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Development (Cambridge, England) Feb 2024Early organogenesis represents a key step in animal development, during which pluripotent cells diversify to initiate organ formation. Here, we sampled 300,000...
Early organogenesis represents a key step in animal development, during which pluripotent cells diversify to initiate organ formation. Here, we sampled 300,000 single-cell transcriptomes from mouse embryos between E8.5 and E9.5 in 6-h intervals and combined this new dataset with our previous atlas (E6.5-E8.5) to produce a densely sampled timecourse of >400,000 cells from early gastrulation to organogenesis. Computational lineage reconstruction identified complex waves of blood and endothelial development, including a new programme for somite-derived endothelium. We also dissected the E7.5 primitive streak into four adjacent regions, performed scRNA-seq and predicted cell fates computationally. Finally, we defined developmental state/fate relationships by combining orthotopic grafting, microscopic analysis and scRNA-seq to transcriptionally determine cell fates of grafted primitive streak regions after 24 h of in vitro embryo culture. Experimentally determined fate outcomes were in good agreement with computationally predicted fates, demonstrating how classical grafting experiments can be revisited to establish high-resolution cell state/fate relationships. Such interdisciplinary approaches will benefit future studies in developmental biology and guide the in vitro production of cells for organ regeneration and repair.
Topics: Mice; Animals; Cell Differentiation; Gastrulation; Organogenesis; Primitive Streak; Endothelium; Embryo, Mammalian; Mammals
PubMed: 37982461
DOI: 10.1242/dev.201867 -
Journal of Environmental Sciences... Mar 2024Ethylene thiourea (ETU) converted from ethylene bisdithiocarbamate (EBDC) fungicides has aroused great concern because of its prevalence and harmful effects. Although...
Ethylene thiourea (ETU) converted from ethylene bisdithiocarbamate (EBDC) fungicides has aroused great concern because of its prevalence and harmful effects. Although ETU-induced neurotoxicity has been reported, the potential mechanisms remain unclear. This study provided insights into its neurotoxic effects at environmentally relevant concentrations in zebrafish. Our findings showed that embryonic exposure to ETU decreased the hatch rate and delayed somite development. Furthermore, ETU treatment significantly reduced the dark velocity in the locomotion assay. The upregulated tendency of the mitogen-activated protein kinases (MAPK) pathway (mknk1, atf4, mapkapk3) screened by transcriptome analysis implied motor neuron degeneration, which was validated by subsequent morphological observation, as axon length and branches were truncated in the 62.5 µg/L ETU group. However, although the rescue experiment with a p38 MAPK inhibitor (SB203580) successfully ameliorated axon degeneration, it failed to reverse the locomotion behaviors. Further exploration of transcriptome data revealed the varied expression of presynaptic scaffold protein-related genes (pcloa, pclob, bsna), whose downregulation might impair the neuromuscular junction (NMJ). Therefore, we reasonably suspected that ETU-induced neurobehavioral deficits might result from the combined effects of the MAPK pathway and presynaptic proteins. Considering this, we highlighted the necessity to take precautions and early interventions for susceptible ETU-exposed populations.
Topics: Animals; Zebrafish; Ethylenethiourea; Fungicides, Industrial; Neuromuscular Junction
PubMed: 37980000
DOI: 10.1016/j.jes.2022.11.012 -
BioRxiv : the Preprint Server For... Nov 2023Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models...
Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models using free-floating culture that recapitulate somite formation. Somitogenesis involves intricately orchestrated bio-chemical and -mechanical events. However, none of the current somitogenesis models controls biochemical gradients or biomechanical signals in the culture, limiting their applicability to untangle complex biochemical-biomechanical interactions that drive somitogenesis. Here we report a new human somitogenesis model by confining hPSC-derived presomitic mesoderm (PSM) tissues in microfabricated trenches. Exogenous microfluidic morphogen gradients imposed on PSM cause axial patterning and trigger spontaneous rostral-to-caudal somite formation. A mechanical theory is developed to explain the size dependency between somites and PSM. The microfluidic somitogenesis model is further exploited to reveal regulatory roles of cellular and tissue biomechanics in somite formation. This study presents a useful microengineered, hPSC-based model for understanding the bio-chemical and -mechanical events that guide somite formation.
PubMed: 37961125
DOI: 10.1101/2023.10.29.564399 -
Acta Parasitologica Dec 2023A new species of the genus Ceratocolax Vervoort, 1965 is described based on specimens collected from the Tomtate grunt Haemulon aurolineatum Cuvier, caught in the coast...
Ceratocolax tavaresi n. sp. (Copepoda: Bomolochidae) parasitic in the Tomtate Grunt Haemulon aurolineatum Cuvier, 1830 (Actinopterygii: Haemulidae) off Rio de Janeiro, southeastern Brazil.
PURPOSE
A new species of the genus Ceratocolax Vervoort, 1965 is described based on specimens collected from the Tomtate grunt Haemulon aurolineatum Cuvier, caught in the coast of Angra dos Reis, off the State of Rio de Janeiro, Brazil.
METHODS
One hundred specimens of H. aurolineatum were purchased from the local fish market and examined for parasitic copepods. Parasites were fixed and preserved in 80% ethanol. Morphological features of the copepods were examined and drawn using an Olympus BX51 equipped with a drawing tube.
RESULTS
Ceratocolax tavaresi n. sp. can be distinguished from all congeners by the following combination of characters in the adult female: (1) second endopodal segment of leg 3 with one seta, (2) lack of stout spinules along outer margins on rami of legs 2-4, (3) genital somite without flaplike structures, (4) terminal exopodal segment of leg 4 with seven elements; and in the adult male: (1) legs 1 to 4 with 3-segmented rami (except endopod of leg 4), (2) presence of a pair of blunt processes on dorsal surface of the third pedigerous somite, (3) second endopodal segment of leg 3 with one seta.
CONCLUSION
The number of species of Ceratocolax reported in the Atlantic Ocean was increased to three, including the new species. This is the forty-second species of copepod found parasitizing haemulid fish in marine waters from the Americas; however, the diversity of parasitic copepods off this continent is still underestimated.
Topics: Female; Male; Animals; Copepoda; Parasites; Brazil; Perciformes; Fishes
PubMed: 37943414
DOI: 10.1007/s11686-023-00733-7 -
Microscopy Research and Technique Mar 2024Sugammadex is a new generation drug that has led to significant changes in the practice of anesthesia. However, its effects on fetal development are not yet fully known....
Sugammadex is a new generation drug that has led to significant changes in the practice of anesthesia. However, its effects on fetal development are not yet fully known. The aim of this study is to investigate the teratogenic effects of sugammadex on neural tube and embryonic development in early chick embryos. In this study, 50 0-day fertile specific non-pathogenic (SPF) eggs were used. Fifty eggs were divided into 5 different groups, each consisting of 10 pieces. While no substance was given to the control group at the 28th hour of the study, 4 different doses of sugammadex were administered to the experimental groups, respectively 2, 4, 8, 16 mg/kg. Cranio-caudal lengths of embryos, somite numbers, average number of argyrophilic nucleolar regulatory regions (AgNOR) per nucleus, total AgNOR area/total nuclear area (TAA/NA) ratios, Caspase-3 H-Score results, and presence of neural tube defect were compared among the groups. While the mean cranio-caudal lengths, somite counts, TAA/NA ratios and AgNOR counts of the embryos were found to be statistically significantly lower than the control group, Caspase-3 H-Score mean results were found to be significantly higher (p < .05). In addition, it was observed that there was an increase in neural tube patency and developmental delay. As a result, sugammadex crossing the placenta was revealed to increase the release of proapopitotic molecules and disrupt the developmental stages of embryos. Thus, it was determined that sugammadex in increased developmental delay and incidence of neural tube defects in early chick embryos with increased dose dependent. Despite these results, the effects of sugammadex on fetal development in in vivo and in vitro environments should be studied with further studies. RESEARCH HIGHLIGHTS: Sugammadex is a new generation drug that has led to significant changes in the practice of anesthesia. However, its effects on fetal development are not yet fully known. It has been observed that different doses of sugammadex increase the risk of neural tube defect development on chick embryos and slow the embryo development in a dose-dependent manner.
Topics: Animals; Chick Embryo; Neural Tube; Caspase 3; Sugammadex; Neural Tube Defects; Embryonic Development
PubMed: 37933747
DOI: 10.1002/jemt.24452 -
Frontiers in Cell and Developmental... 2023Maternal diabetes during pregnancy is well known to be associated with a higher risk for structural birth defects in the offspring. Recent searches for underlying...
Maternal diabetes during pregnancy is well known to be associated with a higher risk for structural birth defects in the offspring. Recent searches for underlying mechanisms have largely focused on aberrant processes in the embryo itself, although prior research in rodent models implicated dysfunction also of the visceral yolk sac. The objective of our research was to investigate both tissues within the conceptus simultaneously. We conducted unbiased transcriptome profiling by RNA sequencing on pairs of individual yolk sacs and their cognate embryos, using the non-obese diabetic (NOD) mouse model. The analysis was performed at gestational day 8.5 on morphologically normal specimen to circumvent confounding by defective development. Even with large sample numbers ( = 33 in each group), we observed considerable variability of gene expression, primarily driven by exposure to maternal diabetes, and secondarily by developmental stage of the embryo. Only a moderate number of genes changed expression in the yolk sac, while in the embryo, the exposure distinctly influenced the relationship of gene expression levels to developmental progression, revealing a possible role for altered cell cycle regulation in the response. Also affected in embryos under diabetic conditions were genes involved in cholesterol biosynthesis and NAD metabolism pathways. Exposure to maternal diabetes during gastrulation changes transcriptomic profiles in embryos to a substantially greater effect than in the corresponding yolk sacs, indicating that despite yolk sac being of embryonic origin, different mechanisms control transcriptional activity in these tissues. The effects of maternal diabetes on expression of many genes that are correlated with developmental progression (i.e. somite stage) highlight the importance of considering developmental maturity in the interpretation of transcriptomic data. Our analyses identified cholesterol biosynthesis and NAD metabolism as novel pathways not previously implicated in diabetic pregnancies. Both NAD and cholesterol availability affect a wide variety of cellular signaling processes, and can be modulated by diet, implying that prevention of adverse outcomes from diabetic pregnancies may require broad interventions, particularly in the early stages of pregnancy.
PubMed: 37928898
DOI: 10.3389/fcell.2023.1273641 -
Disease Models & Mechanisms Nov 2023Sonic hedgehog (Shh) signaling is the morphogen signaling that regulates embryonic craniofacial and neural tube development. G protein-coupled receptor 161 (Gpr161) is...
Sonic hedgehog (Shh) signaling is the morphogen signaling that regulates embryonic craniofacial and neural tube development. G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling, and its inactivation in mice results in embryo lethality associated with craniofacial defects and neural tube defects. However, the structural defects of later embryonic stages and cell lineages underlying abnormalities have not been well characterized due to the limited lifespan of Gpr161 null mice. We found that embryos with Pax3 lineage-specific deletion of Gpr161 presented with tectal hypertrophy (anterior dorsal neuroepithelium), cranial vault and facial bone hypoplasia (cranial neural crest), vertebral abnormalities (somite) and the closed form of spina bifida (posterior dorsal neuroepithelium). In particular, the closed form of spina bifida was partly due to reduced Pax3 and Cdx4 gene expression in the posterior dorsal neural tubes of Gpr161 mutant embryos with decreased Wnt signaling, whereas Shh signaling was increased. We describe a previously unreported role for Gpr161 in the development of posterior neural tubes and confirm its role in cranial neural crest- and somite-derived skeletogenesis and midbrain morphogenesis in mice.
Topics: Mice; Animals; Neural Tube; Hedgehog Proteins; Transcription Factors; Spinal Dysraphism; Embryonic Development; Wnt Signaling Pathway; Neurogenesis; Spine
PubMed: 37885410
DOI: 10.1242/dmm.050277 -
Developmental Dynamics : An Official... Apr 2024Noncanonical Wnts are morphogens that can elevate intracellular Ca, activate the Ca/calmodulin-dependent protein kinase, CaMKII, and promote cell movements during...
BACKGROUND
Noncanonical Wnts are morphogens that can elevate intracellular Ca, activate the Ca/calmodulin-dependent protein kinase, CaMKII, and promote cell movements during vertebrate gastrulation.
