Early Stages Of Embryogenesis Research Articles

Identification and Analysis of Differentially Expressed Genes During Seed Development Using Suppression Subtractive Hybridization (SSH) in Phaseolus vulgaris

Interspecific hybridization in the genus Phaseolus, conducted to introgress desired traits into common bean (Phaseolus vulgaris L.), leads to the abortion of immature embryos, usually at early developmental stages. Little is known about the physiological responses of embryo dysfunction in P. vulgaris during the early stages of embryogenesis and the genes that are involved in these responses. Identification of the genes involved in Phaseolus embryogenesis may provide information that will help to understand the molecular basis of Phaseolus embryo dysfunction. To investigate the genes expressed during Phaseolus seed development, we constructed a suppression subtractive hybridization (SSH) library using cDNA from abortive seed development as the driver and those from normal seed development as tester. The differentially expressed cDNA fragments were identified by differential screening. We identified 72 unique ESTs of which we selected 12 candidates on the basis of their redundancy. These candidates were subjected to a validation procedure based on the study of their expression level by real-time PCR. Sequence analysis revealed that most of the differentially expressed genes are related to metabolism and regulation such as protein synthesis. Some genes also encoded transcription factors. These genes showed high mRNA transcript levels in seed tissues and little or no expression in other tissues (root, stem, flower, leaf, and cotyledon). Seven genes were chosen and their expression profile during seed development in P. vulgaris was analyzed by real-time PCR using RNA preparations originating from different seed development stages. This study revealed hitherto unknown genes putatively involved in dicotyledonous embryogenesis, which serve as a starting point for understanding Phaseolus embryogenesis.

Klippel Trenaunay syndrome

Alias: Angio-osteohypertophy syndrome Note: Various disorders with varying combination of limb hypertrophy, vascular lymphatic malformation and other features like naevi include Klippel Trenaunay syndrome (KTS), Sturge Weber syndrome, Proteus syndrome and isolated hemihypertrophy are clinically distinct entities and need to be approp-riately diagnosed. Inheritance: Most cases of KTW syndrome are sporadic, through familial aggregations are reported. However, a strong doubt has been raised about familial cases as in most of these cases the family members may have isolated vascular naevi or varicose veins which are not uncommon in general population. Presence of cases with variable severity and locations of manifestations in family members can be explained by paradominant inheritance. It means a mutation is passed from one generation to another and heterozygous individuals are normal unless the other copy of the gene gets mutated. The mutation in other copy of the gene in early stages of embryogenesis might be giving rise to a clonal population of cells with homozygous for KTS mutation. This also explains mosaic pattern of lesions.

Innate and adaptive immunity in teleost fish: a review

  The immune system of fish is very similar to vertebrates, although there are some important differences. Fish are free-living organisms from the embryonic stage of life in their aquatic environment. They have mechanisms to protect themselves from a wide variety of microorganisms. Consequently, fish rely on their innate immune system for an extended period of time, beginning at the early stages of embryogenesis. The components of the innate immune response are divided into physical, cellular and humoral factors and include humoral and cellular receptor molecules that are soluble in plasma and other body fluids. The lymphoid organs found in fish include the thymus, spleen and kidney. Immunoglobulins are the principal components of the immune response against pathogenic organisms. Immunomodulatory products, including nucleotides, glucans and probiotics, are increasingly used in aquaculture production. The use of these products reduces the need for therapeutic treatments, enhances the effects of vaccines and, in turn, improves the indicators of production. The aim of this review is to provide a review of the immune system in fish, including the ontogeny, mechanisms of unspecific and acquired immunity and the action of some immunomodulators.

