Multicellular Embryos Research Articles

Zero-shot learning enables instant denoising and super-resolution in optical fluorescence microscopy.

Computational super-resolution methods, including conventional analytical algorithms and deep learning models, have substantially improved optical microscopy. Among them, supervised deep neural networks have demonstrated outstanding performance, however, demanding abundant high-quality training data, which are laborious and even impractical to acquire due to the high dynamics of living cells. Here, we develop zero-shot deconvolution networks (ZS-DeconvNet) that instantly enhance the resolution of microscope images by more than 1.5-fold over the diffraction limit with 10-fold lower fluorescence than ordinary super-resolution imaging conditions, in an unsupervised manner without the need for either ground truths or additional data acquisition. We demonstrate the versatile applicability of ZS-DeconvNet on multiple imaging modalities, including total internal reflection fluorescence microscopy, three-dimensional wide-field microscopy, confocal microscopy, two-photon microscopy, lattice light-sheet microscopy, and multimodal structured illumination microscopy, which enables multi-color, long-term, super-resolution 2D/3D imaging of subcellular bioprocesses from mitotic single cells to multicellular embryos of mouse and C. elegans.

The maternal embrace: the protection of plant embryos.

All land plants-the embryophytes-produce multicellular embryos, as do other multicellular organisms, such as brown algae and animals. A unique characteristic of plant embryos is their immobile and confined nature. Their embedding in maternal tissues may offer protection from the environment, but also physically constrains development. Across the different land plants, a huge discrepancy is present between their reproductive structures whilst leading to similarly complex embryos. Therefore, we review the roles that maternal tissues play in the control of embryogenesis across land plants. These nurturing, constraining, and protective roles include both direct and indirect effects. In this review, we explore how the maternal surroundings affect embryogenesis and which chemical and mechanical barriers are in place. We regard these questions through the lens of evolution, and identify key questions for future research.

The Feeder Effects of Cultured Rice Cells on the Early Development of Rice Zygotes.

Feeder cells and the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) in a culture medium promote mitosis and cell division in cultured cells. These are also added to nutrient medium for the cultivation of highly active in mitosis and dividing zygotes, produced in vitro or isolated from pollinated ovaries. In the study, an in vitro fertilization (IVF) system was used to study the precise effects of feeder cells and 2,4-D on the growth and development of rice (Oryza sativa L.) zygote. The elimination of 2,4-D from the culture medium did not affect the early developmental profiles of the zygotes, but decreased the division rates of multicellular embryos. The omission of feeder cells resulted in defective karyogamy, fusion between male and female nuclei, and the subsequent first division of the cultured zygotes. The culture of zygotes in a conditioned medium corrected developmental disorders. Proteome analyses of the conditioned medium revealed the presence of abundant hydrolases possibly released from the feeder cells. Exogenously applied α-amylase ameliorated karyogamy and promoted zygote development. It is suggested that hydrolytic enzymes, including α-amylase, released from feeder cells may be involved in the progression of zygotic development.

СТАДИЯ ЭМБРИОГЕНЕЗА IN PLANTA ВЛИЯЕТ НА РЕАЛИЗАЦИЮ МОРФОГЕНЕТИЧЕСКОГО ПОТЕНЦИАЛА ЗАРОДЫШЕЙ ПШЕНИЦЫ IN VITRO

Biotechnological elaborations for breeding purposes aimed at creating highly productive wheat varieties require a significant number of plants-full-fledged regenerants. Such regenerants can be obtained under embryo culture in vitro conditions when using embryos at different ages as explants, according to the indirect pathway for the scheme “one embryo → one callus → several regenerants” and the direct pathway for the scheme “one embryo → one regenerant”. As a result of the performed experiments, it was found that the pathway of obtaining regenerants is determined by the stage of embryogenesis in planta of the embryo inoculated on the nutrient medium: the stage of completion of organogenesis and the stage of the formed embryo, respectively. In addition, data on the results of culture in vitro of embryos inoculated at other stages of embryogenesis are presented: four-cell and multicellular embryos, the early organogenesis, the middle organogenesis, the mature embryo.

Resetting of the 24-nt siRNA landscape in rice zygotes.

