Xenopus chip for single-egg trapping, in vitro fertilization, development, and tadpole escape.

Xenopus laevis is highly suitable as a toxicology animal model owing to its advantages in embryogenesis research. For toxicological studies, a large number of embryos must be handled simultaneously because they very rapidly develop into the target stages within a short period of time. To efficiently handle the embryos, a convenient embryo housing device is essential for fast and reliable assessment and statistical evaluation of malformation caused by toxicants. Here, we suggest 3D fabrication of single-egg trapping devices in which Xenopus eggs are fertilized in vitro, and the embryos are cultured. We used manual pipetting to insert the Xenopus eggs inside the trapping sites of the chip. By introducing a liquid circulating system, we connected a sperm-mixed solution with the chip to induce in vitro fertilization of the eggs. After the eggs were fertilized, we observed embryo development involving the formation of egg cleavage, blastula, gastrula, and tadpole. After the tadpoles grew inside the chip, we saved their lives by enabling their escape from the chip through reverse flow of the culture medium. The Xenopus chip can serve as an incubator to induce fertilization and monitor normal and abnormal development of the Xenopus from egg to tadpole.

Rif1 prolongs the embryonic S phase at the Drosophila mid-blastula transition.

In preparation for dramatic morphogenetic events of gastrulation, rapid embryonic cell cycles slow at the mid-blastula transition (MBT). In Drosophila melanogaster embryos, down-regulation of cyclin-dependent kinase 1 (Cdk1) activity initiates this slowing by delaying replication of heterochromatic satellite sequences and extending S phase. We found that Cdk1 activity inhibited the chromatin association of Rap1 interacting factor 1 (Rif1), a candidate repressor of replication. Furthermore, Rif1 bound selectively to satellite sequences following Cdk1 down-regulation at the MBT. In the next S phase, Rif1 dissociated from different satellites in an orderly schedule that anticipated their replication. Rif1 lacking potential phosphorylation sites failed to dissociate and dominantly prevented completion of replication. Loss of Rif1 in mutant embryos shortened the post-MBT S phase and rescued embryonic cell cycles disrupted by depletion of the S phase-promoting kinase, cell division cycle 7 (Cdc7). Our work shows that Rif1 and S phase kinases compose a replication timer controlling first the developmental onset of late replication and then the precise schedule of replication within S phase. In addition, we describe how onset of late replication fits into the progressive maturation of heterochromatin during development.

Synchronizing developmental stages in Neotropical catfishes for application in germ cell transplantation.

SummaryThe aim of this study was to describe the effect of temperature on the fertilization, early developmental stages, and survival rate of two Neotropical catfishes Pimelodus maculatus and Pseudopimelodus mangurus. After fertilization, the eggs were incubated at 22°C, 26°C, and 30°C, which resulted in fertilization rates of 96.95 ± 1.79%, 98.74 ± 0.76%, and 98.44 ± 0.19% for P. maculatus and 96.10 ± 1.58%, 98.00 ± 0.63%, and 94.60 ± 2.09% for P. mangurus, respectively. For P. maculatus, hatching occurred after 22 h 30 min post-fertilization at 22°C, 16 h 30 min at 26°C, and 11 h 20 min at 30°C, and the hatching rates were 43.87 ± 7,46%, 57.57 ± 17.49%, and 53.63 ± 16.27%, respectively. For P. mangurus, hatching occurred after 28 h 30 min post-fertilization at 22°C and 17 h 30 min at 26°C with respective hatching rates of 45.4 ± 21.02% and 68.1 ± 12.67%. For this species, all embryos incubated at 30°C died before hatching. Additionally, for P. maculatus, the larvae from the lower (22°C) and higher temperatures (30°C) presented increased abnormality rates, as observed in the head, tail and yolk regions. The lowest abnormality rate was detected at 26°C, which was considered the optimal incubation temperature for both species. The developed protocol enables the manipulation of embryonic development, which is important for the application of reproductive biotechniques, including chimerism and chromosome-set manipulation. The data obtained here are also important for the surrogate propagation of this species as P. mangurus was recently categorized as an endangered fish species.