Molecular dissection of an adaptive epigenetic memory mechanism in norway spruce

Molecular dissection of an adaptive epigenetic memory mechanism in norway spruce

Important processes during differentiation and early development of somatic embryos of Norway spruce as revealed by changes in global gene expression

The aim of this study has been to identify important processes that regulate early stages of embryo development in conifers. Somatic embryogenesis in Picea abies has become a model system for studying embryology in conifers, providing a well-characterized sequence of developmental stages, resembling zygotic embryogeny, which can be synchronized by specific treatments, making it possible to collect a large number of somatic embryos at specific developmental stages. We have used this model to analyze global changes in gene expression during early stages of embryo development by generating an expression profile of 12,536 complementary DNA clones. This has allowed us to identify molecular events regulating putative processes associated with pattern formation during the earliest stages of embryogenesis which have not been identified on the molecular level in conifers before. We recognize notable changes in the expression of genes involved in regulating auxin biosynthesis and auxin response, gibberellin-mediated signaling, signaling between the embryo and the female gametophyte, tissue specification including the formation of boundary regions, and the switch from embryonic to vegetative development. In addition, our results confirm the involvement of previously described processes, including stress, differentiation of a protoderm, and programmed cell death.

Molecular analysis and its expression of a pou homeobox protein gene during development and in response to salinity stress from brine shrimp, Artemia sinica

Brine shrimps of the genus Artemia are aquatic species of economic importance because of their important significance to aquaculture and are used as a model species in physiology and developmental biology. Research on Artemia POU homeobox gene function will enhance our understanding of the physiological and developmental processes of POU homeobox gene in animals. Herein, a full-length cDNA encoding an Artemia POU homeobox protein gene 1 ( APH-1) from Artemia sinica (designated as As-APH-1) was cloned and characterized by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) method. The As-APH-1 gene encoded a protein of 388 amino acid polypeptide with a calculated molecular mass of 42.85 kDa and an isoelectric point of 6.90 and the protein belongs to the POU III family. Multiple sequence alignments revealed that A. sinica As-APH-1 protein sequence shared a conserved POU homeobox domain with other species. The early and persistent expression of As-APH-1 in the naupliar stages by semi-quantitative RT-PCR and whole-mount embryonic immunohistochemistry suggest that As-APH-1 functions very early in the salt gland and may be required continuously in this organ. Later in development, expression of As-APH-1 begins to dramatically decrease and disappear in salt gland of the sub-adult Artemia. In addition, we also discovered that As-APH-1 increased obviously as the salinity increased, indicating that As-APH-1 might be used as a good indicator of salinity stress. In summary, we are the first to identify the As-APH-1 gene and to determine its gene expression patterns in early embryogenesis stages and in different salinity stress in brine shrimp, A. sinica. The result of expression of As-APH-1 affected by salinity changes will provide us further understanding of the underlying mechanisms of osmoregulation in Artemia early embryonic development.

Atrial natriuretic factor in the developing heart: a signpost for cardiac morphogenesis

The developing heart forms during the early stages of embryogenesis, and misregulated heart development results in congenital heart defects (CHDs). To understand the molecular basis of CHDs, a deep understanding of the morphological and genetic basis of heart development is necessary. Atrial Natriuretic Factor (ANF) is an important and extremely sensitive marker for specific regions of the developing heart, as well as for disturbances in the patterning of the heart. This review summarizes the dynamic expression of ANF in the developing heart and its usefulness in understanding the early molecular defects underlying CHDs.

Cell–cell communication and signalling pathways within the ovule: from its inception to fertilization

Cell-cell communication pervades every aspect of the life of a plant. It is particularly crucial for the development of the gametes and their subtle interaction leading to double fertilization. The ovule is composed of a funiculus, one or two integuments, and a gametophyte surrounded by nucellus tissue. Proper ovule and embryo sac development are critical to reproductive success. To allow fertilization, the correct relative positioning and differentiation of the embryo sac cells are essential. Integument development is also intimately linked with the normal development of the female gametophyte; the sporophyte and gametophyte are not fully independent tissues. Inside the gametophyte, numerous signs of cell-cell communication take place throughout development, including cell fate patterning, fertilization and the early stages of embryogenesis. This review highlights the current evidence of cell-cell communication and signalling elements based on structural and physiological observations as well as the description and characterization of mutants in structurally specific genes. By combining data from different species, models of cell-cell interactions have been built, particularly for the establishment of the germline, for the progression through megagametogenesis and for double fertilization.

