Axis formation in annual killifish: Nodal and ß-catenin regulate morphogenesis without Huluwa prepatterning.

Axis formation in fish and amphibians typically begins with a prepattern of maternal gene products. Annual killifish embryogenesis, however, challenges prepatterning models as blastomeres disperse and then aggregate to form the germ layers and body axes. We show that huluwa, a prepatterning factor thought to break symmetry by stabilizing ß-catenin, is truncated and inactive in Nothobranchius furzeri. Nuclear ß-catenin is not selectively stabilized on one side of the blastula but accumulates in cells forming the aggregate. Blocking ß-catenin activity or Nodal signaling disrupts aggregate formation and germ layer specification. Nodal signaling coordinates cell migration, establishing an early role for this signaling pathway. These results reveal a surprising departure from established mechanisms of axis formation: Huluwa-mediated prepatterning is dispensable, and ß-catenin and Nodal regulate morphogenesis.

BET activity plays an essential role in control of stem cell attributes in Xenopus.

Neural crest cells are a stem cell population unique to vertebrate embryos that retains broad multi-germ layer developmental potential through neurulation. Much remains to be learned about the genetic and epigenetic mechanisms that control the potency of neural crest cells. Here, we examine the role that epigenetic readers of the BET (bromodomain and extra terminal) family play in controlling the potential of pluripotent blastula and neural crest cells. We find that inhibiting BET activity leads to loss of pluripotency at blastula stages and a loss of neural crest at neurula stages. We compare the effects of HDAC (an eraser of acetylation marks) and BET (a reader of acetylation) inhibition and find that they lead to similar cellular outcomes through distinct effects on the transcriptome. Interestingly, loss of BET activity in cells undergoing lineage restriction is coupled to increased expression of genes linked to pluripotency and prolongs the competence of initially pluripotent cells to transit to a neural progenitor state. Together these findings advance our understanding of the epigenetic control of pluripotency and the formation of the vertebrate neural crest.

SoxB1 transcription factors are essential for initiating and maintaining neural plate border gene expression.

SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). Using Xenopus laevis, we set out to determine whether SoxB1 transcription factors have a regulatory function in NPB formation. Here, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq, we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as potential Sox3 partners in regulating the formation of the NPB and show that their combined activity is needed for normal NPB gene expression. Together, these data identify a role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors.

Changes in repair pathways of radiation-induced DNA double-strand breaks at the midblastula transition in Xenopus embryo.

Ionizing radiation (IR) causes DNA damage, particularly DNA double-strand breaks (DSBs), which have significant implications for genome stability. The major pathways of repairing DSBs are homologous recombination (HR) and nonhomologous end joining (NHEJ). However, the repair mechanism of IR-induced DSBs in embryos is not well understood, despite extensive research in somatic cells. The externally developing aquatic organism, Xenopus tropicalis, serves as a valuable model for studying embryo development. A significant increase in zygotic transcription occurs at the midblastula transition (MBT), resulting in a longer cell cycle and asynchronous cell divisions. This study examines the impact of X-ray irradiation on Xenopus embryos before and after the MBT. The findings reveal a heightened X-ray sensitivity in embryos prior to the MBT, indicating a distinct shift in the DNA repair pathway during embryo development. Importantly, we show a transition in the dominant DSB repair pathway from NHEJ to HR before and after the MBT. These results suggest that the MBT plays a crucial role in altering DSB repair mechanisms, thereby influencing the IR sensitivity of developing embryos.

Small molecule-mediated reprogramming of Xenopus blastula stem cells to a neural crest state.