The zygote, a totipotent stem cell, is crucial to the life cycle of sexually reproducing organisms. It is produced by the fusion of two differentiated cells—the egg and sperm, which in plants have radically different siRNA transcriptomes from each other and from multicellular embryos. Owing to technical challenges, the epigenetic changes that accompany the transition from differentiated gametes to totipotent zygote are poorly understood. Because siRNAs serve as both regulators and outputs of the epigenome, we characterized small RNA transcriptomes of zygotes from rice. Zygote small RNAs exhibit extensive maternal carryover and an apparent lack of paternal contribution, indicated by absence of sperm signature siRNAs. Zygote formation is accompanied by widespread redistribution of 24-nt siRNAs relative to gametes, such that ∼70% of the zygote siRNA loci do not overlap any egg cell siRNA loci. Newly detected siRNA loci in zygote are gene-proximal and not associated with centromeric heterochromatin, similar to canonical siRNAs, in sharp contrast to gametic siRNA loci that are gene-distal and heterochromatic. In addition, zygote but not egg siRNA loci are associated with high DNA methylation in the mature embryo. Thus, the zygote begins transitioning before the first embryonic division to an siRNA profile that is associated with future RdDM in embryogenesis. These findings indicate that, in addition to changes in gene expression, the transition to totipotency in the plant zygote is accompanied by resetting of the epigenetic reprogramming that occurred during gamete formation.

Automatic wavelet-based 3D nuclei segmentation and analysis for multicellular embryo quantification

Identification of individual cells in tissues, organs, and in various developing systems is a well-studied problem because it is an essential part of objectively analyzing quantitative images in numerous biological contexts. We developed a size-dependent wavelet-based segmentation method that provides robust segmentation without any preprocessing, filtering or fine-tuning steps, and is robust to the signal-to-noise ratio. The wavelet-based method achieves robust segmentation results with respect to True Positive rate, Precision, and segmentation accuracy compared with other commonly used methods. We applied the segmentation program to zebrafish embryonic development IN TOTO for nuclei segmentation, image registration, and nuclei shape analysis. These new approaches to segmentation provide a means to carry out quantitative patterning analysis with single-cell precision throughout three dimensional tissues and embryos and they have a high tolerance for non-uniform and noisy image data sets.

An Essential Function for Auxin in Embryo Development.

Embryogenesis in seed plants is the process during which a single cell develops into a mature multicellular embryo that encloses all the modules and primary patterns necessary to build the architecture of the new plant after germination. This process involves a series of cell divisions and coordinated cell fate determinations resulting in the formation of an embryonic pattern with a shoot-root axis and cotyledon(s). The phytohormone auxin profoundly controls pattern formation during embryogenesis. Auxin functions in the embryo through its maxima/minima distribution, which acts as an instructive signal for tissue specification and organ initiation. In this review, we describe how disruptions of auxin biosynthesis, transport, and response severely affect embryo development. Also, the mechanism of auxin action in the development of the shoot-root axis and the three-tissue system is discussed with recent findings. Biological tools that can be implemented to study the auxin function during embryo development are presented, as they may be of interest to the reader.

Low cryoprotectant concentration rapid vitrification of mouse oocytes and embryos

Vitrification of mammalian oocytes and embryos is typically a two-step procedure involving two solutions of increasing concentrations of cryoprotectants. In the present study, we report a simple vitrification protocol that uses low cryoprotectant concentration and a single medium (LCSM). This medium, along with the traditional high concentration two media (HCTM) protocol, was used to vitrify mouse oocytes, zygotes, and blastocysts using silica capillary, cryotop, cryolock, and 0.25 ml straws. Survival rates, two-cell rates, and blastocyst formation rates were compared for oocytes and zygotes vitrified using both protocols. Results show that the LCSM protocol was as good as or better than the traditional HCTM protocol for vitrifying mouse MII oocytes and zygotes using silica capillary, cryotop, and cryolock. On the other hand, for blastocysts, only silica capillary using LCSM had comparable results with the traditional HCTM protocol while cryolock and cryotop had significantly lower percentages of re-expanded and hatched blastocysts. Collapsing blastocysts prior to vitrification or longer duration for better cryoprotectant distribution in multicellular embryos may improve the outcome. In conclusion, the LCSM protocol, with one medium of much lower cryoprotectant concentrations and shorter equilibration time, reduces exposure to cryoprotectant toxicity while improves efficiency, consistency and reliability for mammalian oocyte and embryo preservation.

