Blastula Embryos Research Articles

Transgenerational plasticity as a mechanism of response to marine heatwaves in the purple sea urchin, Strongylocentrotus purpuratus

Kelp forests of the California Current System have experienced prolonged marine heatwave (MHW) events that overlap in time with the phenology of life history events (e.g., gametogenesis and spawning) of many benthic marine invertebrates. To study the effect of thermal stress from MHWs during gametogenesis in the purple sea urchin (Strongylocentrotus purpuratus) and further, whether MHWs might induce transgenerational plasticity (TGP) in thermal tolerance of progeny, adult urchins were acclimated to two conditions in the laboratory – a MHW temperature of 18°C and a non-MHW temperature of 13°C. Following a four-month long acclimation period (October–January), adults were spawned and offspring from each parental condition were reared at MHW (18°C) and non-MHW temperatures (13°C), creating a total of four embryo treatment groups. To assess transgenerational effects for each of the four groups, we measured thermal tolerance of hatched blastula embryos in acute thermal tolerance trials. Embryos from MHW-acclimated females were more thermally tolerant with higher LT50 values as compared to progeny from non-MHW-acclimated females. Additionally, there was an effect of female acclimation state on offspring body size at two stages of embryonic development - early gastrulae and prism, an early stage echinopluteus larvae. To assess maternal provisioning as means to also alter embryo performance, we assessed gamete traits from the differentially acclimated females, by measuring size and biochemical composition of eggs. MHW-acclimated females had eggs with higher protein concentrations, while egg size and lipid content showed no differences. Our results indicate that TGP plays a role in altering the performance of progeny as a function of the thermal history of the female, especially when thermal stress coincides with gametogenesis. In addition, the data on egg provisioning show that maternal experience can influence embryo traits via egg protein content. Although this is a laboratory-based study, the results suggest that TGP may play a role in the resistance and tolerance of S. purpuratus early stages in the natural kelp forest setting.

Feeder cell-dependent primary culture of single blastula-derived embryonic cell lines from marine medaka (Oryzias dancena).

Fish embryonic stem cells (ESCs) are derived from blastomeres that have been cultured from blastula embryos. The most widely used method for derivation of fish ESCs is the culture of blastomeres that have been isolated from approximately 10 blastula embryos under feeder-free conditions. However, this method leads to intercellular genetic heterogeneity among the cultured cells, which is a major obstacle to the development of stable ESC culture conditions. In this study, to establish ESC lines with intercellular genetic homogeneity at the early stage of culture, we attempted to derive embryonic cell lines from single blastula-derived blastomeres of marine medaka (Oryzias dancena) in a feeder cell culture system. Using basic fibroblast growth factor-expressing feeder cells during primary culture, we successfully established 22 single blastula-derived embryonic cell lines that could be subcultured more than 20 times. In contrast, we were unable to efficiently derive cell lines using wild-type feeder cells and under feeder-free conditions. The established cell lines exhibited ESC-like cell characteristics in terms of alkaline phosphatase activity, pluripotency-related gene expression, and embryoid body formation. The results of this study will contribute to the development of methods for derivation of fish ESCs.

Nascent transcriptome reveals orchestration of zygotic genome activation in early embryogenesis

