Gene Expression In Mouse Embryos Research Articles

Effect of paternal aging and vitrification on mitochondrial DNA copy number and telomere length of mouse blastocysts.

In this study, we examined the effects of paternal aging on the mitochondrial DNA copy number (mt-cn), telomere length (TL), and gene expression in mouse embryos. The effects of vitrification on the mt-cn and TL of the embryos derived from young and aged male parents (YF and AF, respectively) were examined. C57BL/6N male mice were used for embryo production at 13-23 and 50-55 weeks of age. Two-cell stage embryos were collected from the oviducts of superovulated female mice (8-15 weeks old) and cultured for 24 h until the 8-cell stage, followed by embryo vitrification. Fresh and vitrified-warmed embryos were incubated for 2 days until the blastocyst stage, and mt-cn and TL were investigated. The cell-free mitochondrial DNA copy number (cf-mt-cn) in the spent culture medium (SCM) of the embryos was then investigated. RNA sequencing of blastocysts revealed that metabolic pathways, including oxidative phosphorylation and mTOR pathways, were enriched in differentially expressed genes. The mt-cn and TL of AF-derived blastocysts were lower and shorter, respectively, than those of YF-derived blastocysts. Paternal aging did not affect the blastocyst rate after vitrification. Vitrification of the 8-cell stage embryos did not affect the mt-cn of the blastocysts. However, it increased the cf-mt-cn (cell-free mt-cn) in the SCM of both YF- and AF-derived embryos. Vitrification did not affect the TL of either YF- or AF-derived embryos. Thus, paternal aging affected the mt-cn and TL of the embryos, but vitrification did not affect these parameters in either age groups.

Zona pellucida removal by acid Tyrode's solution affects pre- and post-implantation development and gene expression in mouse embryos†.

The zona pellucida plays a crucial role in the process of fertilization to early embryonic development, including cellular arrangement and communication between blastomeres. However, little is known regarding the role of the zona pellucida in pre- and post-implantation embryonic development associated with gene expression. We investigated the effect of zona pellucida removal on pre- and post-implantation development of mouse embryos. After zona pellucida removal of two-cell stage embryos was performed by acid Tyrode's solution, which is commonly used for zona pellucida treatment, compaction occurred earlier in zona pellucida-free than zona pellucida-intact embryos. In addition, the expression of differentiation-related genes in the inner cell mass and trophectoderm was significantly altered in zona pellucida-free blastocyst compared with zona pellucida-intact embryos. After embryo transfer, the rate of implantation and live fetuses was lower in zona pellucida-free embryos than in control embryos, whereas the fetal weight at E17.5 was not different. However, placental weight significantly increased in zona pellucida-free embryos. RNA-sequencing analysis of the placenta showed that a total of 473 differentially expressed genes significantly influenced the biological process. The present study suggests that zona pellucida removal by acid Tyrode's solution at the two-cell stage not only disturbs the expression pattern of inner cell mass-/trophectoderm-related genes but affects the post-implantation development of mouse embryos. Overall, this study provides deeper insight into the role of the zona pellucida during early embryonic development and the viability of post-implantation development.

G1/S restriction point coordinates phasic gene expression and cell differentiation

Pluripotent embryonic stem cells have a unique cell cycle structure with a suppressed G1/S restriction point and little differential expression across the cell cycle phases. Here, we evaluate the link between G1/S restriction point activation, phasic gene expression, and cellular differentiation. Expression analysis reveals a gain in phasic gene expression across lineages between embryonic days E7.5 and E9.5. Genetic manipulation of the G1/S restriction point regulators miR-302 and P27 respectively accelerates or delays the onset of phasic gene expression in mouse embryos. Loss of miR-302-mediated p21 or p27 suppression expedites embryonic stem cell differentiation, while a constitutive Cyclin E mutant blocks it. Together, these findings uncover a causal relationship between emergence of the G1/S restriction point with a gain in phasic gene expression and cellular differentiation.

Decoding of position in the developing neural tube from antiparallel morphogen gradients.

Like many developing tissues, the vertebrate neural tube is patterned by antiparallel morphogen gradients. To understand how these inputs are interpreted, we measured morphogen signaling and target gene expression in mouse embryos and chick ex vivo assays. From these data, we derived and validated a characteristic decoding map that relates morphogen input to the positional identity of neural progenitors. Analysis of the observed responses indicates that the underlying interpretation strategy minimizes patterning errors in response to the joint input of noisy opposing gradients. We reverse-engineered a transcriptional network that provides a mechanistic basis for the observed cell fate decisions and accounts for the precision and dynamics of pattern formation. Together, our data link opposing gradient dynamics in a growing tissue to precise pattern formation.

