Atlas of Zebrafish Development
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A Nature Research Journal. We are sorry, but there is no personal subscription option available for your country. Reprints and Permissions. Advanced search. Skip to main content. Search Submit My Account Login. Access options Access options Subscribe to Journal Get full journal access for 1 year We are sorry, but there is no personal subscription option available for your country. Welcome to ZebraFish Anatomy Portal. The Z ebra F ish A natomy P ortal ZFAP is an anatomical resource for the zebrafish Danio rerio containing 3D models of zebrafish covering the range of development from 24hrs until adulthood.
Browse in 3D all the images in the database. Search for gene expression based on published literature. Link to other key ressources for zebrafish.
How to use ZFAP? Please visit our "Tutorial" section. Comments and suggestions? Please drop us a line through our feedback form. Search the data using terms in the anatomical ontology. Searches can use multiple anatomical terms and be applied to the complete database or selected developmental stages. Textmining gene expression predictor. This compares with currently annotated in Ensembl for the human genome. Properties of the lincRNAs in terms of numbers of exons, length of transcript and overall expression level are similar to what has been reported previously Cabili et al.
The fact that there is so little overlap between the three sets is probably because of the different methods that were used to define them. Ulitsky et al. Pauli et al. The numbers of transcripts produced by the Ensembl pipeline suggest that it is more sensitive, but less specific than the other two methods. For human and mouse genomes, Ensembl uses chromatin methylation state H3K4me3 and H3K36me3 , but does not yet use this for the zebrafish. The involvement of microRNAs during embryonic development is widespread reviewed in Bartel, ; Mishima, However, the primary transcripts of miRNAs are often poorly annotated.
In agreement with previous studies Chang et al. This diversity of transcripts means that the expression of miRNAs in relatively close proximity e. In some transcripts let-7d-1 and mira-1 are both intronic and in some mira-1 is exonic. There are also transcripts with alternate promoters which exclude the let-7d-1 locus entirely.
These features provide significant flexibility to the transcriptional regulation of miRNAs. It is possible that a proportion of the assembled constructs are fragments. The dataset uncovers new splice isoforms that show differential expression across the 18 stages. When viewed in Ensembl against the Ensembl genebuild, this refines the current gene annotation and ties splice isoforms to their temporal expression patterns. This is of particular relevance to the ongoing debate surrounding reverse genetics approaches in zebrafish Kok et al.
An improved annotation that gives temporal resolution of exon usage is of great value for the identification of critical exons. In accordance with previous work on a subset of stages Collins et al. We show that pdlim5b is expressed as several transcripts, with a single maternal isoform and multiple zygotic ones which would be predicted to produce different proteins.
This type of data provides scope for temporally resolved analysis using transcript-specific knockout targeting strategies. A major aim for this work was to create an accessible reference of normal mRNA expression during zebrafish development. These pre-computed analyses allow an in-depth examination of the data on a gene by gene basis. This makes it easy for researchers to benefit from the data and provides a direct link to the wealth of information present in genomic databases.
Wild-type HLF strain zebrafish Danio rerio were maintained at At the time of mating, breeding males and females were separated overnight before letting them spawn naturally in the morning to allow for synchronisation of developmental stages.
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Samples from 18 different developmental stages from 1 cell to 5 days post-fertilisation were collected by snap freezing the embryos in dry ice. Details of sample names and accession numbers are provided in Supplementary file 1. The lysate was combined with 1. The plate was applied to a plate magnet Invitrogen until the solution cleared and the supernatant was removed without disturbing the beads. RNA was eluted from the beads by applying the plate to the magnetic rack. Strand-specific RNA-seq libraries containing unique index sequences in the adapter were generated simultaneously following the dUTP method.
Libraries were pooled and sequenced on Illumina HiSeq in bp paired-end mode. The data were assessed for technical quality GC-content, insert size, proper-pairs etc. Counts for genes were produced using htseq-count v0. The processed count data are in Supplementary file 2. The sample correlation matrix was produced by calculating the Spearman correlation coefficient between each possible pair of samples. Spearman correlation coefficient was used in preference to Pearson because it is less sensitive to outliers. Principal Component Analysis was carried out using the prcomp function in R.
This projects the data down from 32, dimensions genes to 90 samples. The genes that contribute most to each component were determined by calculating the proportion of variance explained by each gene for each component and selecting those above 0. Orthologues along with the predicted last common ancestor were obtained from the Ensembl Compara database Vilella et al. Genes with no identifiable orthologue in any of the species were classed as Danio -specific.
Those with orthologues in the six fish species but not the mammals examined last common ancestor Neopterygii, Clupeocephala or Otophysi were categorised as Teleost-specific. Those with orthologues also present in the mammalian species last common ancestor Euteleostomi, Vertebrata, Chordata or Bilateria were labelled as Vertebrate. The input to the program is the expression values for the genes in each sample. The Pearson correlation cut-off was set to 0.
Imaging and Bioinformatics
After running MCL, clusters with fewer than six genes were removed genes which produced clusters ranging in size from 6 to genes. The input expression file is provided as Supplementary file 4. Enrichment for particular chromosomes within BioLayout clusters was tested for using a one-tailed binomial test followed by multiple testing correction using the R qvalue package Storey, ; Storey et al. For each cluster, only chromosomes that appeared more than five times were tested.
