scholarly journals Staging of ovine embryos and expression of the T-box genes Brachyury and Eomesodermin around gastrulation

Reproduction ◽  
2004 ◽  
Vol 127 (4) ◽  
pp. 491-501 ◽  
Author(s):  
Michel Guillomot ◽  
Annick Turbe ◽  
Isabelle Hue ◽  
Jean-Paul Renard
Keyword(s):  
Embryonic Development ◽  
Expression Patterns ◽  
Posterior Pole ◽  
Primitive Endoderm ◽  
T Box ◽  
Whole Mount ◽  
Early Failures ◽  
Early Expression

The high rates of embryonic mortalities which follow in vitro production of ruminant embryos have emphasized the need for increased knowledge of early development. It is likely that early failures in embryonic development and placenta formation involve abnormal differentiation of mesoderm. The aim of this study was to investigate the pattern of expression of two T-box genes known to control the gastrulation process, Brachyury and Eomesodermin, by whole-mount in situ hybridization. To allow a more precise comparison of both expression patterns between embryos, we describe a new staging of pre-implanted ovine embryos by gross morphology and histology from pre-gastrulation stages to the beginning of neurulation. In pre-streak embryos primitive mesoderm cells delaminated in between the primitive endoderm and the epiblast. At that stage, no expression of Brachyury or Eomesodermin could be detected in the embryos. Early expression of both T-genes was observed by the early-streak stages in epiblast cells located close to the presumptive posterior pole of the embryos. Later on, during gastrulation both genes followed a pattern of expression similar to the ones described in other mammals. These observations suggest that other genes, which remain to be identified, are responsible for extra-embryonic mesoderm differentiation in ruminant embryos.

Expression of the SMP antigen by oligodendrocytes in the developing avian central nervous system

Development ◽  
1989 ◽  
Vol 107 (4) ◽  
pp. 825-833
Author(s):  
P. Cameron-Curry ◽  
C. Dulac ◽  
N.M. Le Douarin
Keyword(s):  
Monoclonal Antibody ◽  
Schwann Cell ◽  
Basic Protein ◽  
Culture Conditions ◽  
Cell Marker ◽  
Cellular Expression ◽  
Early Expression ◽  
Different Parts

Expression of the avian antigen SMP (Schwann cell Myelin Protein, Mr 75-80000), first characterized in the PNS with a monoclonal antibody as an early and strictly specific Schwann cell marker, was further studied in the CNS. Comparing SMP immunoreactive areas in the different parts of the CNS with those expressing the Myelin Basic Protein (MBP), we showed a strict colocalisation of both phenotypes. In vitro, MBP+ oligodendrocytes express the surface antigen SMP as well. SMP cellular expression was followed in situ and in culture using nervous tissues from embryos at different stages. We were thus able to detect an early expression of this marker by oligodendroblasts before the first appearance of MBP immunoreactivity. We have also identified a subpopulation of SMP+/MBP- and SMP+/GC- cells, which persists under our culture conditions as precursors remaining in an immature state.

mRNA expression patterns of the IGF system during mouse limb bud development, determined by whole mount in situ hybridization

1998 ◽  
Vol 138 (1-2) ◽  
pp. 151-161 ◽  
Author(s):  
Marjolein van Kleffens ◽  
Cora Groffen ◽  
Roberto R. Rosato ◽  
Stefan M. van den Eijnde ◽  
Johan W. van Neck ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Mrna Expression ◽  
Expression Patterns ◽  
Limb Bud ◽  
Bud Development ◽  
Igf System ◽  
Whole Mount ◽  
Mouse Limb

af9 Regulates gata2 Expression During Early Hemangioblast Specification and Vascular Pattern Formation In Zebrafish.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2600-2600
Author(s):  
Giuseppe Germano ◽  
Ilaria Guariento ◽  
Natascia Tiso ◽  
Blaine W. Robinson ◽  
Enrico Moro ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Pattern Formation ◽  
Embryonic Development ◽  
Blood Cells ◽  
Erythroid Cell ◽  
Fusion Partner ◽  
Vascular Pattern ◽  
Cell Stage ◽  
Whole Mount