Characterization of gene expression in mouse embryos at the 1-cell stage.

In mice, transcription from the zygotic genome is initiated at the mid-1-cell stage after fertilization. Although a recent high-throughput sequencing (HTS) analysis revealed that this transcription occurs promiscuously throughout almost the entire genome in 1-cell stage embryos, a detailed investigation of this process has yet to be conducted using protein-coding genes. Thus, the present study utilized previous RNA sequencing (RNAseq) data to determine the characteristics and regulatory regions of genes transcribed at the 1-cell stage. While the expression patterns of protein-coding genes of mouse embryos were very different at the 1-cell stage than at other stages and in various tissues, an analysis for the upstream and downstream regions of actively expressed genes did not reveal any elements that were specific to 1-cell stage embryos. Therefore, the unique gene expression pattern observed at the 1-cell stage in mouse embryos appears to be governed by mechanisms independent of a specific promoter element.

Abnormal Gene Expression in Mouse Embryos Resulting from Excessive Sperm Histone Retention Due to Perturbed Poly(ADP-ribose) Metabolism During Spermiogenesis.

Abnormal Gene Expression in Mouse Embryos Resulting from Excessive Sperm Histone Retention Due to Perturbed Poly(ADP-ribose) Metabolism During Spermiogenesis.

Automatically identifying and annotating mouse embryo gene expression patterns

Motivation: Deciphering the regulatory and developmental mechanisms for multicellular organisms requires detailed knowledge of gene interactions and gene expressions. The availability of large datasets with both spatial and ontological annotation of the spatio-temporal patterns of gene expression in mouse embryo provides a powerful resource to discover the biological function of embryo organization. Ontological annotation of gene expressions consists of labelling images with terms from the anatomy ontology for mouse development. If the spatial genes of an anatomical component are expressed in an image, the image is then tagged with a term of that anatomical component. The current annotation is done manually by domain experts, which is both time consuming and costly. In addition, the level of detail is variable, and inevitably errors arise from the tedious nature of the task. In this article, we present a new method to automatically identify and annotate gene expression patterns in the mouse embryo with anatomical terms.Results: The method takes images from in situ hybridization studies and the ontology for the developing mouse embryo, it then combines machine learning and image processing techniques to produce classifiers that automatically identify and annotate gene expression patterns in these images. We evaluate our method on image data from the EURExpress study, where we use it to automatically classify nine anatomical terms: humerus, handplate, fibula, tibia, femur, ribs, petrous part, scapula and head mesenchyme. The accuracy of our method lies between 70% and 80% with few exceptions. We show that other known methods have lower classification performance than ours. We have investigated the images misclassified by our method and found several cases where the original annotation was not correct. This shows our method is robust against this kind of noise.Availability: The annotation result and the experimental dataset in the article can be freely accessed at http://www2.docm.mmu.ac.uk/STAFF/L.Han/geneannotation/.Contact: l.han@mmu.ac.ukSupplementary Information: Supplementary data are available at Bioinformatics online.

Arsenic- and cadmium-induced toxicogenomic response in mouse embryos undergoing neurulation

Arsenic (As) and cadmium (Cd) are well-characterized teratogens in animal models inducing embryotoxicity and neural tube defects (NTDs) when exposed during neurulation. Toxicological research is needed to resolve the specific biological processes and associated molecular pathways underlying metal-induced toxicity during this timeframe in gestational development. In this study, we investigated the dose-dependent effects of As and Cd on gene expression in C57BL/6J mouse embryos exposed in utero during neurulation (GD8) to identify significantly altered genes and corresponding biological processes associated with embryotoxicity. We quantitatively examined the toxicogenomic dose–response relationship at the gene level. Our results suggest that As and Cd induce dose-dependent gene expression alterations representing shared (cell cycle, response to UV, glutathione metabolism, RNA processing) and unique (alcohol/sugar metabolism) biological processes, which serve as robust indicators of metal-induced developmental toxicity and indicate underlying embryotoxic effects. Our observations also correlate well with previously identified impacts of As and Cd on specific genes associated with metal-induced toxicity ( Cdkn1a, Mt1). In summary, we have identified in a quantitative manner As and Cd induced dose-dependent effects on gene expression in mouse embryos during a peak window of sensitivity to embryotoxicity and NTDs in the sensitive C57BL/6J strain.

