High-throughput sequencing of small RNAs in chicken somite embryos
Ontology highlight
ABSTRACT:
ORGANISM(S): Gallus gallus
PROVIDER: E-GEOD-15513 | BioStudies |
REPOSITORIES: biostudies
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Project description:High-throughput sequencing of small RNAs in chicken somite embryos Overall design: Dissected tissue white leghorn chicken embryos was disaggregated and RNA extracted using the miRVana kit (Ambion). Small RNA fraction between 19 and 24 nt was isolated from 15% denaturing polyacrylamide gel and 15 micro gram was ligated to Solexa adaptors (Illumina) without de-phosphorylating and re-phosphorylating. The short RNAs were converted to DNA by RT-PCR following the Illumina protocol.
2009-04-28 | GSE15513 | GEO
Project description:High-throughput sequencing of small RNAs in chicken somite embryos Dissected tissue white leghorn chicken embryos was disaggregated and RNA extracted using the miRVana kit (Ambion). Small RNA fraction between 19 and 24 nt was isolated from 15% denaturing polyacrylamide gel and 15 micro gram was ligated to Solexa adaptors (Illumina) without de-phosphorylating and re-phosphorylating. The short RNAs were converted to DNA by RT-PCR following the Illumina protocol.
2009-04-27 | E-GEOD-15513 | ArrayExpress
Project description:Anolis carolinensis embryos were collected 0-1 days post egg laying, and total RNA was extracted for RNA-Seq analysis (Illumina Hi-Seq2000). Transcriptome sequence from these stages in the green anole, equivalent to mouse 9.5-10.5 dpc embryos, will help to improve gene annotations in A. carolinensis and provide expression level information for key organogenesis and patterning processes. Anolis carolinensis embryos were collected 0-1 days post egg laying for RNA-Seq analysis. The two embryos collected were at 28 somite-pair (28S) and 38 somite-pair (38S), equivalent to mouse 9.5 dpc and 10.5 dpc embryos, respectively. Total RNA was extracted using the total RNA component of the mirVana (Ambion) kit, RNA-Seq library prep was carried out using the NuGEN Ovation RNA-Seq kit, and sequencing was carried out on an Illumina HiSeq 2000, following the manufacturer's protocol. The untrimmed data was then aligned to the Anolis carolinensis reference genome (Anocar2.0) using tophat. Published: Eckalbar WL, Lasku E, Infante CR, Elsey RM, Markov GJ, Allen AN, Corneveaux JJ, Losos JB, DeNardo DF, Huentelman MJ, Wilson-Rawls J, Rawls A, Kusumi K. Somitogenesis in the anole lizard and alligator reveals evolutionary convergence and divergence in the amniote segmentation clock. Dev Biol. DOI: 10.1016/j.ydbio.2011.11.021
| E-GEOD-34415 | BioStudies
Project description:Vertebrate segmentation is characterized by the periodic formation of epithelial somites from the mesenchymal presomitic mesoderm (PSM). How the rhythmic signaling pulse delivered by the segmentation clock is translated into the periodic morphogenesis of somites remains poorly understood. Here, we focused on the role of paraxial protocadherin (PAPC/Pcdh8) in this process. We showed that in chicken and mouse embryos, PAPC expression is tightly regulated by the clock and wavefront system in the posterior PSM. We observed that PAPC exhibits a striking complementary pattern to N-cadherin (CDH2), marking the interface of the future somite boundary in the anterior PSM. Gain and loss of function of PAPC in chicken embryos disrupted somite segmentation by altering the CDH2-dependent epithelialization of PSM cells. Our data suggest that clathrin-mediated endocytosis is increased in PAPC-expressing cells, subsequently affecting CDH2 internalization in the anterior compartment of the future somite. This in turn generates a differential adhesion interface, allowing formation of the acellular fissure that defines the somite boundary. Thus, periodic expression of PAPC in the anterior PSM triggers rhythmic endocytosis of CDH2, allowing for segmental de-adhesion and individualization of somites.
