Deciphering sex-specific miRNAs as heat-recorders in zebrafish

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression in a wide variety of physiological processes, including those related to the reproductive system. Although in the last decade a plethora of miRNAs has been reported, the miRNA alterations occurred by environmental cues and their biological functions have not yet been elucidated. With the aim to identify epigenetic regulations mediated by miRNAs in the gonads in a climate change scenario, zebrafish (Danio rerio) were subjected to high temperatures during sex differentiation (18-32 days post fertilization, dpf), a treatment that results in male-skewed sex ratios. Once the fish reached adulthood (90 dpf), ovaries and testes were sequenced by high-throughput technologies. About 101 million high-quality reads were obtained from gonadal samples. Analyses of the expression levels of the miRNAs identified a total of 23 and 1 differentially expressed (DE) miRNAs in ovaries and testes, respectively, two months after the heat treatment. Most of the identified miRNAs were involved in human sex-related cancer. After retrieving 3’ UTR regions, ~400 predicted targets of the 24 DE miRNAs were obtained, some with reproduction-related functions. Their synteny in the zebrafish genome was, for more than half of them, in the chromosomes 7, 2, 4, 3 and 11 in the ovaries, chromosome 4 being the place where the predicted sex-associated-region (sar) is localized in wild zebrafish. Further, spatial localization in the gonads of two selected miRNAs (miR-122-5p and miR-146-5p) showed exclusive expression in the ovarian germ cells. The present study expands the catalog of sex-specific miRNAs and deciphers, for the first time, thermosensitive miRNAs in the zebrafish gonads that might be used as potential epimarkers to predict environmental past events.

Long-Read Sequencing of the Zebrafish Genome Reorganizes Genomic Architecture

Nanopore sequencing technology has revolutionized the field of genome biology with its ability to generate extra-long reads that can resolve regions of the genome that were previously inaccessible to short-read sequencing platforms. Although long-read sequencing has been used to resolve several vertebrate genomes, a nanopore-based zebrafish assembly has not yet been released. Over 50% of the zebrafish genome consists of difficult to map, highly repetitive, low complexity elements that pose inherent problems for short-read sequencers and assemblers. We used nanopore sequencing to improve upon and resolve the issues plaguing the current zebrafish reference assembly (GRCz11). Our long-read assembly improved the current resolution of the reference genome by identifying 1,697 novel insertions and deletions over 1Kb in length and placing 106 previously unlocalized scaffolds. We also discovered additional sites of retrotransposon integration previously unreported in GRCz11 and observed their expression in adult zebrafish under physiologic conditions, implying they have active mobility in the zebrafish genome and contribute to the ever-changing genomic landscape.

Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM

The CRISPR/Cas9 system has been widely used for gene editing in zebrafish. However, the required NGG protospacer adjacent motif (PAM) of Streptococcus pyogenes Cas9 (SpCas9) notably restricts the editable range of the zebrafish genome. Recently, Cas9 from S. canis (ScCas9), which has a more relaxed 5′-NNG-3′ PAM, was reported to have activities in human cells and plants. However, the editing ability of ScCas9 has not been tested in zebrafish. Here we characterized and optimized the activity of ScCas9 in zebrafish. Delivered as a ribonucleoprotein complex, ScCas9 can induce mutations in zebrafish. Using the synthetic modified crRNA:tracrRNA duplex instead of in vitro-transcribed single guide RNA, the low activity at some loci were dramatically improved in zebrafish. As far as we know, our work is the first report on the evaluation of ScCas9 in animals. Our work optimized ScCas9 as a new nuclease for targeting relaxed NNG PAMs for zebrafish genome editing, which will further improve genome editing in zebrafish.