Intra-ooplasmic injection of a multiple number of sperm to induce androgenesis and polyploidy in the dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae).

SummaryPolyspermy was initiated by microinjecting a multiple number of sperm into the activated and dechorionated eggs of dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae). A 10-nl sperm suspension from an albino (recessive trait) male (105, 106, 107 or 108 sperm ml -1) was microinjected into eggs from a wild-type female. Although the rates of embryos developing into the blastula stage in the injection group at the highest sperm concentration were similar to that of the control group, the hatching rates of the injection group were much lower. A large proportion of embryos that developed from the injected eggs was haploid and were mosaics containing haploid cells. Most of the haploid and mosaic embryos inherited only paternally derived alleles in the microsatellite markers (i.e. androgenesis was initiated by injecting multiple sperm). In contrast, some haploid embryos contained both paternal and maternal alleles despite haploidy, suggesting that they were mosaics consisting of cells with either paternal or maternal inheritance. The injected eggs displayed diploid, hypotriploid and triploid cells, all of which included both maternally and paternally derived alleles. One albino tetraploid with only paternal alleles was also observed from the injected eggs. These results suggested that part of the sperm microinjected into the ooplasm should form a male pronucleus/pronuclei, which could develop by androgenesis or could fuse with the female pronucleus/pronuclei. Therefore, microinjection of multiple sperm should be considered a potential technique to induce androgenesis and polyploidy.

Developmental transcriptomics in Atlantic haddock: Illuminating pattern formation and organogenesis in non-model vertebrates.

Gadiforms such as Atlantic haddock comprise some of the world's most economically important fisheries. Understanding the early life history of these fish is a prerequisite for predicting effects of a changing environment and increased human activities. Robust assessment of the effects of environmental impacts on the embryos of non-model vertebrates is hampered by a lack of molecular resources and detailed knowledge regarding the regulation of genes and pathways in early development. Here we used mRNA sequencing to link transcriptional changes to developmental processes in haddock, specifically, pattern formation and organogenesis. Temporal expression of key developmental genes was tightly anchored to either the appearance of visible structures or cellular processes characterised in model organisms. These findings demonstrate the high potential of developmental transcriptomics as an analytical tool for improved understanding of pathophysiological mechanisms leading to abnormal development in any vertebrate.

The effect of temperature on the initial development of Brycon amazonicus Spix & Agassiz, 1829 as tool for micromanipulation of embryos.

Primordial germ cell (PGC) transplant is a promising tool in aquaculture; however, successful use of this technique requires in depth knowledge of the early stages of embryo and larval development. The aim of this study was to analyse the effect of different temperatures (22, 26, and 30°C) on the early development of B. amazonicus. The newly fertilized eggs were distributed into tanks with controlled temperature and oxygenation. Samples were collected at pre-established times and analysed under light and fluorescence microscopy. Temperature influenced the speed and duration of each stage of early development, including hatching time. The highest pronuclei fusion rate was observed 8 min post-fertilization (mpf) at 22 and 26°C, and 6 mpf at 30°C. The duration of the 512-1000 blastomeres phase during in the blastocyst stage was 1 h 30 min at 22°C, and 25 min at 26 and 30°C. Hatching occurred at 24 h 30 mpf at 22°C, 16 h post-fertilization (hpf) at 26°C, and 11 h 30 mpf at 30°C. The rate of morphologically normal larvae was 88.34% at 22°C, 90.49% at 26°C, and 73% at 30°C. Malformations of the head, yolk sac, heart, and tail were observed in all temperatures. Nevertheless, B. amazonicus embryos were able to develop satisfactory in all three temperatures tested. These results enable embryo manipulation at different temperatures to optimize the micromanipulation time of embryos and larvae for biotechnological studies.

Cellular mechanism for selective vertical transmission of an obligate insect symbiont at the bacteriocyte-embryo interface.

