Blastula Stage Research Articles

PERKEMBANGAN EMBRIO DAN PERFORMA AWAL LARVA IKAN TAWES (Barbonymus gonionotus)

The research was carried out from November 2021 to January 2022, and the trial period from November 1 to November 12, 2021 at the Germplasm Research Installation for Freshwater Fisheries in Cijeruk, Bogor, West Java. This study aims to determine the embryonic development and early performance of Tawes fish (Barbonymus gonionotus) larvae. This experimental study used 2 male tawes broodstock, 2 female brood fish, and anesthetic drugs. Parameters observed included observation of embryos and early performance of Tawes larvae. The embryo data were analyzed in a qualitative descriptive manner with pictures of each phase of egg development and followed by data on egg hatching time. The results showed that the early embryonic development of Tawes fish generally includes the stages of egg division, morula, blastula, gastrula, segmentation and hatching. The initial performance of Tawes larvae reared at 28oC showed abnormality values ​​of Tawes fish of 5.78±1.52%, average egg diameter of 1.02±0.04 mm, average initial length of larvae of 7.28±0.55 mm.

Toxic, cytotoxic and genotoxic effect of plumbagin in the developmental stages of Biomphalaria glabrata (Say, 1818-intermediate host) and cercaricidal activity against the infectious agent of schistosomiasis mansoni.

Snails of the genus Biomphalaria are intermediate hosts of Schistosoma mansoni, the main etiological agent of schistosomiasis mansoni, which affects about 236.6 million people in tropical and subtropical regions of the world. The World Health Organization recommends the population control of vector snails as one of the strategies to reduce the prevalence and incidence of schistosomiasis. In this study, molluscicidal and antiparasitic activities of plumbagin, a naturally sourced naphthoquinone with a range of biological effects, were evaluated against B. glabrata and cercariae of S. mansoni. After 24 h of exposure, plumbagin demonstrated molluscicidal activity at low concentrations against embryos (LC50 of 0.56, 0.93, 0.68, 0.51 and 0.74 μg mL-1 for the blastula, gastrula, trochophore, veliger and hippo stage, respectively) and adult snails (LC50 of 3.56 μg mL-1 ). There were no changes in exposed snails' fecundity or fertility; however, plumbagin was able to increase the frequency of DNA damage and the number of hemocytes, with apoptosis and binucleation being the main hemocyte alterations. In addition, plumbagin showed death of S. mansoni cercariae in the concentration of 1.5μg mL-1 in 60 min, while showing moderate toxicity to Artemia salina. Plumbagin proved to be a promising substance for the control of B. glabrata population, intermediate host of S. mansoni, as well as the cercariae, infective stage for humans (definitive host), while being moderately toxic to A. salina, a crustacean widely used in ecotoxicity tests. © 2022 Society of Chemical Industry.

Genome-Wide Identification of Laminin Family Related to Follicular Pseudoplacenta Development in Black Rockfish (Sebastes schlegelii).

As major elements of the basement membrane, laminins play a significant role in angiogenesis, migration, and adhesion of various cells. Sebastes schlegelii is a marine viviparous teleost of commercial importance. Previous research has reported abundant blood vessels and connective tissue in the ovary during gestation. In this study, 14 laminin genes of the α, β, and γ subfamilies from genomic data were identified based on zebrafish and human laminins, distributed on 9 chromosomes in S. schlegelii. Analysis of structural domains showed that coiled-coil regions and EGF domains existed in all laminin genes. Moreover, via qPCR, we found that the expression of laminin genes, including lama4, lama5, lamb4, lamc1, and lamc3, gradually increased from the phase III ovary stage and peaked in the early stage of gestation, especially lama4 and lama5 which showed dramatically increased expression at the blastula stage. Accordingly, in situ hybridization of lama4 was conducted. The results revealed that signals became stronger following the phase IV ovary stage, and the strongest signals were located on the follicular pseudoplacenta at the blastula stage. These results suggest that the high expression of laminin genes, especially lama4 after fertilization, may drive cell proliferation, migration, and tissue expansion in the S. schlegelii ovary and ultimately promote follicular pseudoplacenta formation.

