The NICHD Zebrafish Core
- Benjamin Feldman, PhD, Staff Scientist and Director of the NICHD Zebrafish Core
- ChonHwa Tsai-Morris, PhD, Staff Scientist and Assistant Director of the NICHD Zebrafish Core
- Yvonne Rosario, PhD, Postdoctoral Intramural Research Training Award Fellow
- Emily N. Katz, BS, Postbaccalaureate Intramural Research Training Award Fellow
The NICHD Zebrafish Core was established in May 2012 with the goal of providing its clients with consultation, access to equipment and reagents, and service in the area of zebrafish genetics. NICHD investigators as well as investigators from other NIH institutes and from outside the NIH are its clientele. The oversight committee for the Core comprises Harold Burgess, Ajay Chitnis, Brant Weinstein, and Katie Drerup. The Core's activities consist of (1) oversight and support of client-specific projects, (2) custom generation of genetic zebrafish models, (3) troubleshooting of new methodologies with promising application in zebrafish, (4) maintenance and improvement of equipment and infrastructure, and (5) service and educational outreach.
Oversight and support of client-specific projects
Over 2018–2019, the Core engaged in research projects with fourteen labs and two cores.
Porter Lab, NICHD: Genetic dissection and creation of human disease models of sterol metabolism
In previous years, the Core used CRISPR-Cas9 technology to create genetic mutant zebrafish lines for the Porter lab in four genes: npc1, npc2, cln3, and ebp, which have roles in various steps of sterol metabolism and lipid storage. In the previous year, we characterized and published some phenotypes of npc1 mutants and, this year, we investigated other npc1 phenotypes as well as phenotypes of cln3 and ebp mutants. This year, the Core also used CRISPR-Cas9 technology to regenerate mutant alleles of dhcr7 and cln3 in the TAB5 strain of zebrafish, with the goal of reducing the phenotypic variability seen in the more outbred EK strain of zebrafish that was originally used.
Stratakis Lab, NICHD: Function of zebrafish orthologs to human genes implicated in disorders of the pituitary-adrenal axis
In previous years, the Core used CRISPR-Cas9 technology to generate zebrafish carrying loss-of-function mutations in four zebrafish orthologs to human genes implicated by the Stratakis lab in human growth anomalies and eight zebrafish orthologs to human adrenal hyperplasia and Cushing's disease–associated genes. Over the last year, we made considerable progress in the characterization of one of these genes, with new findings of temperature-sensitive maternal effects on viability and central nervous system anatomy.
Blackshear Lab, NIEHS: Functions of a zinc-finger protein gene family in zebrafish
The Core has been engaged over the past several years to use CRISPR-Cas9 technology to generate zebrafish carrying loss-of-function mutations in seven zebrafish orthologs and assist with preliminary phenotype characterization. We observed no abnormal phenotypes for thirteen out of thirteen mutant alleles for these seven genes. This year, the Core sent tissue samples to the Blackshear lab to compare the transcriptomes of mutants with those of control siblings to determine whether subtle molecular consequences of these gene disruptions can illuminate their function and whether unexpected functional splice forms and/or genetic compensation might be responsible for the lack of abnormal phenotypes. Eleven of the thirteen alleles were cryopreserved, and cryopreservation of the remaining three alleles is scheduled in the upcoming months.
Larson Lab, NCI: Transgenic zebrafish carrying mutant and WT Runx1-mKate protein fusions
This year, the Larson lab engaged the Core to generate transgenic zebrafish carrying mutant and wild-type Runx1-mKate protein fusions. Optimal microinjection conditions for three constructs were determined, and a screen for germ-line transmitters of each is ongoing.
Lawal Lab, NINR: Function of ryr1b mutants
Tokunbor Lawal spent the first part of the year continuing previous efforts to optimize an assay to test candidate drugs for their potential to ameliorate muscle defects seen in zebrafish mutants that carry mutations in ryr1b, a gene whose human counterpart is implicated in various myopathies. Owing to an insufficient magnitude of the assay read-out as designed, we are now troubleshooting an alternative assay. We are also launching a new project to test the functionality of ryr1b alleles using a common genetic platform.