Egg formation and the early embryonic development of Aspidogaster limacoides Diesing, 1835 (Aspidogastrea: Aspidogastridae), with comments on their phylogenetic significance

Ultrastructural aspects of the early embryonic development of the aspidogastrean Aspidogaster limacoides are described and their phylogenetic implications discussed. Whereas the proximal regions of the uterine lumen usually contain unembryonated eggs or eggs with early embryos, the posterior or distal regions of the uterus are filled with eggs containing a fully-developed cotylocidium. The eggs of A. limacoides can be classified as polylecithal due to the presence of numerous vitellocytes which accompany each fertilized oocyte or ovum during egg formation. The results of the study are described in details under six headings: (1) general characteristics of the intrauterine eggs; (2) eggshell and operculum formation; (3) unembryonated eggs; (4) zygote formation and early cleavage divisions; (5) embryonic envelope formation; and (6) early degeneration or apoptosis of some blastomeres. The late differentiation of the operculum, possible functions of GER-bodies, and the early degeneration of vitellocytes and some blastomeres in this species are compared, drawn and discussed with corresponding observations reported for other parasitic Platyhelminthes. The most important differences are apparent in the number of egg envelopes and their mode of formation in A. limacoides compared with previous reports for both digeneans and cestodes. The results of the present TEM study indicate that the three macromeres, resulting from two cleavage divisions, take part in the formation of a single embryonic outer envelope in A. limacoides, and that this takes place at a very early stage of embryogenesis. Their fusion results in the formation of a single continuous cytoplasmic layer surrounding the early embryo, which is composed of only a small number of undifferentiated blastomeres. The early separation of the macromeres may indicate an equal cleavage pattern. These results suggest that the systematic position of the Aspidogastrea among the Platyhelminthes still remains somewhat equivocal, and indicate the need for more studies on the embryonic development, larval morphogenesis and molecular phylogeny for the elucidation of the relationships between this enigmatic group and related taxa.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on steroid concentrations in blood and gonads of chicken embryo

The aim of the present study was to examine the effect of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) injected at the early stage of incubation on hormonal activity of the chicken ovary and testis during the second half of embryogenesis measured by steroid concentrations in blood plasma and gonads. The effect of TCDD on hatchability was also evaluated. In the first experiment TCDD was administrated to chicken eggs on day 7 of embryogenesis (E7) at doses of 0 (control), 2.5, 5 and 10ng/egg dissolved in 50μl of peanut oil. Blood plasma for estradiol (E2) and testosterone (T) determination was collected from embryos at E14, E18, E20 and at hatching (D1). In the second experiment TCDD at doses of 0 (control), 2.5, 5 and 10ng/egg dissolved in 50μl of DMSO was injected on E6. Blood plasma and gonads were collected on the day of hatching for progesterone (P4), T and E2 concentrations measured by means of the RIA method. It was found that TCDD injection: (1) increased the level of T and decreased the level of E2 in the plasma of female chicken embryos during the second half of embryogenesis, while on the other hand in male embryos the effect of TCDD was opposite, (2) increased T and E2 concentrations in plasma of newly hatched female chickens with a concomitant decrease of these hormones in the ovary; P4 in the ovary was elevated by TCDD, (3) decreased P4 and T in plasma and testes, whereas increased E2 concentration in the plasma of newly hatched male embryos (E2 concentration in the testes was below the sensitivity of the applied RIA method), and (4) decreased hatchability at all examined doses. The results obtained clearly demonstrate that exposure to TCDD at an early stage of embryogenesis affects steroid production and secretion by chicken gonads. The effect of TCDD depends on (1) the applied dose, (2) the day of embryogenesis, and (3) the sex. It is suggested that dioxins are potent modulators of the process of steroidogenesis in the chicken gonads. Moreover, the data obtained indicate that dioxins exert an effect on embryo development and the hatching process.