Early embryo development requires maternal-to-zygotic transition, during which transcriptionally silent nuclei begin widespread gene expression during zygotic genome activation (ZGA).1-3 ZGA is vital for early cell fating and germ-layer specification,3,4 and ZGA timing is regulated by multiple mechanisms.1-5 However, controversies remain about whether these mechanisms are interrelated and vary among species6-10 and whether the timing of germ-layer-specific gene activation is temporally ordered.11,12 In some embryonic models, widespread ZGA onset is spatiotemporally graded,13,14 yet it is unclear whether the transcriptome follows this pattern. A major challenge in addressing these questions is to accurately measure the timing of each gene activation. Here, we metabolically label and identify the nascent transcriptome using 5-ethynyl uridine (5-EU) in Xenopus blastula embryos. We find that EU-RNA-seq outperforms total RNA-seq in detecting the ZGA transcriptome, which is dominated by transcription from maternal-zygotic genes, enabling improved ZGA timing determination. We uncover discrete spatiotemporal patterns for individual gene activation, a majority following a spatial pattern of ZGA that is correlated with a cell size gradient.14 We further reveal that transcription necessitates a period of developmental progression and that ZGA can be precociously induced by cycloheximide, potentially through elongation of interphase. Finally, most ectodermal genes are activated earlier than endodermal genes, suggesting a temporal orchestration of germ-layer-specific genes, potentially linked to the spatially graded pattern of ZGA. Together, our study provides fundamental new insights into the composition and dynamics of the ZGA transcriptome, mechanisms regulating ZGA timing, and its role in the onset of early cell fating.

Identification and prediction of developmental enhancers in sea urchin embryos

BackgroundThe transcription of developmental regulatory genes is often controlled by multiple cis-regulatory elements. The identification and functional characterization of distal regulatory elements remains challenging, even in tractable model organisms like sea urchins.ResultsWe evaluate the use of chromatin accessibility, transcription and RNA Polymerase II for their ability to predict enhancer activity of genomic regions in sea urchin embryos. ATAC-seq, PRO-seq, and Pol II ChIP-seq from early and late blastula embryos are manually contrasted with experimental cis-regulatory analyses available in sea urchin embryos, with particular attention to common developmental regulatory elements known to have enhancer and silencer functions differentially deployed among embryonic territories. Using the three functional genomic data types, machine learning models are trained and tested to classify and quantitatively predict the enhancer activity of several hundred genomic regions previously validated with reporter constructs in vivo.ConclusionsOverall, chromatin accessibility and transcription have substantial power for predicting enhancer activity. For promoter-overlapping cis-regulatory elements in particular, the distribution of Pol II is the best predictor of enhancer activity in blastula embryos. Furthermore, ATAC- and PRO-seq predictive value is stage dependent for the promoter-overlapping subset. This suggests that the sequence of regulatory mechanisms leading to transcriptional activation have distinct relevance at different levels of the developmental gene regulatory hierarchy deployed during embryogenesis.

Glyconectin Cell Adhesion Epitope, β-d-GlcpNAc3S-(1→3)-α-l-Fucp, Is Involved in Blastulation of Lytechinus pictus Sea Urchin Embryos

Glycans, as the most peripheral cell surface components, are the primary candidates to mediate the initial steps of cell recognition and adhesion via glycan–glycan binding. This molecular mechanism was quantitatively demonstrated by biochemical and biophysical measurements at the cellular and molecular level for the glyconectin 1 β-d-GlcpNAc3S-(1→3)-α-l-Fucp glycan structure (GN1). The use of adhesion blocking monoclonal antibody Block 2 that specifically recognize this epitope showed that, besides Porifera, human colon carcinoma also express this structure in the apical glycocalyx. Here we report that Block 2 selectively immune-precipitate a Mr 580 × 103 (g580) acidic non-glycosaminoglycan glycan from the total protein-free glycans of Lytechinus pictus sea urchin hatched blastula embryos. Immuno-fluorescence confocal light microscopy and immunogold electron microscopy localized the GN1 structure in the apical lamina glycocalyx attachments of ectodermal cells microvilli, and in the Golgi complex. Biochemical and immune-chemical analyses showed that the g580 glycan is carrying about 200 copies of the GN1 epitope. This highly polyvalent g580 glycan is one of the major components of the glycocalyx structure, maximally expressed at hatched blastula and gastrula. The involvement of g580 GN1 epitope in hatched blastula cell adhesion was demonstrated by: (1) enhancement of cell aggregation by g580 and sponge g200 glycans, (2) inhibition of cell reaggregation by Block 2, (3) dissociation of microvilli from the apical lamina matrix by the loss of its gel-like structure resulting in a change of the blastula embryonal form and consequent inhibition of gastrulation at saturating concentration of Block 2, and (4) aggregation of beads coated with the immune-purified g580 protein-free glycan. These results, together with the previous atomic force microscopy measurements of GN1 binding strength, indicated that this highly polyvalent and calcium ion dependent glycan–glycan binding can provide the force of 40 nanonewtons per single ectodermal cell association of microvilli with the apical lamina, and conservation of glycocalyx gel-like structure. This force can hold the weight of 160,000 cells in sea water, thus it is sufficient to establish, maintain and preserve blastula form after hatching, and prior to the complete formation of further stabilizing basal lamina.

