Early Stages Of Embryogenesis Research Articles

Subcellular metabolite and lipid analysis of Xenopus laevis eggs by LAESI mass spectrometry.

Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI), an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen), were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis.

SEUSS and SEUSS-LIKE 2 coordinate auxin distribution and KNOXI activity during embryogenesis.

In Arabidopsis, SEUSS (SEU) and SEUSS-LIKE 2 (SLK2) are components of the LEUNIG (LUG) repressor complex that coordinates various aspects of post-embryonic development. The complex also plays a critical role during embryogenesis, as seu slk2 double mutants have small, narrow cotyledons and lack a shoot apical meristem (SAM). Here we show that seu slk2 double mutant embryos exhibit delayed cotyledon outgrowth and that this is associated with altered PIN-FORMED1 (PIN1) expression and localisation during the early stages of embryogenesis. These observations suggest that SEU and SLK2 promote the transition to bilateral symmetry by modulating auxin distribution in the embryonic shoot. This study also shows that loss of SAM formation in seu slk2 mutants is associated with reduced expression of the class I KNOX (KNOXI) genes SHOOTMERISTEMLESS (STM), BREVIPEDICELLUS and KNAT2. Furthermore, elevating STM expression in seu slk2 mutant embryos was sufficient to restore SAM formation but not post-embryonic activity, while both SAM formation and activity were rescued when SLK2 expression was restored in either the cotyledons or boundary regions. These results demonstrate that SEU and SLK2 function redundantly to promote embryonic shoot development and likely act through a non-cell autonomous pathway to promote KNOXI activity.

Early markers are present in both embryogenesis pathways from microspores and immature zygotic embryos in cork oak, Quercus suber L.

BackgroundIn Quercus suber, cork oak, a Mediterranean forest tree of economic and social interest, rapid production of isogenic lines and clonal propagation of elite genotypes have been achieved by developing in vitro embryogenesis from microspores and zygotic embryos respectively. Despite its high potential in tree breeding strategies, due to their recalcitrancy, the efficiency of embryogenesis in vitro systems in many woody species is still very low since factors responsible for embryogenesis initiation and embryo development are still largely unknown. The search for molecular and cellular markers during early stages of in vitro embryogenesis constitutes an important goal to distinguish, after induction, responsive from non-responsive cells, and to elucidate the mechanisms involved in embryogenesis initiation for their efficient manipulation. In this work, we have performed a comparative analysis of two embryogenesis pathways derived from microspores and immature zygotic embryos in cork oak in order to characterize early markers of reprogrammed cells in both pathways. Rearrangements of the cell structural organization, changes in epigenetic marks, cell wall polymers modifications and endogenous auxin changes were analyzed at early embryogenesis stages of the two in vitro systems by a multidisciplinary approach.ResultsResults showed that early embryo cells exhibited defined changes of cell components which were similar in both embryogenesis in vitro systems, cellular features that were not found in non-embryogenic cells. DNA methylation level and nuclear pattern, proportion of esterified pectins in cell walls, and endogenous auxin levels were different in embryo cells in comparison with microspores and immature zygotic embryo cells from which embryos originated, constituting early embryogenesis markers.ConclusionsThese findings suggest that DNA hypomethylation, cell wall remodeling by pectin esterification and auxin increase are involved in early in vitro embryogenesis in woody species, providing new evidences of the developmental pattern similarity between both embryogenesis pathways, from microspores and immature zygotic embryos, in woody species.

Recovery of dicer-like 1-late flowering phenotype by miR172 expressed by the noncanonical DCL4-dependent biogenesis pathway.

