CytoD was injected laterally at mid-late cellularization with 0.5 mg/ml CytoD in 10 % DMSO (Calbiochem). Double stranded RNA against snail and twist (2 mg/ml) were injected laterally into freshly laid eggs that were incubated 2.5–3 hours before imaging gastrulation.
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Organism Function
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Gastrulation
Gastrulation
Gastrulation is a critical stage of embryonic development where the embryo undergoes dramatic morphological changes, establishing the three primary germ layers: ectoderm, mesoderm, and endoderm.
This complex process involves cell migration, invagination, and differentiation, laying the foundation for the formation of major organ systems.
Studying the molecular and cellular mechanisms governing gastrulation is crucial for understanding early embryogenesis and developmental biology.
PubCompare.ai's AI-driven tools can help researchers streamline their gastrulation studies by optimizing protocol selection, enhancing reproducibility, and improving accuracy - effortlessly identifiying the best protocols and products from literature, pre-prints, and patents.
This complex process involves cell migration, invagination, and differentiation, laying the foundation for the formation of major organ systems.
Studying the molecular and cellular mechanisms governing gastrulation is crucial for understanding early embryogenesis and developmental biology.
PubCompare.ai's AI-driven tools can help researchers streamline their gastrulation studies by optimizing protocol selection, enhancing reproducibility, and improving accuracy - effortlessly identifiying the best protocols and products from literature, pre-prints, and patents.
Most cited protocols related to «Gastrulation»
Eggs
Gastrulation
Helix (Snails)
RNA, Double-Stranded
Sulfoxide, Dimethyl
2-(beta-(4-hydroxyphenyl)ethylaminomethyl)tetralone
Cells
Chromium
Embryo
Euthanasia
Females
Gastrulation
Joint Dislocations
Lanugo
Mice, Inbred C57BL
Mus
Neck
Pregnant Women
Rivers
Serum
Single-Cell RNA-Seq
Strains
Uterus
alexa fluor 488
anti-IgG
Antibodies
Blastomeres
Embryo
Gastrulation
Genes
Goat
Immunofluorescence
Morpholinos
Mus
Protein Biosynthesis
Proteins
RNA, Messenger
Xenopus laevis
To obtain separated count matrices for spliced and unspliced mRNAs, we ran velocyto 0.17.17 [10 (link)] on the .bam files from the mouse atlas in Pijuan-Sala et al. ([25 (link)]; Arrayexpress accession number: E-MTAB-6967). We kept all cells that passed the QC as described in the original publication, but filtered out from downstream analysis the extraembryonic tissues: ExE endoderm, ExE ectoderm, and Parietal endoderm as well as samples with no timepoint allocation (labelled as “mixed gastrulation”). To select highly variable genes (HVGs) we applied both the scanpy v1.5.1 and the scVelo v0.2.1 [14 ] pipelines. That is, we removed genes with less than 20 shared counts between spliced and unspliced counts, before normalizing and log transforming the remaining genes. Then, we selected the top 2500 HVGs from each approach (resulting in a total of 4000, with 1000 overlapping genes) for further calculation of moments, while performing imputation using the top 30 nearest neighbours from the graph connectivities generated with the original UMAP coordinates from Pijuan-Sala et al. [25 (link)]. The velocity vectors were computed in dynamical mode rather than steady state.
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Cells
Cloning Vectors
Ectoderm
Endoderm
Gastrulation
Genes
Genes, Overlapping
Mus
RNA, Messenger
Tissues
FGF4 protein, human
GAPDH protein, human
Gastrulation
Genes
Heart
Mice, Laboratory
POU5F1 protein, human
Reverse Transcriptase Polymerase Chain Reaction
SOX2 protein, human
Most recents protocols related to «Gastrulation»
The inbred lines used in this study and in Wu et al. [31 ] were kind gifts of Dr. Cecelia Miles [77 (link)]: #2.46.4, #2.49.3, #9.17.1 and #9.31.2, representing LΑ, Lλ, SΑ and Sλ, respectively. Embryo collection, FISH and imaging were performed for Lλ and Sλ as previously reported [31 ]. Briefly, 0–4 h embryos were collected from 5 to 10-day-old females under standard conditions at 25°. We performed mRNA FISH using digoxigenin-labelled RNA probes synthesized as before [31 ,78 (link)], and the fluorescence signals were detected by sheep anti-digoxigenin (Roche, 1:400) and goat anti-sheep AlexaFluor 594 (Life technologies, 1:400) as the primary and secondary antibodies, respectively. The nuclei of collected embryos were counterstained with 4,6-diamidino-2-phenylindole (DAPI).
