Embryonic Events Research Articles

Comparative serum proteome analysis reveals potential early pregnancy-specific protein biomarkers in pigs

In this study, the comparative serum proteome profile of Day 5, 12 and 16 of gestation, representing three early embryonic events, namely formation, elongation and implantation of blastocysts, and non-pregnant control were explored by a label-free quantitation-based mass spectrometric approach to identify early pregnancy biomarkers in pigs. A total of 131 proteins were identified with respect to different groups, out of which 105 were found to be differentially expressed proteins (DEPs). Among the DEPs, 54 and 66 proteins were found to be up and downregulated respectively in early pregnancy groups (fold change >2) and the maximum number of upregulated proteins was observed in the Day 12 pregnancy stage. Functional classification and pathway analysis of the DEPs revealed involvement of most of the proteins in complement and coagulation cascades, metabolic processes and immune and inflammatory responses. Proteins such as glutathione peroxidise (GPX), pregnancy zone protein (PZP), thrombospondin-1 (THBS1), α-1-antitrypsin (AAT) and mannose-binding lectin C (MBLC) were differentially expressed during early pregnancy and actively involved in different pregnancy-related activities. To the best of our knowledge, this is the first report on comparative serum protein profiling of different early pregnancy stages in pigs and our results provide a set of proteins that can be used as potential biomarkers for early pregnancy diagnosis in pigs.

Double maternal-effect: duplicated nucleoplasmin 2 genes, npm2a and npm2b, with essential but distinct functions are shared by fish and tetrapods

BackgroundNucleoplasmin 2 (npm2) is an essential maternal-effect gene that mediates early embryonic events through its function as a histone chaperone that remodels chromatin. Recently, two npm2 (npm2a and npm2b) genes have been annotated in zebrafish. Thus, we examined the evolution of npm2a and npm2b in a variety of vertebrates, their potential phylogenetic relationships, and their biological functions using knockout models via the CRISPR/cas9 system.ResultsWe demonstrated that the two npm2 duplicates exist in a wide range of vertebrates, including sharks, ray-finned fish, amphibians, and sauropsids, while npm2a was lost in coelacanth and mammals, as well as some specific teleost lineages. Using phylogeny and synteny analyses, we traced their origins to the early stages of vertebrate evolution. Our findings suggested that npm2a and npm2b resulted from an ancient local gene duplication, and their functions diverged although key protein domains were conserved. We then investigated their functions by examining their tissue distribution in a wide variety of species and found that they shared ovarian-specific expression, a key feature of maternal-effect genes. We also demonstrated that both npm2a and npm2b are maternally-inherited transcripts in vertebrates, and that they play essential, but distinct, roles in early embryogenesis using zebrafish knockout models. Both npm2a and npm2b function early during oogenesis and may play a role in cortical granule function that impact egg activation and fertilization, while npm2b is also involved in early embryogenesis.ConclusionThese novel findings will broaden our knowledge on the evolutionary history of maternal-effect genes and underlying mechanisms that contribute to vertebrate reproductive success. In addition, our results demonstrate the existence of a newly described maternal-effect gene, npm2a, that contributes to egg competence, an area that still requires further comprehension.

Use of chick neural tube for optimizing the PSM and epithelial somites electroporation parameters: A detailed protocol.

Somite myogenesis is one of the crucial early embryonic events that lead to the formation of muscular tissue. A complex of dynamic gene regulatory networks masters this event. To understand and analyze these networks, there remains a genuine need for the use of a reproducible and highly efficient gene transfer technique. In vivo electroporation has proven to be amongst the best approaches in achieving a high level of gene transfer. However, unoptimized electroporation conditions can directly cause varying degrees of cellular damage which may induce abnormal embryonic development as well as changes in the endogenous gene expression. Presegmented mesoderm and epithelial somites are not easy to electroporate. Chick neural tube has served in many functional studies as an ideal experimental model organ which is both robust and easily manipulated. In the current detailed protocol, the neural tube was used as a tool to optimize the electroporation conditions which were subsequently applied in the electroporation of the presegmented mesoderm and epithelial somites. The protocol highlights important notes and hints that enable reproducible results and could be applied in the in vivo electroporation of other chick embryo tissues.