| S-EPMC5312037 | BioStudies
Project description:Somitogenesis is often described using the clock-and-wavefront (CW) model, which does not explain how molecular signaling rearranges the pre-somitic mesoderm (PSM) cells into somites. Our scanning electron microscopy analysis of chicken embryos reveals a caudally-progressing epithelialization front in the dorsal PSM that precedes somite formation. Signs of apical constriction and tissue segmentation appear in this layer 3-4 somite lengths caudal to the last-formed somite. We propose a mechanical instability model in which a steady increase of apical contractility leads to periodic failure of adhesion junctions within the dorsal PSM and positions the future inter-somite boundaries. This model produces spatially periodic segments whose size depends on the speed of the activation front of contraction (<i>F</i>), and the buildup rate of contractility (Λ). The Λ/<i>F</i> ratio determines whether this mechanism produces spatially and temporally regular or irregular segments, and whether segment size increases with the front speed.
| S-EPMC8050378 | BioStudies
Project description:The DMDD Programme (Deciphering the Mechanisms of Developmental Disorders ) provides a free online database of morphological and molecular phenotypes from embryonic-lethal mouse gene knockouts (http://www.dmdd.org.uk/). Embryos are imaged using HREM, placentas are examined by histology and mutant embryo mRNA expression profiles are compared to wild type. To underpin these investigations we have produced a comprehensive time series of gene expression through normal embryo development. Total RNA was extracted from somite number staged wildtype embryos with no history of genetic modification in previous generations from the Mouse Genetics Programme (http://www.sanger.ac.uk/science/collaboration/mouse-resource-portal) and DNase treated. Stranded RNA-seq libraries were constructed using the Illumina TruSeq Stranded RNA protocol with oligo dT pulldown.<br> Notes about samples and libraries:<br> (1) A combination of litter identifier and embryo identifier within a litter will unambiguously identify a single embryo used in this study. </br><br> (2) There is a margin of error for the somite-stage information (+/- 1 somite) because some embryos could be in between somite stages. </br><br> (3) Sex of the embryos was determined post-RNA-seq by looking at the expression of Xist (strong expression in females only). </br><br> (4) Complementary data on genotypically wild-type mice with mixed G0 lineage and with heterozygous mutant parents can also be found in ArrayExpress under accession numbers E-ERAD-352 ( https://www.ebi.ac.uk/arrayexpress/experiments/E-ERAD-352 ) and E-ERAD-401 ( https://www.ebi.ac.uk/arrayexpress/experiments/E-ERAD-401 ).</br>
| E-ERAD-499 | BioStudies
Project description:The DMDD Programme (Deciphering the Mechanisms of Developmental Disorders, https://dmdd.org.uk/) provides a free online database of morphological and molecular phenotypes from embryonic-lethal mouse gene knockouts (http://www.dmdd.org.uk/). Embryos are imaged using HREM, placentas are examined by histology and mutant embryo mRNA expression profiles are compared to wild type. To underpin these investigations we have produced a comprehensive time series of gene expression through normal embryo development. Total RNA was extracted from somite number staged, second generation genotypically wild type, C57BL/6N embryos of mixed G0 linages from the Mouse Genetics Programme (http://www.sanger.ac.uk/science/collaboration/mouse-resource-portal) and DNase treated. Stranded RNA-seq libraries were constructed using the Illumina TruSeq Stranded RNA protocol with oligo dT pulldown.<br> Notes about samples and libraries: </br><br>(1) A combination of litter identifier and embryo identifier within a litter will unambiguously identify a single embryo used in this study. </br><br>(2) There is a margin of error for the somite-stage information (+/- 1 somite) because some embryos could be in between somite stages. </br><br> (3) All knockouts in the parents or grandparents are for embryonic lethal genes. </br><br>(4) There are four biological replicates per somite-stage, with the exception of the 4-somite embryos (three biological replicates only). Altogether 111 embryos were sourced, of which 14 were sequenced twice to generate enough read depth/coverage. No new libraries were prepared for the repeated sequencing, despite the assays being assigned new ERX* accessions. </br><br> (5) Sex of the embryos was determined post-RNA-seq by looking at the expression of Xist (strong expression in females only). </br><br> (6) Library constructon batch refers to batch of sample handling post RNA-extraction (size selection, PCR amplification during library preparataion). </br><br>(7) Superbatch gathers 17 representative embryos out of the 111 embryos post RNA-extraction, and put them through the library construction pipeline as one single batch. The generated data is intended to be used for normalisation using the remove unwanted variation (RUV) method. </br> <br> This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/ .</br>