We performed imaging on Zeiss Imager Z1 ApoTome microscope, and the associated software AxioVision 4.8 was applied to capture images in linear settings without normalization as before [31 ]. Imaging for embryos was focused on the mid-sagittal section and taken with 10× objective, capturing embryos at the stage of interest (nc13 or nc14 before gastrulation) and avoiding embryos with severe morphological distortion and deformations. We adjusted the exposure time through using stained embryo with highest intensity to effectively avoid pixel intensity saturation. To make direct comparisons of expression profiles between lines, all experiments and imaging were conducted side by side.
We performed imaging on Zeiss Imager Z1 ApoTome microscope, and the associated software AxioVision 4.8 was applied to capture images in linear settings without normalization as before [31 ]. Imaging for embryos was focused on the mid-sagittal section and taken with 10× objective, capturing embryos at the stage of interest (nc13 or nc14 before gastrulation) and avoiding embryos with severe morphological distortion and deformations. We adjusted the exposure time through using stained embryo with highest intensity to effectively avoid pixel intensity saturation. To make direct comparisons of expression profiles between lines, all experiments and imaging were conducted side by side.
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Antibodies
Cell Nucleus
Digoxigenin
Domestic Sheep
Embryo
Females
Fishes
Fluorescence
Gastrulation
Gifts
Goat
Microscopy
RNA, Messenger
RNA Probes
Chemical treatments were completed as previously described, with chemicals as listed in Table 1 [34 (link),38 ,39 (link)]. Chemicals were dissolved in DMSO to make a 10 mM stock solution. Stocks were aliquoted and stored at −80 °C. Aliquots were thawed at room temperature and protected from light. Working solutions were diluted in E3 and distributed to 6- or 12-well plates. Chemical treatments were completed beginning at the shield stage (6 h post-fertilization (hpf)) until the 24 hpf stage, unless otherwise noted. We chose to treat at the shield stage, as the animals were already undergoing gastrulation. Thus, this timepoint prevents interference with the onset of gastrulation. The dose for each chemical was decided by treating at doses consistent with previous studies or slightly increased concentrations to maximize penetrance while minimizing morphological defects. Animals treated with E2 exhibited distal segmentation phenotypes at both 20 µM and 25 µM. As most animals had a curved body axis at 25 µM, we proceeded with 20 µM treatments. We treated with 400 µM DPN, 400 µM MPP, and 75 µM PPT, however the animals did not exhibit changes in distal nephron segmentation. When exposed to higher doses, the animals exhibited morphological defects or mortality. PHTPP exhibited the highest penetrance of distal segmentation without morphological defects at 18 µM. Xenoestrogens genistein and ethinylestradiol exhibited the highest penetrance of distal segmentation without morphological defects at 20 µM. Treatments were conducted in triplicate with at least n > 20 embryos per replicate at various doses (Table 2 ). All experiments were conducted with a DMSO vehicle control. DMSO control animals are demarcated as “WT” in all graphics and schematics.