A New Zebrafish Model for Pseudoxanthoma Elasticum

Calcification of various tissues is a significant health issue associated with aging, cancer and autoimmune diseases. There are both environmental and genetic factors behind this phenomenon and understanding them is essential for the development of efficient therapeutic approaches. Pseudoxanthoma elasticum (PXE) is a rare genetic disease, a prototype for calcification disorders, resulting from the dysfunction of ABCC6, a transport protein found in the membranes of cells. It is identified by excess calcification in a variety of tissues (e.g., eyes, skin, arteries) and currently it has no cure, known treatments target the symptoms only. Preclinical studies of PXE have been successful in mice, proving the usefulness of animal models for the study of the disease. Here, we present a new zebrafish (Danio rerio) model for PXE. By resolving some ambiguous assemblies in the zebrafish genome, we show that there are two functional and one non-functional paralogs for ABCC6 in zebrafish (abcc6a, abcc6b.1, and abcc6b.2, respectively). We created single and double mutants for the functional paralogs and characterized their calcification defects with a combination of techniques. Zebrafish deficient in abcc6a show defects in their vertebral calcification and also display ectopic calcification foci in their soft tissues. Our results also suggest that the impairment of abcc6b.1 does not affect this biological process.

Heparan Sulfate Biosynthesis in Zebrafish

The biosynthesis of heparan sulfate (HS) proteoglycans occurs in the Golgi compartment of cells and will determine the sulfation pattern of HS chains, which in turn will have a large impact on the biological activity of the proteoglycans. Earlier studies in mice have demonstrated the importance of HS for embryonic development. In this review, the enzymes participating in zebrafish HS biosynthesis, along with a description of enzyme mutants available for functional studies, are presented. The consequences of the zebrafish genome duplication and maternal transcript contribution are briefly discussed as are the possibilities of CRISPR/Cas9 methodologies to use the zebrafish model system for studies of biosynthesis as well as proteoglycan biology.

Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in ‘mock’ control embryos

ABSTRACTZebrafish have practical features that make them a useful model for higher-throughput tests of gene function using CRISPR/Cas9 editing to create ‘knockout’ models. Due to the large number of available tools to design CRISPR assays and diversity of theories/model systems they were originally built on, we sought to systematically compare computational and empirical approaches for predicting gene-editing efficacy in zebrafish. We subjected zebrafish embryos to CRISPR/Cas9 with 50 different guide RNAs (gRNAs) targeting 14 genes and assayed individual editing efficiencies. We compared our experimental in vivo efficiencies in mosaic G0 embryos with those predicted by seven commonly used gRNA design tools and found large discrepancies between methods. Assessing off-target mutations (predicted in silico and in vitro) found that the majority of tested loci had low in vivo frequencies (<1%). Moreover, understanding that recent segmental duplications in the zebrafish genome could exacerbate CRISPR targeting of individual genes, we cataloged these loci and have made them available as a resource. Lastly, we assessed the transcriptome of negative ‘mock’ control CRISPR larvae injected with Cas9 enzyme or mRNA with no gRNA using RNA-seq and identified differentially expressed genes with high variability between injections. Using these same data, we discovered on average ~60 putative somatic mosaic frameshift mutations impacting genes per pool of injected larvae, potentially due to background cutting of DNA with Cas9 in the absence of gRNA. To verify this previously unreported phenomenon in zebrafish, we validated seven of twelve genes tested carrying low frequency mosaic somatic mutations in the genomes of a separate batch of injected larvae. These results suggest that negative control embryos may carry mutations within genes leading to spurious phenotypes. Overall, our results provide a valuable resource for the zebrafish community for the design and execution of CRISPR/Cas9 experiments.AUTHOR SUMMARYZebrafish have proven to be a powerful model organism for the functional characterization of genes. Development of new workflows targeting individual or multiple genes simultaneously require a thorough understanding of the advantages and limitations of current available methods for CRISPR-editing in zebrafish. Here, we systematically evaluated on- and off-target efficiencies prediction methods of 50 gRNAs by experimentally testing their CRISPR cutting efficiencies in embryos. Moreover, we performed a global assessment of duplicated portions of the zebrafish genome, providing a powerful resource for the design of future CRISPR assays. Lastly, we evaluated the possibility that spurious editing occurs in samples injected with the Cas9 nuclease without a gRNA, which are commonly used as a baseline control. This analysis revealed high variability in gene expression and the presence of frameshift variants in larvae injected solely with Cas9, suggesting that additional caution should be taken when using these samples as baseline controls in functional characterizations of genes.