Development, Innervation, and Involution of a Transient Cement Gland in a Giant Danio (D. cf. Malabaricus): the Role of Retinoic Acid

Cement glands are transient evolutionarily adaptive adhesive organs. They mediate the attachment of a number aquatic larvae to substrates in their environments. There is significant interest in understanding the mechanisms that orchestrate their development and involution, as well as the chemical constituent of the biological adhesive they produce. We have identified and studied a cephalic and ectodermally derived cement gland in a giant danio (GD), by light microscopy, immune and lectin histochemistry. We hypothesized that early inductive embryonic events regulates the development of the gland, and that exogenous retinoic acid may disrupt its proper development. Our studies revealed that the cement gland is readily identified at hatching time, and mediates attachment of larvae until they are able to swim. At the swimming stage, the gland begins its involution and disappears by day 14. Using lectin histochemistry we demonstrate that the first differentiated goblet‐like cells that constitute the gland appear as early as 12 hours post fertilization. Using acetylated tubulin staining we also show that the gland is innervated by branches of the emerging trigeminal nerve. Lectin histochemistry also indicates that the adhesive substance within the gland is composed in part of various glycoconjugated products. Finally, treatment of GD embryos during gastrulation with exogenous retinoic acid (RA) leads to posteriorization and progressive disappearance of the gland in a dose‐dependent manner. This is the first detailed description of the development, maturation, its innervation and involution of the cement gland in a GD. Our results suggest that the cement gland is one of the earliest formed organs in the GD and that its proper development depends on maintenance of a stable RA gradient. Further studies are required for greater insights onto the function the gland.Support or Funding InformationDePauw FDC, SRF.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ploidy has little effect on timing early embryonic events in the haplo-diploid wasp Nasonia.

The nucleocytoplasmic (N/C) ratio plays a prominent role in the maternal-to-zygotic transition (MZT) in many animals. The effect of the N/C ratio on cell-cycle lengthening and zygotic genome activation (ZGA) has been studied extensively in Drosophila, where haploid embryos experience an additional division prior to completing cellularization and triploid embryos cellularize precociously by one division. In this study, we set out to understand how the obligate difference in ploidy in the haplodiploid wasp, Nasonia, affects the MZT and which aspects of the Drosophila MZT are conserved. While subtle differences in early embryonic development were observed in comparisons among haploid, diploid, and triploid embryos, in all cases embryos cellularize at cell cycle 12. When ZGA was inhibited, both diploid female, and haploid male, embryos went through 12 syncytial divisions and failed to cellularize before dying without further divisions. We also found that key players of the Drosophila MZT are conserved in Nasonia but have novel expression patterns. Our results suggest that zygotically expressed genes have a reduced role in determining the timing of cellularization in Nasonia relative to Drosophila, and that a stronger reliance on a maternal timer is more compatible with species where variations in embryonic ploidy are obligatory.

Generation of the germ layers along the animal-vegetal axis in Xenopus laevis.

After completion of gastrulation, typical vertebrate embryos consist of three cell sheets, called germ layers. The outer layer, the ectoderm, which produces the cells of the epidermis and the nervous system; the inner layer, the endoderm, producing the lining of the digestive tube and its associated organs (pancreas, liver, lungs etc.) and the middle layer, the mesoderm, which gives rise to several organs (heart, kidney, gonads), connective tissues (bone, muscles, tendons, blood vessels), and blood cells. The formation of the germ layers is one of the earliest embryonic events to subdivide multicellular embryos into a few compartments. In Xenopus laevis, the spatial domains of three germ layers are largely separated along the animal-vegetal axis even before gastrulation; ectoderm in the animal pole region; mesoderm in the equatorial region and endoderm in the vegetal pole region. In this review, we summarise the recent advances in our understanding of the formation of the germ layers in Xenopus laevis.

A toolkit for GFP-mediated tissue-specific protein degradation in C. elegans.

Proteins that are essential for embryo production, cell division and early embryonic events are frequently reused later in embryogenesis, during organismal development or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that uses expression of a fusion between a GFP-targeting nanobody and a SOCS-box containing ubiquitin ligase adaptor to target GFP-tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or with rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissue-specific protein ablation. We show that this approach works in multiple tissues - the epidermis, intestine, body wall muscle, ciliated sensory neurons and touch receptor neurons - where it recapitulates expected loss-of-function mutant phenotypes. The transgene toolkit and the strain set described here will complement existing approaches to enable routine analysis of the tissue-specific roles of C. elegans proteins.