Neural crest cells are a stem cell population unique to vertebrates that give rise to a diverse array of derivatives, including much of the peripheral nervous system, pigment cells, cartilage, mesenchyme, and bone. Acquisition of these cells drove the evolution of vertebrates and defects in their development underlies a broad set of neurocristopathies. Moreover, studies of neural crest can inform differentiation protocols for pluripotent stem cells and regenerative medicine applications. Xenopus embryos are an important system for studies of the neural crest and have provided numerous insights into the signals and transcription factors that control the formation and later lineage diversification of these stem cells. Pluripotent animal pole explants are a particularly powerful tool in this system as they can be cultured in simple salt solution and instructed to give rise to any cell type including the neural crest. Here we report a protocol for small molecule-mediated induction of the neural crest state from blastula stem cells and validate it using transcriptome analysis and grafting experiments. This is an powerful new tool for generating this important cell type that will facilitate future studies of neural crest development and mutations and variants linked to neurocristopathies.

Induced formation of primordial germ cells from zebrafish blastomeres by germplasm factors.

The combination of genome editing and primordial germ cell (PGC) transplantation has enormous significance in the study of developmental biology and genetic breeding, despite its low efficiency due to limited number of donor PGCs. Here, we employ a combination of germplasm factors to convert blastoderm cells into induced PGCs (iPGCs) in zebrafish and obtain functional gametes either through iPGC transplantation or via the single blastomere overexpression of germplasm factors. Zebrafish-derived germplasm factors convert blastula cells of Gobiocypris rarus into iPGCs, and Gobiocypris rarus spermatozoa can be produced by iPGC-transplanted zebrafish. Moreover, the combination of genome knock-in and iPGC transplantation perfectly resolves the contradiction between high knock-in efficiency and early lethality during embryonic stages and greatly improves the efficiency of genome knock-in. Together, we present an efficient method for generating PGCs in a teleost, a technique that will have a strong impact in basic research and aquaculture.

Measurement of ultrasound velocity in yolk and blastula of fish embryo in vivo.

An acoustic microscopy method for measuring the velocity of ultrasound in the yolk and blastula of bony fish embryos at early stages of development was proposed. The yolk and blastula were approximated as a sphere and a spherical dome, respectively, consisting of a homogeneous liquid. A theoretical model of ultrasonic wave propagation through a spherical liquid drop located on a solid substrate was developed in the ray approximation. The dependence of the wave propagation time on the speed of sound in the drop, its diameter, and the position of the focus of the ultrasonic transducer has been determined. It was shown that the velocity in the drop can be found by solving the inverse problem by minimizing the discrepancy between the experimental and model spatial distributions of the propagation time, assuming that the velocity in the immersion liquid and the radius of the drop are known. The velocities in the yolk and blastula of the loach (Misgurnus fossilis) embryo at the stage of development of the middle blastula were measured in vivo using a pulsed scanning acoustic microscope operating at a central frequency of 50 MHz. The yolk and blastula radii were determined from ultrasound images of the embryo. Acoustic microscopy measurements conducted with four embryos provide velocities of the acoustic longitudinal wave in the yolk and blastula. They were measured to be 1581 ± 5 m/s and 1525 ± 4 m/s when the temperature of the liquid in the water tank was kept at 22 ± 2 °C.

The complete dorsal structure is formed from only the blastocoel roof of Xenopus blastula: insight into the gastrulation movement evolutionarily conserved among chordates.

Gastrulation is a critical event whose molecular mechanisms are thought to be conserved among vertebrates. However, the morphological movement during gastrulation appears to be divergent across species, making it difficult to discuss the evolution of the process. Previously, we proposed a novel amphibian gastrulation model, the "subduction and zippering (S&Z) model". In this model, the organizer and the prospective neuroectoderm are originally localized in the blastula's blastocoel roof, and these embryonic regions move downward to make physical contact of their inner surfaces with each other at the dorsal marginal zone. The developmental stage when contact between the head organizer and the anterior-most neuroectoderm is established is called "anterior contact establishment (ACE)." After ACE, the A-P body axis elongates posteriorly. According to this model, the body axis is derived from limited regions of the dorsal marginal zone at ACE. To investigate this possibility, we conducted stepwise tissue deletions using Xenopus laevis embryos and revealed that the dorsal one-third of the marginal zone had the ability to form the complete dorsal structure by itself. Furthermore, a blastocoel roof explant of the blastula, which should contain the organizer and the prospective neuroectoderm in the S&Z model, autonomously underwent gastrulation and formed the complete dorsal structure. Collectively, these results are consistent with the S&Z gastrulation model and identify the embryonic region sufficient for construction of the complete dorsal structure. Finally, by comparing amphibian gastrulation to gastrulation of protochordates and amniotes, we discuss the gastrulation movement evolutionarily conserved among chordates.

