTOR +/ − mice, embryonic development of homozygous mTOR − / − mice appears to be arrested at E5.5; such embryos are severely runted and display an aberrant developmental phenotype. The ability of these embryos to implant corresponds to a limited level of trophoblast outgrowth in vitro, reflecting a maternal mRNA contribution, which has been shown to persist during preimplantation development. Moreover, mTOR − / − embryos display a lesion in inner cell mass proliferation, consistent with the inability to establish embryonic stem cells from mTOR − / − embryos.

89 INHIBITION OF MAPKK AND GSK3 SIGNALLING PROMOTES DEVELOPMENT AND EPIBLAST-SPECIFIC EXPRESSION OF PLURIPOTENCY MARKERS IN BOVINE BLASTOCYSTS

2013 ◽  
Vol 25 (1) ◽  
pp. 192
Author(s):  
D. Harris ◽  
B. Huang ◽  
B. Oback
Keyword(s):  
Dimethyl Sulfoxide ◽  
Inner Cell Mass ◽  
Glycogen Synthase ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Mitogen Activated Protein Kinase ◽  
Differential Staining ◽  
Blastocyst Development ◽  
Specific Expression

During blastocyst development, the inner cell mass segregates into the epiblast and the hypoblast. These 2 tissues form morphologically and molecularly distinct cell populations that subsequently develop into the embryo proper and some extraembryonic components, respectively. In mouse, isolated epiblast cells can be directly converted into pluripotent embryonic stem cells, capable of differentiating into all cell types of an adult animal. Epiblast pluripotency is promoted by pharmacological inhibition of mitogen-activated protein kinase kinase (Mapkk). This shields epiblast cells from secreted fibroblast growth factor (Fgf), which would otherwise instruct them to exit pluripotency and differentiate into extraembryonic lineages. Indirect stimulation of the Wnt pathway by inhibiting glycogen synthase kinase 3 (GSK3) further antagonises inductive Fgf/Mapkk signalling. Thus the double inhibition (2i) of Mapkk and Gsk3 effectively promotes pluripotency (Q. L. Ying et al. 2008 Nature 453, 519–523; J. Nichols et al. 2009 Development 136, 3215–3222). We investigated the effect of 2i culture on bovine blastocysts. The IVF embryos were cultured in the presence of dimethyl sulfoxide or inhibitors of MAPKK (0.4 µM PD0325901) and GSK3 (3 µM CHIR99021) from the zygote (Day 1) stage onward. Compared to vehicle controls, 2i increased the abundance of cumulus cells in bovine IVF cultures, compromising blastocyst formation in cumulus-intact (248/823 = 30% v. 211/824 = 26%, respectively, n = 10; P < 0.05) but not cumulus-free cultures (546/1653 = 33% v. 572/1674 = 34%, respectively, n = 15; P = 0.51). In all subsequent experiments, we therefore cultured cumulus-free zygotes in 2i v. dimethyl sulfoxide until the blastocyst stage. This treatment increased the proportion of hatching (19/433 = 4% v. 7/416 = 2%, respectively, n = 10; P < 0.05) at the expense of early blastocysts (70/433 = 16% v. 93/416 = 22%, respectively, n = 11; P < 0.05). Differential staining of expanded IETS grade 1 and 2 blastocysts showed that 2i culture increased putative inner cell mass, trophectoderm, and total cell nuclei numbers by about 30% compared with controls (57 v. 43, 89 v. 69, and 146 v. 112, respectively; P < 0.01). Accelerated development and increased cell numbers were accompanied by gene expression changes in grade 1 and 2 blastocysts. Under 2i conditions, mRNA abundance of putative epiblast markers NANOG and SOX2 was >3-fold increased (P < 0.0001 and P < 0.01, respectively), and the putative hypoblast marker GATA4 was 2-fold reduced (P < 0.05). Other lineage-related markers (POU5F1, KLF4, DPPA3, and CDX2) showed no significant changes. Using microsurgical blastocyst dissection, we found that the increase in NANOG and SOX2 levels was specific to the inner cell mass-containing portion (7-fold for NANOG and 3-fold for SOX2; P < 0.00005 and P < 0.05, respectively) and not due to ectopic expression in the trophoblast-containing part, which showed similarly low expression levels for both genes. In summary, 2i treatment primed bovine blastocysts for pluripotency in the epiblast. Supported by MSI C10X1002.