MicroRNAs (miRNAs) act as down-regulators of gene expression, and play a dominant role in eukaryote development. In Arabidopsis thaliana, DICER-LIKE 1 (DCL1) is the main processor in miRNA biogenesis, and dcl1 mutants show various developmental defects at the early stage of embryogenesis or at gamete formation. However, miRNAs responsible for the respective developmental stages of the dcl1 defects have not been identified. Here, we developed a DCL1-independent miRNA expression system using the unique DCL4-dependent miRNA, miR839. By replacing the mature sequence in the miR839 precursor sequence with that of miR172, one of the most widely conserved miRNAs in angiosperms, we succeeded in expressing miR172 from a chimeric miR839 precursor in dcl1-7 plants and observed the repression of miR172 target gene expression. In parallel, the DCL4-dependent miR172 expression rescued the late flowering phenotype of dcl1-7 by acceleration of flowering. We established the DCL1-independent miRNA expression system, and revealed that the reduction of miR172 expression is responsible for the dcl1-7 late flowering phenotype.

Cover Picture: Eur. J. Immunol. 6'14

This month's cover features a macro confocal macroscopy image of a mouse E10.5 embryo. It was stained with Hoechst 33342 (blue), and paternally inherited EGFP fluorescence (green), which are driven from the β‐actin promoter and enable to distinguish hematopoietic cells of maternal and embryonic origin. The image's background shows the expression profiles of different types of macrophages. These images were provided by Balounova et al. (pp. 1491–1502), who describe in this issue that TLRs are functionally expressed on yolk sac–derived macrophages during early mouse embryogenesis and contribute to inflammation‐mediated regulation of embryonic homeostasis via iron metabolism. Taken together, the results show that TLRs are involved in the maintenance of organismal homeostasis through the entire ontogenetic development, including early stages of embryogenesis.

Specific features of embryonic development of whitefishes (Coregonidae) as representatives of the “pagophilous” ecological group of reproduction

Studies of reproduction and embryonic development in six species of coregonid fishes have revealed the ability of their fertilized eggs to develop normally while being embedded in the ice of a spawning water body (optionally). Such ability is facilitated by the extremely low respiratory activity of embryos at early stages of embryogenesis (from the stage of fission to the stage of organogenesis). The low level of oxygen consumption and carbon dioxide emission is an adaptation to the low diffusion gas permeability of the ice. The main factor controlling the rate of coregonids’ embryonic development is not temperature but the intensity and periodicity of isolation. Without sunlight-an obligatory external factor-normal development is just not possible, Under experimental conditions, developing in the water at near zero temperature, or in the ice, normal morphogenesis of Arctic cisco and Sevan whitefish embryos was observed at an illumination of 50–300 lx. Hemoproteid cytochrome β560, the pigment that has been discovered in the water-soluble part of coregonids oocyte yolk and is treated as a biochemical marker for eggs of the family Coregonidae, in all likelihood performs protective (antioxidant) functions, preventing spontaneous oxidation of embryo’s fatty inclusions. In the oxygen shortage inside the ice envelope, cytochrome β560 probably sets the conditions for oxidation processes of embryo’s tissue respiration. Spherome, being kept until the time of hatching, acts as a temporary hydrostatic organ and ensures larvae buyoancy at the stage of postembryonic metamorphosis. It also serves as an energy store after the downstream migration of larvae from the spawning areas until their shift to exogenous feeding on zooplankton. Conforming to the ecological traits of reproduction and development, and also to revealed morphogenetic, physiological, and biochemical features, it is proposed to ascribe all of the currently known 26 species of whitefishes to the “pagophilous” ecological group.

Cover Picture: Eur. J. Immunol. 5'14

This month's cover features amacro confocal macroscopy image of a mouse E10.5 embryo. It was stained with Hoechst 33342 (blue), and paternally inherited EGFP fluorescence (green), which are driven from the β‐actin promoter and enable to distinguish hematopoietic cells of maternal and embryonic origin. The image's background shows the expression profiles of different types of macrophages. These images were provided by Balounova et al. (pp. 1491–1502), who describe in this issue that TLRs are functionally expressed on yolk sac–derived macrophages during early mouse embryogenesis and contribute to inflammation‐mediated regulation of embryonic homeostasis via iron metabolism. Taken together, the results show that TLRs are involved in the maintenance of organismal homeostasis through the entire ontogenetic development, including early stages of embryogenesis.