Fluconazole alters CYP26 gene expression in mouse embryos

Disruption of embryonal retinoic acid homeostasis has been postulated to represent an etiological factor involved in the onset of fluconazole-induced teratogenesis. In the present study the impact of a teratogenic pulse of fluconazole on the gene expression of cytochrome P450 (CYP) 26 isoforms, which plays a central role in maintaining proper retinoic acid levels by mediating its degradation, was investigated. ICR pregnant mice were orally administered with 0 (vehicle) or 700 mg/kg of fluconazole on gestation day 8. Embryos were collected 12, 24 and 48 h after treatment. Quantitative real-time reverse-transcription polymerase chain reaction (quantitative real-time RT-PCR) assay was used to quantify the mRNA expression of CYP26a1, CYP26b1 and CYP26c1 in embryos. As result, fluconazole exposure was associated to an up-regulation of CYP26a1, CYP26b1, whereas no significant change was identified for the CYP26c1 isoform. This study demonstrates the capacity of fluconazole to alter CYP26 gene expression in mouse embryos.

Regulation of Gene Expression in Mouse Embryos and Its Embryonic Cells Through RNAi

The ability to direct cell differentiation of embryo-derived cells should have considerable practical applications. A prerequisite for success is the availability of robust tools that allows efficient knockdown of specific genes to elucidate gene function and direct differentiation. Loss-of-function studies in the mouse have traditionally been carried out using gene knockout animals to obtain mutant embryonic cells. Despite improvements in this technology, it still remains a laborious method. In addition, the use of knockout for studying preimplantation embryonic cells is limited due to maternal contributions. RNA interference (RNAi) is a powerful genetic approach for specific gene silencing of target genes in which 21-23 nt small interfering RNAs work as sequence-specific RNAi mediators. Using RNAi approaches, gene function has been eliminated in mouse embryonic stem cells and carcinoma cells to direct cell differentiation. In mouse oocytes and embryos, RNAi eliminated both maternal and zygotic transcripts and disturbed normal development.

Expression of the endothelial lipase gene in murine embryos and reproductive organs

Endothelial lipase (EL) is a recently discovered member of the triglyceride-lipase family that is involved in plasma HDL metabolism. In this study, we investigated the putative role of EL in mouse reproduction by studying EL gene expression in mouse embryos and adult reproductive organs. PCR analysis revealed that EL mRNA is expressed in mouse embryos on embryonic day 8.5 (E8.5) to E11.5, but not later in development. In situ hybridization studies on E10.5 whole embryos and embryonic sections showed expression of EL mRNA in multiple tissues, although of varying intensity. High expression was found in the neuroepithelium of the brain and the neural tube, the mesenchymal cells between organs, the optic lens and cup, and the otocyst. In adult mice, EL mRNA expression was high in ovaries from pregnant mice but low in ovaries from nonpregnant mice. EL mRNA was also highly expressed in placenta and testes. In situ hybridization studies demonstrated intense EL mRNA staining of lutein cells in corpora lutei in ovaries, of spermatocytes in the late pachytene and diplotene stages in testes, and of principal cells in epididymis. These results suggest that EL, in addition to its effects on plasma lipoprotein metabolism, plays a role in murine reproduction.

Experimental studies on cervical and lumbar ribs in mouse embryos.

In humans, the presence of cervical and lumbar ribs is of particular clinical significance. However, the relevance of their occurrence in the offspring of experimental animals in reproductive toxicologic studies is poorly understood. Maternal toxicity has been implicated in the etiology but conclusive evidence is lacking. The present study was undertaken to determine the incidence of supernumerary ribs (SNR) in mouse fetuses prenatally exposed to valproic acid (VPA) and retinoic acid (RA), and to compare their differential developmental susceptibility and morphological association with other axial skeletal anomalies. Single doses of valproic acid (VPA) or retinoic acid (RA) were administered to groups of mice on one of gestation days (GD) 7-12. Fetuses were collected on GD 18 and their skeletons examined for SNR. VPA treatment on GD 7 and GD 8 resulted in a high incidence of cervical and lumbar ribs, respectively. Cervical neural arch anomalies in the GD 7 group, and eight pairs of sternal ribs and seven sternebrae in the GD 8 group were observed in excess of the background SNR suggesting a direct effect of VPA on the developing mouse skeletal system. In the RA groups, GD 8-12 were susceptible for lumbar rib induction but increased incidence of cervical ribs was observed only from GD 9-12. Peak incidence of cervical ribs was found in the GD 10 and 11 groups and that of the lumbar ribs in the GD 8 and 11 groups. Although SNR incidence generally increased with increasing dose of RA, a strict dose-response relationship was lacking. Cervical arch anomalies were observed in as many embryos as those with cervical ribs, but eight pairs of sternal ribs and seven sternebrae did not correlate well with the lumbar ribs in the peak day groups. Interrupted cervical neural arches correlated well with lumbar ribs. The reduction in the frequency of presacral vertebrae from 26 to 25 in the VPA groups was limited to GD 7 (30%) and 8 (18%) groups. RA-induced reduction in presacral vertebral number extended to GD 9 and was greater in the GD 8 than in the GD 9 groups. Sternal anomalies occurred both in VPA and RA experiments and did not strictly correlate with the frequency of SNR. VPA had a narrow window of susceptibility, whereas RA effects on sternum extended from GD 9-12. The incidence of sternal anomalies generally increased with increasing dose and advancing developmental stage at which RA exposure occurred. These developmental susceptibility windows and associated malformations, when considered in the context of the ability of these drugs to induce alterations in gene expression in mouse embryos suggest that SNR are polygenic in origin and greatly influenced by environmental toxicants.