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Animals
DNA Replication
Embryo
Epistropheus
Ethinyl Estradiol
Fertilization
Gastrulation
Genistein
Human Body
Light
Nephrons
Phenotype
Sulfoxide, Dimethyl
We applied CellOracle to a scRNA-seq atlas of mouse gastrulation and organogenesis by Pijuan-Sala et al.30 (link). This single-cell profiling of WT cells highlighted a continuous differentiation trajectory across the early development of various cell types (Extended Data Fig. 9a ). In addition, the developmental effects of Tal1 KO, a TF known to regulate early haematoendothelial development64 (link),65 (link), were investigated in this study. We validated the CellOracle simulation using these Tal1 KO ground-truth scRNA-seq data. The data were generated from seven chimeric E8.5 embryos of WT and Tal1 KO cells (25,307 cells and 26,311 cells, respectively). We used the R library, MouseGastrulationData (https://github.com/MarioniLab/MouseGastrulationData ), to download the mouse early gastrulation scRNA-seq dataset. This library provides the GEM and metadata. We used the Tal1 chimera GEM and cell-type annotation, “cell type.mapped”, provided by this library. Data were normalized with SCTransform66 (link). The GEM was converted to the AnnData format and processed in the same way as the Paul et al. dataset. For the dimensionality reduction, we used UMAP using the PAGA graph for the initialization (maxiter=500, min_dist=0.6). We removed the extraembryonic ectoderm (ExE), primordial germ cell (PGC) and stripped nuclei clusters which lie outside the main differentiation branch. After removing these clusters, we used the WT cell data for the simulations (24,964 cells). GRN calculations and simulations were performed as described above using the default parameters. We used the base GRN generated from the mouse sci-ATAC-seq atlas dataset. We constructed cluster-wise GRN models for 25 cell states. Then, we simulated Tal1 KO effects using the WT scRNA-seq dataset. For the late-stage-specific Tal1 conditional KO simulation, we set Tal1 expression to be zero in the blood progenitor and erythroid clusters to analyse the role of Tal1 in late erythroid differentiation stages (Extended Data Fig. 9i,j ).
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ATAC-Seq
BLOOD
cDNA Library
Cell Nucleus
Cells
Chimera
Ectoderm
Embryo
Gastrulation
Germ Cells
Mus
Organogenesis
Single-Cell RNA-Seq
TAL1 protein, human
Embryos were imaged using a Zeiss LSM 900 confocal microscope. Plan-Apochromat 40x/1.4 N.A. oil immersion objective was used. Images were acquired with the following settings: 512 × 512 pixels, 16-bit depth, 18 z-slices separated by 0.6 μm, ~16.8 s/frame time-resolution. The fluorescence of GFP, mCherry, and eBFP2 was excited using 488-, 561-, and 405-nm lasers, respectively. Excitation power was measured and calibrated using X-Cite XR2100/XP750 Optical Power Measurement System (EXCELITAS Technologies) to keep the same experimental setting for each set of experiments. Image acquisition was started before the end of nc13 and ended after the onset of gastrulation at nc14. During imaging, data acquisition was occasionally stopped for a few seconds to correct the z-position. Obtained data were concatenated and cropped into 430 × 512 pixels (sna shadow enhancer), 430 × 430 pixels (Ubx BRE), 300 × 512 pixels (rho NEE), or 300 × 350 pixels (hairy enhancer) to remove nuclei outside of the expression domain. One hundred eighty timeframes starting from the entry into nc14 as defined by the progression of prior anaphase were used for subsequent image analysis. The temperature was kept between 22.0 to 23.0 °C during imaging.
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Anaphase
Cell Nucleus
Disease Progression
Embryo
Fluorescence
Gastrulation
Hair
Microscopy, Confocal
Neoplasm Metastasis
Reading Frames
Submersion
Xenopus laevis oocytes were collected using the in vitro fertilization technique. Ovulation of the female frog was previously induced by subcutaneous injection of the hormone Human Chorionic Gonadotropin (hCG). 48 h before fertilization (pre-prime), the female was injected with 50 units of hCG. 24 h later the female was injected again with 250–300 units of hCG (prime). Embryos were maintained in Marc’s modified Ringer’s solution (10% MMR, pH 7.4), containing 1 M NaCl, 20 mM KCl, 10 mM MgSO4, 20 mM CaCl2, and 50 mM HEPES. The male gonads were obtained through abdominal dissection. The testes were stored in 1× MMR medium supplemented with 20% FBS. 12 h after the second injection of hCG, the oocytes were collected in 1× MMR solution, later they were fertilized with X. laevis testis extract and incubated for 1 h at room temperature in 10% MMR solution. Afterward, the medium was replaced by a 2% cysteine solution in 10% MMR, pH 7.8–7.9 for 5 min, to dissolve the gelatinous layer “degelatinization” [22 (link)]. Subsequently, the embryos were washed and incubated in 10% MMR at room temperature and finally harvested at different neurulation stages: 12.5 (early neurulation), 14 (middle neurulation), and stage 20 (late neurulation); and stage 40–45 (tadpole) [2 (link),105 ]. We observed the expression of Cxs in stages 12.5–20 and the analysis of the closure of the neural tube and morphological effects in stages 40–45. Stage 10 (gastrulation) was used as a negative control for Cxs expression and the adult brain as a positive control to validate Cxs expression in molecular biology assays.