The Identification of a Novel Mutant Allele of topoisomerase II in Caenorhabditis elegans Reveals a Unique Role in Chromosome Segregation During Spermatogenesis.

Topoisomerase II alleviates DNA entanglements that are generated during mitotic DNA replication, transcription, and sister chromatid separation. In contrast to mitosis, meiosis has two rounds of chromosome segregation following one round of DNA replication. In meiosis II, sister chromatids segregate from each other, similar to mitosis. Meiosis I, on the other hand, segregates homologs, which requires pairing, synapsis, and recombination. The exact role that topoisomerase II plays during meiosis is unknown. In a screen reexamining Caenorhabditis elegans legacy mutants isolated 30 years ago, we identified a novel allele of the gene encoding topoisomerase II, top-2(it7). In this study, we demonstrate that top-2(it7) males produce dead embryos, even when fertilizing wild-type oocytes. Characterization of early embryonic events indicates that fertilization is successful and sperm components are transmitted to the embryo. However, sperm chromatin is not detected in these fertilized embryos. Examination of top-2(it7) spermatogenic germ lines reveals that the sperm DNA fails to segregate properly during anaphase I of meiosis, resulting in anucleate sperm. top-2(it7) chromosome-segregation defects observed during anaphase I are not due to residual entanglements incurred during meiotic DNA replication and are not dependent on SPO-11-induced double-strand DNA breaks. Finally, we show that TOP-2 associates with chromosomes in meiotic prophase and that chromosome association is disrupted in the germ lines of top-2(it7) mutants.

Sensory evolution in blind cavefish is driven by early embryonic events during gastrulation and neurulation.

Natural variations in sensory systems constitute adaptive responses to the environment. Here, we compared sensory placode development in the blind cave-adapted morph and the eyed river-dwelling morph of Astyanax mexicanus Focusing on the lens and olfactory placodes, we found a trade-off between these two sensory components in the two morphs: from neural plate stage onwards, cavefish have larger olfactory placodes and smaller lens placodes. In a search for developmental mechanisms underlying cavefish sensory evolution, we analyzed the roles of Shh, Fgf8 and Bmp4 signaling, which are known to be fundamental in patterning the vertebrate head and are subtly modulated in space and time during cavefish embryogenesis. Modulating these signaling systems at the end of gastrulation shifted the balance toward a larger olfactory derivative. Olfactory tests to assess potential behavioral outcomes of such developmental evolution revealed that Astyanax cavefish are able to respond to a 105-fold lower concentration of amino acids than their surface-dwelling counterparts. We suggest that similar evolutionary developmental mechanisms may be used throughout vertebrates to drive adaptive sensory specializations according to lifestyle and habitat.

Transient transcription in the early embryo sets an epigenetic state that programs postnatal growth.

The potential for early embryonic events to program epigenetic states that influence adult physiology remains an important question in health and development. Using the imprinted Zdbf2 locus as a paradigm for the early programming of phenotypes, we demonstrate here that chromatin changes that occur in the pluripotent embryo can be dispensable for embryogenesis but instead signal essential regulatory information in the adult. The Liz (long isoform of Zdbf2) transcript is transiently expressed in early embryos and embryonic stem cells (ESCs). This transcription locally promotes de novo DNA methylation upstream of the Zdbf2 promoter, which antagonizes Polycomb-mediated repression of Zdbf2. Strikingly, mouse embryos deficient for Liz develop normally but fail to activate Zdbf2 in the postnatal brain and show indelible growth reduction, implying a crucial role for a Liz-dependent epigenetic switch. This work provides evidence that transcription during an early embryonic timeframe can program a stable epigenetic state with later physiological consequences.

