An improved zebrafish transcriptome annotation for sensitive and comprehensive detection of cell type-specific genes
- University of Massachusetts Medical School, United States
- University of Oxford, United Kingdom
Abstract
The zebrafish is ideal for studying embryogenesis and is increasingly applied to model human disease. In these contexts, RNA-sequencing (RNA-seq) provides mechanistic insights by identifying transcriptome changes between experimental conditions. Application of RNA-seq relies on accurate transcript annotation for a genome of interest. Here, we find discrepancies in analysis from RNA-seq datasets quantified using Ensembl and RefSeq zebrafish annotations. These issues were due, in part, to variably annotated 3' untranslated regions and thousands of gene models missing from each annotation. Since these discrepancies could compromise downstream analyses and biological reproducibility, we built a more comprehensive zebrafish transcriptome annotation that addresses these deficiencies. Our annotation improves detection of cell type-specific genes in both bulk and single cell RNA-seq datasets, where it also improves resolution of cell clustering. Thus, we demonstrate that our new transcriptome annotation can outperform existing annotations, providing an important resource for zebrafish researchers.
Data availability
All data generated in this study are available in accompanying source data files. Transcriptome annotation files described in this study are available for download at zf-transcriptome.umassmed.edu. Raw and processed RNA-seq data generated in this study are available at GEO (GSE152759).
-
Bulk RNA-seq data to assess an improved zebrafish transcriptome annotationNCBI Gene Expression Omnibus, GSE152759.
-
Morphogenesis and differentiation of embryonic vascular smooth muscle cells in zebrafishNCBI Gene Expression Omnibus, GSE119718.
-
Comprehensive identification of long non-coding RNAs expressed during zebrafish embryogenesisNCBI Gene Expression Omnibus, GSE32900.
-
Extensive alternative polyadenylation during zebrafish developmentNCBI Gene Expression Omnibus, GSE37453.
Article and author information
Author details
Funding
National Heart, Lung, and Blood Institute (R35HL140017)
- Nathan D Lawson
National Human Genome Research Institute (U01HG007910)
- Onur Yukselen
- Alper Kucukural
National Center for Advancing Translational Sciences (UL1TR001453)
- Onur Yukselen
- Alper Kucukural
National Institute of Neurological Disorders and Stroke (R21NS105654)
- Nathan D Lawson
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Elisabeth Busch-Nentwich, University of Cambridge
Ethics
Animal experimentation: Zebrafish studies were performed in accordance with protocols #A2613 and #A2632 approved by the University of Massachusetts institutional animal care and use committee (IACUC).
Version history
- Received: February 6, 2020
- Accepted: August 21, 2020
- Accepted Manuscript published: August 24, 2020 (version 1)
- Version of Record published: September 11, 2020 (version 2)
Copyright
© 2020, Lawson et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 9,781
- views
-
- 843
- downloads
-
- 83
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
An improved zebrafish transcriptome annotation for sensitive and comprehensive detection of cell type-specific genes
eLife 9:e55792.
https://doi.org/10.7554/eLife.55792
Further reading
-
- Developmental Biology
Precise developmental timing control is essential for organism formation and function, but its mechanisms are unclear. In C. elegans, the microRNA lin-4 critically regulates developmental timing by post-transcriptionally downregulating the larval-stage-fate controller LIN-14. However, the mechanisms triggering the activation of lin-4 expression toward the end of the first larval stage remain unknown. We demonstrate that the transmembrane transcription factor MYRF-1 is necessary for lin-4 activation. MYRF-1 is initially localized on the cell membrane, and its increased cleavage and nuclear accumulation coincide with lin-4 expression timing. MYRF-1 regulates lin-4 expression cell-autonomously and hyperactive MYRF-1 can prematurely drive lin-4 expression in embryos and young first-stage larvae. The tandem lin-4 promoter DNA recruits MYRF-1GFP to form visible loci in the nucleus, suggesting that MYRF-1 directly binds to the lin-4 promoter. Our findings identify a crucial link in understanding developmental timing regulation and establish MYRF-1 as a key regulator of lin-4 expression.
-
- Developmental Biology
- Structural Biology and Molecular Biophysics
The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, brachydactyly B, and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.
