Dataset Information

Biotagging of Specific Cell Populations in Zebrafish Reveals Gene Regulatory Logic Encoded in the Nuclear Transcriptome.

ABSTRACT: Interrogation of gene regulatory circuits in complex organisms requires precise tools for the selection of individual cell types and robust methods for biochemical profiling of target proteins. We have developed a versatile, tissue-specific binary in vivo biotinylation system in zebrafish termed biotagging that uses genetically encoded components to biotinylate target proteins, enabling in-depth genome-wide analyses of their molecular interactions. Using tissue-specific drivers and cell-compartment-specific effector lines, we demonstrate the specificity of the biotagging toolkit at the biochemical, cellular, and transcriptional levels. We use biotagging to characterize the in vivo transcriptional landscape of migratory neural crest and myocardial cells in different cellular compartments (ribosomes and nucleus). These analyses reveal a comprehensive network of coding and non-coding RNAs and cis-regulatory modules, demonstrating that tissue-specific identity is embedded in the nuclear transcriptomes. By eliminating background inherent to complex embryonic environments, biotagging allows analyses of molecular interactions at high resolution.

SUBMITTER: Trinh LA

PROVIDER: S-EPMC5400779 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Biotagging of Specific Cell Populations in Zebrafish Reveals Gene Regulatory Logic Encoded in the Nuclear Transcriptome.

Trinh Le A LA Chong-Morrison Vanessa V Gavriouchkina Daria D Hochgreb-Hägele Tatiana T Senanayake Upeka U Fraser Scott E SE Sauka-Spengler Tatjana T

Cell reports 20170401 2

Interrogation of gene regulatory circuits in complex organisms requires precise tools for the selection of individual cell types and robust methods for biochemical profiling of target proteins. We have developed a versatile, tissue-specific binary in vivo biotinylation system in zebrafish termed biotagging that uses genetically encoded components to biotinylate target proteins, enabling in-depth genome-wide analyses of their molecular interactions. Using tissue-specific drivers and cell-compartm ...[more]

PMID: 28402863

Similar Datasets

Project description:Large-scale sequencing efforts have documented extensive genetic variation within the human genome. However, our understanding of the origins, global distribution, and functional consequences of this variation is far from complete. While regulatory variation influencing gene expression has been studied within a handful of populations, the breadth of transcriptome differences across diverse human populations has not been systematically analyzed. To better understand the spectrum of gene expression variation, alternative splicing, and the population genetics of regulatory variation in humans, we have sequenced the genomes, exomes, and transcriptomes of EBV transformed lymphoblastoid cell lines derived from 45 individuals in the Human Genome Diversity Panel (HGDP). The populations sampled span the geographic breadth of human migration history and include Namibian San, Mbuti Pygmies of the Democratic Republic of Congo, Algerian Mozabites, Pathan of Pakistan, Cambodians of East Asia, Yakut of Siberia, and Mayans of Mexico. We discover that approximately 25.0% of the variation in gene expression found amongst individuals can be attributed to population differences. However, we find few genes that are systematically differentially expressed among populations. Of this population-specific variation, 75.5% is due to expression rather than splicing variability, and we find few genes with strong evidence for differential splicing across populations. Allelic expression analyses indicate that previously mapped common regulatory variants identified in eight populations from the International Haplotype Map Phase 3 project have similar effects in our seven sampled HGDP populations, suggesting that the cellular effects of common variants are shared across diverse populations. Together, these results provide a resource for studies analyzing functional differences across populations by estimating the degree of shared gene expression, alternative splicing, and regulatory genetics across populations from the broadest points of human migration history yet sampled.

Project description:BackgroundPrevious work aimed at understanding the gene regulatory networks (GRNs) governing caudal hindbrain formation identified morphogens such as Retinoic Acid (RA) and Fibroblast growth factors (FGFs), as well as transcription factors like hoxb1b, hoxb1a, hnf1ba, and valentino as being required for rhombomere (r) r4-r6 formation in zebrafish. Considering that the caudal hindbrain is relatively complex - for instance, unique sets of neurons are formed in each rhombomere segment - it is likely that additional essential genes remain to be identified and integrated into the caudal hindbrain GRN.MethodsBy taking advantage of gene expression data available in the Zebrafish Information Network (ZFIN), we identified 84 uncharacterized genes that are expressed in r4-r6. We selected a representative set of 22 genes and assayed their expression patterns in hoxb1b, hoxb1a, hnf1b, and valentino mutants with the goal of positioning them in the caudal hindbrain GRN. We also investigated the effects of RA and FGF on the expression of this gene set. To examine whether these genes are necessary for r4-r6 development, we analyzed germline mutants for six of the genes (gas6, gbx1, sall4, eglf6, celf2, and greb1l) for defects in hindbrain development.ResultsOur results reveal that r4 gene expression is unaffected by the individual loss of hoxb1b, hoxb1a or RA, but is under the combinatorial regulation of RA together with hoxb1b. In contrast, r5/r6 gene expression is dependent on RA, FGF, hnf1ba and valentino - as individual loss of these factors abolishes r5/r6 gene expression. Our analysis of six mutant lines did not reveal rhombomere or neuronal defects, but transcriptome analysis of one line (gas6 mutant) identified expression changes for genes involved in several developmental processes - suggesting that these genes may have subtle roles in hindbrain development.ConclusionWe conclude that r4-r6 formation is relatively robust, such that very few genes are absolutely required for this process. However, there are mechanistic differences in r4 versus r5/r6, such that no single factor is required for r4 development while several genes are individually required for r5/r6 formation.

Project description:Alternative splicing is an important way of regulating gene functions in eukaryotes. Several key genes involved in sex determination and gonadal differentiation, such as nr5a1 and ddx4, have sex-biased transcripts between males and females, suggesting a potential regulatory role of alternative splicing in gonads. Currently, the sex-specific alternative splicing events and genes have not been comprehensively studied at the genome-wide level in zebrafish. In this study, through global splicing analysis on three independent sets of RNA-seq data from matched zebrafish testes and ovaries, we identified 120 differentially spliced genes shared by the three datasets, most of which haven't been reported before. Functional enrichment analysis showed that the GO terms of mRNA processing, mRNA metabolism and microtubule-based process were strongly enriched. The testis- and ovary-biased alternative splicing genes were identified, and part of them (tp53bp1, tpx2, mapre1a, kif2c, and ncoa5) were further validated by RT-PCR. Sequence characteristics analysis suggested that the lengths, GC contents, and splice site strengths of the alternative exons or introns may have different influences in different types of alternative splicing events. Interestingly, we identified an unexpected high proportion (over 70%) of non-frameshift exon-skipping events, suggesting that in these cases the two protein isoforms derived from alternative splicing may both have functions. Furthermore, as a representative example, we found that the alternative splicing of ncoa5 causes the loss of a conserved RRM domain in the short transcript predominantly produced in testes. Our study discovers novel sex-specific alternative splicing events and genes with high reliabilities in zebrafish testes and ovaries, which would provide attractive targets for follow-up studies to reveal the biological significances of alternative splicing events and genes in sex determination and gonadal differentiation.

Dataset Information

Biotagging of Specific Cell Populations in Zebrafish Reveals Gene Regulatory Logic Encoded in the Nuclear Transcriptome.

Publications

Biotagging of Specific Cell Populations in Zebrafish Reveals Gene Regulatory Logic Encoded in the Nuclear Transcriptome.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets