Stage Embryos Research Articles

The Wnt-dependent master regulator NKX1-2 controls mouse pre-implantation development

Embryo size, specification, and homeostasis are regulated by a complex gene regulatory and signaling network. Here we used gene expression signatures of Wnt-activated mouse embryonic stem cell (mESC) clones to reverse engineer an mESC regulatory network. We identify NKX1-2 as a novel master regulator of preimplantation embryo development. We find that Nkx1-2 inhibition reduces nascent RNA synthesis, downregulates genes controlling ribosome biogenesis, RNA translation, and transport, and induces severe alteration of nucleolus structure, resulting in the exclusion of RNA polymerase I from nucleoli. In turn, NKX1-2 loss of function leads to chromosome missegregation in the 2- to 4-cell embryo stages, severe decrease in blastomere numbers, alterations of tight junctions (TJs), and impairment of microlumen coarsening. Overall, these changes impair the blastocoel expansion-collapse cycle and embryo cavitation, leading to altered lineage specification and developmental arrest.

Effects of Oral Zinc Supplementation on Early Embryonic Development and Neonates of Aged Female Albino Mice

Female reproductive aging is a normal phase of life that eventually leads to menopause and reproductive senescence. The current experimental investigation was intended to study zinc supplementation effects on early embryonic development and neonates in 26-28 weeks old female mice. In this investigation, 80 mature female mice were used that were divided into two groups of forty each. Two control groups received distilled water, while two Zn groups were given 0.115 mg/kg/day Zn orally daily during 2-3 estrous cycles. The female mice were all mated with adult males. Twenty female mice from both the control and Zn groups were euthanized by CO2 gas inhalation, and then a longitudinal incision in the abdomen of each female mouse was performed and uterine horns and oviducts were detached from the body in order to obtain two-cell embryos. On the other hand, 20 pregnant mice from each group were permitted to complete their pregnancy until birth and then their neonates were collected for macroscopic examination. The findings showed that the Zn supplementation significantly (P≤0.01) improved the mean embryonic development and the quality of 2-cell stage embryo grade A. Whereas, it decreased the mean embryonic development of 2-cell stage embryo grade D in aged female mice significantly (P≤0.01) in comparison with aged control groups. Also, the count and quality of neonates from Zn-treated aged female mice enhanced significantly (P≤0.01) as compared to non-treated aged female mice. It was concluded from these findings that the Zn can improve embryonic development and neonate count and quality in aged mice.

Toxicity Detection of Pollutants in the Tallo River Using Simple Biomarkers of Oryzias celebensis Embryo

The current research demonstrated the use of simple biomarkers from Oryzias celebensis embryos to detect the effects of pollutants from the Tallo River. The biomarkers used in this research were somite number, egg biometrics, heart rate, survival rate, and hatching time. Pollutants were sampled from three hot spots in the Tallo River (Station 1 (S1), Station 2 (S2) and Station 3 (S3)). Two exposure models were used in this study: in situ and semi-in situ exposure. In situ exposure was carried out by placing ten embryos (stage 17) at each station for six days. Semi-in situ embryos (10 animals) were exposed to polluted water from each station and embryo rearing media (ERM) as a control at stage 17 until hatching in the laboratory. Biomarker data were analyzed using ANOVA (Analysis of variance) to determine the sensitivity of each biomarker used. Almost all the embryos used as model animals in the in situ experiment died, except for two embryos placed in stations 2 and 3. The embryos from station 2 hatched after 18 days. The embryos from station 3 died after 18 days. The semi in situ experiment showed that the number of somites and heart rate showed statistically significant differences between embryos exposed to water from S1 and control embryos (p < 0.05). Other endpoints did not show potential as biomarkers. This study suggested that the number of somites and heart rate of O. celebensis embryos could potentially be used as biomarkers in effect-based monitoring.

Microcephaly Gene Mcph1 Deficiency Induces p19ARF-Dependent Cell Cycle Arrest and Senescence.

