The NICHD Zebrafish Core
- Benjamin Feldman, PhD, Staff Scientist, Director of the NICHD Zebrafish Core
- Felicia Benoit, BS, Postbaccalaureate Intramural Research Training Award Fellow
The NICHD Zebrafish Core was established in 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, and Brant Weinstein. The Core's activities consist of:
- oversight and support of client-specific projects,
- custom generation of genetic zebrafish models,
- troubleshooting of new methodologies with promising application in zebrafish,
- maintenance and improvement of equipment and infrastructure, and
- service and educational outreach.
Oversight and support of client-specific projects
Over 2021–2022, the Core engaged in research projects with ten labs and other customers.
Translation of distinct RNA populations by elongation initiation factors Eif1 and Eif5 (Dever Lab, NICHD)
Feldman advised Dever and performed several microinjection experiments to explore differences in zebrafish embryo phenotypes resulting from RNA–based over-expression or antisense morpholino-based reduction of expression of either Eif1 or Eif5. Experiments are continuing with a focus on testing the hypothesis that restoration of a balanced Eif1/Eif5 ratio can ameliorate phenotypes resulting from targeting just one of the two.
Functional assessment of tubulin isoforms (Sackett Lab, NICHD)
The degree to which specific tubulin isotypes and/or their post-translational modification are essential for specific aspects of development in any organism remains a surprisingly open question. Feldman assisted Sackett in finding and recruiting two postbaccalaureate students with undergraduate training in reputable zebrafish labs. Since the arrival of these students in the summer of 2022, Feldman has been training and mentoring them through an ambitious project to systematically knock-out each zebrafish alpha and beta tubulin isotype in F0 embryos and determine how their absence affects early development.
Genetic dissection and creation of human disease models of sterol metabolism (Porter Lab, NICHD)
In previous years, the Core used CRISPR-Cas9 technology to create genetic mutant zebrafish lines for the Porter lab in five genes: dhcr7, npc1, npc2, cln3, and ebp, which play roles in various steps of cholesterol metabolism. Feldman is in the process of cryopreserving these lines for future use.
Function of zebrafish orthologs to human genes implicated in disorders of the pituitary-adrenal axis (Stratakis Lab, Ex-NICHD)
In previous years, the Core used CRISPR-Cas9 technology to generate zebrafish carrying loss-of-function mutations in four zebrafish orthologs of human genes, implicated by the Stratakis lab in human growth anomalies, and eight zebrafish orthologs of human adrenal hyperplasia and Cushing's disease–associated genes. Feldman is in the process of cryopreserving these lines for future use.
Function of zebrafish rca2.1 (Kemper lab, NHLBI)
The Kemper lab is interested in zebrafish rca2.1s function, because it has certain similarities to human CD46 that are not found in the mouse genome. The Core previously generated two mutant rca2.1 alleles, revealing essential roles in growth and cardiac function. Phenotypic characterization is ongoing.
Function of zebrafish cacna1c, whose human orthologue is affected in Timothy Syndrome (Golden lab, NIDDK)
The Core previously generated a targeted AA-alteration in the cacna1c gene. Feldman found that recessive mutants have profound developmental anomalies, including heart edema and reduction in red blood cells. Characterization of this phenotype by trainees from the Golden lab is ongoing.
Role of minerals in bone health (LaVerne Brown, Office of Dietary Supplements)
The NICHD Zebrafish Core is working with LaVerne Brown in planning and implementing a nutritional study to explore how certain minerals influence bone health in the presence or absence of adequate vitamin D, a topic of relevance to human bone health. This year, the study parameters were refined, including acquisition of a forced-swimming exercise tunnel, to be used to stimulate bone remodeling.
Independent research by the NICHD Zebrafish Core
Strategies for CRISPR-Cas9–based homology-directed repair (HDR)
Over the past several years, Feldman and NICHD Zebrafish Core Staff explored several approaches to generating zebrafish lines with amino-acid substitutions cognate to human disease alleles of interest and generated three such alleles in house: atp7a, cacna1c and satb1. This past year, Feldman worked to devise a less labor-intensive pipeline that will feature outsourcing of CRISPR–based design and reagent steps to In Vivo Biosciences, followed by microinjection and allele recovery in-house via high-throughput sequencing of extruded gametes from candidate carriers.
Cryopreservation and in vitro fertilization of zebrafish sperm
Over the last year, Feldman, assisted by Felicia Benoit, has continued to focus on improving quality control measures to ensure viability of cryopreserved zebrafish lines and minimize variability in viability. This year, they developed an approach of pre-assessing the number and activity of sperm from individual males and only cryopreserving when yields exceeding two million active sperm are obtained.
Service
ACUC Membership
Feldman has served on the NICHD ACUC since 2015 and continued in this capacity this year, meeting monthly to evaluate and decide upon animal-study proposals, renewals and amendments, and ad hoc issues relevant to animal welfare.
Faculty Reports
As a broader contribution to the scientific community, Feldman regularly pens recommendations for scientific articles of interest as a member of Faculty Opinions in the Developmental Biology/Pattern Formation Section. This year, he contributed two recommendations.
Additional Funding
- One-year salary support for Postbaccalaureate Fellow Felicia Benoit from the Office of Intramural Training & Education, NIH
Publications
- Tseng WC, Johnson-Escauriza AJ, Tsai-Morris CH, Feldman B, Dale RK, Wassif CA, Porter FD. The role of Niemann-Pick type C2 in zebrafish embryonic development. Development 2021 148(7):dev194258.
- Trivellin G, Tirosh A, Hernández-Ramírez LC, Gupta T, Tsai-Morris C-H, Faucz FR, Burgess HA, Feldman B, Stratakis CA. The X-linked acrogigantism-associated gene gpr101 is a maternal regulator of early embryonic development and somatic growth in zebrafish. Mol Cell Endocrinol 2021 520:111091.
Collaborators
- LaVerne L. Brown, PhD, Office of Dietary Supplements, Office of the Director, NIH, Rockville, MD
- Harold Burgess, PhD, Section on Behavioral Neurogenetics, NICHD, Bethesda, MD
- Thomas Dever, PhD, Section on Protein Biosynthesis, NICHD, Bethesda, MD
- Andy Golden, PhD, Laboratory of Biochemistry and Genetics, NIDDK, Bethesda, MD
- Claudia Kemper, PhD, Laboratory for Complement and Inflammation Research, NHLBI, Bethesda, MD
- Tokunbor Lawal, PhD, Tissue Injury Branch, NINR, Bethesda, MD
- Kenneth Olivier, MPH, MD, Laboratory of Chronic Airway Infection, NHLBI, Bethesda, MD
- Forbes D. Porter, MD, PhD, Section on Molecular Dysmorphology, NICHD, Bethesda, MD
- Daniel Sackett, PhD, Division of Basic and Translational Biophysics, NICHD, Bethesda, MD
- Constantine Stratakis, MD, D(med)Sci, Section on Endocrinology and Genetics, NICHD, Bethesda, MD
Contact
For more information, email bfeldman@mail.nih.gov or visit https://zcore.nichd.nih.gov.