Many insects are associated with obligate symbiotic bacteria, which are localized in specialized cells called bacteriocytes, vertically transmitted through host generations via ovarial passage, and essential for growth and reproduction of their hosts. Although vertical transmission is pivotal for maintenance of such intimate host-symbiont associations, molecular and cellular mechanisms underlying the process are largely unknown. Here we report a cellular mechanism for vertical transmission of the obligate symbiont Buchnera in the pea aphid Acyrthosiphon pisum. In the aphid body, Buchnera cells are transmitted from maternal bacteriocytes to adjacent blastulae at the ovariole tips in a highly coordinated manner. By making use of symbiont-manipulated strains of A. pisum, we demonstrated that the facultative symbiont Serratia is, unlike Buchnera, not transmitted from maternal bacteriocytes to blastulae, suggesting a specific mechanism for Buchnera transmission. EM observations revealed a series of exo-/endocytotic processes operating at the bacteriocyte-blastula interface: Buchnera cells are exocytosed from the maternal bacteriocyte, temporarily released to the extracellular space, and endocytosed by the posterior syncytial cytoplasm of the blastula. These results suggest that the selective Buchnera transmission is likely attributable to Buchnera-specific exocytosis by the maternal bacteriocyte, whereas both Buchnera and Serratia are nonselectively incorporated by the endocytotic activity of the posterior region of the blastula. The sophisticated cellular mechanism for vertical transmission of Buchnera must have evolved to ensure the obligate host-symbiont association, whereas facultative symbionts like Serratia may coopt the endocytotic component of the mechanism for their entry into the host embryos.

Possible regulation of Oct60 transcription by a positive feedback loop in Xenopus oocytes.

The POU family subclass V (POU-V) proteins have important roles in maintaining cells in an undifferentiated state. In Xenopus, expression of the POU-V protein Oct60 was detected in oocytes and was found to decrease in blastula- to gastrula-stage embryos. In addition, Oct60 overexpression inhibits some signals in early embryogenesis, including Activin/Nodal, BMP, and Wnt signalling. In this report, we analysed mechanisms of Oct60 promoter activation and discovered that Oct60 transcription was activated ectopically in somatic nuclei by oocyte extract treatment. Promoter assays demonstrated that Oct60 transcription was activated in oocytes specifically and that this activation was dependent on an Octamer-Sox binding motif. ChIP assays showed that the Oct60 protein binds the motif. These results suggest that Oct60 transcription is regulated by a positive-feedback loop in Xenopus oocytes.

Earliest ciliary swimming effects vertical transport of planktonic embryos in turbulence and shear flow.

Eggs released by broadcast-spawning marine invertebrates are often negatively buoyant. Blastulae and gastrulae of these species are commonly motile, with passive stability that leads to upward swimming in still water. The earliest occurrence of swimming in developing embryos of diverse invertebrates may therefore permit vertical migration in nature. I used turbulent and laminar shear flows to investigate: (1) the speed and direction of transport of non-motile and newly swimming stages of the echinoids Dendraster excentricus and Strongylocentrotus purpuratus in turbulence, and (2) the limit of stable vertical orientation in swimming blastulae of D. excentricus. Swimming contributed significantly to the rate of upward transport of D. excentricus in turbulence experiments where the kinetic energy dissipation rate (ε) was ∼10(-2) cm(2) s(-3). However, swimming significantly reduced the rate of upward transport of S. purpuratus blastulae in turbulence, suggesting that passively stable swimmers of this species were turned from the vertical, crossed flow-lines, and migrated into downwelling. Observations of swimming in laminar shear indicate that D. excentricus swimming blastulae maintain a vertical orientation until shear approaches 0.26 s(-1), equivalent to sub-microscale shear in turbulence where ε is ∼10(-3) cm(2) s(-3). Swimming speeds of D. excentricus showed an unexpected dependence on shear, indicating that greater shear (within limits) can enhance speed of ciliary swimming. In D. excentricus, swimming by newly hatched blastulae should support upward migration in turbulence characteristic of coastal surface waters, whereas species differences in passive stability and swimming responses to shear may lead to differences in vertical transport and subsequent dispersal.