Producción de embriones de búfalo por fertilización in vitro luego de la maduración de los ovocitos durante el transporte prolongado

El búfalo <i>(Bubalus bubalis)</i> es una especie con excelente adaptación a sectores inundables. El mejoramiento genético a través de superovulación y transferencia embrionaria ha tenido escasos resultados debido a dificultades en la detección de celo, pobre respuesta ovárica y limitada recuperación de embriones post-lavaje. La técnica de fertilización in vitro de embriones (FIV) es una biotecnología de gran impacto en el progreso genético. El objetivo del presente trabajo fue estudiar los eventos tempranos de la FIV, analizando la tasa de maduración y desarrollo embrionario post-fertilización de ovocitos madurados in vitro (IVM) durante el transporte. Ovocitos bovinos y bubalinos fueron obtenidos por punción folicular de ovarios post-mortem e IVM durante el transporte por un período de 18 h. Se realizó la FIV con toros de fertilidad comprobada, con una concentración en microgotas de inseminación de 3-4 x 106 espermatozoides motiles/ml por un período de 6 horas. Los embriones fueron cultivados en medio oviductal sintético SOFaa en incubadora gaseada y ambiente humidificado a 38,5ºC durante 9 días. Se evaluaron las tasas de IVM, clivaje (día 2 post-fertilización) y blastocisto (días 7 a 9). Los resultados fueron analizados estadísticamente utilizando Fischer’s Exact Test (p<0,05). No se observaron diferencias significativas en la tasa de maduración de ovocitos bubalinos de buena calidad respecto al control sin transporte (72 vs 88%), pero se registró una reducción significativa en la maduración de los ovocitos bubalinos de mala calidad (35%). Asimismo, se lograron producir los primeros embriones bubalinos luego de FIV, aunque las tasas de clivaje (34 vs 70 y 78%) y blastocisto (3 vs 27 y 31%) fueron significativamente menores en búfalos que en bovinos con y sin transporte, respectivamente. Los datos del presente trabajo constituirían el primer informe de FIV en búfalos y producción in vitro de embriones luego de IVM de ovocitos durante el transporte.

Blastomere biopsy for PGD delays embryo compaction and blastulation: a time-lapse microscopic analysis.

The purpose of the study was to explore the effect of blastomere biopsy for preimplantation genetic diagnosis (PGD) on the embryos' dynamics, further cleavage, development, and implantation. The study group included 366 embryos from all PGD treatments (September 2012 to June 2014) cultured in the EmbryoScope™ time-lapse monitoring system. The control group included all intracytoplasmic sperm injection (ICSI) embryos cultured in EmbryoScope™ until day 5 during the same time period (385 embryos). Time points of key embryonic events were analyzed with an EmbryoViewer™. Most (88%) of the embryos were biopsied at ≥8 cells. These results summarize the further dynamic development of the largest cohort of biopsied embryos and demonstrate that blastomere biopsy of cleavage-stage embryos significantly delayed compaction and blastulation compared to the control non-biopsied embryos. This delay in preimplanation developmental events also affected postimplantation development as observed when the dynamics of non-implanted embryos (known implantation data (KID) negative) were compared to those of implanted embryos (KID positive). Analysis of morphokinetic parameters enabled us to explore how blastomere biopsy interferes with the dynamic sequence of developmental events. Our results show that biopsy delays the compaction and the blastulation of the embryos, leading to a decrease in implantation.

A novel small molecule that disrupts a key event during the oocyte-to-embryo transition in C. elegans.

The complex cellular events that occur in response to fertilization are essential for mediating the oocyte-to-embryo transition. Here, we describe a comprehensive small-molecule screen focused on identifying compounds that affect early embryonic events in Caenorhabditis elegans We identify a single novel compound that disrupts early embryogenesis with remarkable stage and species specificity. The compound, named C22, primarily impairs eggshell integrity, leading to osmotic sensitivity and embryonic lethality. The C22-induced phenotype is dependent upon the upregulation of the LET-607/CREBH transcription factor and its candidate target genes, which primarily encode factors involved in diverse aspects of protein trafficking. Together, our data suggest that in the presence of C22, one or more key components of the eggshell are inappropriately processed, leading to permeable, inviable embryos. The remarkable specificity and reversibility of this compound will facilitate further investigation into the role and regulation of protein trafficking in the early embryo, as well as serve as a tool for manipulating the life cycle for other studies such as those involving aging.