82 Stage-specific expression of lineage marker genes and pluripotency marker distribution in porcine pre-implantation embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 167
Author(s):  
J. Oh ◽  
M. Lee ◽  
D. Lee ◽  
K. Choi ◽  
S. Kim ◽  
...  
Keyword(s):  
Internal Control ◽  
Inner Cell Mass ◽  
Electric Pulse ◽  
Cell Mass ◽  
Expression Level ◽  
Marker Genes ◽  
Lineage Specification ◽  
Cell Stage ◽  
Specific Expression ◽  
Pluripotency Marker

Lineage specification in pre-implantation embryos has been revealed, and it was expedited recently by single cell studies. However, data on expression marker genes and proteins in porcine embryos were still missing. We aimed to investigate the expression and distribution of marker genes and proteins, respectively, in IVF and parthenogenetically activated (PA) embryos. For this, cumulus-free oocytes were co-incubated with sperm in modified Tris-buffered medium (mTBM) for 5h and PA was performed using an electric pulse in activation medium. Following this, the embryos were incubated in porcine zygote medium 3 (PZM3). We first tested gene expression level of lineage candidates (internal control; β-actin). In IVF embryos (30, 25, 20, 15, 10, and 5 embryos pooled on Day 2, 3, 4, 5, 6, and 7; replicated 3 times), trophectoderm (TE)-specific genes (Dab2, Gata3) showed peaks on Day 4-5. Within the 2 genes, Dab2 had an earlier peak than Gata3. Inner cell mass (ICM) marker candidates (Nanog, Sox2, and Hnf4a) had diverse patterns. The Nanog and Sox2 genes had peak expression on Day 3. The Nanog expression dropped gradually, but Sox2 dropped suddenly on Day 4. Otherwise, Hnf4a expressed little in Day 3 and expression was sustained from Day 4 to 7. Primitive endoderm markers showed the highest expression on Day 4. We also checked expression level of ICM markers (Sox2, Oct4, and Nanog) in PA embryos (20, 20, 20, 10, and 5 embryos were pooled in 2, 4, 6-8 cells, morula, early, and late blastocyst stages; replicated 3 times). Expression of markers was similar (the highest in the 6-8-cell stage; at least 7.3-, 4.5-, and 3.7-fold compared with the other stages in Sox2, Oct4, and Nanog). We used analysis of variance and Tukey's test for statistical analysis. Following this, we conducted immunocytochemistry with both IVF and PA embryos (20 in each condition). Primary antibodies were treated overnight at 4°C and appropriate secondary antibodies were treated 1h at room temperature. In the case of IVF, well-known ICM markers (SOX2, OCT4, NANOG, and SOX17) showed restricted distribution in nuclei of ICM cells. However, DAB2 was distributed in the cytoplasm of TE cells. In PA embryos, SOX2 and NANOG distributions were similar to IVF. The OCT4 in ICM cells from morula to early blastocyst was restricted, but not in Day 7 embryos. In conclusion, marker genes showed diverse expression pattern in IVF, but all ICM-specific genes had a similar pattern in PA. Also, ICM marker proteins were restricted in nuclei of ICM cells only except Day 7 PA. Our results provide eye-opening information on marker contribution to lineage specification of porcine embryos. This work was supported by the National Research Foundation in Korea (NRF) funded by the Ministry of Education (NRF-2017R1D1A1B03032256, NRF-2019R1C1C1004514).

scholarly journals Targeted Deletion Reveals an Essential Function for the Telomere Length Regulator Trf1

2003 ◽  
Vol 23 (18) ◽  
pp. 6533-6541 ◽  
Author(s):  
Jan Karlseder ◽  
Leili Kachatrian ◽  
Hiroyuki Takai ◽  
Kim Mercer ◽  
Sunil Hingorani ◽  
...  
Keyword(s):  
Telomere Length ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Growth Defect ◽  
Telomeric Dna ◽  
Gene Encoding ◽  
Targeted Deletion ◽  
Exon 1 ◽  
Essential Function ◽  
Tankyrase 1

