Early Stages Of Embryogenesis Research Articles

The development of embryos in Brassica rapa L. in microgravity

In this work we present the results of the comparative cytoembryological research on embryos of an identified age that were produced in microgravity and in the ground laboratory control. A considerable similarity was found between the rate of embryo development and degree of their differentiation in both variants. Abnormalities in the process of embryo formation and acceleration in development of the endosperm were revealed at the early stages of embryogenesis in microgravity.

Targeted Inactivation of p12Cdk2ap1, CDK2 Associating Protein 1, Leads to Early Embryonic Lethality

Targeted disruption of murine Cdk2ap1, an inhibitor of CDK2 function and hence G1/S transition, results in the embryonic lethality with a high penetration rate. Detailed timed pregnancy analysis of embryos showed that the lethality occurred between embryonic day 3.5 pc and 5.5 pc, a period of implantation and early development of implanted embryos. Two homozygous knockout mice that survived to term showed identical craniofacial defect, including a short snout and a round forehead. Examination of craniofacial morphology by measuring Snout Length (SL) vs. Face Width (FW) showed that the Cdk2ap1+/− mice were born with a reduced SL/FW ratio compared to the Cdk2ap1+/+ and the reduction was more pronounced in Cdk2ap1−/− mice. A transgenic rescue of the lethality was attempted by crossing Cdk2ap1+/− animals with K14-Cdk2ap1 transgenic mice. Resulting Cdk2ap1+/−:K14-Cdk2ap1 transgenic mice showed an improved incidence of full term animals (16.7% from 0.5%) on a Cdk2ap1−/− background. Transgenic expression of Cdk2ap1 in Cdk2ap1−/−:K14-Cdk2ap1 animals restored SL/FW ratio to the level of Cdk2ap1+/−:K14-Cdk2ap1 mice, but not to that of the Cdk2ap1+/+:K14-Cdk2ap1 mice. Teratoma formation analysis using mESCs showed an abrogated in vivo pluripotency of Cdk2ap1−/− mESCs towards a restricted mesoderm lineage specification. This study demonstrates that Cdk2ap1 plays an essential role in the early stage of embryogenesis and has a potential role during craniofacial morphogenesis.

Mechanical Computation in Neurons

Motility is a major function of cells, playing a fundamental role in development and embryogenesis. Growth cones are the major motile structures usually located at the tip of neurites and are composed of a lamellipodium from which thin filopodia emerge. We have analyzed the kinetics and dynamics of growth cones from a computational point of view with the aim to understand two major issues: firstly, the strategy used by filopodia and lamellipodia during their exploration and navigation; secondly, which kind of mechanical problems neurons need to solve during their operation. Filopodia grow and retract following statistical patterns nearly optimal for an efficient exploration of the environment. This exploratory motion is at the basis of contact formation and the establishment of appropriate synaptic connections. Filopodia and lamellipodia can also avoid obstacles and occasionally lamellipodia can displace them. From this point of view, filopodial and lamellipodial motion can be described as a random process in which errors are corrected by efficient feed-back loops. We argue that neurons not only process sensory signals, but also solve mechanical problems throughout their entire lifespan, from the early stages of embryogenesis to adulthood.

The promoter of the Arabidopsis thaliana BAN gene is active in proanthocyanidin-accumulating cells of the Brassica napus seed coat

As part of an ongoing research program dedicated to the understanding of proanthocyanidin (PA) accumulation in Brassica napus seed coat, transgenic rapeseed plants carrying a 2.3-kb fragment of the Arabidopsis thaliana BAN promoter (ProAtBAN) fused to the uidA reporter gene (GUS) were generated. Analysis of these plants revealed that ProAtBAN was activated in B. napus seed coat, following a spatio-temporal pattern that was very similar to the PA deposition profile in rapeseed and also to the one previously described in Arabidopsis. ProAtBAN activity occurred as soon as the early stages of embryogenesis and was restricted to the cells where PAs were shown to accumulate. Therefore, the Arabidopsis BAN promoter can be used to trigger gene expression in B. napus seed coat for both genetic engineering and functional validation of candidate genes. In addition, these data strongly suggest that the transcriptional regulatory network of the BAN gene is conserved between Arabidopsis and rapeseed. This is consistent with the fact that similarity searches of the public rapeseed sequence databases allowed recovering the rapeseed homologs for several BAN regulators, namely TT1, TT2, TT8, TT16 and TTG1, which have been previously described in Arabidopsis.