Nuclease Footprints in Sperm Project Past and Future Chromatin Regulatory Events

Nuclear remodeling to a condensed state is a hallmark of spermatogenesis. This is achieved by replacement of histones with protamines. Regions retaining nucleosomes may be of functional significance. To determine their potential roles, sperm from wild type and transgenic mice harboring a single copy insert of the human protamine cluster were subjected to Micrococcal Nuclease-seq. CENTIPEDE, a hierarchical Bayesian model, was used to identify multiple spatial patterns, "footprints", of MNase-seq reads along the sperm genome. Regions predicted by CENTIPEDE analysis to be bound by a regulatory factor in sperm were correlated with genomic landmarks and higher order chromatin structure datasets to identify potential roles for these factors in regulating either prior or post spermatogenic, i.e., early embryonic events. This approach linked robust endogenous protamine transcription and transgene suppression to its chromatin environment within topologically associated domains. Of the candidate enhancer-bound regulatory proteins, Ctcf, was associated with chromatin domain boundaries in testes and embryonic stem cells. The continuity of Ctcf binding through the murine germline may permit rapid reconstitution of chromatin organization following fertilization. This likely reflects its preparation for early zygotic genome activation and comparatively accelerated preimplantation embryonic development program observed in mouse as compared to human and bull.

HES-Mediated Repression of Pten in Caenorhabditis elegans.

The hairy/enhancer-of-split (HES) group of transcription factors controls embryonic development, often by acting downstream of the Notch signaling pathway; however, little is known about postembryonic roles of these proteins. In Caenorhabditis elegans, the six proteins that make up the REF-1 family are considered to be HES orthologs that act in both Notch-dependent and Notch-independent pathways to regulate embryonic events. To further our understanding of how the REF-1 family works to coordinate postembryonic cellular events, we performed a functional characterization of the REF-1 family member, HLH-25. We show that, after embryogenesis, hlh-25 expression persists throughout every developmental stage, including dauer, into adulthood. Like animals that carry loss-of-function alleles in genes required for normal cell-cycle progression, the phenotypes of hlh-25 animals include reduced brood size, unfertilized oocytes, and abnormal gonad morphology. Using gene expression microarray, we show that the HLH-25 transcriptional network correlates with the phenotypes of hlh-25 animals and that the C. elegans Pten ortholog, daf-18, is one major hub in the network. Finally, we show that HLH-25 regulates C. elegans lifespan and dauer recovery, which correlates with a role in the transcriptional repression of daf-18 activity. Collectively, these data provide the first genetic evidence that HLH-25 may be a functional ortholog of mammalian HES1, which represses PTEN activity in mice and human cells.

An in vitro gastrulation model recapitulates the morphogenetic impact of pharmacological inhibitors of developmental signaling pathways.

Certain chemical agents act as teratogens, causing birth defects and fetal deaths when pregnant women are exposed to them. The establishment of in vitro models that recapitulate crucial embryonic events is therefore vital to facilitate screening of potential teratogens. Previously, we created a three-dimensional culture method for mouse P19C5 embryonal carcinoma stem cells that, when cultured as embryoid bodies, display elongation morphogenesis resembling gastrulation, which is the critical event resulting in the germ layers and major body axes. Determination of how well this in vitro morphogenesis represents in vivo gastrulation is essential to assess its applicability as well as to identify limitations of the model for detecting teratogenic agents. Here, we investigated the morphological and molecular characteristics of P19C5 morphogenesis using pharmacological agents that are known to cause abnormal patterning in the embryo in vivo by inhibiting major developmental signaling--e.g., involving Wnt, Nodal, Bone morphogenic protein (Bmp), Fibroblast growth factor (Fgf), Retinoic acid, Notch, and Hedgehog pathways. Inhibitors of Wnt, Nodal, Bmp, Fgf, and Retinoic acid signaling caused distinct changes in P19C5 morphogenesis that were quantifiable using morphometric parameters. These five inhibitors, plus the Notch inhibitor, also altered temporal expression profiles of developmental regulator genes in a manner consistent with the in vivo roles of the corresponding signaling pathways. In contrast, the Hedgehog inhibitor did not have any impact on the process, suggesting an absence of active Hedgehog signaling in these embryoid bodies. These results indicate that the P19C5 in vitro gastrulation model is a promising tool to screen for teratogenic agents that interfere with many of the key developmental signals.