A new alternative splice variant of ecdysteroid receptor ( EcR ) transcript differentially expressed during embryogenesis in the oriental river prawn Macrobrachium nipponense

Ecdysteroids are well known as a class of steroid hormones, which not only mediate moulting in insects and crustaceans but also are involved in the reproduction and embryogenesis as well. To characterize the role of ecdysteroid receptor (EcR) in embryogenesis, a novel alternative splicing of EcR (MnEcR) was identified from the oriental river prawn Macrobrachium nipponense, and its spatio-temporal expression profile was examined during embryogenesis. The MnEcR cDNA is 2246 bp in length and encodes a protein with 487 amino acids. Quantitative real-time PCR analysis revealed that the relative amount of MnEcR transcripts was low at cleavage stage, gradually increased from blastula stage, and reached at a peak at the metanauplius when the Y-organ appeared and, thereafter, dramatically decreased at the protozoea and back to a low level at the zoea. Such a dynamic expression profile of MnEcR mRNA is paralleled by fluctuation of ecdysteroids during morphogenesis in the prawn M. rosenbergii and Palaemon serratus. Hybridization in situ demonstrated MnEcR transcripts were localized to the epidermis of appendage rudiments in metanauplius and the cuticle membrane of the thoracic legs and telson in zoeas, suggesting a role for MnEcR in the development of appendages. These results indicated that MnEcR associated with ecdysteroids might be essential for morphogenesis during embryogenesis in the prawn.

Molecular cloning and expression analysis of CDK genes during selective and wild populations of juvenile Meretrix meretrix

The saltwater hard clam Meretrix meretrix is an important commercially bivalve in China. Therefore, identification of key genes involved in growth regulation are the primary prerequisites for further development of phenotype-selective breeding of the clams and understanding molecular mechanisms of growth regulation. In the present study, we cloned the full-length cDNA sequences of the growth-related genes cyclin-dependent kinase 1 and 7 in M. meretrix (MmCDK1 and MmCDK7) and then investigated their gene expression characteristics and associations with growth traits. The full-length cDNA of MmCDK1 was 1623 base pairs (bp), with a 900 bp open reading frame (ORF) encoding 299 amino acids. The full-length MmCDK7 cDNA was 1296 bp, with a 1017 bp ORF encoding 338 amino acids. In adult clams, expression levels of MmCDK1 and MmCDK7 were significantly higher in the gonad than other tissues (p < 0.05). In the eight developmental stages of M. meretrix, MmCDK1 and MmCDK7 had the highest expression levels in the multicellular stage, followed by oosperm, blastula stage and gastrulae stage. The BLAST searches showed that MmCDK1 has high similarity to the cyclin-dependent kinases from Crassostrea gigas (89.26%), and the protein structure of MmCDK7 was similar to the Mizuhopecten yessoensis (75.00%). In addition, the growth trait (shell length) of clams was tested in different cultivation conditions. A 2 × 2 layout was designed including four treatment groups were abbreviated as LHFH (high light and high food), LHFL (high light and low food), LLFH (low light and high food) and LLFL (low light and low food). The shell length of the selective and wild clams in the LHFH group was higher than those in other groups on the 75th day. Moreover, the shell length and MmCDK7 expression levels of the selective clams was higher in the LHFH, LLFH and LHFL groups than the wild clams on the 75th day, significantly (p < 0.05). However, the shell length and relative expression levels of MmCDK7 were no significant differences between the selective clams and the wild clams in the LLFL group (p > 0.05). MmCDK1 expression levels of the selective clams were higher than the wild clams in four treatment groups on the 75th day, significantly (p < 0.05). Further analysis showed that there was a significant linear correlation between shell length and relative expression levels of MmCDKs genes (MmCDK1 and MmCDK7). These results suggest that MmCDK1 and MmCDK7 are key growth-related genes, and they may have potential value in selective breeding to improve growth performance of M. meretrix.

MiR-202-3p determines embryo viability during mid-blastula transition.

Developmental growth is an intricate process involving the coordinated regulation of the expression of various genes, and microRNAs (miRNAs) play crucial roles in diverse processes throughout animal development. The mid-blastula transition (MBT) is a developmental milestone when maternal RNAs are cleared and the zygotic genome programmed asynchronous cell division begins to drive embryogenesis. While mechanisms underlying MBT have been intensively revealed, factors regulating cell proliferation at the transition remain largely unknown. We report here a microRNA, miR-202-3p to be a key factor that determines embryonic fate during MBT in zebrafish. A miR-202-3p antagomir specifically terminated embryo development at the mid-blastula stage. In vivo deletion of the miR-202 locus recapitulated the fatal phenotypes, which were rescued only by miR-202-3p or its precursor. Transcriptome comparison revealed >250 RNAs including both maternal and zygotic origins were dysregulated at MBT in the miR-202−/− embryos, corresponding with arrays of homeostatic disorders leading to massive apoptosis. A trio of genes: nfkbiaa, perp and mgll, known to be intimately involved with cell proliferation and survival, were identified as direct targets of miR-202-3p. Importantly, over- or under-expression of any of the trio led to developmental delay or termination at the blastula or gastrula stages. Furthermore, nfkbiaa and perp were shown to inter-regulate each other. Thus, miR-202-3p mediates a regulatory network whose components interact closely during MBT to determine embryonic viability and development.