Cooperative epithelial phagocytosis enables error correction in the early embryo.

Errors in early embryogenesis are a cause of sporadic cell death and developmental failure1,2. Phagocytic activity has a central role in scavenging apoptotic cells in differentiated tissues3-6. However, how apoptotic cells are cleared in the blastula embryo in the absence of specialized immune cells remains unknown. Here we show that the surface epithelium of zebrafish and mouseembryos, which is the first tissue formed during vertebrate development, performs efficient phagocytic clearance of apoptotic cells through phosphatidylserine-mediated target recognition. Quantitative four-dimensional in vivo imaging analyses reveal a collective epithelial clearance mechanism that is based on mechanical cooperation by two types of Rac1-dependent basal epithelial protrusions. The first type of protrusion, phagocytic cups, mediates apoptotic target uptake. The second, a previously undescribed type of fast and extended actin-based protrusion that we call 'epithelial arms', promotes the rapid dispersal of apoptotic targets through Arp2/3-dependent mechanical pushing. On the basis of experimental data and modelling, we show that mechanical load-sharing enables the long-range cooperative uptake of apoptotic cells by multiple epithelial cells. This optimizes the efficiency of tissue clearance by extending the limited spatial exploration range and local uptake capacity of non-motile epithelial cells. Our findings show that epithelial tissue clearance facilitates error correction that is relevant to the developmental robustness and survival of the embryo, revealing the presence of an innate immune function in the earliest stages of embryonic development.

Collimated Microbeam Reveals that the Proportion of Non-Damaged Cells in Irradiated Blastoderm Determines the Success of Development in Medaka (Oryzias latipes) Embryos.

Simple SummaryStudies on teratogenesis in mammals have revealed that exposure to ionizing radiation (IR) during the pre-implantation period induces a high frequency of lethality instead of teratogenesis. Here, to elucidate the IR-induced disturbance of embryonic development when IR exposure occurs during the pre-implantation period, we utilized medaka as a vertebrate model for clear observation of developmental process for its transparency. Blastula embryos exposed to IR with a lower lethal dose (gamma-rays) transiently exhibited smaller brains than those of sham-controls, however, their brain size restored equally to those of controls until hatching. We then conducted targeting irradiation, which allowed various proportions of blastoderm cells to be exposed to IR (carbon-ions), and identified that the loss of approximately 10% or less of blastoderm was compensated by the remaining non-damaged blastoderm cells even though they transiently exhibited smaller brains. In contrast, when IR exposure occurred during the late embryogenesis period, 3 days post fertilization, the brain size was not completely restored until hatching even with a lower lethal dose. Collectively, blastoderm cells with IR-induced injury undergo transient delays in brain development, however, can avoid teratogenesis at hatching presumably for their pluripotency whereas embryos during the late embryogenesis period lack the ability to do so.It has been widely accepted that prenatal exposure to ionizing radiation (IR) can affect embryonic and fetal development in mammals, depending on dose and gestational age of the exposure, however, the precise machinery underlying the IR-induced disturbance of embryonic development is still remained elusive. In this study, we examined the effects of gamma-ray irradiation on blastula embryos of medaka and found transient delay of brain development even when they hatched normally with low dose irradiation (2 and 5 Gy). In contrast, irradiation of higher dose of gamma-rays (10 Gy) killed the embryos with malformations before hatching. We then conducted targeted irradiation of blastoderm with a collimated carbon-ion microbeam. When a part (about 4, 10 and 25%) of blastoderm cells were injured by lethal dose (50 Gy) of carbon-ion microbeam irradiation, loss of about 10% or less of blastoderm cells induced only the transient delay of brain development and the embryos hatched normally, whereas embryos with about 25% of their blastoderm cells were irradiated stopped development at neurula stage and died. These findings strongly suggest that the developmental disturbance in the IR irradiated embryos is determined by the proportion of severely injured cells in the blastoderm.