Caldovic Lab, Children's National Medical Center: Neuroprotective drugs to mitigate hyperammonemia
Exposure of the brain to high ammonia, a consequence of urea cycle defects and liver failure, causes neurocognitive deficits, intellectual disabilities, coma, and death. Since 2012, the Core has helped this lab use zebrafish embryos to identify small molecules that can diminish the effects of hyperammonemia. In the first few years, a library of hundreds of small molecules with known safety profiles for humans was screened, and several promising candidates were identified for follow-up validation studies in zebrafish and other animal models. A manuscript summarizing this work is currently being drafted. Over the past three years, the Core has supported a reimplementation of this screen, using a larger library of 10,000 compounds, bolstered by additional personnel from the Caldovic lab. The screen was completed during the last year, and the team is writing up the data and conducting validation assays.
Swartz Lab (NINDS)
This year the Swartz lab engaged the Core to provide consultation and access to zebrafish embryos, equipment, and reagents, enabling them to determine that zebrafish tmem266 is expressed in the cerebellum. Follow-on studies are currently being planned.
Olivier Lab (NHBLI)
The Core began providing embryos to the Olivier lab this year, enabling them to conduct experiments on infection in their own laboratory and on their own animal protocol.
Kemper (NHLBI) and Afzalii (NIDDK) Labs
These labs are interested in the human immune system and the role of zebrafish rca2.1, a CD-46 ortholog, in development and health. Our initial goal was to generate rca2.1 null zebrafish using CRISPR/Cas9. Based on concerns that novel translational start sites and/or decay of otherwise nonfunctional RNAs can elicit genetic compensation, we sought to disrupt all rca2.1 transcription. To identify transcriptional start sites (TSSs) and determine which predicted alternate transcripts are expressed during early development, we identified public databases of zebrafish RNA sequences and locations of their 5′ termini and, with help from the NICHD Computer Support Services Core (Dale), integrated them into a searchable private track on the UCSC Genome Browser. We used the technique track to select gRNA (guide RNA) combinations aimed at cooperatively deleting the TSS target and downstream spans of rca2.1. Fragment analysis of coinjected embryos indicated the presence of diagnostic peaks arising from the desired 20 Kb deletion in close to 20% of F0 embryos. Germline transmission from seven out of eight F0 adults was subsequently achieved. Thus, using several gRNAs, we efficiently recovered large genomic rca2.1 deletion alleles. Unlike classic CRISPR/Cas9 insertion-deletion (in-del) alleles, interpretation of phenotypes observed should be uncomplicated by concerns of extant or novel alternative transcripts or genetic compensation linked to nonsense-mediated decay.
Additional Funding
- Director’s Award for year 2 of 2
Publications
- Tseng WC, Loeb HE, Pei W, Tsai-Morris CH, Xu L, Cluzeau CV, Wassif CA, Feldman B, Burgess SM, Pavan WJ, Porter FD. Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for screening of candidate therapeutic compounds. Dis Model Mech 2018;11(9):034165.
Collaborators
- Behdad Afzali, MD, PhD, Kidney Diseases Branch, NIDDK, Bethesda, MD
- Perry Blackshear, PhD, Signal Transduction Laboratory, NIEHS, Research Triangle Park, NC
- Ljubica Caldovic, PhD, Children's National Medical Center, Washington, DC
- Steven Coon, PhD, Molecular Genomics Core, NICHD, Bethesda, MD
- Ryan Dale, MS, PhD, Bioinformatics and Scientific Programming Core; Computer Support Services Core, NICHD, Bethesda, MD
- Andy Golden, PhD, Laboratory of Biochemistry and Genetics, NIDDK, Bethesda, MD
- Stephen Kaler, MD, Section on Translational Neuroscience, NICHD, Bethesda, MD
- Claudia Kemper, PhD, Laboratory for Complement and Inflammation Research, NHLBI, Bethesda, MD
- Dan Larson, PhD, Center for Cancer Research, NCI, Bethesda, MD
- Tokunbor Lawal, PhD, Neuromuscular Symptoms Unit, NINR, Bethesda, MD
- Joan Marini, MD, PhD, Section on Heritable Disorders of Bone and Extracellular Matrix, NICHD, Bethesda, MD
- Katy Meilleur, PhD, Neuromuscular Symptoms Unit, NINR, Bethesda, MD
- Forbes D. Porter, MD, PhD, Section on Molecular Dysmorphology, NICHD, Bethesda, MD
- Constantine Stratakis, MD, D(med)Sci, Section on Endocrinology and Genetics, NICHD, Bethesda, MD
- Kenton Swartz, PhD, Molecular Physiology and Biophysics Section, NINDS, Bethesda, MD
- Brant Weinstein, PhD, Section on Vertebrate Organogenesis, NICHD, Bethesda, MD
Contact
For more information, email bfeldman@mail.nih.gov or visit http://zcore.nichd.nih.gov.