Effect of Bauhinia monandra Kurz Leaf Preparations on Embryonic Stages and Adult Snails of Biomphalaria glabrata (Say, 1818), Schistosoma mansoni Cercariae and Toxicity in Artemia salina.

Biomphalaria glabrata snails constitute the main vector of schistosomiasis in Brazil, and Bauhinia monandra Kurz, the leaves of which contain BmoLL lectin with biocidal action, is a plant widely found on continents in which the disease is endemic. This work describes the composition of B. monandra preparations and the effect on embryos and adult snails, their reproduction parameters and hemocytes. We also describe the results of a comet assay after B. glabrata exposure to sublethal concentrations of the preparations. Additionally, the effects of the preparations on S. mansoni cercariae and environmental monitoring with Artemia salina are described. In the chemical evaluation, cinnamic, flavonoid and saponin derivatives were detected in the two preparations assessed, namely the saline extract and the fraction. Both preparations were toxic to embryos in the blastula, gastrula, trochophore, veliger and hippo stages (LC50 of 0.042 and 0.0478; 0.0417 and 0.0419; 0.0897 and 0.1582; 0.3734 and 0.0974; 0.397 and 0.0970 mg/mL, respectively) and to adult snails (LC50 of 6.6 and 0.87 mg/mL, respectively), which were reproductively affected with decreased egg deposition. In blood cell analysis, characteristic cells for apoptosis, micronucleus and binucleation were detected, while for comet analysis, different degrees of nuclear damage were detected. The fraction was able to cause total mortality of the cercariae and did not present environmental toxicity. Therefore, B. monandra preparations are promising in combating schistosomiasis since they can control both the intermediate host and eliminate the infectious agent, besides being safe to the environment.

Establishment of diploid hybrid strains derived from female Larimichthys crocea × male Larimichthys polyactis and transmission of parental mtDNA in hybrid progenies

In this study, a new type of diploid hybrid fish (LCP) was obtained successfully using interspecific hybridization of female Larimichthys crocea (LC, 2n = 48) and male Larimichthys polyactis (LP, 2n = 48). The embryonic development showed that the hatching rate of the hybrid embryos was the highest at 24 °C, with 35.75% of embryos hatching normally. A polymerase chain reaction (PCR) with specific primers (5S−F/−R) was used to distinguish LCP from their parents (LC and LP). Results indicate that LCP was hybrids with a hybrid genome that originated from original parents. Based on the chromosome number, LCP was a diploid hybrid fish with 48 chromosomes (2n = 48). Histological observations of the gonads revealed that both female and male LCP individuals developed normally and were potentially fertile. The analysis of mitochondrial genes showed that the late elimination and transcriptional silencing of paternal mitochondrial genome (mtDNA) may occur in hybrid progenies. The paternal mitochondrial gene of cox1 was detected at the blastula and tail bud stages, diminished at the heart-beating stage, then completely disappeared. This suggests that late elimination leads to mitochondrial heteroplasmy. The transcriptional status showed that cox1 mRNA was not detected at any of the stages examined in hybrid progenies, indicating that the paternal mtDNA may have been transcriptionally silenced and the influence on phenotype during embryonic development may have been excluded. This study provides basis for the establishment of diploid hybrid fish as a new germplasm resource, which in turn, can promote the formation of a new hybrid fish line and contribute in future fish genetic breeding.

Molecular characterization of four innate immune genes in Tor putitora and their comparative transcriptional abundance during wild- and captive-bred ontogenetic developmental stages

