Cleavage Of Mouse Embryos Research Articles

Overexpression of KAT8 induces a failure in early embryonic development in mice

Embryo quality is strongly associated with subsequent embryonic developmental efficiency. However, the detailed function of lysine acetyltransferase 8 (KAT8) during early embryonic development in mice remains elusive. In this study, we reported that KAT8 played a pivotal role in the first cleavage of mouse embryos. Immunostaining results revealed that KAT8 predominantly accumulated in the nucleus throughout the entire embryonic developmental process. Kat8 overexpression (Kat8-OE) was correlated with early developmental potential of embryos to the blastocyst stage. We also found that Kat8-OE embryos showed spindle-assembly defects and chromosomal misalignment, and that Kat8-OE in embryos led to increased levels of reactive oxygen species (ROS), accumulation of phosphorylated γH2AX by affecting the expression of critical genes related to mitochondrial respiratory chain and antioxidation pathways. Subsequently, cellular apoptosis was activated as confirmed by TUNEL (Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling) assay. Furthermore, we revealed that KAT8 was related to regulating the acetylation status of H4K16 in mouse embryos, and Kat8-OE induced the hyperacetylation of H4K16, which might be a key factor for the defective spindle/chromosome apparatus. Collectively, our data suggest that KAT8 constitutes an important regulator of spindle assembly and redox homeostasis during early embryonic development in mice.

Toxicity of the Mycotoxin Deoxynivalenol on Early Cleavage of Mouse Embryos by Fluorescence Intensity Analysis.

Deoxynivalenol is a mycotoxin, produced by Fusarium from contaminated corn, wheat, and other grains, that induces multiple effects in humans and animals, including cytotoxic, genotoxic, immunotoxic, and carcinogenic effects. Recent studies show that deoxynivalenol also affects the reproductive system of mammals, including oocyte quality. However, the effects of deoxynivalenol on early embryonic development have not been reported. In this study, fluorescence intensity analysis was used to show that deoxynivalenol disrupted the first cleavage of the zygote. The high deoxynivalenol dose disturbed the movement of the pronucleus after fertilization, while the low deoxynivalenol dose caused aberrant spindle morphology during the metaphase of the first cleavage. Further analysis showed that the reactive oxygen species level increased in the deoxynivalenol-exposed two-cell embryos, indicating oxidative stress. Moreover, deoxynivalenol caused DNA damage in the embryos, as positive γH2A.X signals were detected in the nucleus. These events led to the early apoptosis of mouse embryos, which was confirmed by autophagy. Taken together, our study provides evidence for the toxicity of deoxynivalenol during early embryonic development in the mouse model.

An approach for live imaging of first cleavage in mouse embryos using fluorescent chemical probes for DNA, microtubules, and microfilaments.

Dynamic morphological changes in the chromosome and cytoskeleton occur in mammals and humans during early embryonic development, and abnormalities such as embryonic chromosomal aneuploidy occur when development does not proceed normally. Visualization of the intracellular organelles and cytoskeleton allows elucidation of the development of early mammalian embryos. The behavior of the DNA and cytoskeleton in early mammalian embryos has conventionally been observed by injecting target molecule mRNAs, incorporating a fluorescent substance-expressing gene, into embryos. In this study, we visualized the chronological behavior of male and female chromosome condensation in mouse embryos, beginning in the two-pronuclear zygote, through the first division to the two-cell stage, using fluorescent chemical probes to visualize the behavior of DNA, microtubules, and microfilaments. Mouse two-pronuclear stage embryo were immersed in medium containing fluorescent chemical probes to visualize DNA, microtubules, and microfilaments. Observation was performed with a confocal microscope. This method allowed us to observe how chromosome segregation errors in first somatic cell divisions in mouse embryos and enabled dynamic analysis of a phenomenon called lagging chromosomes. By applying this method, we can observe any stage of embryonic development, which may provide new insights into embryonic development in other mammals.