Huntingtin protein is essential for mitochondrial metabolism, bioenergetics and structure in murine embryonic stem cells

Mutations in the Huntington locus (htt) have devastating consequences. Gain-of-poly-Q repeats in Htt protein causes Huntington׳s disease (HD), while htt−/− mutants display early embryonic lethality. Despite its importance, the function of Htt remains elusive. To address this, we compared more than 3700 compounds in three syngeneic mouse embryonic stem cell (mESC) lines: htt−/−, extended poly-Q (Htt-Q140/7), and wild-type mESCs (Htt-Q7/7) using untargeted metabolite profiling. While Htt-Q140/7 cells did not show major differences in cellular bioenergetics, we find extensive metabolic aberrations in htt−/− mESCs, including (i) complete failure of ATP production despite preservation of the mitochondrial membrane potential; (ii) near-maximal glycolysis, with little or no glycolytic reserve; (iii) marked ketogenesis; (iv) depletion of intracellular NTPs; (v) accelerated purine biosynthesis and salvage; and (vi) loss of mitochondrial structural integrity. Together, our findings reveal that Htt is necessary for mitochondrial structure and function from the earliest stages of embryogenesis, providing a molecular explanation for htt−/− early embryonic lethality.

A bioinformatic analysis of the vertebrate Twist protein family (778.5)

Twist protein family members are expressed in different tissues during early stages of embryogenesis and their presence is essential for proper development and survival. Though the highly conserved basic helix‐loop‐helix (bHLH) and carboxy‐terminal domains of Twist proteins have received a lot of attention, the amino‐terminal region and the evolution of such proteins merit further study, particularly among mammals. We analyzed the conservation of the amino‐terminal region of different vertebrate Twist proteins by sequence comparisons. We identified two putative de novo motifs (SSSPVSP and SEEE), specifically in mammals. In addition, a number of amino acid substitutions present in the nuclear localization signals (NLSs) of a few species demonstrate that there could be other possible residues influencing the nuclear localization of such proteins, with the G residue of the second NLS being a potential target. By phylogenetic analysis, we investigated the origin and evolution of the two paralog twist proteins. First, a gene duplication event occurred to produce the two twist paralogs. Second, among the two twist paralog branches, twist1 is the most distant in vertebrates, suggesting it evolved first due an abrupt increase of substitutions in a short period of time due to functional divergence. Also, in mammals, twist1 seems to have a higher rate of evolution than twist2 since only twist1 sequences contain two glycine‐rich motifs not present in twist2. Our findings shed light on the relationship of twist1 and twist2 paralogs among mammals in vertebrates.Grant Funding Source: Supported by NIH grants No. T36GM008789 and P41 RR06009

Coming together is a beginning: The making of an intervertebral disc

The intervertebral disc (IVD) is a complex fibrocartilaginous structure located between the vertebral bodies that allows for movement and acts as a shock absorber in our spine for daily activities. It is composed of three components: the nucleus pulposus (NP), annulus fibrosus, and cartilaginous endplate. The characteristics of these cells are different, as they produce specific extracellular matrix (ECM) for tissue function and the niche in supporting the differentiation status of the cells in the IVD. Furthermore, cell heterogeneities exist in each compartment. The cells and the supporting ECM change as we age, leading to degenerative outcomes that often lead to pathological symptoms such as back pain and sciatica. There are speculations as to the potential of cell therapy or the use of tissue engineering as treatments. However, the nature of the cells present in the IVD that support tissue function is not clear. This review looks at the origin of cells in the making of an IVD, from the earliest stages of embryogenesis in the formation of the notochord, and its role as a signaling center, guiding the formation of spine, and in its journey to become the NP at the center of the IVD. While our current understanding of the molecular signatures of IVD cells is still limited, the field is moving fast and the potential is enormous as we begin to understand the progenitor and differentiated cells present, their molecular signatures, and signals that we could harness in directing the appropriate in vitro and in vivo cellular responses in our quest to regain or maintain a healthy IVD as we age.