Inactivating the spindle checkpoint kinase Bub1 during embryonic development results in a global shutdown of proliferation.

BackgroundBub1 is a component of the spindle assembly checkpoint, a surveillance mechanism that maintains chromosome stability during M-phase. Bub1 is essential during the early stages of embryogenesis, with homozygous BUB1-null mice dying shortly after day E3.5. Bub1 is also required later during embryogenesis; inactivation of BUB1 on day E10.5 appears to rapidly block all further development. However, the mechanism(s) responsible for this phenotype remain unclear.FindingsHere we show that inactivating BUB1 on day E10.5 stalls embryogenesis within 48 hours. This is accompanied by a global shutdown of proliferation, widespread apoptosis and haemorrhaging.ConclusionOur results suggest that Bub1 is required throughout the developing embryo for cellular proliferation. Therefore, Bub1 has been shown to be essential in all scenarios analyzed thus far in mice: proliferation of cultured fibroblasts, spermatogenesis, oogenesis and both early and late embryonic development. This likely reflects the fact that Bub1 has dual functions during mitosis, being required for both SAC function and chromosome alignment.

New Aspects for the Treatment of Cardiac Diseases Based on the Diversity of Functional Controls on Cardiac Muscles: The Regulatory Mechanisms of Cardiac Innervation and Their Critical Roles in Cardiac Performance

The heart is abundantly innervated, and the nervous system precisely controls the function of this organ. The density of cardiac innervation is altered in diseased hearts, which can lead to unbalanced neural activation and lethal arrhythmia. For example, diabetic sensory neuropathy causes silent myocardial ischemia, characterized by loss of pain perception during myocardial ischemia, and it is a major cause of sudden cardiac death in diabetes mellitus. Despite the clinical importance of cardiac innervation, the mechanisms underlying the control of this process remain poorly understood. We demonstrate that cardiac innervation is determined by the balance between neural chemoattractants and chemorepellents within the heart. Nerve growth factor (NGF), a potent chemoattractant, is synthesized abundantly by cardiomyocytes, and is induced by the upregulation of endothelin-1 during development. By comparison, the neural chemorepellent Sema3a is expressed at high levels in the subendocardium in the early stage of embryogenesis and is downregulated as development progresses, leading to epicardial-to-endocardial transmural sympathetic innervation patterning. We also show that the downregulation of cardiac NGF is a cause of diabetic neuropathy and that NGF supplementation prevents silent myocardial ischemia in diabetes mellitus. Both Sema3a-targeted and Sema3a-overexpressing mice display sudden cardiac death or lethal arrhythmias due to disruption of innervation patterning. The present review focuses on the regulatory mechanisms controlling cardiac innervation and the critical roles of these processes in cardiac performance.

A Vanishing Act, Explained

In a paper in this issue of Developmental Cell, Shibutani et al. (2008) uncover the mechanism that underlies tightly regulated S-phase degradation of Drosophila E2F1 during development. They show that dE2F1 is degraded by the Cul4(Cdt2) ubiquitin ligase in a manner that resembles the DNA replication-dependent turnover of Cdt1.