Gametic embryogenesis and callogenesis in Isolated microspore culture of Jatropha curcas L. a recalcitrant bioenergy crop

Jatropha curcas is an undomesticated crop and its plantations did not meet commercial expectation due to absence of high yielding commercial line with desired agronomic characters. Earlier, breeding efforts did not pay much attention on yield and disease improving traits due to lack of desired genetic variability. Isolated microspore culture is one of the most widely used in vitro techniques to induce gametic embryogenesis with wide degree of genetic variability for the development of haploids and doubled haploids. In this study, an efficient isolated microspore culture system was established for different genotypes with the optimization of various factors that affect microspore embryogenesis. After 15–20 days of culture at 25 °C, tetrads, mid, early un-vacuolated and vacuolated late stage uninucleate microspores, which were preincubated at 4 °C for 7 days under shaking conditions, induced the formation of embryo like structures (ELSs) in a modified MS medium with 2.0 mg/l, 2,4-d, 0.1 mg/l kinetin, 300 mg/l casein hydrolysate, 1 g/l glutamine, 0.5 mg/l folic acid, 0.05 mg/l biotin and 5% sucrose. It is observed that the shock at 4 °C for 7 days, subsequently incubation of microspore cultures at 25 °C for 15 days and then at 15 °C for 10 days played a significant role in induction and accelerated creation of ELSs in three different genotypes. Microscopic analyses confirmed the different developmental stages of microspore embryogenesis including cell division and multicellular embryo like structures. Additionally, flow cytometric analyses verified the calli and ELSs were haploids in nature and strengthened that the origin was from microspores. This study supports the evidence of gametic embryogenesis in Jatropha microspore culture. Development of haploid and doubled haploids in Jatropha Microspore culture.

Culture of the brown seaweed Sargassum siliquosum J. Agardh (Phaeophyceae, Ochrophyta): from hatchery to out-planting

The brown seaweed Sargassum siliquosum is among the most common species with economic potential in the Philippines. However, reports on its culture are non-existent. This study describes the culture of S. siliquosum from land-based hatchery to out-planting in the open sea. Zygotes released from female receptacles can be recruited in large quantities unto artificial substrates and within 24 h produce multicellular embryos characterized by multiple bundles of rhizoids, and after 48–72 h elongated into germlings. After 2.5 months, germlings formed cauline leaves, and after 4 months, developed branches as seedlings. Recruits favored clay substrate than limestone and nylon in terms of density, growth, and longevity of seedlings. Desiccation treatment at different periods resulted to lower recruit density compared with control, had inferior growth and shorter longevity in all substrate types. For a period of 16 months, growth of S. siliquosum in the hatchery tanks was very slow, but became faster when out-planted in open sea using suspension culture method. Cultivars developed more branches after 4 months, at the same time producing receptacles. Maximum growth was achieved after 6 months, reaching a mean length of 42.8 ± 30.9 cm (max recorded was 1 m), some cultivars starting to show signs of senescence. In contrast, seedlings deployed using the bottom culture method survived for 3 months only without signs of further growth mainly due to epiphytism, siltation, and grazing. In conclusion, this study demonstrated the feasibility of farming S. siliquosum from hatchery-produced seedlings using suspension culture method to produce biomass for commercial use.

Gliding into diapause: early embryo development in roe deer (Capreolus capreolus)

During mammalian diapause, the embryo is arrested at the blastocyst stage. In diapausing carnivores and marsupials, growth arrest of the blastocyst is coincident with a quiescent corpus luteum (CL). In the roe deer, progesterone is produced throughout diapause and the blastocyst exhibits very slow growth. To date, early roe deer embryos of known age have not been described. Therefore, it is not known at which stage and at what pace the roe deer embryo enters diapause. To collect multiple embryos of known gestational age, we performed cycle synchronization and superovulation in captive roe deer prior to artificial insemination (AI) and natural breeding (NB), respectively. Superovulatory responses were evaluated based on ultrasonographic detection of CL formation on the day of in vivo embryo flushing and on plasma progesterone (P4) analysis. In total, 33 superovulation treatments resulted in 6.6 ± 2.2 (mean ± SD) CL per doe on the day of embryo flushing. At days 6 and 7 after AI, 37 multicellular embryos up to the morula stage were retrieved from 8 does. At day 12 and 13 after natural breeding, 28 embryos, mainly at the blastocyst stage (n=20), were collected from 7 does. Multiple embryos could be collected from single animals, proving the feasibility of superovulation in this species. Compared to other diapausing and non-diapausing species where blastocysts have formed at days 6 – 8, roe deer embryo development appeared to be already decelerated prior to the blastocyst stage. The roe deer might therefore be well suited as a model to investigate mechanisms regulating embryonic growth velocity with respect to cell cycle control.