Abstract Abstract 2600 Background AF9 is a transcription factor that plays an essential role in hematopoiesis and embryonic development. The alteration of AF9 is principally associated in acute myeloid leukemia as fusion partner of human MLL (mixed-lineage leukemia) gene rearrangements. Zebrafish is an excellent model organism to study embryonic development and hematopoiesis. We have previously shown that zebrafish af9 is expressed within the intermediate cell mass (ICM), a site of primitive hematopoiesis in zebrafish. Here we study the loss of af9 in zebrafish development to further understand how af9 modulates early hematopoietic and embryonic development. Methods and results Two morpholino antisense oligos (MOs), designed to block translation and inhibit pre-mRNA splicing of af9, were co-injected in embryos at 1–2 cell stage. To control for off-target effects, two morpholino mismatch oligos were designed and co-injected. Efficacy of MOs was demonstrated by Western blot analysis and RT-PCR in controls and MO-injected embryos (morphants). In vivo monitoring of both morphants and control embryos was carried out by microscopy. Effects of af9 depletion on vasculature and erythropoiesis were evaluated in Tg(fli1:eGFP) and Tg(gata1:DsRed) transgenic lines, respectively. Whole-mount in situ hybridization of known hematopoietic markers was used to decipher the developmental time-points in which af9 regulates blood development. Following injection of two MOs at 1–2 cell stage, we compared the morphological features of the morphants with control embryos at about 24 hours post-fertilization (hpf). The af9 morphants showed small head and eyes, disruption of tail development and pronounced swelling in the posterior ICM. Circulating blood cells were reduced from 26 hpf to later stages of development. At 48 hpf the heart was enlarged, showed a paucity of blood-cells and pericardial edema. Decreased number of blood cells in morphant embryos was further confirmed by o-dianisidine staining at 48 hpf and 72 hpf and in living af9-knockdown gata1:DsRed transgenic animals, suggesting that the differentiation of erythroblasts remains insufficient or impaired. Concordant with this observation, we examined the expression of specific markers for early hematopoiesis (scl, lmo2 and gata2) and primitive erythropoiesis (gata1, hbbe, and band3) using whole-mount in situ hybridization (WISH). At the 5-somite stage, the early hematopoietic precursor marker gata2 was markedly increased while scl and lmo2 remained unaffected in af9 morphants. Interestingly, by 24 hpf gata2 was found to be specifically over-expressed in ICM while no change was observed for scl and lmo2 markers. Besides, the erythroid progenitors and mature erythrocyte markers gata1, band3 and hbbe displayed nearly normal expression. To further confirm the role af9 in early hematopoiesis, we examined its expression in moonshine, a mutant zebrafish with defects in erythroid maturation due to deficiency of tif1γ, a key regulator of hematopoietic gene expression. WISH analysis in moonshine showed loss of af9 expression in the ICM at 24 hpf, suggesting that af9 functions genetically downstream of tif1γ in normal erythroid cell development. To determine the effect of af9 on endothelial and vascular development, we performed knockdown of af9 in fli1:eGFP transgenic line. By 24 hpf, these morphants showed significant increase of fluorescence intensity in the posterior ICM and a clear perturbation in the inter-segmental vessels (ISV) of the trunk at 30 hpf, indicating that af9 is required for early steps in hemangioblast specification and vascular pattern formation in zebrafish. Conclusion af9 regulates gata2 expression during early hemangioblast specification and vascular pattern formation in zebrafish. af9 may also be involved in caudal segment morphogenesis. Taken together, these data provide the initial framework of a pathway that can be used to further integrate the molecular events regulating hemangioblast differentiation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals SOX2 Plays a Critical Role in the Pituitary, Forebrain, and Eye during Human Embryonic Development

2008 ◽  
Vol 93 (5) ◽  
pp. 1865-1873 ◽  
Author(s):  
Daniel Kelberman ◽  
Sandra C. P. de Castro ◽  
Shuwen Huang ◽  
John A. Crolla ◽  
Rodger Palmer ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Anterior Pituitary ◽  
De Novo ◽  
Hypogonadotropic Hypogonadism ◽  
Expression Patterns ◽  
Critical Role ◽  
Loss Of Function ◽  
De Novo Mutations ◽  
Direct Role

Abstract Context: Heterozygous, de novo mutations in the transcription factor SOX2 are associated with bilateral anophthalmia or severe microphthalmia and hypopituitarism. Variable additional abnormalities include defects of the corpus callosum and hippocampus. Objective: We have ascertained a further three patients with severe eye defects and pituitary abnormalities who were screened for mutations in SOX2. To provide further evidence of a direct role for SOX2 in hypothalamo-pituitary development, we have studied the expression of the gene in human embryonic tissues. Results: All three patients harbored heterozygous SOX2 mutations: a deletion encompassing the entire gene, an intragenic deletion (c.70_89del), and a novel nonsense mutation (p.Q61X) within the DNA binding domain that results in impaired transactivation. We also show that human SOX2 can inhibit β-catenin-driven reporter gene expression in vitro, whereas mutant SOX2 proteins are unable to repress efficiently this activity. Furthermore, we show that SOX2 is expressed throughout the human brain, including the developing hypothalamus, as well as Rathke’s pouch, the developing anterior pituitary, and the eye. Conclusions: Patients with SOX2 mutations often manifest the unusual phenotype of hypogonadotropic hypogonadism, with sparing of other pituitary hormones despite anterior pituitary hypoplasia. SOX2 expression patterns in human embryonic development support a direct involvement of the protein during development of tissues affected in these individuals. Given the critical role of Wnt-signaling in the development of most of these tissues, our data suggest that a failure to repress the Wnt-β-catenin pathway could be one of the underlying pathogenic mechanisms associated with loss-of-function mutations in SOX2.