Medaka oct4 is essential for gastrulation, central nervous system development and angiogenesis

Gene oct4 (also called oct3/4 or pou5f1) encodes an octamer-binding transcription factor and is best known for its pluripotency-specific expression and pluripotency-maintaining role in early embryos and embryonic stem cells of mouse and human. Its fish paralog oct4 (also called pou2 or pou5f3) plays divergent roles in embryos and stem cells development. Here the expression and function of the medaka oct4 (Oloct4) during gastrulation and organogenesis were analysed. Oloct4 RNA was abundant in pluripotent cells and differentiated extraembryonic cells of blastula embryos. It was also detectable in primordial germ cells, brain, eye and tail bud at advanced stages. Importantly, oct4 depletion at high dosages severely affected gastrulation and axis formation. Surprisingly, Oloct4 depletion at low dosages also led to embryos that either had defective brain, eye and/or blood vessels or completely lacked them. Oloct4 depletion in transgenic embryos caused the loss of rx2-positive retinal stem cells in the developing eye. Therefore, Oloct4 is essential for gastrulation, central nervous system development as well as angiogenesis in medaka besides its role in pluripotency maintenance. These results together with previous studies suggest that Oloct4 play pleiotropic roles and represent the ancestral prototype of vertebrate oct4 and pou2 genes.

Evaluation of BMP-mediated patterning in a 3D mathematical model of the zebrafish blastula embryo.

Bone Morphogenetic Proteins (BMPs) play an important role in dorsal-ventral (DV) patterning of the early zebrafish embryo. BMP signaling is regulated by a network of extracellular and intracellular factors that impact the range and signaling of BMP ligands. Recent advances in understanding the mechanism of pattern formation support a source-sink mechanism, however it is not clear how the source-sink mechanism shapes patterns in 3D, nor how sensitive the pattern is to biophysical rates and boundary conditions along both the anteroposterior (AP) and DV axes of the embryo. We propose a new three-dimensional growing Partial Differential Equation (PDE)-based model to simulate the BMP patterning process during the blastula stage. This model provides a starting point to elucidate how different mechanisms and components work together in 3D to create and maintain the BMP gradient in the embryo. We also show how the 3D model fits the BMP signaling gradient data at multiple time points along both axes. Furthermore, sensitivity analysis of the model suggests that the spatiotemporal patterns of Chordin and BMP ligand gene expression are dominant drivers of shape in 3D and more work is needed to quantify the spatiotemporal profiles of gene and protein expression to further refine the models.

Transcriptome analysis of regeneration during Xenopus laevis experimental twinning