Poly(I:C) exposure during in vitro fertilization disrupts first cleavage of mouse embryos and subsequent blastocyst development

The reproductive system can be infected by a variety of double-stranded RNA viruses, which disrupt ovary function and pregnancy. However, whether viral infection directly affects early embryonic development remains unknown. Here we show that Poly(I:C), which mimics a double-stranded RNA virus, significantly impaired mouse early embryonic development in vitro, and up-regulated TLR3 and IFNα at the two cells embryo stage. Further studies indicated that Poly(I:C)-treatment caused DNA damage and abnormal spindle morphology at the first cleavage. Moreover, CDX2 and SOX2 expression was decreased while blastocyst cell apoptosis was increased. Altogether, Poly(I:C) decreased the rate of successful in vitro fertilization via DNA damage and abnormal spindle morphology at the first cleavage and inhibited early embryonic development by inducing immune response and promoting blastocyst cell apoptosis. This study provides an implication for exploring the causes of reproductive disorders in mammals and humans caused by infection of double-stranded RNA virus.

Exposure to citrinin induces DNA damage, autophagy, and mitochondria dysfunction during first cleavage of mouse embryos.

Citrinin (CTN) is a mycotoxin, which is isolated from Penicillium citrinum and widely existed in the contaminated feeds. It is reported that CTN is toxic to heart, liver, and reproductive system. Previous studies indicated that CTN induced apoptosis in oocytes and embryos. In this study, we reported the potential causes of CTN on embryo development. Our results showed that 40 μM CTN exposure significantly reduced the first cleavage of mouse embryos, showing with the low rate of 2-cell embryos. We found that CTN induced DNA damage, showing the higher positive γH2A.X signals. Autophagy was occurred since more LC3 positive autophagosomes were found in the cytoplasm. This could be confirmed by the enhanced lysosome function, since higher accumulated lysosome distribution were found and LAMP2 was also increased under CTN exposure. Besides, we showed that mitochondria distribution was disturbed, indicating that CTN could disrupt mitochondria function, which could be the possible reason for the oxidative stress and apoptosis in CTN-exposed embryos. In conclusion, our study showed that CTN exposure had adverse effects on the early embryo development during first cleavage through its effects on the induction of DNA damage, autophagy, and mitochondria dysfunction.

Podophyllotoxin Exposure Causes Spindle Defects and DNA Damage-Induced Apoptosis in Mouse Fertilized Oocytes and Early Embryos.

Podophyllotoxin (PPT) is a kind of lignans extracted from the roots and stems of the genus Podophyllum from the tiller family, and it has been widely used in the treatment of condyloma acuminatum, multiple superficial epithelioma in the clinics. However, PPT has been reported to be toxic and can cause liver defects and other organ poisoning. In addition, emerging evidences also indicate that PPT has reproductive toxicity and causes female reproduction disorders. In this study, we used fertilized oocytes and tried to explore the effects of PPT on the early embryonic development with the mouse model. The results showed that exposure to PPT had negative effects on the cleavage of zygotes. Further analysis indicated that PPT could disrupt the organization of spindle and chromosome arrangement at the metaphase of first cleavage. We also found that PPT exposure to the zygotes induced excessive reactive oxygen species (ROS), suggesting the occurrence of oxidative stress. Moreover, in the PPT-exposed embryos, there was positive γH2A.X and Annexin-V signals, indicating that PPT induced embryonic DNA damage and early apoptosis. In conclusion, our results suggested that PPT could affect spindle formation and chromosome alignment during the first cleavage of mouse embryos, and its exposure induced DNA damage-mediated oxidative stress which eventually led to embryonic apoptosis, indicating the toxic effects of PPT on the early embryo development.

Mps1 controls spindle assembly, SAC, and DNA repair in the first cleavage of mouse early embryos.

Monopolar spindle-1 (Mps1) is a critical interphase regulator that also involves into the spindle assembly checkpoint for the cell cycle control in both mitosis and meiosis. However, the functions of Mps1 during mouse early embryo development is still unclear. In this study, we reported the important roles of Mps1 in the first cleavage of mouse embryos. Our data indicated that the loss of Mps1 activity caused precocious cleavage of zygotes to 2-cell embryos; however, prolonged culture disturbed the early embryo development to the blastocyst. We found that the spindle organization was disrupted after Mps1 inhibition, and the chromosomes were misaligned in the first cleavage. Moreover, the kinetochore-microtubule attachment was lost and Aurora B failed to accumulate to the kinetochores, indicating that the spindle assembly checkpoint (SAC) was activated. Furthermore, the inhibition of Mps1 activity resulted in an increase of DNA damage, which further induced oxidative stress, showing with positive γ-H2A.X signal and increased reactive oxygen species level. Ultimately, irreparable DNA damage and oxidative stress-activated apoptosis and autophagy, which was confirmed by the positive Annexin-V signal and increased autophagosomes. Taken together, our data indicated that Mps1 played important roles in the control of SAC and DNA repair during mouse early embryo development.