Self-organization of developing embryo using scale-invariant approach

BackgroundSelf-organization is a fundamental feature of living organisms at all hierarchical levels from molecule to organ. It has also been documented in developing embryos.MethodsIn this study, a scale-invariant power law (SIPL) method has been used to study self-organization in developing embryos. The SIPL coefficient was calculated using a centro-axial skew symmetrical matrix (CSSM) generated by entering the components of the Cartesian coordinates; for each component, one CSSM was generated. A basic square matrix (BSM) was constructed and the determinant was calculated in order to estimate the SIPL coefficient. This was applied to developing C. elegans during early stages of embryogenesis. The power law property of the method was evaluated using the straight line and Koch curve and the results were consistent with fractal dimensions (fd). Diffusion-limited aggregation (DLA) was used to validate the SIPL method.Results and conclusionThe fractal dimensions of both the straight line and Koch curve showed consistency with the SIPL coefficients, which indicated the power law behavior of the SIPL method. The results showed that the ABp sublineage had a higher SIPL coefficient than EMS, indicating that ABp is more organized than EMS. The fd determined using DLA was higher in ABp than in EMS and its value was consistent with type 1 cluster formation, while that in EMS was consistent with type 2.

Embryonic stem cell bioprinting for uniform and controlled size embryoid body formation

Embryonic stem cells (ESCs) are pluripotent with multilineage potential to differentiate into virtually all cell types in the organism and thus hold a great promise for cell therapy and regenerative medicine. In vitro differentiation of ESCs starts with a phase known as embryoid body (EB) formation. EB mimics the early stages of embryogenesis and plays an essential role in ESC differentiation in vitro. EB uniformity and size are critical parameters that directly influence the phenotype expression of ESCs. Various methods have been developed to form EBs, which involve natural aggregation of cells. However, challenges persist to form EBs with controlled size, shape, and uniformity in a reproducible manner. The current hanging-drop methods are labor intensive and time consuming. In this study, we report an approach to form controllable, uniform-sized EBs by integrating bioprinting technologies with the existing hanging-drop method. The approach presented here is simple, robust, and rapid. We present significantly enhanced EB size uniformity compared to the conventional manual hanging-drop method.

Production of loach (Misgurnus anguillicaudatus) germ-line chimera using transplantation of primordial germ cells isolated from cryopreserved blastomeres1

An efficient procedure for the cryopreservation of fish blastomeres followed by restoration through germ-line chimera formation was established. Blastomeres of the loach (Misgurnus anguillicaudatus) were cryopreserved in 250-µL straws in Eagle's minimum essential medium with various concentrations of dimethyl-sulfoxide (0, 5, 10, 15, and 20%), and the best concentration was combined with glycerol (1, 2, and 4%) and external cryoprotectants (1 or 2% sucrose; 2, 5, or 10% fetal bovine serum; 1 or 2% BSA). Postthaw viability of the blastomeres was used to optimize cryopreservation conditions. Donor blastomeres were injected with zebrafish green fluorescence protein-nos1 3' untranslated region mRNA and biotin dextran before cryopreservation in the optimal freeze medium. Host embryos were injected with zebrafish DsRed-nos1 3' untranslated region mRNA and reared to the blastula stage. Donor blastomeres were thawed at 25 °C for 10 s and transplanted to the host embryos either immediately or after incubation for 16 h at 20 °C. Donor and host primordial germ cell migration was visualized with fluorescent imaging during the early stages of embryogenesis, and also by histology in 4-d-old embryos. Transplantation of blastomeres immediately after thawing gave decreased hatching rates (approximately 3%) and generated a smaller percentage of germ-line chimeras (approximately 1.1%). In contrast, incubation of a cryopreserved sample for 16 h followed by transplantation of the green fluorescence protein-positive blastomeres improved the hatching rate to 90%, and successfully produced presumable germ-line chimeras at a rate of 16.5%. The improved survival rates and germ-line chimerism may be an effective method for gene banking and subsequent reconstitution of endangered fish genotypes.