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Abdomen
Adult
Biological Assay
Brain
CCL7 protein, human
Cysteine
Dissection
Embryo
Females
Fertilization
Fertilization in Vitro
Gastrulation
Gelatins
HEPES
Hormones
Human Chorionic Gonadotropin
In Vitro Techniques
Neurulation
Ovulation
Ovum
Rana
Ringer's Solution
Sodium Chloride
Subcutaneous Injections
Sulfate, Magnesium
Tadpole
Testis
Tube, Neural
Xenopus laevis
Top products related to «Gastrulation»
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The LSM 900 is a confocal laser scanning microscope designed for high-resolution imaging. It utilizes laser excitation and a pinhole system to achieve optical sectioning, allowing for the visualization of three-dimensional structures within samples.
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More about "Gastrulation"
Gastrulation is a crucial stage of embryonic development where the embryo undergoes remarkable morphological changes, establishing the three primary germ layers: ectoderm, mesoderm, and endoderm.
This complex process involves cell migration, invagination, and differentiation, laying the foundation for the formation of major organ systems.
Studying the molecular and cellular mechanisms governing gastrulation is vital for understanding early embryogenesis and developmental biology.
PubCompare.ai's AI-driven tools can help researchers streamline their gastrulation studies by optimizing protocol selection, enhancing reproducibility, and improving accuracy.
Researchers can effortlessly identify the best protocols and products from literature, pre-prints, and patents, including techniques such as MMessage mMachine kit, MEGAscript T7 Transcription Kit, TRIzol, LSM 900, StepOnePlus real-time PCR machine, Megascript T7, NBT/BCIP solution, TaqMan probes, and Nicotine.
The RNeasy Mini Kit can also be used for RNA extraction and purification during gastrulation studies.
By leveraging PubCompare.ai's powerful tools, researchers can enhance the reproducibility and accuracy of their gastrulation experiments, leading to more reliable and impactful findings in the field of developmental biology.
Whether you're studying the intricacies of cell migration, germ layer formation, or organogenesis, PubCompare.ai can help you navigate the vast landscape of scientific literature and identify the most effective protocols and products to streamline your gastrulation research.
This complex process involves cell migration, invagination, and differentiation, laying the foundation for the formation of major organ systems.
Studying the molecular and cellular mechanisms governing gastrulation is vital for understanding early embryogenesis and developmental biology.
PubCompare.ai's AI-driven tools can help researchers streamline their gastrulation studies by optimizing protocol selection, enhancing reproducibility, and improving accuracy.
Researchers can effortlessly identify the best protocols and products from literature, pre-prints, and patents, including techniques such as MMessage mMachine kit, MEGAscript T7 Transcription Kit, TRIzol, LSM 900, StepOnePlus real-time PCR machine, Megascript T7, NBT/BCIP solution, TaqMan probes, and Nicotine.
The RNeasy Mini Kit can also be used for RNA extraction and purification during gastrulation studies.
By leveraging PubCompare.ai's powerful tools, researchers can enhance the reproducibility and accuracy of their gastrulation experiments, leading to more reliable and impactful findings in the field of developmental biology.
Whether you're studying the intricacies of cell migration, germ layer formation, or organogenesis, PubCompare.ai can help you navigate the vast landscape of scientific literature and identify the most effective protocols and products to streamline your gastrulation research.