To calculate a similarity measure for groups of genes in a chromosomal region the genome was split up into overlapping windows of 10 genes. For each window, the Pearson correlation coefficient was calculated for every combination of pairs of genes 45 pairs for a group of 10 genes and averaged. Windows with a mean correlation coefficient greater than 0.
One-to-one paralogues were identified from the Ensembl Compara database Vilella et al. Transcript level counts were produced using Salmon Patro et al. Differentially spliced genes and differentially expressed transcripts were called using the R package maSigPro Conesa et al. DeTCT libraries were generated, sequenced and analysed as described previously Collins et al.
For each mutant allele — drosha sa rnasen ; dgcr8 sa pasha — embryos were collected at 5 dpf and genotyped by KASP genotyping as previously described Dooley et al. We produced and sequenced stranded RNA-seq libraries from single homozygous, heterozygous and wild-type embryos 5 replicates of each. Ensembl v86 zebrafish annotation was provided as a junction file.
FishNet: an online database of zebrafish anatomy
Aligned reads were sorted, indexed and mate pairings were fixed using Samtools v1. Alignments for the homozygous, heterozygous and wild-type samples for both the drosha and dgcr8 lines were merged into two line-specific datasets with Samtools. Each set of merged alignments was then used to assemble a transcriptome with Cufflinks v2.
The drosha and dgcr8 assemblies were then merged with Cuffmerge. For the purpose of this analysis we defined pri-miRNAs as any spliced transcript overlapping at least one of these loci. Ensembl and Havana v87 annotation was selected as the baseline for novel pri-miRNA prediction.
Pri-miRNAs were identified by removing single exon transcripts from the merged assembly and Ensembl annotations and comparing the remainder to annotated miRNA loci using bedtools v2. In the first stage, this method traverses each toplevel sequence e. These potential non-coding models are filtered by looking for Pfam Finn et al. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses.
A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included. Thank you for submitting your article "A high-resolution mRNA expression time course of embryonic development in zebrafish" for consideration by eLife.
Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Didier Stainier as the Senior Editor and Reviewing Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Shawn Burgess Reviewer 1 and Tatjana Sauka-Spengler Reviewer 3. The reviewers have discussed the reviews with one another and the editor has drafted this decision to help you prepare a revised submission.
This manuscript represents a large effort to generate a major genomic resource for the zebrafish community. The work comprises quality, strand-specific RNA-seq across 18 early developmental time points. Having high-resolution transcription data is an essential aspect of developing zebrafish as an essential model organism, and a major strength of the paper is that these data will all be easily accessed from Ensembl. Without a doubt, this work will be a valuable resource not only for zebrafish researchers, but also for developmental biologists in general. The following issues should be addressed in terms of analysis and discussion of the data :.
In any case, this should be clarified and discussed. Hence, fine detail and developmental programmes are important but less predominant embryonic populations will be missed. This point and the limitations of the analysis should be discussed; suggesting that less represented developmental populations should be studied in isolation. The authors could do a more thorough study of uncharacterised genes, analyse types of proteins encoded by those and discuss the groups that are conserved across species or species specific; otherwise, this part should be attenuated and cut down.
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Hence the discussion of the shortcoming of the analysis should be added. Given that the two previous studies found very few overlapping candidates, the authors should discuss how many of previously identified lincRNAs they confirmed. This is important info as the lincRNA discovery in this study nearly doubles what was previously annotated. This part should be better integrated into the paper e. We believe this represents a real phenomenon, which reflects part of the maternal to zygotic transition.
To check, we have downsampled each sample to 2 million read pairs per sample and recounted. The number of genes detected by the same criteria in this set is slightly smaller for all samples, but follows the same pattern of a large drop at gastrula stages. This effect is even larger at Shield stage. Our interpretation of this is that maternally expressed genes are being cleared from the embryo by miR and other pathways and the number of newly expressed zygotic genes does not compensate for this loss.
We agree. The maximum stage groupings were used primarily to order the heatmap. As outlined below we have removed the GO and ZFA enrichment to make the manuscript more compact as it confirms what was already known rather than being genuinely novel. The BioLayout analysis provides more detail with respect to expression correlation and potential functional groups. We have added text to the discussion to acknowledge the limitations of the whole embryo approach and emphasize the need for future detailed tissue- and cell-specific studies Discussion section.
We agree and have examined the distribution of Pfam domains within these sets. The largest set of domains within the unnamed genes are zinc finger domains. These belong to the zinc finger genes in BioLayout clusters 7, 23, 26 and We retrieved Pfam domains annotated to genes from the Ensembl database and counted the number of genes for each domain. We placed domains which appear less than ten times for a stage in the Other category yellow and counted genes with no annotated Pfam domain as NA grey.
Three different ZnF domains were aggregated into a single ZnF category blue. As would be expected, the named genes with vertebrate orthologues top right have a lot of different Pfam domains associated with them. In contrast, the unnamed genes bottom panels are mostly in the Other category. At gastrulation stages the largest sets are the ZnF domains across all three orthology levels.
We agree and have expanded the section in the discussion on the caveats of this analysis and mention it in the Results section as well.
ZFIN Atlas of Zebrafish Anatomy
We would like to thank the reviewers for this suggestion. Furthermore, we have attempted a promoter analysis. This is due to the lack of supporting evidence in the form of CAGE, H3K4me3 and H3K36me3 data which are used in human and mouse lincRNA calling pipeline to define the genomic promoter and gene body features. We have moved the section and expanded the Introduction regarding the roles of miRNAs in zebrafish development. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.