Impacts of Acidic Seawater on Early Developmental Stages of Fucus gardneri at Burrard Inlet, British Columbia

Increases in stressors associated with climate change such as ocean acidification and warming temperatures pose a serious threat to intertidal ecosystems. Of crucial importance are the effects on foundational species, such as fucoid algae, a critical component of rocky intertidal shorelines around the world. The impact of climate change on adult fronds of fucoid algae has been documented but effects on early developmental stages is not as well understood. In particular, ocean acidification stands to impact these stages because zygotes and embryos are known to maintain internal pH and develop a cytosolic pH gradient during development. To assess the effects of seawater acidification on early development, zygotes were exposed to conditions that approximate current global averages and extend beyond future projections. Exposure to acidic seawater had significant effects on embryonic growth. Specifically, rhizoid elongation, which occurs by a process known as tip growth, was significantly reduced with each 0.5 unit drop in pH. Even a moderate decrease in pH from 8.0 to 7.5, which is similar to levels observed in the field, was associated with a nearly 20% reduction in rhizoid growth rate. Under more extreme conditions, at pH 6, rhizoid growth rates were reduced by 64% in comparison to embryos exposed to seawater at pH 8.0. On the other hand, acidic seawater had no effect on earlier processes; zygotes became multicellular embryos with well-formed rhizoids on a similar time course within the first 24 hours of development, even when exposed to pH 6, an extreme pH well below what is expected in the future. This suggests that zygotes can maintain an internal pH that allows germination and cell division to occur. Tip growth however, depends on the maintenance of an internal pH gradient. It is therefore possible that disruptions to this gradient could account for the observed reductions in rhizoid elongation. Under acidic conditions proton influx into the cell becomes energetically more favorable than at pH 8, and expulsion would be more difficult. This could disrupt the cytosolic pH gradient and in turn affect rhizoid growth.

Signaling events regulating embryonic polarity and formation of the primitive streak in the chick embryo.

The avian embryo is a key experimental model system for early development of amniotes. One key difference with invertebrates and "lower" vertebrates like fish and amphibians is that amniotes do not rely so heavily on maternal messages because the zygotic genome is activated very early. Early development also involves considerable growth in volume and mass of the embryo, with cell cycles that include G1 and G2 phases from very early cleavage. The very early maternal to zygotic transition also allows the embryo to establish its own polarity without relying heavily on maternal determinants. In many amniotes including avians and non-rodent mammals, this enables an ability of the embryo to "regulate": a single multicellular embryo can give rise to more than one individual-monozygotic twins. Here we discuss the embryological, cellular, molecular and evolutionary underpinnings of gastrulation in avian embryos as a model amniote embryo. Many of these properties are shared by human embryos.

Electroporation in Ascidians: History, Theory and Protocols.

Embryonic development depends on the orchestration of hundreds of regulatory and structural genes to initiate expression at the proper time, in the correct spatial domain(s), and in the amounts required for cells and tissues to become specified, determined, and ultimately to differentiate into a multicellular embryo. One of the key approaches to studying embryonic development is the generation of transgenic animals in which recombinant DNA molecules are transiently or stably introduced into embryos to alter gene expression, to manipulate gene function or to serve as reporters for specific cell types or subcellular compartments. In some model systems, such as the mouse, well-defined approaches for generating transgenic animals have been developed. In other systems, particularly non-model systems, a key challenge is to find a way of introducing molecules or other reagents into cells that produces large numbers of embryos with a minimal effect on normal development. A variety of methods have been developed, including the use of viral vectors, microinjection, and electroporation. Here, I describe how electroporation was adapted to generate transgenic embryos in the ascidian, a nontraditional invertebrate chordate model that is particularly well-suited for studying gene regulatory activity during development. I present a review of the electroporation process, describe how electroporation was first implemented in the ascidian, and provide a series of protocols describing the electroporation process, as implemented in our laboratory.

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

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

Live birth rates and miscarriage rates with the use of calcium ionophore in patients with prior failed or poor fertilization

Live birth rates and miscarriage rates with the use of calcium ionophore in patients with prior failed or poor fertilization

Auxin Biosynthesis, Accumulation, Action and Transport are Involved in Stress-Induced Microspore Embryogenesis Initiation and Progression in Brassica napus.