The current study was designed to characterize immune genes and compare their expression during ontogenetic developmental stages in progenies of wild-collected and captive-matured T. putitora. The full-length cDNA sequences of Tptlr2, Tpmyd88, Tpcd36, and Tpil8 were 2814, 1545, 1807, and 653 bp in length, with ORFs of 2379 bp, 855 bp, 1410 bp, and 297 bp, encoding for putative peptides of 793, 284, 469 and 98 amino acids, respectively. The predicted peptide sequences of the genes had high sequence homology and structural conservation with other teleost fishes, especially cyprinids. The expression of Tptlr2 was relatively low in both wild- and captive-bred offsprings during the early embryonic stages, but significantly increased later in development. The mRNA abundance of the Tpmyd88 gene was significantly low until the blastula stage, then increased notably from the gastrula stage to the advanced fry stage. The Tpcd36 expression elevated during later developmental stages, peaking at advanced fry stage in both. On the other hand, expression of Tpil8 was relatively low until the blastula stage and showed a moderate increase from the organogenesis stage onwards in wild-bred offsprings, whereas a significant upregulation was seen in the captive-bred offsprings from the organogenesis stage until the advanced fry stage, with its maximum expression at the pre-metamorphosis stage. Overall, the findings suggest the crucial role of Tpmyd88, Tptlr2, Tpcd36, and Tpil8 in inducing innate immunity in embryonic and larval stages of T. putitora. Further, the considerably higher expression of the immune genes in the embryonic and larval stages of captive-bred offsprings may indicate a stronger immune system.

New insights on folliculogenesis and follicular placentation in marine viviparous fish black rockfish (Sebastes schlegelii).

In viviparous fish, a considerable degree of variation in placental structures have been described. However, no distinct structures are reported in Scorpaenidae. In this study, we demonstrate a new type of folliculogenesis and follicular placentation in Sebastes schlegelii. Before copulation, the germinal epithelium gradually surrounds the oocytes and develops into individually follicles with a stalk-like structure hanging on the ovigerous lamella, which ensures each follicle have access to spermatozoa after copulation. From V to early gestation stage, the cyp17-I highly expressesaccompanied by cyp19a1a signals disappearance, and 11-ketotestosterone level keeps rising and peaks at blastula stage, while 17β-estradiol declines to the bottom. Meanwhile, the theca cells rapidly proliferate and invade outwards forming a highly hypertrophied and folded microvillous placenta. This unbalance of hormone might be an important factor driving the theca cells proliferation and invasion. Additionally, some conserved genes related to mammalian placentation are significantly high expression in follicular placenta suggesting the high convergence in vertebrate placenta evolution.

Gene identification and functional analysis of a D-type cyclin (CCND2) in freshwater pearl mussel (Hyriopsis cumingii).

Cyclin D (CCND) plays an important role in the cell cycle and is a rate-limiting factor that facilitates the G1/S transition. In this study, the full-length cDNA of Hc-CCND2 was isolated from freshwater pearl mussel (Hyriopsis cumingii; Hc) and amplified using the 3´/5´ RACE system. The Hc-CCND2 expression profiles were analysed by quantitative real-time PCR. Functional analysis of the Hc-CCND2 genes was examined by both RNA interference (RNAi) and overexpression in H. cumingii. Hc-CCND2 protein sequences were 295 amino acids long, possessed D-type cyclin signature motifs and contained conserved cyclin box domains. Hc-CCND2 was expressed in all examined tissues (adductor, foot, visceral mass, gill, outer mantle, inner mantle and gonad), with the highest expression levels found in the gill (P < 0.05). During the different developmental periods of the embryo, the relative expression of Hc-CCND2 increased with embryonic development, peaking at the blastula stage and decreasing significantly in the gastrula stage. After knockdown of Hc-CCND2 by RNAi, a significant decrease in CDK6 expression levels was found, while the percentage of cells in the G0/G1 phase significantly increased. Overexpression of Hc-CCND2 in mantle cells led to increased proliferation of cultured cells (P < 0.05). Our results demonstrated that Hc-CCND2 may promote cell cycle progression in H. cumingii, and that overexpression of Hc-CCND2 promotes mantle cell proliferation. These findings may provide a novel approach for improving the slow proliferation rate of shellfish cells in in vitro cultures.

The TALE never ends: A comprehensive overview of the role of PBX1, a TALE transcription factor, in human developmental defects.

PBX1 is a highly conserved atypical homeodomain transcription factor (TF) belonging to the TALE (three amino acid loop extension) family. Dimerized with other TALE proteins, it can interact with numerous partners and reach dozens of regulating sequences, suggesting its role as a pioneer factor. PBX1 is expressed throughout the embryonic stages (as early as the blastula stage) in vertebrates. In human, PBX1 germline variations are linked to syndromic renal anomalies (CAKUTHED). In this review, we summarized available data on PBX1 functions, PBX1-deficient animal models, and PBX1 germline variations in humans. Two types of genetic alterations were identified in PBX1 gene. PBX1 missense variations generate a severe phenotype including lung hypoplasia, cardiac malformations, and sexual development defects (DSDs). Conversely, truncating variants generate milder phenotypes (mainly cryptorchidism and deafness). We suggest that defects in PBX1 interactions with various partners, including proteins from the HOX (HOXA7, HOXA10, etc.), WNT (WNT9B, WNT3), and Polycomb (BMI1, EED) families are responsible for abnormal proliferation and differentiation of the embryonic mesenchyme. These alterations could explain most of the defects observed in humans. However, some phenotype variability (especially DSDs) remains poorly understood. Further studies are needed to explore the TALE family in greater depth.