Cooperative interaction of Etv2 and Gata2 regulates the development of endothelial and hematopoietic lineages

Regulatory mechanisms that govern lineage specification of the mesodermal progenitors to become endothelial and hematopoietic cells remain an area of intense interest. Both Ets and Gata factors have been shown to have important roles in the transcriptional regulation in endothelial and hematopoietic cells. We previously reported Etv2 as an essential regulator of vasculogenesis and hematopoiesis. In the present study, we demonstrate that Gata2 is co-expressed and interacts with Etv2 in the endothelial and hematopoietic cells in the early stages of embryogenesis. Our studies reveal that Etv2 interacts with Gata2 in vitro and in vivo. The protein–protein interaction between Etv2 and Gata2 is mediated by the Ets and Gata domains. Using the embryoid body differentiation system, we demonstrate that co-expression of Gata2 augments the activity of Etv2 in promoting endothelial and hematopoietic lineage differentiation. We also identify Spi1 as a common downstream target gene of Etv2 and Gata2. We provide evidence that Etv2 and Gata2 bind to the Spi1 promoter in vitro and in vivo. In summary, we propose that Gata2 functions as a cofactor of Etv2 in the transcriptional regulation of mesodermal progenitors during embryogenesis.

Developmental roles of D-bifunctional protein-A zebrafish model of peroxisome dysfunction.

The peroxisome is an intracellular organelle that responds dynamically to environmental changes. Various model organisms have been used to study the roles of peroxisomal proteins in maintaining cellular homeostasis. By taking advantage of the zebrafish model whose early stage of embryogenesis is dependent on yolk components, we examined the developmental roles of the D-bifunctional protein (Dbp), an essential enzyme in the peroxisomal β-oxidation. The knockdown of dbp in zebrafish phenocopied clinical manifestations of its deficiency in human, including defective craniofacial morphogenesis, growth retardation, and abnormal neuronal development. Overexpression of murine Dbp rescued the morphological phenotypes induced by dbp knockdown, indicative of conserved roles of Dbp during zebrafish and mammalian development. Knockdown of dbp impaired normal development of blood, blood vessels, and most strikingly, endoderm-derived organs including the liver and pancreas - a phenotype not reported elsewhere in connection with peroxisome dysfunction. Taken together, our results demonstrate for the first time that zebrafish might be a useful model animal to study the role of peroxisomes during vertebrate development.

Buckling of a Physically-Constrained Growing Epithelium

In growing tissue, cell proliferation and tensile/compressive forces generated by growth are essential for controlling shape and size of organs during development (Mammoto T and Ingber DE 2010). One example of a system where these parameters may have a critical influence is an early stage of embryogenesis, namely gastrulation. During gastrulation, the spherically shaped epithelium invaginates and forms a tube in the lumena of the embryo that later will be the digestive tube. The invagination of the epithelia cells is promoted by genetic factors. However, recent theoretical models proposed that compression of the embryo in a spherical shell could promote gastrulation by inward buckling (Hannezo E et al. 2011, Tamulonis C et al. 2011). As embryo is not easily amenable to forces and shape measurements, to study the coupling between mechanical forces/confinement and epithelium shape and proliferation, we create an epithelial spherical monolayer in the elastic shell using a technique of cell encapsulation in the shell made out of alginate. As the elastic modulus of alginate is known, small deformation of the shell will allow us to measure compression within the epithelium (Alessandri K et al. 2013). We then follow the growth of the epithelium through time and observe its buckling. We address the questions of how the pressure builds up in the growing epithelia enclosed in a confined environment, what are the cell tension fields in the epithelial monolayer under these conditions, how the cells adapt to the pressure and the shape and rigidity of the confinement, and how the whole monolayer react to the accumulated lateral pressure due to cell proliferation. These will let me decipher how epithelium deformation is driven by cell proliferation and tension during development.