CHK2 is essential for spindle assembly and DNA repair during the first cleavage of mouse embryos

The quality of the early embryo is critical for embryonic development and implantation. Errors during cleavage lead to aneuploidy in embryos. As a cell cycle checkpoint protein, CHK2 participates in DNA replication, cell cycle arrest and spindle assembly. However, the functions of CHK2 in early development of the mouse embryo remain largely unknown. In this study, we show that CHK2 is localized on the spindle in metaphase and mainly accumulates at spindle poles in anaphase/telophase during the first cleavage of the mouse embryo. CHK2 inhibition led to cleavage failure in early embryonic development, accompanied by abnormal spindle assembly and misaligned chromosomes. Moreover, the loss of CHK2 activity increased the level of cellular DNA damage, which resulted in oxidative stress. Then, apoptosis and autophagy were found to be active in these embryos. In summary, our results suggest that CHK2 is an essential regulator of spindle assembly and DNA repair during early embryonic development in mice.

The dynamics of DAXX protein distribution in the nucleus of mouse early embryos

Nuclear distribution of Death-associated protein 6 (Daxx) was studied using fluorescent and electron microscopy in mouse embryos at different stages of development in vivo, from zygote to morula. Daxx was found in association with transcriptionally silent chromatin predominantly with a heterochromatin rim surrounding the nucleolus precursor bodies (NPBs) at all stages studied. At the zygote stage, Daxx was detected only at the periphery of NPBs both in male and female pronuclei. At the late two-cell stage, Daxx was localized not only in the heterochromatin rim at the periphery of NPBs but also in heterochromatin zones not associated with NPBs. At the morula stage, a diffuse distribution of Daxx prevailed. Scarce Daxx-positive zones were detected only in some embryos at the nucleolar periphery. Thus, Daxx is noticeably redistributed during mouse embryo cleavage, and the most conspicuous areas of Daxx concentration are observed at the end of two-cell stage. Daxx is found colocalized with the chromatin-remodeling protein ATRX exclusively in two-cell embryos, but the heterochromatin areas containing either Daxx or ATRX individually are also observed at this stage. However, most zones containing both Daxx and ATRX demonstrated a low FRET-efficiency. This suggest that two molecules are not approached sufficiently close for molecular interactions to occur. Our data suggests that Daxx may function without cooperation with ATRX at least at some stages of early mouse development.

Removal of blastomere from cleaving mouse embryos programs adult energy balance phenotype

Removal of blastomere from cleaving mouse embryos programs adult energy balance phenotype

The effect of incubator type on mouse embryo development: conventional box versus top-load bench incubators

Multiple embryo incubator models are available for in vitro fertilization; however there is limited scientific data supporting use of one type over another. Our goal was to determine the effect of a conventional box incubator versus a top-load bench incubator in a non-humidity setting on mouse embryo cleavage rates, blastocyst development, and embryo hatching.

Comparison of Mouse Embryo Cleavage Rate from One Cell Stage to Blastocyst Stage in Co-culture with Vero Cells and Without Co-culture

Comparison of Mouse Embryo Cleavage Rate from One Cell Stage to Blastocyst Stage in Co-culture with Vero Cells and Without Co-culture

Two-photon fluorescence real-time imaging on the development of early mouse embryo by stages

Mouse zygotes are widely used in developmental biology and transgenic animal research. Two-photon laser scanning microscopy is particularly useful in four-dimensional observation of big and thick biological samples, such as mouse embryo. The early mouse embryo development from zygote to 8-cell stage compaction was observed in real-time by stages using two-photon laser scanning microscopy in this paper. During our experiment, several scanning parameters were optimized in different development stages. The initial cleavages of mouse embryo, from the zygote to 2-cell, 2-cell to 4-cell, 4-cell to 8-cell, and the compaction at 8-cell stage were observed. During the first stage, localized intracellular calcium elevation along with the cleavage furrow and the asymmetric zygote cytokinesis was detected. The relation between the asymmetry and the location of the second polar body was investigated. The rotational cleavage of mouse embryo was also observed in the experiment. These results would be helpful to further research on mammalian embryonic development.