ABSTRACT The human telomeric DNA binding factor TRF1 (hTRF1) and its interacting proteins TIN2, tankyrase 1 and 2, and PINX1 have been implicated in the regulation of telomerase-dependent telomere length maintenance. Here we show that targeted deletion of exon 1 of the mouse gene encoding Trf1 causes early (day 5 to 6 postcoitus) embryonic lethality. The absence of telomerase did not alter the Terf1ex1Δ/ex1Δ lethality, indicating that the phenotype was not due to inappropriate telomere elongation by telomerase. Terf1ex1Δ/ex1Δ blastocysts had a severe growth defect of the inner cell mass that was accompanied by apoptosis. However, no evidence was found for telomere uncapping causing this cell death; chromosome spreads of Terf1ex1Δ/ex1Δ blastocysts did not reveal chromosome end-to-end fusions, and p53 deficiency only briefly delayed Terf1ex1Δ/ex1Δ lethality. These data suggest that murine Trf1 has an essential function that is independent of telomere length regulation.

scholarly journals Inner Cell Mass-Specific Expression of a Cell Adhesion Molecule (PECAM-1/CD31) in the Mouse Blastocyst

2001 ◽  
Vol 234 (2) ◽  
pp. 317-329 ◽  
Author(s):  
Paul Robson ◽  
Paula Stein ◽  
Bin Zhou ◽  
Richard M. Schultz ◽  
H.Scott Baldwin
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Blastocyst ◽  
Specific Expression ◽  
A Cell

Use of F9 teratocarcinoma STEM cells to study cell-matrix interactions in the early mouse embryo

1992 ◽  
Vol 50 (1) ◽  
pp. 602-603
Author(s):  
Marc Lenburg ◽  
Rulang Jiang ◽  
Lengya Cheng ◽  
Laura Grabel
Keyword(s):  
Mouse Embryo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Embryoid Bodies ◽  
F9 Cells ◽  
Cell Matrix ◽  
Matrix Interactions ◽  
Cell Matrix Interactions ◽  
F9 Teratocarcinoma

We are interested in defining the cell-cell and cell-matrix interactions that help direct the differentiation of extraembryonic endoderm in the peri-implantation mouse embryo. At the blastocyst stage the mouse embryo consists of an outer layer of trophectoderm surrounding the fluid-filled blastocoel cavity and an eccentrically located inner cell mass. On the free surface of the inner cell mass, facing the blastocoel cavity, a layer of primitive endoderm forms. Primitive endoderm then generates two distinct cell types; parietal endoderm (PE) which migrates along the inner surface of the trophectoderm and secretes large amounts of basement membrane components as well as tissue-type plasminogen activator (tPA), and visceral endoderm (VE), a columnar epithelial layer characterized by tight junctions, microvilli, and the synthesis and secretion of α-fetoprotein. As these events occur after implantation, we have turned to the F9 teratocarcinoma system as an in vitro model for examining the differentiation of these cell types. When F9 cells are treated in monolayer with retinoic acid plus cyclic-AMP, they differentiate into PE. In contrast, when F9 cells are treated in suspension with retinoic acid, they form embryoid bodies (EBs) which consist of an outer layer of VE and an inner core of undifferentiated stem cells. In addition, we have established that when VE containing embryoid bodies are plated on a fibronectin coated substrate, PE migrates onto the matrix and this interaction is inhibited by RGDS as well as antibodies directed against the β1 integrin subunit. This transition is accompanied by a significant increase in the level of tPA in the PE cells. Thus, the outgrowth system provides a spatially appropriate model for studying the differentiation and migration of PE from a VE precursor.

Decreased inner cell mass proportion in blastocysts from diabetic rats

Diabetes ◽  
1990 ◽  
Vol 39 (4) ◽  
pp. 471-476 ◽  
Author(s):  
S. Pampfer ◽  
R. de Hertogh ◽  
I. Vanderheyden ◽  
B. Michiels ◽  
M. Vercheval
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Diabetic Rats ◽  
Mass Proportion
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