MCPH1 has been identified as the causal gene for primary microcephaly type 1, a neurodevelopmental disorder characterized by reduced brain size and delayed growth. As a multifunction protein, MCPH1 has been reported to repress the expression of TERT and interact with transcriptional regulator E2F1. However, it remains unclear whether MCPH1 regulates brain development through its transcriptional regulation function. This study showed that the knockout of Mcph1 in mice leads to delayed growth as early as the embryo stage E11.5. Transcriptome analysis (RNA-seq) revealed that the deletion of Mcph1 resulted in changes in the expression levels of a limited number of genes. Although the expression of some of E2F1 targets, such as Satb2 and Cdkn1c, was affected, the differentially expressed genes (DEGs) were not significantly enriched as E2F1 target genes. Further investigations showed that primary and immortalized Mcph1 knockout mouse embryonic fibroblasts (MEFs) exhibited cell cycle arrest and cellular senescence phenotype. Interestingly, the upregulation of p19ARF was detected in Mcph1 knockout MEFs, and silencing p19Arf restored the cell cycle and growth arrest to wild-type levels. Our findings suggested it is unlikely that MCPH1 regulates neurodevelopment through E2F1-mediated transcriptional regulation, and p19ARF-dependent cell cycle arrest and cellular senescence may contribute to the developmental abnormalities observed in primary microcephaly.

Single-cell transcriptome atlas in C57BL/6 mice encodes morphological phenotypes in the aging kidneys

C57BL/6 mice are frequently utilized as murine models with the desired genetic background for altertion in multiple research contexts. So far, there is still a lack of comprehensive kidney morphology and single-cell transcriptome atlas at all stages of growth of C57BL/6 mice. To provide an interactive set of reference standards for the scientific community, we performed the current study to investigate the kidney’s development throughout the capillary-loop stage until senescence. Eight groups, with five to six mice each, represented embryonic stage (embryos 18.5 days), suckling period (1 day after birth), juvenile stage (1 month old), adulthood (containing 3 months old, 6 months old and 10 months old), reproductive senescence stage (20 months old), and post-senescence stage (30 months old), respectively. With age, the thickness of the glomerular basement membrane (GBM) was increased. Notably, GBM knobs appeared at three months and became frequent with age. Using single-cell transcriptome data, we evaluated how various biological process appear in particular cell types and investigated the potential mechanism of formation of GBM konbs. In conclusion, having access to detailed kidney morphology and single-cell transcriptome maps from C57BL/6 mice at various developmental stages of C57BL/6 mice would be a novel and major resource for biological research and testing of prospective therapeutic approaches.

Maternal supplementation with mulberry-leaf flavonoids improves the development of skeletal muscle in the offspring of chickens

The development of skeletal muscle is a crucial factor in determining the meat yield and economic benefits of broiler production. Recent research has shown that mulberry leaves and their extracts can be used to significantly improve the growth performance of livestock and poultry. The present study aims to elucidate the mechanisms involved in the regulation of skeletal muscle development in broiler offspring by dietary mulberry-leaf flavonoids (MLF) supplementation from the perspective of maternal effect theory. A total of 270 Qiling broiler breeder hens were randomly assigned to 3 treatments with different doses of MLF (0, 30, 60 mg/kg) for 8 weeks before collecting their fertilized eggs. The chicken offspring at 13 and 19 d of embryonic stage, and from 1 to 28 d old after hatching were included in this study. The results showed that maternal supplementation increased the breast muscle weight and body weight of the offspring at the embryo and chick stages (P < 0.05). This was followed by increased cross-sectional area of pectoral muscle fibres at 14 d (P < 0.05). Further determination revealed a tendency towards increased serum levels of insulin-like growth factor 1 (IGF-1) (P = 0.092) and muscle fibre count (P = 0.167) at 1 d post-hatching following maternal MLF treatment, while serum uric acid (UA) was decreased at 14 d after hatching (P < 0.05). Moreover, maternal MLF supplementation significantly up-regulated the mRNA expression of the myogenic regulatory factor Myf5 in skeletal muscle at the both embryonic and growth stages (P < 0.05). The relative abundance of the downstream protein of BMPR2, Smad1 and p-Smad1/5/9 in the TGFβ signalling pathway was significantly increased by maternal MLF treatment. Meanwhile, the increased expression of the target protein p-mTOR in the breast muscle of the offspring chicks is in accordance with the improved growth rate of the breast and the body. In conclusion, maternal MLF supplementation can promote muscle protein metabolism and muscle fibre development of chick embryos through upregulation of Myf5 expression and BMP/p-Smad1/5/9 axis, thereby improving growth performance of slow growing broiler.

Developmental toxicity of short-chain chlorinated paraffins on early-stage chicken embryos in a shell-less (ex-ovo) incubation system

Short-chain chlorinated paraffins (SCCPs) are listed as a category of globally controlled persistent organic pollutants (POPs) by the Stockholm Convention in 2017. However, SCCP toxicity, particularly their developmental toxicity in avian embryos, has not been well studied. In this study, we observed the early development of chicken embryos (Gallus gallus domesticus) by applying a shell-less (ex-ovo) incubation system developed in our previous studies. After exposing embryos at Hamburger Hamilton stage (HHS) 1 to SCCPs (control, 0.1% DMSO; SCCPs-L, 200 ng/g; SCCPs-M, 2000 ng/g; SCCPs-H, 20,000 ng/g), we observed the development of embryos from the 3rd to 9th incubation day. Exposure to SCCPs-M and -H induced a significant reduction in survival, with an LD50 of 3100 ng/g on the 9th incubation day. Significant dose-dependent decreases in body length were observed from days 4–9. We also found that SCCPs-H decreased the blood vessel length and branch number on the 4th incubation day. Additionally, SCCPs-H significantly reduced the heart rate on the 4th and 5th incubation days. These findings suggest that SCCPs may have potential of developmental and cardiovascular toxicity during the early stages of chicken embryos. Quantitative PCR of the mRNA of genes related to embryonic development showed that SLC16A10 (a triiodothyronine transporter) level decreased in the SCCPs-H group, showing a significant positive correlation with the body length of embryos. THRA level, a thyroid hormone receptor, was significantly decreased in the SCCPs-H group, whereas that of DIO3 level, a deiodinase was significantly increased. These results suggest that SCCPs exposure induces developmental delays via the thyroxine signaling pathway. Analysis of thyroid hormones (THs) in blood plasma also indicated a significant reduction in thyroxine (T4) levels in the SCCPs-H group on the 9th incubation day of embryos. In conclusion, SCCPs induce developmental toxicity by disrupting thyroid functions at the early-life stage of chicken embryos.

Aberrant methylation of placental development genes in chorionic villi of spontaneous abortions with trisomy 16.

In humans, aneuploidy is incompatible with the birth of healthy children and mainly leads to the death of embryos in the early stages of development in the first trimester of pregnancy. Trisomy 16 is the most common aneuploidy among spontaneous abortions of the first trimester of pregnancy. However, the mechanisms leading to the death of embryos with trisomy 16 remain insufficiently investigated. One of these potential mechanisms is abnormal placental development, including aberrant remodeling of spiral arteries. Spiral artery remodeling involves the migration of trophoblast cells into the maternal spiral arteries, replacing their endothelium and remodeling to ensure a stable embryonic nutrition and oxygen supply. This is a complex process which depends on many factors from both the embryo and the mother. We analyzed the methylation level of seven genes (ADORA2B, NPR3, PRDM1, PSG2, PHTLH, SV2C, and TICAM2) involved in placental development in the chorionic villi of spontaneous abortions with trisomy 16 (n = 14), compared with spontaneous abortions with a normal karyotype (n = 31) and the control group of induced abortions (n = 10). To obtain sequencing libraries, targeted amplification of individual gene regions using designed oligonucleotide primers for bisulfite-converted DNA was used. The analysis was carried out using targeted bisulfite massive parallel sequencing. In the group of spontaneous abortions with trisomy 16, the level of methylation of the PRDM1 and PSG2 genes was significantly increased compared to induced abortions (p = 0.0004 and p = 0.0015, respectively). In the group of spontaneous abortions, there was no increase in the level of methylation of the PRDM1 and PSG2 genes, but the level of methylation of the ADORA2B gene was significantly increased compared to the induced abortions (p = 0.032). The results obtained indicate the potential mechanisms of the pathogenetic effect of trisomy 16 on the placental development with the participation of the studied genes.

Deoxynivalenol leads to endoplasmic reticulum stress-mediated apoptosis via the IRE1/JNK/CHOP pathways in porcine embryos

The cytotoxic mycotoxin deoxynivalenol (DON) reportedly has adverse effects on oocyte maturation and embryonic development in pigs. Recently, the interplay between cell apoptosis and endoplasmic reticulum (ER) stress has garnered increasing attention in embryogenesis. However, the involvement of the inositol-requiring enzyme 1 (IRE1)/c-jun N-terminal kinase (JNK)/C/EBP homologous protein (CHOP) pathways of unfolded protein response (UPR) signaling in DON-induced apoptosis in porcine embryos remains unknown. In this study, we revealed that exposure to DON (0.25 μM) substantially decreased cell viability until the blastocyst stage in porcine embryos, concomitant with initiation of cell apoptosis through the IRE1/JNK/CHOP pathways in response to ER stress. Quantitative PCR confirmed that UPR signaling-related transcription factors were upregulated in DON-treated porcine blastocysts. Western blot analysis showed that IRE1/JNK/CHOP signaling was activated in DON-exposed porcine embryos, indicating that ER stress-associated apoptosis was instigated. The ER stress inhibitor tauroursodeoxycholic acid protected against DON-induced ER stress in porcine embryos, indicating that the toxic effects of DON on early developmental competence of porcine embryos can be prevented. In conclusion, DON exposure impairs the developmental ability of porcine embryos by inducing ER stress-mediated apoptosis via IRE1/JNK/CHOP signaling.

Effects of Mixed Pasture Legume Phytoestrogens on Superovulatory Response and Embryo Quality in Angus Cows.

Ovulation and artificial insemination rates have been observed to decrease in sheep and cows when exposed to dietary phytoestrogens at concentrations greater than 25 mg/kg DM. A grazing trial was undertaken to investigate the effects of coumestrol and other key phytoestrogens on the superovulatory response, embryo numbers and quality in beef cows grazing legume pastures. A 7-week controlled grazing trial was conducted with legume and ryegrass pasture treatments, with cows exposed to legumes at two timed treatments, 4 and 7 weeks. Twenty Angus cows were subjected to a conventional estrus synchronization and superovulation protocol. Embryos were recovered via conventional uterine body flushing 7 days post artificial insemination (AI). Numerous phytoestrogens were identified in both pasture and plasma samples, including coumestrol and formononetin. Concentrations of phytoestrogens in the pasture ranged from 0.001 to 47.5 mg/kg DM and 0 to 2.6 ng/mL in plasma. Approximately 50% of cows produced viable embryos 7 days post AI. A significant interaction between the effect of treatment groups on the embryo stage was observed (p < 0.05). The results suggest that concentrations of >25 mg/kg DM of phytoestrogens less than 20 days preceding AI may negatively affect oocyte developmental competence, reduce progesterone production and thus contribute to early embryonic loss.

Bifenox induces hepatotoxicity and vascular toxicity in zebrafish embryos via ROS production and alterations in signaling pathways

Existing evidence shows that currently used pesticides pose toxicological risks to exposed wildlife. Chemically, bifenox belongs to diphenyl ethers, a well-known group of herbicides. Its mechanism of action primarily involves inducing lipid peroxidation and blocking protoporphyrinogen oxidases. Toxicity of diphenyl ether herbicides has been elucidated in animal cells; however, in vivo toxicological evaluations of bifenox are required to determine its unexpected effects. This study aimed to determine the negative effects of bifenox, and its effects on higher eukaryotes. We found that early stages of zebrafish embryo exposed to bifenox demonstrated increased mortality and physiological defects, based on the LC50 value. Bifenox severely inhibited blood vessel growth by reducing key elements of complex connectivity; fluorescently tagged transgenic lines (fli1a:EGFP) showed morphological changes. Additionally, transgenic lines that selectively identified hepatocytes (fabp10a:DsRed) showed reduced fluorescence, indicating that bifenox may inhibit liver development. To evaluate the level of oxidative stress, we used 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probes in zebrafish embryos to identify the underlying mechanisms causing developmental damage. Our findings demonstrate that exposure to bifenox causes abnormalities in the hepatic and cardiovascular systems during zebrafish embryogenesis. Therefore, this study provides new information for the evaluation of toxicological risks of bifenox in vertebrates.

The effects of culture media and media components on the development of rat embryos

After in vitro culture of rat embryos, blastocyst rates are lower than the other species because of the embryonic block observed in the 2- or 4-cell stages in vitro. Optimal culture media and systems that provide variable physiologic needs in the different stages of rat embryos. The modifications of rat embryo culture media could have a positive effect on increasing the blastocyst rates. However, since the results of rat embryo studies are changed depending on factors like strains preferred, maintenance conditions and different commercial products added to the culture media, the success rate of producing healthy newborns for reproductive biotechnological studies has not yet reached the desired level by using current embryo culture media. Understanding the needs of rat embryos cultured from zygote to blastocyst stage in vitro is important for successful advanced studies such as cloning and transgenesis. The purpose of this review is the effects of different culture media and media components on the preimplantation stages of rat embryos and get a perspective for developing the culture media

Local intracellular stiffening of ascidian embryo in cleavage developmental stage observed by atomic force microscopy

During the cleavage stage in ascidian embryos, cells undergo repeated and regular cell division in the animal hemisphere. We investigated how the intracellular stiffness, that is, the apparent Young’s modulus of cells in the animal hemisphere changes in the cleavage stages from the 16-cell to 76-cell stages using atomic force microscopy (AFM). The AFM force mapping experiments showed that a pair of dot-like stiffening clearly appeared in the intracellular region, which involves the cortical and cytoplasmic regions indented by the AFM probe, in a period of the interphase after cell division. The dot-like structures disappeared before beginning to increase the whole cell stiffening towards the proceeding mitotic phase. We found that the positioning of a pair of dots was located so as to intersect the daughter cells formed in the proceeding cell division. These results suggest that the local intracellular stiffening observed in the interphase may play an important role in controlling the orientation of the mitotic spindle that determines the direction of cell division.

Triploid brown trout, Salmo trutta, develop functional gonads with age and are able to interbreed with diploid counterparts.

The study investigated if gonad maturation in triploid brown trout, Salmo trutta, was entirely suppressed or only delayed, and if triploids could interbreed with diploid counterparts. Ten percent of the total number of 3-year-old triploid S. trutta, 15% of 4-year-old fish, and 17% of 5-year-old fish produced semen. Three and 4 years old triploid fish did not produce eggs, but 15% of the 5-year-old fish did so. The quantity and sperm motility of triploid semen did not differ from diploids, but the sperm concentration was significantly lower. When diploid eggs were fertilized with triploid semen (3n × 2n crosses), the percentage of eyed stage embryos, of hatched larvae, and of normal-shaped larvae did not differ from the diploid controls. Circa 90% of 3n × 2n crosses had a ploidy level of 2.4n. In the remaining percentage of 3n × 2n crosses, the ploidy level was ≥2n and <2.4n. In sperm competition experiments where diploid eggs were fertilized with a mixture of diploid and triploid semen, 52% of the originating larvae had a ploidy level of 2n, 43% of 2.4n, and 5% of the fish were not exactly classified. From the start of feeding to an age of 248 days, the mortality rate of 3n × 2n interploid crosses and of 2n × 2n controls was similar. The growth of interploid crosses was significantly higher than that of controls. In triploid mature females, the egg mass per kilogram of body weight was significantly lower than in diploids. The mass of the non-hardened eggs and the percentile weight increase during hardening did not differ from diploid eggs. When triploid eggs were fertilized with diploid semen (2n × 3n crosses), the development rate to normal hatched larvae was less than 10%. All originating larvae had a ploidy level of 3n. From the start of feeding to an age of 248 days, 2n × 3n crosses had a higher mortality rate (15%) than diploid controls (<5%). Growth of this type of interploid crosses was reduced in comparison to controls. Therefore, triploids introduced into natural waters for recreational fisheries or escaping from farms may interbreed with diploid counterparts. This not only alters the genotypes of local populations but also changes the ploidy levels.

Lessons learned from 64,071 embryos subjected to PGT for aneuploidies: results, recurrence pattern and indications analysis

Research questionWhat is the influence of biological, technical and clinical factors on embryo outcomes in preimplantation genetic testing for aneuploidies (PGT-A) and what is the recurrence pattern? DesignThis retrospective study included 64,071 embryos undergoing PGT-A in the same laboratory between 2011 and 2019. Biopsies were performed at the day 3 embryo stage (48.32%) or blastocyst stage (51.70%). Advanced maternal age (AMA) was the main indication (65.62%). ResultsThe aneuploidy rate was 67.75%, higher in women aged over 35 years than in women aged 35 years or less (71.76% versus 47.44%), and higher in day 3 embryo versus blastocyst biopsies (77.51% versus 58.62%). The trisomy:monosomy ratio was 1.01 for blastocysts versus 0.84 for day 3 embryos. Trisomy 21 was present in 4.9% of embryos. In aneuploid embryos, the probability of having one or more involved chromosomes followed a decreasing exponential pattern. The probability of an embryo being euploid was constant at around 30% (40% in blastocysts, 20% in day 3 embryos). The cumulative probability of having one or more euploid embryos after 10 biopsied embryos was 94.79% in blastocysts and 80.61% in day 3 embryos. AMA was associated with a much higher aneuploidy rate than all other indications, which among them had similar aneuploidy rate and chromosomal involvement. ConclusionsThere is a considerably lower aneuploidy rate in blastocysts than day 3 embryos, which is most notable for monosomies. While AMA shows an increased aneuploidy rate and a specific chromosomal pattern of involvement, the remaining indications showed a similar aneuploidy rate and chromosomal pattern. Even after producing many consecutive aneuploid embryos, the possibility of obtaining a euploid embryo is not negligible.

Identification and characterization of the WOX Gene Family revealed two WUS Clade Members associated with embryo development in Cunninghamia lanceolata

The WUSCHEL-related homeobox (WOX) gene family is vital for plant development and stress response. In this study, we conducted a comprehensive analysis of WOX genes in Cunninghamia lanceolata (C. lanceolata) and subsequently explored the potential roles of two ClWOX genes within the WUS clade. In total, six ClWOX genes were identified through a full-length transcriptome analysis. These genes, exhibiting conserved structural and functional motifs, were assigned to the ancient clade and Modern/WUS clade, respectively, through a phylogenetic analysis. Our expression analysis indicated that these ClWOX genes were highly expressed in the middle and late developmental stages of zygotic embryos in C. lanceolata. Moreover, only ClWOX5 and ClWOX6 within the Modern/WUS clade exhibited transcriptional activity, and their expressions were also induced in response to auxin and wounding. Overexpression of ClWOX5 and ClWOX6 in Arabidopsis caused a partially sterile phenotype, resulting in a very low seed setting rate. Transcriptomic analysis revealed that expressions of many embryo-defective (EMB) genes, phytohormone-related genes, and transcription factors (TFs) were dramatically altered in ClWOX5 and ClWOX6 transgenic plants, which suggested that ClWOX5 and ClWOX6 may play specific important roles in embryo development via complex gene networks. In addition, overexpression of ClWOX5 and ClWOX6 in leaf segments promoted shoot regeneration in tobacco, indicating that ClWOX5 and ClWOX6 can promote plant regeneration and could be used to improve genetic transformation. In conclusion, these results help to elucidate the function of the WOX gene and provide a valuable basis for future studies of the developmental regulation and applications of WOX genes in C. lanceolata.

Identification and Analysis of Expression Patterns of the Caleosin Genes in Hickory (Carya cathayensis Sarg.)

The deciduous tree hickory (Carya cathayensis) holds economic significance in China due to its high oil content, particularly in unsaturated fatty acids. Oil bodies are crucial for storing triacylglycerol (TAG), with caleosin serving as a predominant oil body protein that aids in oil body formation and stability maintenance. Our study utilized bioinformatics techniques to identify caleosin genes within Carya cathayensis, Carya illinoinensis, and Juglans regia. Three caleosin genes were discovered in the genomes of Carya cathayensis, Carya illi-noinensis, and Juglans regia. These genes encode hydrophilic proteins. Additionally, all caleosin proteins feature a single Ca2+-binding EF-hand, a conserved “proline knot” motif, and a C-terminal hydrophilic region with four potential phosphorylation sites. The caleosin proteins in Carya cathayensis consist of α-helix, β-corner, extended chain, and random curl structures. Cis-acting elements related to stress response and hormone signaling were identified in Carya cathayensis, Carya illinoinensis, and Juglans regia, with distinct cis-acting elements implicated in seed-specific regulation in Carya cathayensis. Additionally, subcellular localization analysis confirmed that CcaCLO1 and CcaCLO2 were localized within oil bodies. Transcriptome analysis and quantitative real-time polymerase chain reaction (qRT-PCR) data demonstrated a significant up-regulation of CcaCLO1 expression during the developmental stages of the Carya cathayensis embryo. Furthermore, qPCR findings indicated that caleosins from Carya cathayensis were responsive to salt stress, with a significant up-regulation of CcaCLO1 following exposure to salt stress treatment. Consequently, caleosin genes in Carya cathayensis, Carya illinoinensis, and Juglans regia share similar physicochemical characteristics and conserved motifs. Specifically, CcaCLO1 in Carya cathayensis primarily responds to embryo development and salt stress. These findings offer foundational insights for future investigations into the regulatory mechanisms of oil accumulation and response to salt stress in hickory.