Maternal mRNA deadenylation and allocation via Rbm14 condensates facilitate vertebrate blastula development.

Early embryonic development depends on proper utilization and clearance of maternal transcriptomes. How these processes are spatiotemporally regulated remains unclear. Here we show that nuclear RNA-binding protein Rbm14 and maternal mRNAs co-phase separate into cytoplasmic condensates to facilitate vertebrate blastula-to-gastrula development. In zebrafish, Rbm14 condensates were highly abundant in blastomeres and markedly reduced after prominent activation of zygotic transcription. They concentrated at spindle poles by associating with centrosomal γ-tubulin puncta and displayed mainly asymmetric divisions with a global symmetry across embryonic midline in 8- and 16-cell embryos. Their formation was dose-dependently stimulated by m6 A, but repressed by m5 C modification of the maternal mRNA. Furthermore, deadenylase Parn co-phase separated with these condensates, and this was required for deadenylation of the mRNAs in early blastomeres. Depletion of Rbm14 impaired embryonic cell differentiations and full activations of the zygotic genome in both zebrafish and mouse and resulted in developmental arrest at the blastula stage. Our results suggest that cytoplasmic Rbm14 condensate formation regulates early embryogenesis by facilitating deadenylation, protection, and mitotic allocation of m6 A-modified maternal mRNAs, and by releasing the poly(A)-less transcripts upon regulated disassembly to allow their re-polyadenylation and translation or clearance.

Nile tilapia (Oreochromis niloticus) Nanog co-expression with Pou5f3, transcriptional regulation and biological activity in embyonic development and embryonic cells.

Mammalian Nanog is critical in pluripotency acquisition and maintenance. Nonetheless, a recent report from zebrafish (Danio rerio) suggests that Nanog is not required for embryonic cells which is not like the mammalian homologs, but is necessary for the proper formation of the extra-embryonic yolk syncytial layer (YSL). However, whether its biological function in other fishes is conservative remains to be investigated. Our previous work shows that Nanog from Nile tilapia (Oreochromis niloticus) (termed as Ong thereafter) displays differential spatiotemporal expression patterns from the other teleost fishes including zebrafish. In this study, Ong co-expression with Pou5f3 (another core pluripotent transcription factor), transcriptional regulation and its biological functions during embryonic development and in the survival and proliferation of embryonic cells were investigated. At the blastula stage, both Ong and Pou5f3 were highly expressed in embryonic cells and co-located in the nucleus. After that, the expression of both Ong and Pou5f3 began to decrease at the gastrula stage (24 haf) and then exhibited a differential expression profile at the segmentation stage (28-36 haf). Ong disappeared in embryonic cells and was limited to YSL, whilst Pou5f3 was highly expressed in embryonic cells even some with obvious cytoplasmic distribution. Luciferase assay indicated that Ong was negatively regulated by Pou5f3 and positively regulated by androgen and itself. Ong depletion in fertilized one-cell embryos through CRISPR/Cas9 led to blastula blockage or death, and the survival and proliferation of blastula-derived embryonic cells in vitro failed. Collectively, Ong has similar expression and biological function to Pou5f3 at the blastula stage, which is similar to mammalian homolog but different from zebrafish homolog. These data suggest that the expression patterns and functions of Nanog are not conservative in fishes and vary from species to species. This study enriches our understanding about Nanog and its evolution.

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.