Animal embryos have the remarkable property of self-organization. Over 125 years ago, Hans Driesch separated the two blastomeres of sea urchin embryos and obtained twins, in what was the foundation of experimental embryology. Since then, embryonic twinning has been obtained experimentally in many animals. In a recent study, we developed bisection methods that generate identical twins reliably from Xenopus blastula embryos. In the present study, we have investigated the transcriptome of regenerating half-embryos after sagittal and dorsal-ventral (D-V) bisections. Individual embryos were operated at midblastula (stage 8) with an eyelash hair and cultured until early gastrula (stage 10.5) or late gastrula (stage 12) and the transcriptome of both halves were analyzed by RNA-seq. Since many genes are activated by wound healing in Xenopus embryos, we resorted to stringent sequence analyses and identified genes up-regulated in identical twins but not in either dorsal or ventral fragments. At early gastrula, cell division-related transcripts such as histones were elevated, whereas at late gastrula, pluripotency genes (such as sox2) and germ layer determination genes (such as eomesodermin, ripply2 and activin receptor ACVRI) were identified. Among the down-regulated transcripts, sizzled, a regulator of Chordin stability, was prominent. These findings are consistent with a model in which cell division is required to heal damage, while maintaining pluripotency to allow formation of the organizer with a displacement of 90 0 from its original site. The extensive transcriptomic data presented here provides a valuable resource for data mining of gene expression during early vertebrate development.

Ketamine induction of p53-dependent apoptosis and oxidative stress in zebrafish (Danio rerio) embryos

Ketamine is a widely used pharmaceutical that has been detected in water sources worldwide. Zebrafish embryos were used in this study to investigate the oxidative stress and apoptotic signals following a 24h exposure to different ketamine concentrations (0, 50, 70 and 90 mg L−1). Early blastula embryos (∼2 h post fertilisation-hpf) were exposed for 24 h and analysed at 8 and 26 hpf. Reactive oxygen species and apoptotic cells were identified in vivo, at 26 hpf. Enzymatic activities (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), lactate dehydrogenase (LDH) and acetylcholinesterase (AChE)), glutathione levels (oxidised (GSSG) and reduced (GSH)), oxidative damage (lipid peroxidation (LPO) and protein carbonyls (CO)) as well as oxidative stress (gclc, gstp1, sod1 and cat), apoptosis (casp3a, casp6, casp8, casp9, aifm1 and tp53) and cell proliferation (pcna) related-genes were evaluated at 8 and 26 hpf. Caspase (3 and 9) activity was also determined at both time-points by colorimetric methods. Superoxide dismutase (SOD), catalase (CAT), glutathione levels (GSSG), caspase-9 and reactive oxygen species (ROS) were shown to be affected by ketamine exposure while in vivo analysis showed no difference in ROS. A significant up-regulation of superoxide dismutase (sod1) and catalase (cat) genes expression was also perceived. Ketamine-induced apoptosis was observed in vivo and confirmed by the apoptotic-related genes up-regulation. The overall results suggest that ketamine induced oxidative stress and apoptosis through the involvement of p53-dependent pathways in zebrafish embryos which could be important for the evaluation of the overall risk of ketamine in aquatic environments.

First steps towards Echinometra lucunter embryo cryopreservation

We have studied the sensitivity to cryoprotecting agents of different embryos of the local sea urchin, Echinometra lucunter which is the species used for embryo-larval bioassays in Brazil. We have located significant differences between both species sensitivity to cryoprotecting agents; while for P. lividus propylene glycol was the less toxic compound for most development stages, whereas for E. lucunter is was the most toxic and the least toxic was Dimethyl sulfoxide. There is a significant difference between development stages as well; in the case of P. lividus, the blastula embryo was the most resistant to the cryoprotecting agents, meanwhile for E. lucunter, it was the fertilized oocyte. This is a very promising result, a really early embryo that is not extremely sensitive to Me2SO. Our next aim is to develop a cryopreservation protocol for E. lucunter early embryos and test them in an embryo-larval bioassay.

Establishment and growth responses of Nile tilapia embryonic stem-like cell lines under feeder-free condition.