144 Oct-4 EXPRESSION PATTERN IN THE EQUINE EMBRYO

2007 ◽  
Vol 19 (1) ◽  
pp. 189
Author(s):  
Y. H. Choi ◽  
H. D. Harding ◽  
A. D. Obermiller ◽  
K. Hinrichs
Keyword(s):  
Embryonic Development ◽  
Expression Pattern ◽  
Embryo Development ◽  
Inner Cell Mass ◽  
Ex Vivo ◽  
Expression Patterns ◽  
Cell Mass ◽  
Population Variation ◽  
Early Embryonic Development

Oct-4 is a key transcription factor in the control of early embryonic development and maintenance of a pluripotent cell population. Variation in Oct-4 expression patterns during embryo development have been reported among species, and have been related to the time of placental development in those species. This study was conducted to investigate Oct-4 expression pattern during early embryonic development in the horse, a species with relatively delayed placentation. In vitro-produced embryos were obtained from in vitro-matured oocytes via fertilization by intracytoplasmic sperm injection. Ex vivo blastocysts were recovered from mares that had been artificially inseminated. Oct-4 status was determined by immunocytochemistry; photomicrographs were taken at 4 standardized settings to aid in qualitative comparison of the amount of fluorescence. A total of 106 oocytes and embryos were evaluated. Immature oocytes showed Oct-4 expression in the nucleus and cytoplasm, as did early-cleaved embryos (2 to 5 cells, 1 to 2 days). Oct-4 expression in embryos at 3 to 4 days (6 to 12 cells) decreased and was restricted to the cytoplasm. From 5 to 6 days (15 cells to morulae), Oct-4 intensity increased and was exclusively found in the nuclei. In vitro-produced blastocysts (7 to 8 days) expressed Oct-4 equivalently in the trophectoderm and inner cell mass nuclei; culture for 2 to 3 more days (10 to 11 days) did not alter Oct-4 expression. However, when in vitro-produced blastocysts were transferred to the uteri of mares and recovered after 2 to 3 days (IVP-ET), the embryos showed strong expression of Oct-4 within the inner cell mass and limited expression in the trophectoderm, and a similar pattern was seen for ex vivo-recovered embryos. In bigger embryos (such as a 1779-�m ex vivo embryo and a 1121-�m IVP-ET embryo), the trophectoderm lost staining completely. These results suggest that Oct-4 expression is present in both nucleus and cytoplasm in equine oocytes and early-cleaved embryos as a result of maternal mRNA accumulation. Oct-4 protein decreases over the first few days of embryonic development as these stores are used. The shift to greater expression, in the nucleus only, during further embryo development suggests embryonic genome activation. Oct-4 expression in the trophectoderm of in vitro-produced blastocysts was different from that in blastocysts that had been exposed to the uterus (both ex vivo and IVP-ET); this indicates that differentiation of the trophectoderm is dependent upon factors present in the uterine environment. The Oct-4 expression in the trophectoderm of in vitro-produced equine blastocysts thus appears to be an artifact due to in vitro culture; this finding may be applicable to the reported patterns of Oct-4 expression in embryos of other species. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University.

scholarly journals Cytochrome P450 Expression and Chemical Metabolic Activity before Full Liver Development in Zebrafish

2020 ◽  
Vol 13 (12) ◽  
pp. 456
Author(s):  
Tasuku Nawaji ◽  
Natsumi Yamashita ◽  
Haruka Umeda ◽  
Shuangyi Zhang ◽  
Naohiro Mizoguchi ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
In Situ Hybridization ◽  
Metabolic Activity ◽  
Expression Patterns ◽  
Xenobiotic Metabolism ◽  
Liver Development ◽  
P450 Expression ◽  
Whole Mount ◽  
Metabolic Activities

Zebrafish are used widely in biomedical, toxicological, and developmental research, but information on their xenobiotic metabolism is limited. Here, we characterized the expression of 14 xenobiotic cytochrome P450 (CYP) subtypes in whole embryos and larvae of zebrafish (4 to 144 h post-fertilization (hpf)) and the metabolic activities of several representative human CYP substrates. The 14 CYPs showed various changes in expression patterns during development. Many CYP transcripts abruptly increased at about 96 hpf, when the hepatic outgrowth progresses; however, the expression of some cyp1s (1b1, 1c1, 1c2, 1d1) and cyp2r1 peaked at 48 or 72 hpf, before full liver development. Whole-mount in situ hybridization revealed cyp2y3, 2r1, and 3a65 transcripts in larvae at 55 hpf after exposure to rifampicin, phenobarbital, or 2,3,7,8-tetrachlorodibenzo-p-dioxin from 30 hpf onward. Marked conversions of diclofenac to 4′-hydroxydiclofenac and 5-hydroxydiclofenac, and of caffeine to 1,7-dimethylxanthine, were detected as early as 24 or 50 hpf. The rate of metabolism to 4’-hydroxydiclofenac was more marked at 48 and 72 hpf than at 120 hpf, after the liver had become almost fully developed. These findings reveal the expression of various CYPs involved in chemical metabolism in developing zebrafish, even before full liver development.

Knockdown of regulator of G-protein signalling 2 (Rgs2) leads to abnormal early mouse embryo development in vitro

2015 ◽  
Vol 27 (3) ◽  
pp. 557 ◽  
Author(s):  
Yan Zhu ◽  
Ya-Hong Jiang ◽  
Ya-Ping He ◽  
Xuan Zhang ◽  
Zhao-Gui Sun ◽  
...  
Keyword(s):  
Embryonic Development ◽  
G Protein ◽  
Embryo Development ◽  
Expression Patterns ◽  
Critical Role ◽  
Α Subunit ◽  
G Protein Signalling ◽  
Early Embryos ◽  
Development In Vitro

Regulator of G-protein signalling 2 (Rgs2) is involved in G-protein-mediated signalling by negatively regulating the activity of the G-protein α-subunit. In the present study, the expression patterns of Rgs2 in mouse ovarian tissues and early embryos were determined by semiquantitative reverse transcription–polymerase chain reaction, immunohistochemistry and immunofluorescent analyses. Rgs2 expression was observed in the ovarian tissues of adult female mice, with an almost equal expression levels during different stages of the oestrous cycle. Rgs2 was abundant in the cytoplasm, membrane, nuclei and spindles of intact polar bodies in mouse early embryos at different developmental stages from the zygote to blastocyst. The effect of Rgs2 knockdown on early embryonic development in vitro was examined by microinjecting Rgs2-specific short interfering (si) RNAs into mouse zygotes. Knockdown of endogenous Rgs2 expression led to abnormal embryonic development in vitro, with a considerable number of early embryos arrested at the 2- or 4-cell stage. Moreover, mRNA expression of three zygotic gene activation-related genes (i.e. Zscan4, Tcstv1 and MuERV-L) was decreased significantly in 2-cell arrested embryos. These results suggest that Rgs2 plays a critical role in early embryo development.

scholarly journals How to distinguish between different cell lineages sharing common markers using combinations of double in-situ-hybridization and immunostaining in avian embryos: CXCR4-positive mesodermal and neural crest-derived cells

2020 ◽  
Vol 155 (1) ◽  
pp. 145-155 ◽  
Author(s):  
Imadeldin Yahya ◽  
Marion Böing ◽  
Beate Brand-Saberi ◽  
Gabriela Morosan-Puopolo
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Embryonic Development ◽  
Neural Crest ◽  
Expression Patterns ◽  
Simultaneous Detection ◽  
Neural Crest Cells ◽  
Cell Lineages ◽  
Wide Range

AbstractCell migration plays a crucial role in early embryonic development. The chemokine receptor CXCR4 has been reported to guide migration of neural crest cells (NCCs) to form the dorsal root ganglia (DRG) and sympathetic ganglia (SG). CXCR4 also plays an important part during the formation of limb and cloacal muscles. NCCs migration and muscle formation during embryonic development are usually considered separately, although both cell lineages migrate in close neighbourhood and have markers in common. In this study, we present a new method for the simultaneous detection of CXCR4, mesodermal markers and NCCs markers during chicken embryo developmental stages HH18–HH25 by combining double whole-mount in situ hybridization (ISH) and immunostaining on floating vibratome sections. The simultaneous detection of CXCR4 and markers for the mesodermal and neural crest cells in multiple labelling allowed us to compare complex gene expression patterns and it could be easily used for a wide range of gene expression pattern analyses of other chicken embryonic tissues. All steps of the procedure, including the preparation of probes and embryos, prehybridization, hybridization, visualization of the double labelled transcripts and immunostaining, are described in detail.

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