Structural chromosomal aberrations, aneuploidy, and mosaicism in early cleavage mouse embryos derived from spermatozoa exposed to γ-rays

Purpose: To quantitatively and qualitatively investigate the changes in chromosomal aberrations during early cleavage in mouse embryos derived from γ-irradiated spermatozoa.Materials and methods: Mature males were exposed to 2 Gy or 4 Gy of 137Cs γ-rays, and their spermatozoa were used to produce embryos via in vitro fertilisation (IVF). The metaphase chromosomes were prepared from one-cell, two-cell, and four-cell embryos. In the chromosome preparations from two-cell and four-cell embryos, the separation of the sister blastomeres was precluded by treatment of the embryos with concanavalin A. The incidence of embryos with structural chromosomal aberrations, aneuploidy, or mosaicism was estimated. The fates of the different types of γ-ray-induced structural chromosomal aberrations were also investigated in those embryos.Results: The exposure of spermatozoa to 2 Gy or 4 Gy γ-rays caused structural chromosomal aberrations in 25.9% and 35.7% of the resultant one-cell embryos, respectively. At two-cell embryonic stage, the incidence of structural chromosomal aberrations was 17.4% in the 2 Gy group and 27.1% in the 4 Gy group. At the four-cell embryonic stage, although the incidence of control embryos with structural chromosomal aberrations was considerably high, the net incidence of embryos with radiation-induced structural chromosomal aberrations was similar to that at the one-cell stage. The incidence of aneuploidy was high in two-cell and four-cell embryos after both doses of γ-rays. The incidence of mosaicism increased significantly in dose- and embryonic-stage-dependent manners. Anaphase lag, and the degeneration and non-disjunction of the aberrant chromosomes were frequently observed in aneuploid and mosaic embryos.Conclusions: Mouse sperm DNA is highly vulnerable to γ-rays. The structural chromosomal aberrations of sperm origin are unstable in their behaviour and structure during cleavage, and therefore cause secondary aneuploidy and mosaicism in the early cleavage embryos.

Effect of medium conditioned with rat hepatoma BRL cells on ‘2-cell block’ of random-bred mouse embryos culturedin vitro

The phenomenon of developmental arrest at the 2-cell stage of zygotes obtained from certain mouse strains during in vitro culture is known as the 2-cell block. The effect of conditioned medium (CM) with rat hepatoma BRL cells on the 2-cell block of CD-1 mouse zygotes was investigated in comparison with that of CM with rat hepatoma Reuber H-35 cells. In control medium with EDTA, 75.4% of 2-cell embryos developed to the 4- to 8-cell stages. In the same conditions, the BRL Mr <10000 fraction inhibited the development of 2-cell embryos to the 4- to 8-cell stages (57.7%), although the inhibition by this fraction was weaker than by the Reuber Mr <10000 fraction (19.8%). As a result of reversed-phase column chromatography, a 2-cell stage specific inhibitor of the cleavage of mouse embryos (Fr.B-25), which separated into the Mr <10000 fraction of the Reuber CM, was detected at a low level in the BRL Mr <10000 fraction. On the other hand, the Mr >10000 fraction of BRL CM accelerated the development of the embryos (90.3%). This beneficial effect was also evident even in the absence of EDTA. RT-PCR analysis revealed that mRNAs encoding the beta-A or beta-B subunit of activins (Mr ~29000), which are well characterized cytokines that act as releasers of the 2-cell block, were expressed in BRL cells. These results indicate that BRL cells synthesize Fr.B-25 at low levels, and that activins contained in the BRL CM probably contributed to overcoming the 2-cell block of CD-1 zygotes cultured in vitro.