RESULTS
Zebrafish express seven CaMKII genes during embryogenesis; two of these, camk2b1 and camk2g1, are necessary for convergent extension (CE) cell movements. CaMKII morphant phenotypes were observed as early as epiboly. At the 1-3 somite stage, neuroectoderm and paraxial cells remained unconverged in both morphants. Later, somites lacked their stereotypical shape and were wider, more closely spaced, and body gap angles increased. At 24hpf, somite compression and notochord undulation coincided with a shorter and broader body axis. A camk2b1 crispant was generated which phenocopied the camk2b1 morphant. The levels of cell proliferation, apoptosis and paraxial and neuroectodermal markers were unchanged in morphants. Hyperactivation of CaMKII during gastrulation by transient pharmacological intervention (thapsigargin) also caused CE defects. Mosaically expressed dominant-negative CaMKII recapitulated these phenotypes and showed significant midline bifurcation. Finally, the introduction of CaMKII partially rescued Wnt11 morphant phenotypes.
CONCLUSIONS
Overall, these data support a model whereby cyclically activated CaMKII encoded from two genes enables cell migration during the process of CE.
Topics: Animals; Zebrafish; Zebrafish Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Gastrulation; Cell Movement
PubMed: 37860955
DOI: 10.1002/dvdy.665 -
Comparative Biochemistry and... Jan 2024The mixture of agrochemicals can be made to improve pest control or accidentally. In this way, the effects on non-target organisms are a critical aspect of the...
The mixture of agrochemicals can be made to improve pest control or accidentally. In this way, the effects on non-target organisms are a critical aspect of the environment and heath. Thus, this work aimed to show how a mixture of pyriproxyfen, and glyphosate can impair biochemical routes and embryonic development. Zebrafish embryos 0-72 hpf were exposed to 0.001-1 μg/mL of pyriproxyfen, glyphosate, and a mixture of both pesticides. The ADMETox was evaluated in silico. The FET-test was used to estimate teratogenic effects. The biochemical effects were estimated using AChE, SOD, and CAT as parameters. ROS generation was estimated using 30 μM HDCF-DA and 5 μM DHE. The ADMETox reveals that intestinal absorption and P-glycoprotein are the main sites for PPx and Gly adsorption. The distribution parameters were diverse. PPx + Gly at 0.1 μg/mL leads to 50 % of lethality and at 1 μg/mL 100 % of lethality. PPx + Gly leads to a 22 % of lack of somite formation at 1 μg/mL. The heart rate was reduced by >10 % in all concentrations tested. The AChE has a decrease with IC 19.6 μM and IC 261.5 μM. SOD showed a reduction of 28 % to PPx and CAT was reduced by 58 % to PPx + Gly and Gly at 1 μg/mL. Glyphosate does not increase unspecific ROS generation. The superoxide generation was 2× higher in the PPx + Gly at 1 μg/mL. Summarily, was observed that the mixture of PPx + Gly potentiated the toxic effects. This finding suggests a possible synergism between the PPx and Gly even at lower concentrations.
Topics: Animals; Zebrafish; Reactive Oxygen Species; Superoxide Dismutase; Glyphosate
PubMed: 37844749
DOI: 10.1016/j.cbpc.2023.109766 -
Methods in Molecular Biology (Clifton,... 2024Paraxial mesoderm in the early embryo is segmented into epithelial blocks called somites that establish the metameric organization of the vertebrate body plan. Somites...
Paraxial mesoderm in the early embryo is segmented into epithelial blocks called somites that establish the metameric organization of the vertebrate body plan. Somites are sequentially formed from head to tail in a rhythmic manner controlled by an oscillating gene regulatory network known as the segmentation clock. We know very little about this important process during human development due to limited access to human embryos and ethical concerns. To bypass these difficulties, model systems derived from human pluripotent stem cells have been established. Here, we detail three protocols modeling different aspects of human paraxial mesoderm development in vitro: a 2D cell monolayer system recapitulating dynamics of the human segmentation clock, a 3D organoid system called "somitoid" supporting the simultaneous formation of somite-like structures, and another organoid system called "segmentoid" reconstituting in vivo-like hallmarks of somitogenesis. Together, these complementary model systems provide an excellent platform to decode somitogenesis and advance human developmental biology.
Topics: Animals; Humans; Mesoderm; Somites; Vertebrates; Pluripotent Stem Cells; Embryonic Development; Gene Expression Regulation, Developmental; Body Patterning
PubMed: 37843773
DOI: 10.1007/7651_2023_507