Two distinct Omp85 paralogs in the chloroplast outer envelope membrane are essential for embryogenesis in Arabidopsis thaliana

Homologs of a bacterial β-barrel protein, Omp85, ubiquitously exist in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. Those in non-photosynthetic bacteria and mitochondria are responsible for β-barrel protein sorting to the outer membranes, and thus are essential for viability of the organisms. There are two distinct Omp85 homologs in chloroplasts of the model plant, Arabidopsis thaliana. One of them, Toc75, functions as the main protein import translocation channel, and was shown to be indispensable from a very early stage of embryogenesis. By contrast, the role of another homolog, OEP80, remains elusive. Recently, we showed that disruption of the OEP80 gene causes embryo abortion in A. thaliana at a stage later than that affected by TOC75 knockout. This indicates that the two chloroplastic Omp85 homologs are both essential for viability of plants from very early stages of development, but may have distinct functions. Defining the functional and evolutionary relationships of Toc75 and OEP80 by further studies should advance our understanding of the importance of plastids during embryogenesis, as well as that of the molecular details of plastid biogenesis.Addendum to: Patel R, Hsu SC, Bédard J, Inoue K, Jarvis P. The Omp85-related chloroplast outer envelope protein, OEP80, is essential for viability in Arabidopsis. Plant Physiol 2008; 148:235-45; DOI: 10.1104/pp.108.122754.

Comparative transcriptomics and proteomics of fetal and adult human liver

The comparative transcriptome and proteome analysis of the fetal liver (10.5 weeks of gestation) and adult liver was carried out. Data on differential gene expression were obtained using two-color fluorescent probe competitive hybridization of fluorescently labeled cRNA and Agilent’s whole human genome microarrays. Protein profiles were obtained by means of two dimensional gel electrophoresis (2D-GE) of cell extracts within the range of pI from 3 to 10. Proteins isolated from gels were identified by MALDI-TOF mass spectrometry. The list of identified proteins was then compared with the list of genes preferentially expressed in fetal liver. The research object, fetal liver, is a unique organ because it combines both hepatic and hematopoietic tissues and during embryogenesis liver changes its main functional activity from hematopoietic (at early stages of development) towards metabolic functions (including detoxification and secretion of various serum proteins). Data obtained show that transcriptome analysis adequately reflects the main hepatic function during early stages of embryogenesis. Hemoglobins of both embryonic and adult types were identified. Results of proteomic analysis were basically confirmed during mRNA analysis, however, some protein products of highly expressed genes were not detected during 2D-GE.

Genetic Variation and De Novo Mutations in the Parthenogenetic Caucasian Rock Lizard Darevskia unisexualis

Unisexual all-female lizards of the genus Darevskia that are well adapted to various habitats are known to reproduce normally by true parthenogenesis. Although they consist of unisexual lineages and lack effective genetic recombination, they are characterized by some level of genetic polymorphism. To reveal the mutational contribution to overall genetic variability, the most straightforward and conclusive way is the direct detection of mutation events in pedigree genotyping. Earlier we selected from genomic library of D. unisexualis two polymorphic microsatellite containg loci Du281 and Du215. In this study, these two loci were analyzed to detect possible de novo mutations in 168 parthenogenetic offspring of 49 D. unisexualis mothers and in 147 offspring of 50 D. armeniaca mothers . No mutant alleles were detected in D. armeniaca offspring at both loci, and in D. unisexualis offspring at the Du215 locus. There were a total of seven mutational events in the germ lines of four of the 49 D. unisexualis mothers at the Du281 locus, yielding the mutation rate of 0.1428 events per germ line tissue. Sequencing of the mutant alleles has shown that most mutations occur via deletion or insertion of single microsatellite repeat being identical in all offspring of the family. This indicates that such mutations emerge at the early stages of embryogenesis. In this study we characterized single highly unstable (GATA)n containing locus in parthenogenetic lizard species D. unisexualis. Besides, we characterized various types of mutant alleles of this locus found in the D. unisexualis offspring of the first generation. Our data has shown that microsatellite mutations at highly unstable loci can make a significant contribution to population variability of parthenogenetic lizards.