| E-ERAD-401 | BioStudies
Project description:The DMDD Programme (Deciphering the Mechanisms of Developmental Disorders ) provides a free online database of morphological and molecular phenotypes from embryonic-lethal mouse gene knockouts (http://www.dmdd.org.uk/). Embryos are imaged using high-resolution episcopic microscopy (HREM), placentas are examined by histology and mutant embryo mRNA expression profiles are compared to wild type. To underpin these investigations we have produced a comprehensive time series of gene expression through normal embryo development<br></br>In this study, yotal RNA was extracted from wildtype C57BL/6N sibling embryos with heterozygous parentage from the Mouse Genetics Project (http://www.dmdd.org.uk/) and DNase treated. Stranded RNAseq libraries were constructed using the Illumina TruSeq Stranded RNA protocol with oligo dT pulldown. <br></br> Notes about samples and libraries:<br></br> (1) A combination of litter identifier and embryo identifier within a litter will unambiguously identify a single embryo used in this study. <br></br> (2) There is a margin of error for the somite-stage information (+/- 1 somite) because some embryos could be in between somite stages. <br></br>(3) Sex of the embryos was determined post-RNA-seq by looking at the expression of Xist (strong expression in females only). <br></br>(4) All knockouts in the parents (and hence grandparents) are for embryonic lethal genes. <br></br> (5) There are four biological replicates per somite-stage. <br></br>(6) Complementary data on genotypically wild-type mice with mixed G0 lineage and no history of genetic modification can also be found in ArrayExpress under accession numbers E-ERAD-401 ( https://www.ebi.ac.uk/arrayexpress/experiments/E-ERAD-401 ) and E-ERAD-499 ( https://www.ebi.ac.uk/arrayexpress/experiments/E-ERAD-499 ) respectively.
| E-ERAD-352 | BioStudies
Project description:A set of genes in the posterior end of developing mouse embryos shows oscillatory expression, thereby regulating periodic somite segmentation. Although the mechanism for generating oscillation has extensively been clarified, what regulates the oscillation period is still unclear. We attempted to elongate the oscillation period by increasing the time to transcribe Hes7 in this research. We generated knock-in mice, in which a large intron was inserted into Hes7 3'UTR. The exogenous intron was unexpectedly not properly spliced out and the transcripts were prematurely terminated. Consequently, Hes7 mRNA lost its 3'UTR, thereby reducing the amount of Hes7 protein. Oscillation was damped in the knock-in embryos and periodic somite segmentation does not occur properly. Thus, we demonstrated that Hes7 3'UTR is essential to accumulate adequate amounts of Hes7 protein for the somite segmentation clock that orchestrates periodic somite formation.
| S-EPMC4173035 | BioStudies
Project description:The reproducibility of embryonic development is remarkable, although molecular processes are intrinsically stochastic at the single-cell level. How the multicellular system resists the inevitable noise to acquire developmental reproducibility constitutes a fundamental question in developmental biology. Toward this end, we focused on vertebrate somitogenesis as a representative system, because somites are repeatedly reproduced within a single embryo whereas such reproducibility is lost in segmentation clock gene-deficient embryos. However, the effect of noise on developmental reproducibility has not been fully investigated, because of the technical difficulty in manipulating the noise intensity in experiments. In this study, we developed a computational model of ERK-mediated somitogenesis, in which bistable ERK activity is regulated by an FGF gradient, cell-cell communication, and the segmentation clock, subject to the intrinsic noise. The model simulation generated our previous in vivo observation that the ERK activity was distributed in a step-like gradient in the presomitic mesoderm, and its boundary was posteriorly shifted by the clock in a stepwise manner, leading to regular somite formation. Here, we showed that this somite regularity was robustly maintained against the noise. Removing the clock from the model predicted that the stepwise shift of the ERK activity occurs at irregular timing with irregular distance owing to the noise, resulting in somite size variation. This model prediction was recently confirmed by live imaging of ERK activity in zebrafish embryos. Through theoretical analysis, we presented a mechanism by which the clock reduces the inherent somite irregularity observed in clock-deficient embryos. Therefore, this study indicates a novel role of the segmentation clock in noise-resistant developmental reproducibility.
| S-EPMC6361423 | BioStudies