Embryonic stem (ES) cells provide an invaluable tool for molecular analysis of vertebrate development and a bridge linking genomic manipulations invitro and functional analysis of target genes invivo. Work towards fish ES cells so far has focused on zebrafish (Danio renio) and medaka (Oryzias latipes). Here we describe the derivation, pluripotency, differentiation and growth responses of ES cell lines from Nile tilapia (Oreochromis niloticus), a world-wide commercial farmed fish. These cell lines, designated as TES1-3, were initiated from blastomeres of Nile tilapia middle blastula embryos (MBE). One representative line, TES1, showed stable growth and phenotypic characteristics of ES cells over 200 days of culture with more than 59 passages under feeder-free conditions. They exhibited high alkaline phosphatase activity and expression of pluripotency genes including pou5f3 (the pou5f1/oct4 homologue), sox2, myc and klf4. In suspension culture together with retinoic acid treatment, TES1 cells formed embryoid bodies, which exhibited expression profile of differentiation genes characteristics of all three germ cell layers. Notably, PKH26-labeled TES1 cells introduced into Nile tilapia MBE could contribute to body compartment development and led to hatched chimera formation with an efficacy of 13%. These results suggest that TES1 cells have pluripotency and differentiation potential invitro and invivo. In the conditioned DMEM, all of the supplements including the fetal bovine serum, fish embryonic extract, fish serum, basic fibroblast growth factor and non-protein supplement combination 5N were mitogenic for TES1 cell growth. This study will promote ES-based biotechnology in commercial fish.

Scaling of pattern formations and morphogen gradients.

The concentration gradient of morphogens provides positional information for an embryo and plays a pivotal role in pattern formation of tissues during the developmental processes. Morphogen-dependent pattern formations show robustness despite various perturbations. Although tissues usually grow and dynamically change their size during histogenesis, proper patterns are formed without the influence of size variations. Furthermore, even when the blastula embryo of Xenopus laevis is bisected into dorsal and ventral halves, the dorsal half of the embryo leads to proportionally patterned half-sized embryos. This robustness of pattern formation despite size variations is termed as scaling. In this review, I focused on the morphogen-dependent dorsal-ventral axis formation in Xenopus and described how morphogens form a proper gradient shape according to the embryo size.

Amino acid transporters implicated in endocytosis of Buchnera during symbiont transmission in the pea aphid.

BackgroundMany insects host their obligate, maternally transmitted symbiotic bacteria in specialized cells called bacteriocytes. One of the best-studied insect nutritional endosymbioses is that of the aphid and its endosymbiont, Buchnera aphidicola. Aphids and Buchnera are metabolically and developmentally integrated, but the molecular mechanisms underlying Buchnera transmission and coordination with aphid development remain largely unknown. Previous work using electron microscopy to study aphid asexual embryogenesis has revealed that Buchnera transmission involves exocytosis from a maternal bacteriocyte followed by endocytotic uptake by a blastula. While the importance of exo- and endocytic cellular processes for symbiont transmission is clear, the molecular mechanisms that regulate these processes are not known. Here, we shed light on the molecular mechanisms that regulate Buchnera transmission and developmental integration.ResultsWe present the developmental atlas of ACYPI000536 and ACYPI008904 mRNAs during asexual embryogenesis in the pea aphid, Acyrthosiphon pisum. Immediately before Buchnera invasion, transcripts of both genes were detected by whole-mount in situ hybridization in the posterior syncytial nuclei of late blastula embryos. Following Buchnera invasion, expression of both genes was identified in the region occupied by Buchnera throughout embryogenesis. Notably during Buchnera migration, expression of both genes was not concomitant with the entirety of the bacterial mass but rather expression colocalized with Buchnera in the anterior region of the bacterial mass. In addition, we found that ACYPI000536 was expressed in nuclei at the leading edge of the bacterial mass, joining the bacterial mass in subsequent developmental stages. Finally, quantitative reverse transcription real-time PCR suggested that early in development both transcripts were maternally provisioned to embryos.ConclusionsWe venture that ACYPI000536 and ACYPI008904 function as nutrient sensors at the site of symbiont invasion to facilitate TOR-pathway-mediated endocytosis of Buchnera by the aphid blastula. Our data support earlier reports of bacteriocyte determination involving a two-step recruitment process but suggest that the second wave of recruitment occurs earlier than previously described. Finally, our work highlights that bacteriocyte-enriched amino acid transporter paralogs have additionally been retained to play novel developmental roles in both symbiont recruitment and bacteriome development.Electronic supplementary materialThe online version of this article (doi:10.1186/s13227-016-0061-7) contains supplementary material, which is available to authorized users.