Exogastrulation and interference with the expression of major yolk protein by estrogens administered to sea urchins

Although estrogens have been detected in some echinoderm species, their role is not clearly understood; so we examined the effects of estrogens administered to sea urchin embryos and larvae. A typical malformation was exogastrulation, induced by the exposure to ethynylestradiol (EER) in a defined period of 12 h from 12 h after fertilization (HAF). Morphogenesis for gastrulation was delayed in the treated embryos: protrusion of the archenteron started at 30 HAF when gastrulation had already finished in normal embryos. Exogastrulation induced by EER was cancelled by the antiestrogen chemical, ICI182,780. Feeding larvae were less sensitive to estrogens than those in early embryogenesis and, at certain concentrations, developed without abnormal morphology. The effect of estrogens was examined at the level of gene expression of the major yolk protein (MYP). MYP expression started during the larval stage and was suppressed by estrone at the six-armed stage, but not by beta-estradiol, and in later stage larvae, the expression was not affected by treatment with either estrogen. Estrogens affect sea urchins in the early stage of embryogenesis, leading to abnormal morphogenesis and interference with gene expression.

The Roles of E2F6 and DNA Methylation in the Regulation of Gene Expression During Germ Cell Development.

Mammalian primordial germ cells (PGCs), the embryonic progenitors of mature gametes, are specified in the extraembryonic mesoderm. These cells actively migrate to the developing genital ridge where they subsequently differentiate into gonocytes. Colonization of the gonad coincides with a widespread DNA demethylation event in the germ cell genome, leading to the upregulation of a subset of gonocyte-specific genes. DNA methylation contributes to silencing these gonocyte genes in migratory PGCs and in somatic tissues. Additionally, recent studies suggest a role for the transcription factor E2F6 in silencing germ cell-specific genes in somatic tissues. A subset of germ cell-specific genes, including Tuba3, Stag3, and Smc1β, is ectopically expressed in somatic tissues lacking E2f6. Furthermore, DNA hypomethylation is found at the promoter region of at least one of these genes, Tuba3, in E2f6-deficient tissues. These observations have led us to investigate a potential relationship between E2F6 and DNA methylation in the temporal regulation of gonocyte-specific gene expression in the developing germ line. We have focused our initial studies on the Tuba3 locus to gain further knowledge about the regulation of germ cell-specific, E2F6 regulated genes. Here we provide evidence that: 1) Members of the E2f family, including E2f6, are expressed in PGCs, 2) The Tuba3 promoter contains two CpG islands within 1kb of exon 1 and the downstream island is highly methylated in PGCs. Remarkably, unlike other known genes, this region of methylation appears to be stable through major stages of early embryogenesis and germ cell development, 3) Similar to several other gonocyte-specific genes, Tuba3 expression is activated as gonocytes differentiate and undergo genome-wide DNA demethylation. This activation coincides with demethylation of the upstream CpG island at the Tuba3 locus, however, the downstream island retains its methylation, 4) E2f6-deficient somatic tissue displays hypomethylation at both CpG islands on the Tuba3 promoter. From these data, we conclude that E2f6 is required for establishing or maintaining DNA methylation at the Tuba3 promoter in certain tissues. Current efforts are focused on creating an E2f6 gene trap mouse line to precisely determine the expression pattern of E2f6 in early development and its contribution to stability of DNA methylation in the germ line.

Giant café-au-lait macule in neurofibromatosis 1: A type 2 segmental manifestation of neurofibromatosis 1?

Type 2 segmental manifestation of autosomal dominant dermatoses refers to pronounced segmental lesions superimposed on the ordinary nonsegmental phenotype, indicating loss of heterozygosity occurring at an early stage of embryogenesis. We describe a 20-year-old Taiwanese woman with typical lesions of neurofibromatosis type 1 (NF1) in the form of characteristic café-au-lait spots, neurofibromas, axillary freckling and Lisch nodules. In addition, a giant garment-like or "bathing-trunk" café-au-lait macule involved the lower half of the trunk, the buttocks, and parts of the thighs, being superimposed on the ordinary smaller spots of NF1. This large café-au-lait macule may be best explained as an example of type 2 segmental NF1. A novel mutation (3009delG) in exon 23 was also identified in this patient, which has not yet been described in sporadic and familial NF1.