Zygotic Genome Activation: Critical Prelude to the Most Important Time of Your Life.

Activation of the embryonic genome during development represents a major developmental transition in all species. The history of its exploration began in the 1950s-1960s, when this idea was put forward and proven experimentally by Alexander Neyfakh. He observed the aberrant development of fish embryos upon X-ray irradiation and noted the different developmental outcomes depending on the stage when fertilized eggs were subjected to irradiation. Neyfakh also discriminated a regional difference of X-irradiation between the nucleus and the cytoplasm. By selecting the X-ray dose causing nuclear damage, he determined the beginning of zygotic transcription, which at that time became known as the morphogenetic function of nuclei. His team defined the link of zygotic transcription with the asynchronization of cell division and cell migration, the two other hallmarks, which along with the morphogenetic function (or the zygotic genome activation), are at the core of the mid-blastula transition during development. Within this framework, current studies using maternal mutants and application of modern methods of whole-embryo and single-cell transcriptomics begin to decipher the molecular mechanisms of the mid-blastula transition (or the maternal-zygotic transition).

G2 acquisition by transcription-independent mechanism at the zebrafish midblastula transition.

Following fertilization of many animal embryos, rapid synchronous cleavage divisions give way to longer, asynchronous cell cycles at the midblastula transition (MBT). The cell cycle changes at the MBT, including the addition of gap phases and checkpoint controls, are accompanied by activation of the zygotic genome and the onset of cell motility. Whereas the biochemical changes accompanying the MBT in the vertebrate embryo have been extensively documented, the cellular events are not well understood. We show that cell cycle remodeling during the zebrafish MBT includes the transcription-independent acquisition of a G2 phase that is essential for preventing entry into mitosis before S-phase completion in cycles 11-13. We provide evidence from high-resolution imaging that inhibition of Cdc25a and Cdk1 activity, but not Cdk2 activity, is essential for cell cycle lengthening and asynchrony between cycles 9 and 12. We demonstrate that lengthening is not required for initiation of zygotic transcription. Our results are consistent with findings from Drosophila and Xenopus that indicate the central importance of G2 addition in checkpoint establishment, and point to similar mechanisms governing the MBT in diverse species.

Development of a dopaminergic system in sea urchin embryos and larvae.

The mechanisms that regulate the organized swimming movements of sea urchin blastulae are largely unknown. Using immunohistochemistry, we found that dopamine (DA) and the Hemicentrotus pulcherrimus homolog of the dopamine receptor D1 (Hp-DRD1) were strongly co-localized in 1-2 microm diameter granules (DA/DRD1 granules). Furthermore, these granules were arranged across the entire surface of blastulae as they developed locomotory cilia before hatching, and remained evident until metamorphosis. DA/DRD1 granules were associated with the basal bodies of cilia, and were densely packed in the ciliary band by the eight-arm pluteus stage. The transcription of Hp-DRD1 was detected from the unfertilized egg stage throughout the period of larval development. Treatment with S-(-)-carbidopa, an inhibitor of aromatic-l-amino acid decarboxylase, for 20-24 h (i) from soon after insemination until the 20 h post-fertilization (20 hpf) early gastrula stage and (ii) from the 24 hpf prism larva stage until the 48 hpf pluteus stage, inhibited the formation of DA granules and decreased the swimming activity of blastulae and larvae in a dose-dependent manner. Exogenous DA rescued these deprivations. The formation of DRD1 granules was not affected. However, in 48 hpf plutei, the serotonergic nervous system (5HT-NS) developed normally. Morpholino antisense oligonucleotides directed against Hp-DRD1 inhibited the formation of DRD1 granules and the swimming of larvae, but did not disturb the formation of DA granules. Thus, the formation of DRD1 granules and DA granules occurs chronologically closely but mechanically independently and the swimming of blastulae is regulated by the dopaminergic system. In plutei, the 5HT-NS closely surrounded the ciliary bands, suggesting the functional collaboration with the dopaminergic system in larvae.