Isolated microspores are reprogrammed in vitro by stress, becoming totipotent cells and producing embryos and plants via a process known as microspore embryogenesis. Despite the abundance of data on auxin involvement in plant development and embryogenesis, no data are available regarding the dynamics of auxin concentration, cellular localization and the expression of biosynthesis genes during microspore embryogenesis. This work involved the analysis of auxin concentration and cellular accumulation; expression of TAA1 and NIT2 encoding enzymes of two auxin biosynthetic pathways; expression of the PIN1-like efflux carrier; and the effects of inhibition of auxin transport and action by N-1-naphthylphthalamic acid (NPA) and α-(p-chlorophenoxy) isobutyric acid (PCIB) during Brassica napus microspore embryogenesis. The results indicated de novo auxin synthesis after stress-induced microspore reprogramming and embryogenesis initiation, accompanying the first cell divisions. The progressive increase of auxin concentration during progression of embryogenesis correlated with the expression patterns of TAA1 and NIT2 genes of auxin biosynthetic pathways. Auxin was evenly distributed in early embryos, whereas in heart/torpedo embryos auxin was accumulated in apical and basal embryo regions. Auxin efflux carrier PIN1-like gene expression was induced in early multicellular embryos and increased at the globular/torpedo embryo stages. Inhibition of polar auxin transport (PAT) and action, by NPA and PCIB, impaired embryo development, indicating that PAT and auxin action are required for microspore embryo progression. NPA also modified auxin embryo accumulation patterns. These findings indicate that endogenous auxin biosynthesis, action and polar transport are required in stress-induced microspore reprogramming, embryogenesis initiation and progression.

Effects of prenatal exposure to electromagnetic fields on biomarkers of oxidative stress and fertility of F1 generation In-vitro

The aim of the present study was to evaluate the effects of Electromagnetic Field (EMF) in the prenatal period on biomarkers of oxidative stress and fertility of F1 generation. Material and Methods: The pregnant rats divided to 3 group’s treatment (EMF exposure), Sham (off -EMF) and control. After delivery the blood sample of mothers provided and pups were kept until maturity age, and then their fertility was investigated. Results: CAT in treatment group was significantly decreased but no differences in GPX were observed. In treatment group decrease in multicellular embryos and abnormality in cleavage pattern was found. Conclusion: Exposure in EMF cause oxidative stress and reduce fertility.

Module 8: Development

Development encompasses the programme of events that begins with fertilization and culminates in complex multicellular organisms like ourselves. Every cell in the adult organism has a distinct life history (Module 8: Figure cellular life history) that can be traced back to the process of maturation that is responsible for the formation of the two gametes that then fuse during fertilization to form the zygote. Fertilization provides a potent mitogenic stimulus, and the zygote enters the cell cycle repeatedly and is rapidly transformed into a multicellular embryo. As development proceeds and the number of cells in the embryo increases, groups of cells are progressively apportioned distinct fates through a process of cell specification. During the final process of cell differentiation, each cell begins to express its unique specialized cell function that enables it to contribute to the operation of different organ systems. This process of differentiation is associated with the cessation of growth, which may be either permanent (e.g. nerves and muscle) or temporary (e.g. lymphocytes, fibroblasts, liver cells, salivary glands, endothelial cells and smooth muscle). In the latter case, the cells that have their growth arrested temporarily retain the ability to return to the cell cycle when stimulated with the appropriate growth factor. Finally, cells die and this can occur either through some catastrophic event, such as necrosis, or through a more controlled programme of cell death known as apoptosis. The typical life history of a cell thus comprises a series of processes: Maturation–the formation of mature gametes (spermatozoa and oocytes). Fertilization–the fusion of gametes and the initiation of development. Cell proliferation–rapid rounds of cell growth and cell division. Cell specification–cell determination and pattern formation. Differentiation–the formation of specialized cells.

Ultrastructural and structural characterization of zygotes and embryos during development in Sargassum cymosum (Phaeophyceae, Fucales).

This study investigates the pattern and performance of cellular structures during the early development of zygotes and embryos of Sargassum cymosum. The early development S. cymosum germlings has already been characterized and compared with the pattern of development established for all fucoid algae, in which the zygote remains attached to the receptacle by mucilage during the establishment of polarity and early cell division. As in the algae Fucus and Silvetia, the first division is transverse across the longer axis of the zygote of S. cymosum. However, the cell that will give rise to the rhizoids is not determined in the first division; rather, the formation of this cell occurs with the second division, forming a small cell in the embryo shaded site. Stabilizing polarity during the process of forming a multicellular embryo occurs rapidly. During development, significant cytoplasmic alterations take place. Initially, the cytoplasm shows large clusters of phenolic compounds located in specific parts, but later, in the course of development, these compounds are dispersed in the cytoplasm, although a significant amount remains confined to the nucleus. Moreover, to produce more zygotes and higher growth rates for the germlings, the best conditions found for the species S. cymosum were 22 and 26 °C, respectively.