Germline replacement by blastula cell transplantation in the fish medaka.

Primordial germ cell (PGC) specification early in development establishes the germline for reproduction and reproductive technologies. Germline replacement (GR) is a powerful tool for conservation of valuable or endangered animals. GR is achievable by germ cell transplantation into the PGC migration pathway or gonads. Blastula cell transplantation (BCT) can also lead to the chimeric germline containing PGCs of both donor and host origins. It has remained largely unknown whether BCT is able to achieve GR at a high efficiency. Here we report efficient GR by BCT into blastula embryos in the fish medaka (Oryzias latipes). Specifically, dnd depletion completely ablated host PGCs and fertility, and dnd overexpression remarkably boosted PGCs in donor blastulae. BCT between normal donor and host produced a germline transmission rate of ~4%. This rate was enhanced up to ~30% upon PGC boosting in donors. Most importantly, BCT between PGC-boosted donors and PGC-ablated hosts led to more than 90% fertility restoration and 100% GR. Therefore, BCT features an extremely high efficiency of fertility recovery and GR in medaka. This finding makes medaka an ideal model to analyze genetic and physiological donor-host compatibilities for BCT-mediated surrogate production and propagation of endangered lower vertebrates and biodiversity.

Cell differentiation and germ-soma separation in Ediacaran animal embryo-like fossils.

Phosphorites of the Ediacaran Doushantuo Formation (∼600 million years old) yield spheroidal microfossils with a palintomic cell cleavage pattern. These fossils have been variously interpreted as sulphur-oxidizing bacteria, unicellular protists, mesomycetozoean-like holozoans, green algae akin to Volvox, and blastula embryos of early metazoans or bilaterian animals. However, their complete life cycle is unknown and it is uncertain whether they had a cellularly differentiated ontogenetic stage, making it difficult to test their various phylogenetic interpretations. Here we describe new spheroidal fossils from black phosphorites of the Doushantuo Formation that have been overlooked in previous studies. These fossils represent later developmental stages of previously published blastula-like fossils, and they show evidence for cell differentiation, germ-soma separation, and programmed cell death. Their complex multicellularity is inconsistent with a phylogenetic affinity with bacteria, unicellular protists, or mesomycetozoean-like holozoans. Available evidence also indicates that the Doushantuo fossils are unlikely crown-group animals or volvocine green algae. We conclude that an affinity with cellularly differentiated multicellular eukaryotes, including stem-group animals or algae, is likely but more data are needed to constrain further the exact phylogenetic affinity of the Doushantuo fossils.

Derivation of stable zebrafish ES-like cells in feeder-free culture.

Zebrafish offers an excellent opportunity to combine embryological, genetic and molecular analyses of vertebrate development in vivo. Embryonic stem (ES) cells have enormous potential to study developmental potency and differentiation in vitro and thus to complement in vivo approaches. Zebrafish ES-like cells have been produced on a feeder cell layer. Here, we report the derivation of Z428, a zebrafish ES-like cell line, from blastula embryos in feeder-free culture. Fetal bovine serum, fish serum, fish embryo extract, basic fibroblast growth factor, non-essential amino acids and 2-mercaptoethanol were found to be important for Z428 growth. After more than 120 passages and many freezing/thawing cycles over a period of 20years, Z428 exhibits stable growth and manifests many ES cell features including an ES cell phenotype, high alkaline phosphatase activity and spontaneous differentiation in culture. Most importantly, Z428 was transplantable to blastula hosts and capable of contributing to embryonic tissues and organ systems of the three germ layers. Therefore, Z428 is a stable cell line and contains ES-like cells with pluripotency in vitro and in vivo, and a feeder layer is dispensable for ES-like cell derivation in zebrafish. The derivation and easy maintenance of zebrafish ES-like cells under feeder-free conditions provide a useful extension of the present toolbox for studying development and differentiation in the zebrafish model.