Analysis of NUAK1 and NUAK2 expression during early chick development reveals specific patterns in the developing head

Several human diseases are associated with the NUAK1 and NUAK2 genes. These genes encode kinases, members of the AMPK-related kinases (ARK) gene family. Both NUAK1 and NUAK2 are known targets of the serine threonine kinase LKB1, a tumor suppressor involved in regulating cell polarity. While much is known about their functions in disease, their expression pattern in normal development has not been extensively studied. Here, we present the expression patterns for NUAK1 and NUAK2 in the chick during early-stage embryogenesis, until day 3 (Hamburger and Hamilton stage HH20). Several embryonic structures, in particular the nascent head, showed distinct expression levels. NUAK1 expression was first detected at stage HH6 in the rostral neural folds. It was then expressed (HH7-11) throughout the encephalalon, predominantly in the telencephalon and mesencephalon. NUAK1 expression was also detected in the splanchnic endoderm area at HH8-10, and in the vitellin vein derived from this area, but not in the heart. NUAK2 expression was first detected at stage HH6 in the neural folds. It was then found throughout the encephalon at stage HH20. Particular attention was paid in this study to the dorsal ectoderm at stages HH7 and HH8, where a local deficit or accumulation of NUAK2 mRNA were found to correlate with the direction of curvature of the neural plate. This is the first description of NUAK1 and NUAK2 expression patterns in the chick during early development; it reveals non-identical expression profiles for both genes in neural development.

Course of hatch and developmental changes in thyroid hormone concentration in blood of chicken embryo following in ovo riboflavin supplementation

The influence of riboflavin on the function of the hypothalamo-pituitary-thyroid axis during chicken embryogenesis is poorly understood. Therefore, examination of the effects of in ovo riboflavin supplementation on the possible linkage between the thyroid gland function and chick embryonic development seems to be interesting. Eggs on the sixth day of incubation were injected with 0 (control), 60, or 600 µg of riboflavin/egg. Blood samples were collected on the 12th, 15th, 18th, and 20th days of embryogenesis. Thyroxine (T4) and triiodothyronine (T3) concentrations in plasma samples were determined by radioimmunoassay method. The time of external pipping and hatching of each chick as well as the body weights of sampled and newly hatched chicks were recorded. Generally, riboflavin supplied in ovo did not affect chicken embryo mortality and hatchability; however, a dose of 600 µg of riboflavin/egg had a tendency to reduce embryo body weight. Chicks exposed to 60 µg of riboflavin/egg hatched 3.7 h earlier in comparison with controls and were characterized by a higher synchronization degree of hatching. Both applied doses of riboflavin significantly elevated T4 concentrations in blood plasma of the chicken embryos; however, on day 20 of embryogenesis, both applied doses of riboflavin decreased T3 levels in blood circulation. The data presented here suggest that riboflavin supplementation at the early stages of embryogenesis markedly affects embryonic development and influences thyroid hormone metabolism during the second half of embryogenesis.

Automated annotation of developmental stages of Drosophila embryos in images containing spatial patterns of expression

Motivation: Drosophila melanogaster is a major model organism for investigating the function and interconnection of animal genes in the earliest stages of embryogenesis. Today, images capturing Drosophila gene expression patterns are being produced at a higher throughput than ever before. The analysis of spatial patterns of gene expression is most biologically meaningful when images from a similar time point during development are compared. Thus, the critical first step is to determine the developmental stage of an embryo. This information is also needed to observe and analyze expression changes over developmental time. Currently, developmental stages (time) of embryos in images capturing spatial expression pattern are annotated manually, which is time- and labor-intensive. Embryos are often designated into stage ranges, making the information on developmental time course. This makes downstream analyses inefficient and biological interpretations of similarities and differences in spatial expression patterns challenging, particularly when using automated tools for analyzing expression patterns of large number of images.Results: Here, we present a new computational approach to annotate developmental stage for Drosophila embryos in the gene expression images. In an analysis of 3724 images, the new approach shows high accuracy in predicting the developmental stage correctly (79%). In addition, it provides a stage score that enables one to more finely annotate each embryo so that they are divided into early and late periods of development within standard stage demarcations. Stage scores for all images containing expression patterns of the same gene enable a direct way to view expression changes over developmental time for any gene. We show that the genomewide-expression-maps generated using images from embryos in refined stages illuminate global gene activities and changes much better, and more refined stage annotations improve our ability to better interpret results when expression pattern matches are discovered between genes.Availability and implementation: The software package is available for download at: http://www.public.asu.edu/∼jye02/Software/Fly-Project/.Contact: jieping.ye@asu.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Silencing of Maternal Heme-binding Protein Causes Embryonic Mitochondrial Dysfunction and Impairs Embryogenesis in the Blood Sucking Insect Rhodnius prolixus