171 THE EFFECT OF TRICHOSTATIN A ON THE DEVELOPMENT AND THE GLOBAL GENE EXPRESSION PATTERNS IN MOUSE EMBRYOS DEVELOPING IN VITRO

Trichostatin A (TSA) is an inhibitor of histone deacetylase and is able to alter gene expression patterns by interfering with the removal of acetyl groups from histones. The aim of this study was to determine the effect of TSA treatment on the development and gene expression patterns of mouse zygotes developing in vitro. The addition of 100 nm TSA to the culture medium did not affect the cleavage of mouse embryos (TSA treatment, 148/150 (99%) v. control, 107/107 (100%)); however, embryos that were treated with TSA arrested at the 2-cell stage (145/148, 98%). We estimated the number of nuclei in control and TSA-treated embryos by propidium iodide staining, taking into account the presence of any cells with two or more nuclei. At 62–63 h post-hCG stimulation, control zygotes had developed to the 4-cell stage and exhibited one nucleus in each blastomere, indicative of normal development. In contrast, we observed tetraploid nuclei in at least one blastomere in 20.8% (11/53) of the embryos that had been treated with TSA. At 28–29 h post-hCG stimulation (metaphase of the 1-cell stage), there was no difference in the mitotic index (as determined by analyzing the microtubule configuration) in the TSA group compared to the control group. At the 2-cell stage, however, we did not observe mitotic spindles and metaphase chromatin in embryos in the TSA treatment group compared to the controls. Interestingly, when embryos were cultured in TSA-free medium from 35 h post-hCG stimulation (S- or early G2-phase of the 2-cell stage) onward, almost all of them (47/50) developed to the blastocyst stage. In contrast, when embryos were cultured in TSA-free medium from 42 h post-hCG stimulation (middle G2-phase of the 2-cell stage) onward, they did not develop to the 4-cell stage. We used Illumina microarray technology to analyze the gene expression profiles in control and TSA-treated late 2-cell-stage embryos. Applied Biosystems Expression System software was used to extract assay signals and assay signal-to-noise ratio values from the microarray images. Our data showed that 897 genes were significantly (P &lt; 0.05; 2-sample t-test) up- or down-regulated by TSA treatment compared to controls. Analysis using the PANTHER classification system (https://panther.appliedbiosystems.com) revealed that the 575 genes that were differentially expressed in the TSA group compared to the control were classified as being associated with putative biological processes or molecular function. Overall, in terms of putative biological processes, more nucleoside, nucleotide, and nucleic acid metabolism, protein metabolism and modification, signal transduction, developmental process, and cell cycle genes were differentially expressed between the TSA and control groups. In terms of putative molecular function, more nucleic acid-binding transcription factor and transferase genes were differentially expressed between the groups. The results collectively suggest that inhibition of histone acetylation in mouse embryos affects gene expression profiles at the time of zygotic genome activation, and this subsequently affects further development.

Foetal fibroblasts introduced to cleaving mouse embryos contribute to full-term development

Foetal fibroblasts (FFs) labelled with vital fluorescent dye were microsurgically introduced into eight-cell mouse embryos, three cells to each embryo. FFs were first identified in the inner cell mass (ICM) in about one-third of embryos, whereas in three quarters of embryos FFs were located among trophoblast cells. Some elimination of FFs from trophoblast occurred later on. Eventually, in blastocysts' outgrowths, an equally high contribution from FFs progeny (60%) was found in both ICM and trophoblast. Three days after manipulation, FFs resumed proliferation in vitro. More than three FFs were found in 46.2% of embryos on day 4. On the 7th day in vitro in 70% of embryos more than 12 FFs were found, proving at least three cell divisions. To study postimplantation development, the embryos with FFs were transferred to pseudopregnant recipients a day after manipulation. After implantation, FFs were identified by electrophoresis for isozymes of glucose phosphate isomerase (GPI). A single 11-day embryo delayed to day 8 proved chimeric by expressing both donor isozyme GPI-1B and recipient GPI-1A. Similar chimerism was found in the extraembryonic lineage of 11% of embryos by day 12. Starting from day 11 onwards, in 32% of normal embryos and in 57% of foetal membranes, hybrid GPI-1AB isozyme, as well as recipient isozyme, was present. Hybrid GPI-1AB can only be produced in hybrid cells derived by cell fusion, therefore, we suggest that during postimplantation development, FFs are rescued by fusion with recipient cells. In the mice born, hybrid isozyme was found in several tissues, including brain, lung, gut and kidney. We conclude that somatic cells (FFs) can proliferate in early embryonic environment until early postimplantation stages. Foetuses and the mice born are chimeras between recipient cells and hybrid cells with contributions from the donor FFs. Transdifferentiation as opposed to reprogramming by cell fusion can be considered as underlying cellular processes in these chimeras.