Simulations of sea urchin early development delineate the role of oriented cell division in the morula-to-blastula transition.

The sea urchin morula to blastula transition has long been thought to require oriented cell divisions and blastomere adherence to the enveloping hyaline layer. In a computer simulation model, cell divisions constrained by a surface plane division rule are adequate to effect morphological transition. The hyaline membrane acts as an enhancer but is not essential. The model is consistent with the orientation of micromere divisions and the open blastulae of direct developing species. The surface plane division rule precedes overt epithelization of surface cells and acts to organize the developing epithelium. It is a universal feature of early metazoan development and simulations of non-echinoid cleavage patterns support its role throughout Metazoa. The surface plane division rule requires only local cues and cells need not reference global positional information or embryonic axes.

Overlapping functions of argonaute proteins in patterning and morphogenesis of Drosophila embryos.

Argonaute proteins are essential components of the molecular machinery that drives RNA silencing. In Drosophila, different members of the Argonaute family of proteins have been assigned to distinct RNA silencing pathways. While Ago1 is required for microRNA function, Ago2 is a crucial component of the RNA-induced silencing complex in siRNA-triggered RNA interference. Drosophila Ago2 contains an unusual amino-terminus with two types of imperfect glutamine-rich repeats (GRRs) of unknown function. Here we show that the GRRs of Ago2 are essential for the normal function of the protein. Alleles with reduced numbers of GRRs cause specific disruptions in two morphogenetic processes associated with the midblastula transition: membrane growth and microtubule-based organelle transport. These defects do not appear to result from disruption of siRNA-dependent processes but rather suggest an interference of the mutant Ago2 proteins in an Ago1-dependent pathway. Using loss-of-function alleles, we further demonstrate that Ago1 and Ago2 act in a partially redundant manner to control the expression of the segment-polarity gene wingless in the early embryo. Our findings argue against a strict separation of Ago1 and Ago2 functions and suggest that these proteins act in concert to control key steps of the midblastula transition and of segmental patterning.

Retinoid signaling can repress blastula Wnt signaling and impair dorsal development in Xenopus embryo.

Vitamin A derivatives (retinoids) are actively involved during vertebrate embryogenesis. However, exogenous retinoids have also long been known as potent teratogens. The defects caused by retinoid treatment are complex. Here, we provided evidence that RAR-mediated retinoid signaling can repress Xenopus blastula Wnt signaling and impair dorsal development. Exogenous retinoic acid (RA) could antagonize the dorsalizing effects of lithium chloride-mediated Wnt activation in blastula embryos. The Wnt-responsive reporter gene transgenesis and luciferase assay showed that excess RA can repress the Wnt signaling in blastula embryos. In addition, the downstream target genes of the Wnt signaling that direct embryonic dorsal development, were also down-regulated in the RA-treated embryos. Mechanically, RA did not interfere with the stability of beta-catenin, but promoted its nuclear accumulation. The inverse agonist of retinoic acid receptors (RAR) rescued the Wnt signaling repression by RA and relieved the RA-induced nuclear accumulation of beta-catenin. Our results explain one of the reasons for the complicated teratogenic effects of retinoids and shed light on the endogenous way of interactions between two developmentally important signaling pathways.

From zygote to implantation: morphological and molecular dynamics during embryo development in the pig.