The role of the hot foot gene in the fertility of The in vitro fertilization and embryonic development of young adult and old mice as a model for assisted reproductive technology

The hotfoot mutation first occurred in strain of mice C57BL/Ks in 1964. The homozygous (ho/ho) hotfoot mutation show a quick pattern motion that produces a progressive neuromuscular disability of the hind legs. The (ho/ho) is an autosomal recessive mutation affects fertility and neuromuscular system in mice. ObjectiveThis study was implemented to determine the nature and causes of infertility, to ascertain potential value as an animal model due to the presence of similar mutation in human and other mammals. MethodsThe experimental design to evaluate the fertility and sterility of young and old adult female and male hotfoot mice by utilizing the assisted reproduction technology (ART) via the in vitro fertilization, the in vitro embryo development, and the normal matting, the growth of the new born hotfoot mice. ResultsDemonstrated that the young adult and the old hotfoot females produce about similar rate of ova number following superovulation. In addition, they yielded similar rate of embryo development in vitro form 1 cell to the 16 cell stage, morula and blastula stage compared to normal females. The old hotfoot females show better rate in the in vitro fertilization IVF and embryo development 53.6%, and the rate of the degenerating ova (46.4%) compared to the young adult females 38.5%, 61.35, respectively. The hotfoot males show sterility (22%) of the homozygous mutant males compared to phenotypically normal males. Regarding the normal matting, the hotfoot female mating with males had less litter size compared to normal mice mating. ConclusionThe hotfoot gene had a noticeable effect on the in vitro fertilization IVF of young adult compared to old adult hotfoot female, also the body weight growth and litter size of hotfoot less than normal mice.

The role of passage numbers of donor cells in the development of Arabian Oryx – Cow interspecific somatic cell nuclear transfer embryos

Abstract The cloning between different animals known as interspecific somatic cell nuclear transfer (iSCNT) was carried out for endangered species. The iSCNT has been characterized by a poor success rate due to several factors that influence the formation of the SCNT in various cytoplasms. The cell cycle of the transferred somatic cell, the passage number of the cultured somatic cell, the mitochondria oocytes, and their capabilities are among these factors. This study investigates the role of the passage number of the Arabian Oryx somatic cell culture when transplanted to an enucleated domestic cow oocyte and embryo development in vitro. The fibroblast somatic cell of the Arabian Oryx was cultured for several passage lanes (3–13). The optimal passage cell number was found to be 10–13 Oryx cell lines that progressed to various cell stages up to the blastula stage. There was some variation between the different passage numbers of the oryx cell line. The 3–9 cell line did not show a good developmental stage. These could be attributed to several factors that control the iSCNT as stated by several investigators. More investigation is needed to clarify the role of factors that affect the success rate for the iSCNT.

Characterization of nanog in Nile tilapia (Oreochromis niloticus) and its spatiotemporal expression patterns during embryonic and gonadal development.

Nanog is one of the well-characterized core transcription factors in pluripotency maintenance network. So far, studies on fishes indicate that the Nanog expression occurs from embryonic 1-cell stage to blastula stage, and is restricted to the gonadal germline cells in adult tissues, which is strikingly different from that in mammals. However, whether this expression profile is conservative in fishes remains to be investigated. Here Nile tilapia (Oreochromis niloticus) nanog (named as Ong) was identified and its spatiotemporal expression patterns during embryonic and gonadal development were investigated. The Ong cDNA contains an open reading frame of 678bp, encoding 226 amino acids. The anti-Ong antibody was prepared through prokaryotic protein expression and its specificity was validated. The Ong expression in embryonic 1-cell stage did not appear until the early stage of blastocyst and continued to the late stage of blastocyst. In adult tissues, its expression was limited to gonads. Its expression patterns during gonadal development were further investigated by in situ hybridization and immunohistochemical staining. In testis, Ong was not expressed at 30 dah (days after hatching), but highly expressed in spermatogonia and spermatocytes at 150 dah; in ovaries at 30 and 150 dah, it was not expressed in germline cells but in all somatic cells. This expression profile is strikingly different from reports in fishes to date. Our study firstly indicates that the Nanog expression profile is not conservative in fishes. This study is valuable for further functional and evolutionary study of this gene.