Chromosome activities in cleaving mouse embryos to construct prenucleolar bodies orienting an intranuclear chromosome-chromosome configuration.

Most chromosomes of cleaving mouse embryos formed spherical structures called as prenucleolar bodies. Embryos stained with fluorescing dyes selective for AT-rich regions of DNA indicated that chromosome centromeres were organized by prenucleolar bodies and that chromosomes at pre-prophase of the cleavage division started to condense with their centromere regions aligned towards the peripheries of spheres. Embryo chromosomes as well as prenucleolar bodies were shown to bind a monoclonal antibody to spliceosomal snRNAs, which are contrasted to the nucleoli or chromosomes of somatic cells. More than 10 prenucleolar bodies were found in the pronuclei at the earliest stage after fertilization and 40 of them at most, diploid number of chromosomes of the mouse, in the nuclei of the 2-cell embryos. The number was decreased later in the cell cycles and at the 4-cell stage. Hence, every chromosome of cleaving mouse embryos is highly active in constructing prenucleolar bodies and behaves during the interphase until mitosis as a unit of the body; either as a single unit or as a member of the units fused. In other words, the prenucleolar bodies function as centers orienting chromosome-chromosome configuration in the fertilized eggs.

LagoZ et LagZ, deux gènes appauvris en dinucléotides CpG dérivés du gène LacZ pour l'étude des contrôles épigénétiques

The methylation of 5'CpG 3' dinucleotides within genes creates potential targets for protein complexes that bind to methylated DNA sequences and to histone deacetylases (MBD-HDAC). This can lead to transcriptional repression by modification of chromatin. To test the importance of this repression in vivo and to determine when during development these epigenetic controls are placed on genes, two novel genes have been engineered by directed mutagenesis of the CpG-rich LacZ gene that are depleted of ( LagZ) or completely lacking ( LagoZ) CpG sequences. We report that the expression (transcriptional and translational) of the three genes is indistinguishable in transient assays in cleaving mouse embryos. Therefore, the complete absence of CpG sequences within three kilobases of coding sequence is compatible with its maintenance in the nucleus and with its expression. These molecules can now be used to study the ontogenesis of the CpG-dependent repressive system in intact organisms.

Expression of human immunodeficiency virus long terminal repeat-coupled genes in early cleaving embryos

Early studies suggested that exogenous retroviral genes are not expressed by mouse embryos infected at early cleavage stages. The general view has therefore been that, since human eggs are non-dividing cells, they could not be infected with HIV before fertilization, nor would HIV be expressed if infection occurred during early cleavage stages following fertilization. In contrast with this view, more recent work has shown that genes coupled to the long terminal repeat (LTR) promotor elements in Rous Sarcoma Virus are readily expressed in early cleaving mouse embryos; in addition, pHIV-LTR, a plasmid construct with HIV-LTR coupled to the reporter gene, lacZ (which encodes β-galactosidase), is expressed when transfected into a human embryonic carcinoma cell line. To determine if HIV LTR coupled genes would be expressed in early cleaving embryos, one nucleus of mouse zygotes were microinjected with ≈5000 copies of the pHIV-LTR plasmid. Following microinjection, zygotes were cultured for 48 h to the four cell stage and stained for expression of β-galactosidase. A plasmid construct containing lacZ without HIV LTR was microinjected as control. A total of 25 of the 111 mouse zygotes microinjected with pHIV-LTR stained positively for β-galactosidase activity. Blastomeres within individual embryos were not uniformly stained, suggesting a mosaic pattern of distribution of the microinjected plasmids among the cleaving blastomeres. None of the 22 mouse zygotes microinjected with control plasmid stained positively. A total of six polypronuclear human eggs were obtained as discarded by-products of a human IVF program. All four human eggs microinjected with ≈6000 copies of HIV-LTR reporter gene construct and cultured for 48 h stained highly positive for β-galactasidase. Staining intensity varied among blastomeres, suggesting a mosaic pattern similar to the mouse embryos. The two human eggs microinjected with control plasmid were negative for β-galactosidase activity. These results suggest that HIV-LTR is active in both early cleaving mouse embryos and in early cleaving polypronuclear human eggs. These findings indicate that human eggs infected at the time of fertilization with HIV could express viral proteins during early embryonic development.