Wolbachia action in the sperm produces developmentally deferred chromosome segregation defects during the Drosophila mid-blastula transition.

Wolbachia, a vertically transmitted endosymbiont infecting many insects, spreads rapidly through uninfected populations by a mechanism known as cytoplasmic incompatibility (CI). In CI, a paternally delivered modification of the sperm leads to chromatin defects and lethality during and after the first mitosis of embryonic development in multiple species. However, whether CI-induced defects in later stage embryos are a consequence of the first division errors or caused by independent defects remains unresolved. To address this question, we focused on ~1/3 of embryos from CI crosses in Drosophila simulans that develop apparently normally through the first and subsequent pre-blastoderm divisions before exhibiting mitotic errors during the mid-blastula transition and gastrulation. We performed single embryo PCR and whole genome sequencing to find a large percentage of these developed CI-derived embryos bypass the first division defect. Using fluorescence in situ hybridization, we find increased chromosome segregation errors in gastrulating CI-derived embryos that had avoided the first division defect. Thus, Wolbachia action in the sperm induces developmentally deferred defects that are not a consequence of the first division errors. Like the immediate defect, the delayed defect is rescued through crosses to infected females. These studies inform current models on the molecular and cellular basis of CI.

Single-cell Sequencing Reveals Clearance of Blastula Chromosomal Mosaicism in In Vitro Fertilization Babies.

Although chromosomal mosaic embryos detected by trophectoderm (TE) biopsy offer healthy embryos available for transfer, high-resolution postnatal karyotyping and chromosome testing of the transferred embryos are insufficient. Here, we applied single-cell multi-omics sequencing for seven infants with blastula chromosomal mosaicism detected by TE biopsy. The chromosome ploidy was examined by single-cell genome analysis, with the cellular identity being identified by single-cell transcriptome analysis. A total of 1616 peripheral leukocytes from seven infants with embryonic chromosomal mosaicism and three control ones with euploid TE biopsy were analyzed. A small number of blood cells showed copy number alterations (CNAs) on seemingly random locations at a frequency of 0%-2.5% per infant. However, none of the cells showed CNAs that were the same as those of the corresponding TE biopsies. The blastula chromosomal mosaicism may be fully self-corrected, probably through the selective loss of the aneuploid cells during development, and the transferred embryos can be born as euploid infants without mosaic CNAs corresponding to the TE biopsies. The results provide a new reference for the evaluations of transferring chromosomal mosaic embryos in certain situations.

The Effects of Low-Level Helium-Neon (He-Ne) Laser Irradiation on Lipids and Fatty Acids, and the Activity of Energetic Metabolism Enzymes and Proteome in the Blastula Stage and Underyearlings of the Atlantic Salmon Salmo salar: A Novel Approach in Salmonid Restoration Procedures in the North.

The effect of He-Ne laser irradiation on fishery parameters as well as on biochemical state, including the lipids and fatty acids, the activity of energy metabolism enzymes and the proteome in the blastula stage and in underyearlings of wild Atlantic salmon after irradiation at the cleavage stage/early blastula (considered as the stages when the cell has a high potential for differentiation) was studied. Low mortality rates of eggs were determined during embryogenesis, as well as increased weight gain and lower morality rates of underyearlings in the experimental group. This is confirmed by changes in a number of interrelated indicators of lipid metabolism: a decrease in total lipids content, including diacylglycerols, triacylglycerols, cholesterol esters, and the phospholipids content remained unchanged. The embryos in the blastula stage (experimental group) had higher aerobic capacity and an increase in pentose phosphate pathway activity. The proteome profiles of eggs in the blastula stage were 131 proteins, of which 48 were significantly identified. The major protein was found to be phosvitin. The proteomes of underyearlings were represented by 2018 proteins, of which 49 were unique for the control and 39 for the experimental group. He-Ne laser irradiation had a strong effect on the contents of histone proteins.