Skin diseases following a Christmas tree pattern

Pattern analysis of skin lesions is an art and a key competence of every dermatologist. Three major line patterns cover the human body—the dermatomes or Head zones, the nevoid lines of Blaschko, and the relaxed skin tension lines, or Langer lines. Head zones represent skin areas innervated from the same sensory neuronal segment or spinal nerve zone. Blaschko lines are borderlines of epidermal aberration caused by genetic mosaicism occurring in the early stages of embryogenesis. Langer lines show the direction of the lowest naturally occurring skin tension, and its thoracodorsal manifestation is the Christmas tree pattern. Here we review clinical aspects of pityriasis rosea, mycosis fungoides, stage 2 syphilis, exanthematic Kaposi sarcoma, exanthematic psoriasis, Leser-Trelat syndrome, and other primary skin diseases with a Christmas tree pattern. Secondary skin diseases, such as herpes zoster or indeterminate cell histiocytosis, may follow this pattern if they are linked to a primary skin disease by the Wolf isotopic response.

The charms of sex chromosomes in snakes

determined and the 1:1 sex ratio is maintained. In many species of reptiles, environmental factors such as temperature play an important role in determining the sex ratio (Head et al. 1987). For example, in alligators, crocodiles and turtles, during the early stages of embryogenesis, temperature has a determining effect on the sex ratio. In amphibians, the sex ratio is usually determined genetically. Thus, the existence of specialized sex-determining chromosomes poses a range of fascinating and fundamental questions, although few of them have been satisfactorily answered. Why, for example, do some species show morphological differentiation of sex chromosomes but not others? If the sex ratio can be maintained by a set of genes, as in the case of amphibians, then what is the need for specializing one entire chromosome exclusively for this singular sex determination function and maintenance of sex ratio at such an enormous cost that most genes, barring a few specifically involved in sex determination, have been lost from the sex-determining chromosome? And to compensate for this, why did yet another mechanism of dosage compensation have to be invented? What is the mechanism involved? Can we answer these questions?

The Dnmt3b Splice Variant is Specifically Expressed in In Vitro-manipulated Blastocysts and Their Derivative ES Cells

Manipulation of preimplantation embryos in vitro, such as in vitro fertilization (IVF), in vitro culture (IVC), intracytoplasmic sperm injection (ICSI), somatic cell nuclear transfer (SCNT) and other assisted reproduction technologies (ART), has contributed to the development of infertility treatment and new animal reproduction methods. However, such embryos often exhibit abnormal DNA methylation patterns in imprinted genes and centromeric satellite repeats. These DNA methylation patterns are established and maintained by three DNA methyltransferases: Dnmt1, Dnmt3a and Dnmt3b. Dnmt3b is responsible for the creation of methylation patterns during the early stage of embryogenesis and consists of many alternative splice variants that affect methylation activity; nevertheless, the roles of these variants have not yet been identified. In this study, we found an alternatively spliced variant of Dnmt3b lacking exon 6 (Dnmt3bΔ6) that is specific to mouse IVC embryos. Dnmt3bΔ6 also showed prominent expression in embryonic stem (ES) cells derived from in vitro manipulated embryos. Interestingly, IVC blastocysts were hypomethylated in centromeric satellite repeat regions that could be susceptible to methylation by Dnmt3b. In vitro methylation activity assays showed that Dnmt3bΔ6 had lower activity than normal Dnmt3b. Our findings suggest that Dnmt3bΔ6 could induce a hypomethylation status especially in in vitro manipulated embryos.

Tissue and developmental expression of SRAM, an unconventional Rel‐family protein

Previously we have reported the purification and cDNA cloning of a novel Rel/Ankyrin-family protein named SRAM from the flesh fly, Sarcophaga peregrina. Rel proteins generally translocate into the nucleus upon immune stimuli by dissociating from an inhibitory ankyrin domain, while SRAM is unique in terms of its constitutive nuclear localization with its internal ankyrin domain accompanied, at least in a Sarcophaga cell line and fat body cells. Although SRAM had been originally identified as a sole factor that binds to the κB motif of the inducible Sarcophaga lectin gene promoter, its transcriptional activity remained controversial. Moreover, homologues of SRAM have not been found in any other established model organisms including Drosophila. Here we report that the developmental expression of SRAM was up-regulated at the early stages of embryogenesis and metamorphosis. Furthermore, SRAM expression was prominent in the digestive tracts of the third instar larvae. We argue the hypothesis that SRAM has evolved as a quite unconventional Rel-family protein in Sarcophaga.