An independent origin of an annual life cycle in a North American killifish species

Abstract An annual life cycle is characterized by short-lived adults that exploit seasonally productive but temporary habitats, paired with a hardy embryo stage that survives unfavourable environmental conditions, such as winter or the dry season. This life cycle is rare amongst vertebrates but has evolved independently several times in African and South American killifishes adapted to life in seasonally ephemeral aquatic pools. A single species of North American annual killifish, with a limited geographical distribution in Mexico, also exhibits this seasonal life cycle. This enigmatic species, Millerichthys robustus, is geographically separated from the nearest South American annual killifish species by &amp;gt;2000 km. Millerichthys robustus exhibits morphological features that have led to several competing phylogenetic hypotheses. To date, its phylogenetic relationships are unclear because no molecular data have been available. Here, we sequence the mitochondrial genome of Millerichthys robustus and generate a molecular phylogeny of killifishes that includes this species. Our results indicate that, rather than being most closely related to South American annual killifishes, this species is sister to two non-annual killifish species from Cuba (Rivulus cylindraceus and Rivulus berovidesi). Ancestral state reconstruction strongly supports an independent origin of an annual life cycle and embryonic diapause in Millerichthys robustus.

GCN2-eIF2α signaling pathway negatively regulates the growth of triploid crucian carp

GCN2-eIF2α signaling pathway plays crucial roles in cell growth,development, and protein synthesis. However, in polyploid fish, the function of this pathway is rarely understood. In this study, genes associated with the GCN2-eIF2α pathway (pkr, pek, gcn2, eif2α) are founded lower expression levels in the triploid crucian carp (3nCC) muscle compared to that of the red crucian carp (RCC). In muscle effect stage embryos of the 3nCC, the mRNA levels of this pathway genes are generally lower than those of RCC, excluding hri and fgf21. Inhibiting gcn2 in 3nCC embryos downregulates downstream gene expression (eif2α, atf4, fgf21), accelerating embryonic development. In contrast, overexpressing of eif2α can alter the expression levels of downstream genes (atf4 and fgf21), and decelerates the embryonic development. These results demonstrate the GCN2-eIF2α pathway's regulatory impact on 3nCC growth, advancing understanding of fish rapid growth genetics and offering useful molecular markers for breeding of excellent strains.

The homeobox transcription factor DUXBL controls exit from totipotency.

In mice, exit from the totipotent two-cell (2C) stage embryo requires silencing of the 2C-associated transcriptional program. However, the molecular mechanisms involved in this process remain poorly understood. Here we demonstrate that the 2C-specific transcription factor double homeobox protein (DUX) mediates an essential negative feedback loop by inducing the expression of DUXBL to promote this silencing. We show that DUXBL gains accessibility to DUX-bound regions specifically upon DUX expression. Furthermore, we determine that DUXBL interacts with TRIM24 and TRIM33, members of the TRIM superfamily involved in gene silencing, and colocalizes with them in nuclear foci upon DUX expression. Importantly, DUXBL overexpression impairs 2C-associated transcription, whereas Duxbl inactivation in mouse embryonic stem cells increases DUX-dependent induction of the 2C-transcriptional program. Consequently, DUXBL deficiency in embryos results in sustained expression of 2C-associated transcripts leading to early developmental arrest. Our study identifies DUXBL as an essential regulator of totipotency exit enabling the first divergence of cell fates.