A single-cell RNA-seq analysis of Brachyury-expressing cell clusters suggests a morphogenesis-associated signal center of oral ectoderm in sea urchin embryos.

Brachyury is a T-box family transcription factor and plays pivotal roles in morphogenesis. In sea urchin embryos, Brachyury is expressed in the invaginating endoderm, and in the oral ectoderm of the invaginating mouth opening. The oral ectoderm is hypothesized to serve as a signaling center for oral (ventral)-aboral (dorsal) axis formation and to function as a ventral organizer. Our previous results of a single-cell RNA-seq (scRNA-seq) atlas of early Strongylocentrotus purpuratus embryos categorized the constituent cells into 22 clusters, in which the endoderm consists of three clusters and the oral ectoderm four clusters (Foster et al., 2020). Here we examined which clusters of cells expressed Brachyury in relation to the morphogenesis and the identity of the ventral organizer. Our results showed that cells of all three endoderm clusters expressed Brachyury in blastulae. Based on expression profiles of genes involved in the gene regulatory networks (GRNs) of sea urchin embryos, the three clusters are distinguishable, two likely derived from the Veg2 tier and one from the Veg1 tier. On the other hand, of the four oral-ectoderm clusters, cells of two clusters expressed Brachyury at the gastrula stage and genes that are responsible for the ventral organizer at the late blastula stage, but the other two clusters did not. At a single-cell level, most cells of the two oral-ectoderm clusters expressed organizer-related genes, nearly a half of which coincidently expressed Brachyury. This suggests that the ventral organizer contains Brachyury-positive cells which invaginate to form the stomodeum. This scRNA-seq study therefore highlights significant roles of Brachyury-expressing cells in body-plan formation of early sea urchin embryos, though cellular and molecular mechanisms for how Brachyury functions in these processes remain to be elucidated in future studies.

Stages of embryonic development in the live-bearing fish, Gambusia holbrooki.

BACKGROUND: Divergent morphology and placentation of Poeciliids make them suitable model for investigating how evolutionary selection has altered and conserved the developmental mechanisms. However, there is limited description of their embryonic staging, despite representing a key evolutionary node that shares developmental strategy with placental vertebrates. Here, we describe the embryonic developmental stages of Gambusia holbrooki from zygote to parturition using freshly harvested embryos. RESULTS: We defined 40 embryonic stages using a numbered (stages 0-39; zygote to parturition, respectively) and named (grouped into seven periods, ie, zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and parturition) staging system. Two sets of quantitative (ie, egg diameter, embryonic total length, otic vesicle closure index, heart rates, the number of caudal fin rays and elements) and qualitative (ie, three-dimensional analysis of images and key morphological criteria) data were acquired and used in combination to describe each stage. All 40 stages are separated by well-defined morphological traits, revealing developmental novelties that are influenced by narrow perivitelline space, placentation, internal gestation, and sex differentiation. CONCLUSIONS: The principal diagnostic features described are quick, reliable, and easy to apply. This system will benefit researchers investigating molecular ontogeny, particularly sexual differentiation mechanisms in G. holbrooki.

The Development of Nomograms to Predict Blastulation Rate Following Cycles of In Vitro Fertilization in Patients With Tubal Factor Infertility, Polycystic Ovary Syndrome, or Endometriosis.

It is well known that the transfer of embryos at the blastocyst stage is superior to the transfer of embryos at the cleavage stage in many respects. However, the rate of blastocyst formation remains low in clinical practice. To reduce the possibility of wasting embryos and to accurately predict the possibility of blastocyst formation, we constructed a nomogram based on range of clinical characteristics to predict blastocyst formation rates in patients with different types of infertility. We divided patients into three groups based on female etiology: a tubal factor group, a polycystic ovary syndrome group, and an endometriosis group. Multiple logistic regression was used to analyze the relationship between patient characteristics and blastocyst formation. Each group of patients was divided into a training set and a validation set. The training set was used to construct the nomogram, while the validation set was used to test the performance of the model by using discrimination and calibration. The area under the curve (AUC) for the three groups indicated that the models performed fairly and that calibration was acceptable in each model.