Δ40p53 controls the switch from pluripotency to differentiation by regulating IGF signaling in ESCs

Δ40p53 is a transactivation-deficient isoform of the tumor suppressor p53. We discovered that Δ40p53, in addition to being highly expressed in embryonic stem cells (ESCs), is the major p53 isoform during early stages of embryogenesis in the mouse. By altering the dose of Δ40p53 in ESCs, we identified a critical role for this isoform in maintaining the ESC state. Haploinsufficiency for Δ40p53 causes a loss of pluripotency in ESCs and acquisition of a somatic cell cycle, while increased dosage of Δ40p53 prolongs pluripotency and inhibits progression to a more differentiated state. Δ40p53 controls the switch from pluripotent ESCs to differentiated somatic cells by controlling the activity of full-length p53 at critical targets such as Nanog and the IGF-1 receptor (IGF-1R). The IGF axis plays a central role in the switch between pluripotency and differentiation in ESCs-and Δ40p53, by controlling the level of the IGF-1R, acts as a master regulator of this switch. We propose that this is the primary function of Δ40p53 in cells of the early embryo and stem cells, which are the only normal cells in which this isoform is expressed.

Important processes during differentiation and early development of somatic embryos of Norway spruce as revealed by changes in global gene expression

The aim of this study has been to identify important processes that regulate early stages of embryo development in conifers. Somatic embryogenesis in Picea abies has become a model system for studying embryology in conifers, providing a well-characterized sequence of developmental stages, resembling zygotic embryogeny, which can be synchronized by specific treatments, making it possible to collect a large number of somatic embryos at specific developmental stages. We have used this model to analyze global changes in gene expression during early stages of embryo development by generating an expression profile of 12,536 complementary DNA clones. This has allowed us to identify molecular events regulating putative processes associated with pattern formation during the earliest stages of embryogenesis which have not been identified on the molecular level in conifers before. We recognize notable changes in the expression of genes involved in regulating auxin biosynthesis and auxin response, gibberellin-mediated signaling, signaling between the embryo and the female gametophyte, tissue specification including the formation of boundary regions, and the switch from embryonic to vegetative development. In addition, our results confirm the involvement of previously described processes, including stress, differentiation of a protoderm, and programmed cell death.

Growth differences and differential expression analysis of pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH) between the sexes in half-smooth tongue sole Cynoglossus semilaevis

Pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH) are regulators of growth hormone secretion. In this article, we examined the difference in growth and mRNA expression of PACAP and GHRH between the sexes in half-smooth tongue sole, an important cultured fish species indicating sexually growth dimorphism in China. Firstly, a significant body weight difference between females and males was first observed at 7 months ( P < 0.05) and at 18 months the mean body weight of the females (771.0 ± 44.3 g) was as much as 4.9 times higher than that of males (130.6 ± 6.0 g). As a result, half-smooth tongue sole, Cynoglossus semilaevis, is a good model to investigate the effects of growth-related genes expression on sexual growth dimorphism. Secondly, the cDNAs encoding PRP/PACAP and GHRH were isolated. Two differently processed mRNA transcripts of PRP/PACAP (PRP-encoding and PRP splice variant) were found. PACAP and GHRH mRNA was highly abundant in brain and less abundant in other tissues. However, PACAP mRNA was expressed in most brain regions, and was lower in the cerebellum. GHRH mRNA was predominantly expressed in the hypothalamus and weakly expressed in all areas of the brain examined. Ontogenetic expression analysis indicated that PACAP and GHRH mRNA was detected in the early stages of embryogenesis. Finally, differential expression showed that there was no significant difference of the expression level of PACAP or GHRH between the sexes before 8 months of age. However, between 9 and 12 months of age, the GHRH mRNA expression level in males was significantly higher than in females ( P < 0.05), which might be associated with GH deficiency in males. In contrast, the male PACAP mRNA expression level was not significantly higher than that in females even at 9 and 12 months of age. The present results provide important clues for understanding the sexual growth dimorphism mechanisms in half-smooth tongue sole.