The heme molecule is the prosthetic group of many hemeproteins involved in essential physiological processes, such as electron transfer, transport of gases, signal transduction, and gene expression modulation. However, heme is a pro-oxidant molecule capable of propagating reactions leading to the generation of reactive oxygen species. The blood-feeding insect Rhodnius prolixus releases enormous amounts of heme during host blood digestion in the midgut lumen when it is exposed to a physiological oxidative challenge. Additionally, this organism produces a hemolymphatic heme-binding protein (RHBP) that transports heme to pericardial cells for detoxification and to growing oocytes for yolk granules and as a source of heme for embryo development. Here, we show that silencing of RHBP expression in female fat bodies reduced total RHBP circulating in the hemolymph, promoting oxidative damage to hemolymphatic proteins. Moreover, RHBP knockdown did not cause reduction in oviposition but led to the production of heme-depleted eggs (white eggs). A lack of RHBP did not alter oocyte fecundation. However, produced white eggs were nonviable. Embryo development cellularization and vitellin yolk protein degradation, processes that normally occur in early stages of embryogenesis, were compromised in white eggs. Total cytochrome c content, cytochrome c oxidase activity, citrate synthase activity, and oxygen consumption, parameters that indicate mitochondrial function, were significantly reduced in white eggs compared with normal dark red eggs. Our results showed that reduction of heme transport from females to growing oocytes by RHBP leads to embryonic mitochondrial dysfunction and impaired embryogenesis.

MORPHOLOGICAL FEATURES AND LOCALIZATION OF VIRCHOW-ROBEN SPACES IN BRAIN

Virchow-Roben spaces (EPVS) are canals that surround intracerebral blood vessels. They are formed at the early stages of embryogenesis due to ingrowth into the nervous tissue of blood vessels that draw in the vascular folium of soft brain tunic. In the present scientific articles there are a lot of contradictions about localization of these spaces around capillaries, venules and veins, about their histological structure in the different areas of central nervous system. EPVSs are known to play a big role in the circulation of cerebral spinal fluid, in immune regulation, and are a compound part of the hematologic encephalitic barrier, which become wider in various pathologic processes. The study of the localization and structure of these spaces with the use of up-to-date methods of research will help to understand their function and clinical significance.

The Central Nervous System of Heterosymelic Individuals of the Spider Tegenaria atrica

This paper presents the results of research on the nervous system in individuals of Tegenaria atrica with heterosymely of walking legs, a pedipalp and the first walking leg. Temperatures alternating between 14 degrees and 32 degrees C every 12 hours were applied as a teratogenic factor in the early stage of embryogenesis. From the thus obtained specimens, histological sections were taken using the paraffin method and then stained. Analysis of the nervous system in individuals with partial heterosymely of walking legs showed no significant changes in the structure of subesophageal nervous mass. In most of the studied individuals with partial and total heterosymely of a pedipalp and walking leg no changes were observed in the relevant parts of the nervous system either, although in two cases a fusion of nerve ganglia was noted, from which the nerves branched off towards the conjoined legs.

Symely, a seldom occurring developmental anomaly in the spider Tegenaria atrica

This article presents the results of teratological studies on the spider Tegenaria atrica. Temperatures alternating between 14°C and 32°C every 12 h were applied as a teratogenic factor in the early stage of embryogenesis. This treatment resulted in specimens with various developmental malformations, including symely of the walking legs. Symely, which is one of the anomalies that results from disturbances during the formation of metameres to the germ band, usually affects the third and fourth pair of walking legs. Fusion of legs from the same pair entails changes in the symmetry of the whole body, particularly in the structure of prosoma and its position in relation to the opisthosoma. These changes are the cause of the exceptionally high mortality among embryos.