Temporal Transcriptome Analysis Reveals Dynamic Expression Profiles of Gametes and Embryonic Development in Japanese Flounder (Paralichthys olivaceus).

The maternal-to-zygotic transition (MZT) is a crucial event in embryo development. While the features of the MZT across species are shared, the stage of this transition is different among species. We characterized MZT in a flatfish species, Japanese flounder (Paralichthys olivaceus). In this study, we analyzed the 551.57 GB transcriptome data of two types of gametes (sperms and eggs) and 10 embryo developmental stages in Japanese flounder. We identified 2512 maternal factor-related genes and found that most of those maternal factor-related genes expression decreased at the low blastula (LB) stage and remained silent in the subsequent embryonic development period. Meanwhile, we verified that the zygotic genome transcription might occur at the 128-cell stage and large-scale transcription began at the LB stage, which indicates the LB stage is the major wave zygotic genome activation (ZGA) occurs. In addition, we indicated that the Wnt signaling pathway, playing a diverse role in embryonic development, was involved in the ZGA and the axis formation. The results reported the list of the maternal genes in Japanese flounder and defined the stage of MZT, contributing to the understanding of the details of MZT during Japanese flounder embryonic development.

Control of Developmental Speed in Zebrafish Embryos Using Different Incubation Temperatures.

The zebrafish is a valuable model organism that is widely used in studies of vertebrate development. In the laboratory, zebrafish embryonic development is normally carried out at 28.5°C. In this study, we sought to determine whether it was possible to modify the speed of embryonic development through the use of short- and long-term variations in incubation temperature. After incubation at 20°C-32°C, most early-stage embryos survived to the epiboly stage, whereas more than half of the embryos died at <20°C or >32°C. The rate of development differed between embryos incubated at the lowest (18°C) and highest (34°C) temperatures: a difference of 60 min was observed at the 2-cell stage and 290 min at the 1k-cell stage. When blastulae that had developed at 28°C were transferred to a temperature lower than 18°C for one or more hours, they developed normally after being returned to the original 28°C. Analyses using green fluorescent protein-buckyball mRNA and in situ hybridization against vasa mRNA showed that primordial germ cells increase under low-temperature culture; this response may be of use for studies involving heterochronic germ cell transplantation. Our study shows that embryonic developmental speed can be slowed, which will be of value for performing time-consuming, complicated, and delicate microsurgical operations.

Extracellular matrix mediates moruloid-blastuloid morphodynamics in malignant ovarian spheroids.

Ovarian cancer metastasizes into peritoneum through dissemination of transformed epithelia as multicellular spheroids. Harvested from the malignant ascites of patients, spheroids exhibit startling features of organization typical to homeostatic glandular tissues: lumen surrounded by smoothly contoured and adhered epithelia. Herein, we demonstrate that cells of specific ovarian cancer lines in suspension, aggregate into dysmorphic solid "moruloid" clusters that permit intercellular movement, cell penetration, and interspheroidal coalescence. Moruloid clusters subsequently mature into "blastuloid" spheroids with smooth contours, a temporally dynamic lumen and immotile cells. Blastuloid spheroids neither coalesce nor allow cell penetration. Ultrastructural examination reveals a basement membrane-like extracellular matrix coat on the surface of blastuloid, but not moruloid, spheroids. Quantitative proteomics reveals down-regulation in ECM protein Fibronectin-1 associated with the moruloid-blastuloid transition; immunocytochemistry also confirms the relocalization of basement membrane ECM proteins: collagen IV and laminin to the surface of blastuloid spheroids. Fibronectin depletion accelerates, and enzymatic basement membrane debridement impairs, lumen formation, respectively. The regulation by ECM dynamics of the morphogenesis of cancer spheroids potentially influences the progression of the disease.