ORIGINAL ARTICLE: Antibody to the Chlamydia trachomatis 60 kDa Heat Shock Protein in Follicular Fluid and In Vitro Fertilization Outcome

ProblemThe association between 60 kDa Chlamydia trachomatis heat shock protein (CHSP60) antibodies and the etiology and outcomes of in vitro fertilization (IVF) outcomes is not well known.Method of studyA retrospective study with a double‐blind analysis of follicular fluid from 253 IVF patients for IgG antibodies to CHSP60.ResultsThe CHSP60 antibodies were detected in 74.1% of women without embryo implantation and in 47.9% of women with 1–3 implantations per IVF cycle (P = 0.0004). CHSP60 antibodies were detected in 69.5% of women with tubal occlusion and 49.7% of women with other causes of infertility (P = 0.01). CHSP60 antibody detection was unrelated to maternal age, number of oocytes collected, or percentage of oocytes fertilized.ConclusionDetection of IgG antibody to CHSP60 may indicate persistence of C. trachomatis in the upper genital tract with low implantation rates resulting from a chronic inflammatory reaction. Alternatively, as human hsp60 is expressed in early stage embryogenesis, a cross‐reacting antibody may induce destruction of the embryo.

In vivo long-term continuous observation of gene expression in zebrafish embryo nerve systems by using harmonic generation microscopy and morphant technology

Gene expression plays an important role in embryo development and organ function. Previous studies have shown that harmonic generation microscopy (HGM) can be used as a fluorescence signal-independent, minimally invasive method with a subcellular 3-D resolution and a penetration depth in the order of millimeters for long-term continuous imaging of vertebrate embryos. We show that it is ideal to combine in vivo HGM with the morphant technology for minimally invasive, long-term continuous observation of gene expression in the nervous system of vertebrate embryos. Since second- and third-harmonic generations (SHG, THG) are virtual-state-transition-based systems that depend only on the structure of the organisms, they are not temporally limited by the expression of the fluorescence proteins. We successfully identified the expression of the zarnt2a and the hif-1alpha, 2alpha, and 3alpha genes in the nervous system of zebrafish embryos with specific knockdown genes by microscopically observing the embryos from the early stages of embryogenesis. The results from a combination of the two different modalities, i.e., SHG microscopy and THG microscopy, successfully revealed the weak cell adhesion, cell apoptosis, nerve formation reduction, and neural tube distortion in the morphant zebrafish embryos.

EpCAM in morphogenesis

Embryonic development is one of the most complex biological phenomena that involves the appropriate expression and synchronized interactions of a plethora of proteins, including cell adhesion molecules (CAMs). Many members of the diverse family of CAMs have been shown to be critically involved in the correct execution of embryonic development. The Epithelial Cell Adhesion Molecule (EpCAM) is an atypical cell adhesion molecule originally identified as a marker for carcinoma. However, recent insights have revealed that EpCAM participates in not only cell adhesion, but also in proliferation, migration and differentiation of cells. All of these processes are known to be fundamental for morphogenesis. Here, we review the current literature that establishes EpCAM as a protein involved in morphogenesis, starting from the earliest stages of embryogenesis and ending in organogenesis. In addition, we provide directions for further elucidation of the role of EpCAM in embryogenesis.