The effect of temperature on the embryonic development of barramundi, the Australian strain of Lates calcarifer (Bloch) using current hatchery practices

Lates calcarifer (barramundi or Asian seabass) has been farmed since the 1970s, yet despite its widespread culture little has been documented on the species’ embryonic development and particularly how development relates to temperature. This is particularly the case for the Australian L. calcarifer genetic strain. Accordingly, embryonic development of fertilised barramundi eggs incubated at 26, 28, 30, 32, 34 and 36°C were followed from the time of incubation until hatching and the timing to reach key developmental stages and temperature-induced hatching success established. Eggs incubated at 26 and 36°C did not survive past the first two hours post-fertilisation. Development of the Australian strain of L. calcarifer was observed to proceed similarly to those documented from Asia, however, differences were observed in the timing of major embryonic events among the two strains. Incubation trials showed that eggs maintained at 30°C had the highest hatch rate (86.7%). The findings of this study are discussed and put in a commercial context with potential future research to further improve practices at the hatchery level.

A Histological Study of Postnatal Development of Clavicle Articular Ends

The clavicle is the first bone to begin the process of ossification and the last one to complete it. Whilst histological studies of the clavicle have been focused mainly on embryonic events, our study focused on post natal clavicle development. The objective of this study was to perform a qualitative description of the clavicle’s epiphyseal growth to further shed light on the postnatal ossification process. Histological studies performed on clavicles obtained from cadavers confirmed medial and lateral extremities as true physes. Unlike the development of other long bones, no secondary ossification centre was present at the acromial end. Furthermore, appearance of the sternal end was observed after the age of 18, as evidenced in clavicles from a 19-year old individual. The articular surface of the clavicle’s acromial end was fibrocartilage. No histological studies have been performed before describing postnatal development of clavicular ends. Our data confirm the absence of an acromial secondary ossification centre, the late development of a secondary ossification centre at the sternal end, and the presence of fibrocartilage at the acromial end.

Micropilar and embryonic events during hydration of Melanoxylon brauna Schott seeds

Abstract:Germination is a complex process that involves molecules properties that make up the cell walls, hydrolytic enzymes that break the bonds between the polymers and action of reactive oxygen substance. Melanoxylon braunais a forest species of high economic value. In order to evaluate the physiological and biochemical changes that occur in the embryonic axis during germination, fresh matter, length, activities of the enzymes pectin methylesterase, polygalacturonase, superoxide dismutase, catalase, peroxidase and hydrogen peroxide levels were quantified in the embryonic axis. Furthermore, in the micropyle area the composition of carbohydrates and micropyle physical resistance were evaluated with and without drying. During soaking, if there are increases in fresh matter and length of the embryonic axis, there is the same trend of polygalacturonase and pectin methylesterase enzymes. The hydrogen peroxide content was reduced during the soaking, as well as the puncture force of the micropylar area. It is concluded that the seed coat and the cotyledons are responsible for 90% of the water soaked by the seeds. The events in the micropyle and embryonic axis occur independently in the first 16 hours. The weakening of the micropyle features an elastic step and a plastic one. Enzymes pectin methylesterase and polygalacturonase act in cellular expansion of the embryonic axis.

A Fresh Look on T-Box Factor Action in Early Embryogenesis (T-Box Factors in Early Development)

T-Box transcription factors are expressed throughout the gestational period and coordinate a variety of embryonic events that enable proper development, from the first differentiation of embryonic and extraembryonic tissues until final organogenesis. Although the T-Box gene family comprises essential roles in early cellular differentiation, in adult tissues it has also been associated with cancer development. In spite of their common T-Box regulatory binding domain, T-Box family members utilize different cofactors and different spatiotemporal expression patterns to confer their specificity in diverse developmental processes. The earliest expression note of T-Box factors can be observed even before fertilization in primordial germ cells and just after zygotic gene activation (around the eight-cell blastomere stage). Thus, particularly the early stages of development are highly influenced by these key regulators in line with the notion that T-Box mutations lead to developmental disorders and even lethality. In this review, we summarize recently acquired findings on T-Box factors to provide a comprehensive overview on their role during early embryogenesis.