The increasing focus on the pig as a biomedical model calls for studies which investigate morphological and molecular mechanisms during initial embryonic development in this species. In the pig, the paternal genome is actively demethylated in the zygote, whereas the maternal genome remains methylated. The major genome activation occurs at the four-cell stage, when prominent ribosome-synthesizing nucleoli develop in the blastomeres, allowing for trophectoderm and inner cell mass (ICM) differentiation. Unlike in mice, the pluripotency gene OCT4 is initially expressed in both compartments. The ICM differentiates into epiblast and hypoblast approximately at the time of hatching from the zona pellucida, and subsequently the loss of the Rauber's layer results in an uncovered epiblast establishing the embryonic disc again in contrast to mice. This particular and protracted ICM/epiblast biology may contribute to the lack of success in culturing porcine embryonic stem cells. The embryonic disc subsequently becomes polarized by a posterior thickening, which includes ingression of the first extra-embryonic mesoderm. Thereafter, the primitive streak forms and gastrulation results in formation of the somatic germ layers and germline, i.e. the primordial germ cells. The latter remain pluripotent for a period and may be isolated and cultured as embryonic germ cells in vitro.

Genome wide decrease of DNA replication eye density at the midblastula transition of Xenopus laevis.

During the first rapid divisions of early development in many species, the DNA:cytoplasm ratio increases until the midblastula transition (MBT) when transcription resumes and cell cycles lengthen. S phase is very rapid in early embryos, about 20-30 times faster than in differentiated cells. Using a combination of DNA fiber studies and a Xenopus laevis embryonic in vitro replication system, we show that S phase slows down shortly after the MBT owing to a genome wide decrease of replication eye density. Increasing the dNTP pool did not accelerate S phase or increase replication eye density implying that dNTPs are not rate limiting for DNA replication at the Xenopus MBT. Increasing the ratio of DNA:cytoplasm in egg extracts faithfully recapitulates changes in the spatial replication program in embryos, supporting the hypothesis that titration of soluble limiting factors could explain the observed changes in the DNA replication program at the MBT in Xenopus laevis.

A cell cycle-coordinated Polymerase II transcription compartment encompasses gene expression before global genome activation.

Most metazoan embryos commence development with rapid, transcriptionally silent cell divisions, with genome activation delayed until the mid-blastula transition (MBT). However, a set of genes escapes global repression and gets activated before MBT. Here we describe the formation and the spatio-temporal dynamics of a pair of distinct transcription compartments, which encompasses the earliest gene expression in zebrafish. 4D imaging of pri-miR430 and zinc-finger-gene activities by a novel, native transcription imaging approach reveals transcriptional sharing of nuclear compartments, which are regulated by homologous chromosome organisation. These compartments carry the majority of nascent-RNAs and active Polymerase II, are chromatin-depleted and represent the main sites of detectable transcription before MBT. Transcription occurs during the S-phase of increasingly permissive cleavage cycles. It is proposed, that the transcription compartment is part of the regulatory architecture of embryonic nuclei and offers a transcriptionally competent environment to facilitate early escape from repression before global genome activation.

The influence of embryo presence on prostaglandins synthesis and prostaglandin E2 and F2alpha content in corpora lutea during periimplantation period in the pig.

We determined the expression of PGE2 synthase (mPGES-1), PGF synthase (PGFS), carbonyl reductase/prostaglandin 9-ketoreductase (CBR1) genes and the content of PGE2, PGF2alpha in porcine corpora lutea on Days 12-14 of pregnancy and Days 12-14 of the estrous cycle. For this study we used a surgically-generated model in which one of the uterine horns was cut transversely and a part of this horn was detached from the uterine corpus. The expression of mPGES-1, PGFS, and CBR1 genes and mPGES-1/PGFS ratio were significantly higher in corpora lutea of the pregnant gilts compared to the corpora lutea from the parallel ovaries of the cyclic gilts. There was no difference in mPGES-1, PGFS, CBR1 genes expression and mPGES-1/PGFS ratio between corpora lutea ipsi-(CL1) and contralateral (CL2) to the uterine horn with the developing embryos. The highest content of PGE2 was found in CL1 of the pregnant gilts. The PGE2/PGF2alpha ratio was significantly higher in CL1 of the pregnant gilts compared to corpora lutea from parallel ovary of the cyclic gilts. We suggest that the activity of the investigated genes is induced by compounds of embryonic origin which are not distributed only to the ipsilateral ovary but are transported within the mesometrium to both ovaries in a more systemic manner.