Genetic evidence for Amh modulation of gonadotropin actions to control gonadal homeostasis and gametogenesis in zebrafish and its noncanonical signaling through Bmpr2a receptor

ABSTRACTAnti-Müllerian hormone (Amh) plays an important role in gonadal function. Amh deficiency causes severe gonadal dysgenesis and dysfunction in zebrafish, with gonadal hypertrophy in both sexes. However, its mechanism of action remains unknown. Intriguingly, the Amh cognate type II receptor (Amhr2) is missing in the zebrafish genome, in sharp contrast to other species. Using a series of zebrafish mutants (amh, fshb, fshr and lhcgr), we provided unequivocal evidence for actions of Amh, via modulation of gonadotropin signaling, on both germ cell proliferation and differentiation. The gonadal hypertrophy in amh mutants was abolished in the absence of Fshr in females or Fshr/Lhcgr in males. Furthermore, we demonstrated that knockout of bmpr2a, but not bmpr2b, phenocopied all phenotypes of the amh mutant in both sexes, including gonadal hypertrophy, hyperproliferation of germ cells, retarded gametogenesis and reduced fshb expression. In summary, the present study provided comprehensive genetic evidence for an intimate interaction of gonadotropin and Amh pathways in gonadal homeostasis and gametogenesis and for Bmpr2a as the possible missing link for Amh signaling in zebrafish.

ZFARED: A Database of the Antioxidant Response Elements in Zebrafish

Background: Antioxidant Response Elements (ARE) play a key role in the expression of Nrf2 target genes by regulating the Keap1-Nrf2-ARE pathway, which offers protection against toxic agents and oxidative stress-induced diseases. Objective: To develop a database of putative AREs for all the genes in the zebrafish genome. This database will be helpful for researchers to investigate Nrf2 regulatory mechanisms in detail. Methods: To facilitate researchers functionally characterize zebrafish AREs, we have developed a database of AREs, Zebrafish Antioxidant Response Element Database (ZFARED), for all the protein-coding genes including antioxidant and mitochondrial genes in the zebrafish genome. The front end of the database was developed using HTML, JavaScript, and CSS and tested in different browsers. The back end of the database was developed using Perl scripts and Perl-CGI and Perl- DBI modules. Results: ZFARED is the first database on the AREs in zebrafish, which facilitates fast and efficient searching of AREs. AREs were identified using the in-house developed Perl algorithms and the database was developed using HTML, JavaScript, and Perl-CGI scripts. From this database, researchers can access the AREs based on chromosome number (1 to 25 and M for mitochondria), strand (positive or negative), ARE pattern and keywords. Users can also specify the size of the upstream/promoter regions (5 to 30 kb) from transcription start site to access the AREs located in those specific regions. Conclusion: ZFARED will be useful in the investigation of the Keap1-Nrf2-ARE pathway and its gene regulation. ZFARED is freely available at http://zfared.buc.edu.in/.

Calculating the Most Likely Intron Splicing Orders in S. pombe, Fruit Fly, Zebrafish, and Humans

Background Introns have been shown to be spliced in a defined order, and this order influences both alternative splicing regulation and splicing fidelity, but previous studies have only considered neighbouring introns. The detailed intron splicing order remains unknown. Results In this work, I developed a method that can calculate the intron splicing orders of all introns in each transcript. A simulation study showed that this method can accurately calculate intron splicing orders. I further applied this method to real pombe, fruit fly, zebrafish and human sequencing datasets and found that intron splicing orders change from gene to gene and that humans contain more not in-order spliced transcripts than S. pombe, fruit fly and zebrafish. In addition, I reconfirmed that the first introns in humans are spliced slower than those in S. pombe, fruit fly and zebrafish genome-widely. Both the calculated most likely orders and the method developed here are available on the web. Conclusions I developed a novel computational method to calculate the intron splicing orders and applied the method to real sequencing datasets. I obtained intron splicing orders for hundreds or thousands of genes in four organisms. I found humans contain more number of not in-order spliced transcripts. Keywords : Splicing; Intron splicing order; Most likely order; Bayesian network