250SPECIFIC GENE KNOCK DOWN OF OCT-4 IN MOUSE PREIMPLANTATION EMBRYOS USING SHORT INTERFERENCE RNA

RNA interference is used to specifically and effectively inhibit the expression of cognate genes. In the present study we investigated the inhibitory effect of gene expression in mouse embryos developing in vitro by injecting short interference RNA (siRNA). Fertilized mouse zygotes were obtained from mated females 20–24h after hCG injection. Chemically synthesized 21-nt siRNA was commercially obtained and injected into mouse zygotes. The zygotes were then cultured in KSOM medium supplemented with 4% BSA at 37°C. Semi-quantitative RT-PCR was used to examine Octamer-binding transcription factor (Oct-4) gene expression in a single mouse embryo developing in vitro following siRNA-injection. In order to determine the expression and distribution of Oct-4 in mouse embryos, the mouse embryos were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.2% triton x-100 for 10min. Embryos were then incubated with rabbit Oct-4 polyclonal antibody for 1h and with FITC-labeled goat anti-rabbit antibody. Propidium iodide was used for DNA staining. siRNA injection did not retard the development of mouse zygotes. The number of blastocyst cells and the ICM/TE ratio did not differ in the siRNA injected blastocysts and the non-injected control group. Semi-quantitative RT-PCR revealed that Oct-4 expression was decreased at the 4-cell embryo stage and was significantly high at the morula and blastocyst stages. Injection of siRNA into oocytes inhibited RNA expression of Oct-4 and Nanog, but not of E-cadherin and Heat shock protein 70.1. Immunocytochemical staining showed inhibition of Oct-4 synthesis of the morulae and blastocysts following injection of siRNA. After culture of the embryos in the ES cell-derived conditioned medium, the embryos were stained for alkaline phosphatase (AP), a marker specific to pluripotent cells. AP was not detected in the inner cell mass of blastocysts following siRNA injection. These results suggest that siRNA injection into a mouse zygote specifically inactivates Oct-4 in mouse embryos developing in vitro.

Mouse latent TGF-β binding protein-2: molecular cloning and developmental expression

The molecular cloning and developmental expression of mouse LTBP-2 are presented here. We established the identity of the cDNA by sequence comparison (80% identity with human LTBP-2) and by chromosome localization (mouse chromosome 12, band D, a region of conserved synteny with the human LTBP-2 gene). In contrast to LTBP-1 and LTBP-3, mouse LTBP-2 apparently is a more modular protein, with proline/glycine-rich sequences always alternating with clusters of cysteine-rich structural motifs. We found for the first time that LTBP-2 gene expression in mouse embryos was restricted to cartilage perichondrium and blood vessels, a somewhat surprising result since other LTBP genes are widely expressed in rodent tissues. Therefore, mouse LTBP-2 may play a critical role in the assembly of latent TGF-β complexes in developing elastic tissues such as cartilage and blood vessel.