Changes in intracapsular fluid nutritional composition during the early development of the marine gastropod Hemifusus tuba Gmelin

The composition and function of intracapsular biochemical substances are intrinsically correlated; thus, it is important to quantify intracapsular nutritional composition, including protein, glycogen and lipid content, which are required for embryonic development. To explore the intracapsular fluid nutritional composition of the marine gastropod Hemifusus tuba Gmelin during early developmental stages, the embryonic development was divided into seven stages: fertilized egg, cleavage, morula, blastula, gastrula, veliger and juvenile. Results showed that the amino acids contents ranged from 44.98 to 71.52/100 g. Furthermore, 13 amino acids were identified, including five essential amino acids and eight non-essential amino acids. The contents of aspartic acid, proline, and valine were higher, while the contents of the other ten amino acids were relatively lower. Notably, aspartic acid and proline content varied across embryonic development. In addition, saturated fatty acids (SFAs) were dominant fatty acid components in the intracapsular fluid, in which C18:0 was the main fatty acid. Monounsaturated fatty acids (MUFA) primarily comprised C20:1(n − 9), accounting for 30% of the total fatty acids. At the same time, the content of C18:2(n − 6) decreased significantly from the fertilized egg to the blastula stage (p < 0.05). The content of C20:4(n − 6) from fertilized egg to gastrula stage was significantly reduced (p < 0.05). C22:6(n − 3) was significantly lower in the gastrula than in other stages (p < 0.05). Overall, this study provides new information on the intracapsular fluid nutritional composition of H. tuba, helping enrich the biological knowledge of the early developmental stages of marine gastropods.

Revealing the cells fated to form the cell mass in embryos of temnopleurid sea urchins

Larvae of temnopleurid sea urchins form a cell mass (CM) instead of an amniotic cavity on the left side at the early developmental stage for formation of the adult rudiment. However, the cell lineage and the mechanisms that form the CM are still unknown. We analyzed the potential to form a CM in partial embryos resulting from microsurgeries, using two temnopleurid species, Mespilia globulus (L.) and Temnopleurus toreumaticus (Leske). CM formation was completed 28-34 h after fertilization at 24°C, corresponding to the period from the late prism to the two-armed pluteus larval stages in both species. In the case of specimens dissected horizontally during the mesenchyme blastula to prism stages, the CM was formed in partial embryos containing enough of the an2 region, a part of the precursor cells that differentiate the ectoderm. The proportion of specimens with a CM was higher in T. toreumaticus than in M. globulus. Additionally, all larvae derived from half embryos obtained from dissection along the animal-vegetal axis at the mesenchyme blastula stage formed the CM. Transplantation of a stained animal or vegetal hemisphere at the 16-cell stage into a nonstained vegetal or animal embryo indicated that the CM derives from the animal half. Exogastrulae vegetalized by lithium chloride treatment did not form the CM. These results indicate that the CM formation is dependent not only on the an2 region but also on signals from the vegetal region after the mesenchyme blastula stage.

Retinal toxicity of isoflucypram to zebrafish (Danio rerio).

Isoflucypram is an active succinate dehydrogenase inhibitor (SDHI) fungicide. Recent studies have demonstrated that isoflucypram is toxic to non-target aquatic organisms such as zebrafish, Danio rerio. However, current knowledge of the potential risks presented by the SDHI to non-target aquatic organism remains limited. To investigate the teratogenic effects of isoflucypram on retinogenesis, zebrafish embryos were exposed to isoflucypram (0.025, 0.25, and 2.5μM) from the blastula stage (3h post-fertilization, hpf) to the larval stage (96 hpf). Prolonged exposure to isoflucypram induced abnormalities in retinal development in zebrafish larvae, resulted in the expression of a microphthalmic phenotype, disrupted retinal lamination, and altered the expression levels of retinal markers (opn1sw1, opn1sw2, opn1mw1, opn1lw1, rho, atoh7, vsx1, prox1a, and sox2). Retinal cell apoptosis was also significantly higher in the isoflucypram-exposed larvae than in the control larvae. Catalase activity decreased significantly and malondialdehyde content increased markedly after exposure to isoflucypram. Thus, isoflucypram should be regarded as having retinal neurotoxicity in zebrafish.

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.