Thermal stress alters expression of genes involved in one carbon and DNA methylation pathways in Atlantic cod embryos

One-carbon (1-C) metabolism is essential for normal embryonic development through its regulation of DNA methylation and cell proliferation. With consideration to the potential future anthropogenic oceanic warming, we studied the effects of both acute thermal stress and continuous thermal stress (10°C) during Atlantic cod embryo development on the expression levels of genes associated with the 1-C metabolism, including DNA methyltransferases. We conducted a phylogenetic analysis of vertebrate DNA methyltransferases to determine the number and similarity of DNMT found in Atlantic cod. This analysis revealed that Atlantic cod have one maintenance dnmt (dnmt1) and five de novo DNMTs (dnmt4, dnmt3, dnmt3b, dnmt3aa, dnmt3ab). Stage specific changes in expression levels occurred for all genes analyzed. The effect of acute thermal stress was evaluated during early development. Compared to controls these experiments showed significant alterations in expression levels of several genes, that in some instances were reversed at later stages of development. A significant effect of continuous thermal stress was found in gastrula embryos where lower mRNA expression levels of 1-C metabolism, de novo DNMTs and cell proliferation genes were detected. One exception was the maintenance DNMT, which was only sensitive to acute and not continuous thermal stress. DNA methylation status indicated that blastula embryos were hypomethylated compared to spermatozoa and late gastrula stages. In post-gastrula stage, however, continuous thermal stress resulted in a higher degree of DNA methylation compared to controls. These data reveal that the regulation of epigenetically important transcripts in the 1-C metabolism of Atlantic cod embryos is sensitive to thermal stress.

Possible Animal Embryos from the Lower Cambrian (Stage 3) Shuijingtuo Formation, Hubei Province, South China

Fossilized animal embryos from lower Cambrian rocks provide a rare opportunity to study the ontogeny and developmental biology of early animals during the Cambrian explosion. This paper reports possible animal embryos, along with sponge spicules, hyolithelminths, and linguliformean brachiopods, from the upper Shuijingtuo Formation limestone (Cambrian Stage 3) at Changyang, Hubei Province, South China. This limestone unit has carbonate carbon and oxygen isotopic compositions similar to the upper Shuijingtuo limestone in the Yangtze Gorges area. The Shuijingtuo embryo fossils were exposed by physical fracturing, extracted with acetic acid maceration, and observed in thin sections. They were examined using light microscopy, scanning electron microscopy, energy dispersive X-ray spectroscopic elemental mapping, and micro-focus X-ray computed tomography. Most of them are poorly preserved, with a phosphatic envelope (interpreted as a chorion or fertilization envelope) surrounding sparitic calcite. In some specimens, a polygonal pattern is present on the surface, and these are interpreted as multicelled blastula embryos. In others, sets of grooves are present on the surface of a calcitic spheroidal structure, presumably representing the calcitic interior within the chorion; these grooves are superficially similar to annulations ofMarkueliaembryos, but their biological significance is unknown. Although their phylogenetic and taxonomic placement is largely unconstrained, the Shuijingtuo animal embryos indicate that chorions are taphonomically more robust and are selectively phosphatized. Embryos within the chorions, on the other hand, can be completely lost or entirely replaced by calcite, with only poorly preserved surficial structures. This style of preservation can be explained by a taphonomic switch from early phosphatization to later calcitization. This study illustrates the importance of combining physical fracturing with widely used acid digestion methods in the study of calcitized animal embryos, and it alludes to the possibility that many empty phosphatic vesicles recovered by acid digestion from Cambrian carbonates may be fossilized chorions.