Placenta Growth Factor and Vascular Endothelial Growth Factor are Co-Expressed During Early Embryonic Development

We have used the polymerase chain reaction to identify mouse proteins similar in primary structure to the endothelial cell mitogen Vascular Endothelial Growth Factor (VEGF). One amplified product encoded mouse Placenta Growth Factor (P1GF). The pattern of P1GF gene expression in mouse embryos was studied by in situ hybridization. Transcripts encoding mouse P1GF were abundant in trophoblastic giant cells associated with the parietal yolk sac at early stages of embryogenesis. VEGF transcripts were also detected in trophoblastic giant cells raising the possibility that these cells may secrete heterodimers consisting of one P1GF subunit and one VEGF subunit. The secretion of P1GF and VEGF by trophoblastic giant cells is likely to be the signal which initiates and co-ordinates vascularization in the deciduum and placenta during early embryogenesis.

Class I and class IV alcohol dehydrogenase (retinol dehydrogenase) gene expression in mouse embryos.

Mammalian alcohol dehydrogenase (ADH) exists as several classes of related enzymes, all linked evolutionarily to the medium-chain ADHs present in bacteria, fungi, plants, and animals (Kaiser et al., 1993). One distinctive feature of the mammalian ADH family is the ability of some members to act as retinol dehydrogenases as well as ethanol dehydrogenases (Zachman and Olson, 1961; Mezey and Holt, 1971; Boleda et al., 1993). ADH classes I and IV have been demonstrated to function in this manner in both humans and rodents (Yang et al., 1993; Boleda et al., 1993). This feature may be important since it implies that these classes of ADH may participate in the conversion of retinol (vitamin A alcohol) to its active forms including retinal and retinoic acid, the latter functioning as a transcriptional regulatory ligand controlling cellular differentiation (De Luca, 1991). It further implies that excessive ethanol consumption may competitively inhibit retinol oxidation by ADH, thus leading to under-utilization of vitamin A. This point is particularly critical when one considers a potential connection between vitamin A utilization by developing embryos and fetal alcohol syndrome, a form of teratogenesis characterized by ethanol-in-duced brain and craniofacial abnormalities (Duester, 1991).

The Ras/Raf Signaling Pathway Is Required for Progression of Mouse Embryos Through the Two-Cell Stage

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos.

The Ras/Raf signaling pathway is required for progression of mouse embryos through the two-cell stage.

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos.

Exposure to retinoic acid before or after the onset of somitogenesis reveals separate effects on rhombomeric segmentation and 3′ HoxB gene expression domains

We have compared the relationship between the patterns of altered morphogenesis and of altered gene expression in mouse embryos exposed to excess retinoic acid (RA) (a) just before and (b) just after the onset of somitic segmentation (day 7.75 to day 8.25). Exposure to RA prior to the onset of somitic segmentation results in suppression of rhombomeric (but not somitic) segmentation, and conversion of the genetic identity of the whole preotic hindbrain to that of rhombomere 4. In contrast, exposure to RA at early somite stages results in near-normal rhombomeric segmentation; rhombomeric gene expression domains indicate that only rhombomere 2 has changed its genetic identity to that of rhombomere 4, the other preotic segments showing normal expression patterns for HoxB genes and Krox-20. The results indicate that RA has separable effects (1) on the genes mediating the process of rhombomeric segmentation per se, such as Krox-20, and (2) on the genes that influence the nature of the structures that subsequently develop from the individual rhombomeres, such as the Hox genes.

The mouse NCAM gene displays a biphasic expression pattern during neural tube development.

The neural cell adhesion molecule (NCAM) is one of the most abundant cell adhesion molecules expressed in vertebrates and it is thought to play important roles as a regulator of morphogenetic processes, but little is known of its expression pattern in mammalian embryos. In this study, we have examined the developmental profile of NCAM gene expression in mouse embryos from gestational day 7.5 to 12.5, focusing on the developing neural tube. NCAM transcripts were first detected around day 8.5 in the somites and the forming neural tube. At this stage, NCAM transcripts were expressed in the neuroepithelium throughout the width of the neural groove and tube up to a rostral boundary within the hindbrain, whereas NCAM mRNA levels were very low or undetectable in the neuroepithelium of the head region. The positional restriction of NCAM expression was confirmed by immunohistochemistry at the protein, and by polymerase chain reaction analysis at the RNA level. Expression in the neuroepithelium was transient as the level of NCAM transcripts declined in the germinal layer beyond day 8.5. By day 9.5, strong NCAM expression had appeared on the earliest postmitotic neurones along the entire neuraxis, and this pattern of expression in all regions with differentiating neurones was maintained until day 12.5. We conclude that NCAM expression in the neural tube occurs in two spatiotemporal distinct waves: a first wave in the proliferating neuroepithelium showing positional dependence along the rostrocaudal axis, and a second wave on essentially all neurones that have become postmitotic.