ERK1 and ERK2 MAPK are key regulators of distinct gene sets in zebrafish embryogenesis

BackgroundThe MAPK signaling proteins are involved in many eukaryotic cellular processes and signaling networks. However, specific functions of most of these proteins in vertebrate development remain elusive because of potential redundancies. For instance, the upstream activation pathways for ERK1 and ERK2 are highly similar, and also many of their known downstream targets are common. In contrast, mice and zebrafish studies indicate distinct roles for both ERKs in cellular proliferation, oncogenic transformation and development. A major bottleneck for further studies is that relatively little is known of in vivo downstream signaling specific for these kinases.ResultsMicroarray based gene expression profiling of ERK1 and ERK2 knockdown zebrafish embryos at various stages of early embryogenesis resulted in specific gene expression signature sets that showed pronounced differences in gene ontology analyses. In order to predict functions of these genes, zebrafish specific in silico signaling pathways involved in early embryogenesis were constructed using the GenMAPP program. The obtained transcriptome signatures were analyzed in the BMP, FGF, Nodal and Wnt pathways. Predicted downstream effects of ERK1 and ERK2 knockdown treatments on key pathways responsible for mesendoderm development were confirmed by whole mount in situ hybridization experiments.ConclusionThe gene ontology analyses showed that ERK1 and ERK2 target common and distinct gene sets, confirming the difference in knockdown phenotypes and diverse roles for these kinases during embryogenesis. For ERK1 we identified specific genes involved in dorsal-ventral patterning and subsequent embryonic cell migration. For ERK2 we identified genes involved in cell-migration, mesendoderm differentiation and patterning.The specific function of ERK2 in the initiation, maintenance and patterning of mesoderm and endoderm formation was biologically confirmed.

Paternally and maternally transmitted GAL4 transcripts contribute to UAS transgene expression in early Drosophila embryos

The GAL4/UAS binary system with its recent modifications provides a powerful tool to study gene function in Drosophila enabling control over the timing, tissue specificity, and magnitude of gene expression. GAL4 expression during early embryonic stages has been well determined for certain driver lines, but for some of the commonly used in Drosophila research it is unknown, or partially determined. By monitoring the developmental kinetics of GAL4 expression and transgene transcription, we show that particular GAL4 drivers transiently direct ectopic expression of UAS-linked transgenes at early stages of embryogenesis in a GAL4- dependent manner via a mechanism that involves parental transmission of Gal4 transcripts.

Glucose-6-phosphate dehydrogenase Buenos Aires: A novel de novo missense mutation associated with severe enzyme deficiency

Objective: Glucose 6-phosphate dehydrogenase (G6PD) catalyzes the first committed steps in the pentose phosphate pathway: the generation of NADPH by this enzyme is essential for protection against oxidative stress. The human enzyme is in a dimer ↔ tetramer equilibrium and its stability depends on NADP + concentration. Herein, we report a case of a symptomatic baby affected by severe deficiency of G6PD activity due to a novel de novo genetic mutation (g1465C>T) in the thirteenth exon of its gene. Methods: Clinical, biochemical and genetic evaluations of the affected baby and his mother were performed. Results: We found the g1465C>T novel mutation, in the thirteenth exon of G6PD gene (named “G6PD Buenos Aires variant”). This g1465C>T mutation produce a P489S substitution at protein level. The P489S mutation was absent in his mother, suggesting that G6PD Buenos Aires resulted from a de novo mutation. Conclusions: The absence of mosaicism in the baby’s DNA (from saliva and blood samples) suggests that a de novo mutation event may occur in the very early stages in embryogenesis or in the mother’s germ cell lines.

New insights into purinergic receptor signaling in neuronal differentiation, neuroprotection, and brain disorders

Ionotropic P2X and metabotropic P2Y purinergic receptors are expressed in the central nervous system and participate in the synaptic process particularly associated with acetylcholine, GABA, and glutamate neurotransmission. As a result of activation, the P2 receptors promote the elevation of free intracellular calcium concentration as the main signaling pathway. Purinergic signaling is present in early stages of embryogenesis and is involved in processes of cell proliferation, migration, and differentiation. The use of new techniques such as knockout animals, in vitro models of neuronal differentiation, antisense oligonucleotides to induce downregulation of purinergic receptor gene expression, and the development of selective inhibitors for purinergic receptor subtypes contribute to the comprehension of the role of purinergic signaling during neurogenesis. In this review, we shall discuss the participation of purinergic receptors in developmental processes and in brain physiology, including neuron-glia interactions and pathophysiology.