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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

The NICHD Zebrafish Core was established in May 2012. The goal of the Facility is to provide its clients with consultation, access to equipment and reagents, and service in the area of zebrafish genetics. To maximize the efficiency of our services, we implemented project management software. 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, Igor Dawid, and Brant Weinstein. The Core's activities consist of (1) oversight and support of client-specific projects, (2) introduction and troubleshooting of new methodologies with promising application in zebrafish and research in the field of developmental biology, (3) maintenance and improvement of equipment and infrastructure, and (4) service and educational outreach.

Oversight and support of client-specific projects

Over 2016-17, Feldman engaged in research projects with six labs: three from NICHD, one from NIEHS, one from NINR, and one from the Children's National Medical Center.

Porter Lab (NICHD): genetic dissection and creation of human disease models of sterol metabolism

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive, multiple malformation syndrome with pediatric onset characterized by intellectual disability and aberrant behavior. Phenotypic characterization is ongoing of zebrafish carrying mutant alleles of dhcr7, the zebrafish ortholog to the human SLOS gene DHCR7, which were generated with support from the Core in previous years. The Core also used Crispr-Cas9 technology to create additional genetic mutant lines for the Porter lab in genes with roles in other steps in cholesterol metabolism. Phenotypic characterization by the Porter lab is ongoing.

Stratakis Lab (NICHD): function of zebrafish orthologs to human genes implicated in disorders of the pituitary-adrenal axis

(1) Gigantism is the result of excess growth hormone (GH) secretion during childhood, before the growth plates close. Since 2012, the Core has supported this lab's investigation of the zebrafish ortholog to a human gene implicated as a driver of gigantism. Feldman is co-author on a paper (Reference 2) that included a description of this gene's developmental expression in zebrafish. This year, the Stratakis lab also began to test the effect on growth and development of zebrafish in which this gene is chronically overexpressed in tissue-specific or ubiquitous locations, using the Gal4/UAS transgene system. Last year, the Core used Crispr-Cas9 methods to generate, for the Stratakis lab, zebrafish carrying loss-of-function mutations in the above gene and three other zebrafish orthologs to genes implicated human growth anomalies. Characterization of the resulting phenotypes is ongoing. Since 2012, the Core has also supported this lab's investigation into the function of two zebrafish orthologs to human adrenal hyperplasia– and Cushing's disease–associated genes. Over previous years, the Core helped Stratakis's lab generate and acquire, respectively, zebrafish carrying loss-of-function mutation for each of these orthologs. Phenotypic characterization found notable effects on juvenile growth in the case of one gene and on early embryogenesis in the case of the other, and these efforts will continue. This year, we generated mutants for six new genes whose human orthologs are implicated in adrenal hyperplasia and/or Cushing's disease, and we plan phenotypic characterization of these mutations.

Kaler Lab (NICHD): modeling copper deficiency-associated distal motoneuropathy

The Menkes gene encodes ATP7A, a copper-binding ATPase localized to the plasma membrane and the trans-Golgi network (TGN); ATP7A is critical for proper intracellular copper distribution. Complete loss of ATP7A causes Menkes' disease, a severe human disease, leading to childhood death without early intervention with copper therapy. Two ATP7A missense mutations cause a milder syndrome than Menkes' disease, a distal motoneuropathy that is nevertheless debilitating for children and young adults. Since 2013, the Core has supported a project to clarify the structure-function relationship of ATP7A with motor neuron defects from the perspective of these missense mutations. Over previous years, the Core supported the Kaler lab's work to visualize and compare motor neuron growth during embryogenesis of wild-type zebrafish embryos and embryos homozygous for null mutations in their ATP7A ortholog atp7a, work that will continue. In parallel with these studies, Feldman and Tsai-Morris are making a concerted effort to establish genome-editing technology in the Core, using CRISPR-Cas9 technology in combination with donor DNA and by comparing two general approaches: double-stranded donor DNA for homologous recombination–based genome editing and single-stranded DNA for homology-directed, repair-based genome editing. The initial goal is to induce formation of zebrafish atp7a point mutations cognate to one of the human ATP7A motoneuropathy alleles.

Blackshear Lab (NIEHS): assessing functions of a zinc-finger protein gene family in zebrafish hematopoiesis

The Blackshear lab is interested in dissecting the connections between a family of zinc-finger proteins and blood development. They contracted the NICHD Zebrafish Core to create null mutations in each of the seven zebrafish orthologs to this family, to determine the viability or fertility of such mutants and, if viable, to provide blood samples from mutants for the Blackshear lab to analyze. This year, we created null alleles for six out of the seven genes. Phenotypic analysis has commenced, with no aberrations in homozygous null fish yet identified for three of the seven genes that have been tested.

Meilleur Lab (NINR): ability of small molecules to mitigate myopathy in zebrafish ryr1b mutants

For this new project, we helped Katy Meilleur and colleagues formulate a plan to test candidate drugs they are currently identifying for their ability to potentially ameliorate muscle defects seen in zebrafish mutants that carry mutations in the gene ryr1b; mutations in the gene's human counterpart are implicated in various myopathies. We have acquired larvae carrying the ryr1b mutation from an outside source and are currently raising them to adulthood.

Tuchman Lab (Children's National Medical Center): neuroprotective drugs to mitigate hyperammonemia, a consequence of urea cycle defects and liver failure

Exposure of the brain to high ammonia levels causes neurocognitive deficits, intellectual disabilities, coma, and death. Since 2012, the Core has helped this lab use zebrafish embryos to identify small molecules capable of diminishing the effects of hyperammonemia. Over previous 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. Last year, the Core supported the Tuchman lab to increase the throughput of this screen, bolstered by additional personnel from the Tuchman lab and the Core’s implementation of NICHDs massive embryo production systems (MEPS; see below) as a source for embryos, enabling them to complete a screen of an additional 10,000 compounds last year. Additional candidate compounds were thus identified and secondary screens and dose-response studies on lead compounds will continue.

Basic gene knockouts

The Core continues to produce new mutant lines using CRISPR/Cas9 technology. We optimized the process so successfully in 2015–16 that, last year, we were able to offer the creation of at least two novel CRISPR/Cas9 frame-shifting alleles per gene on a fee-for-service basis. Using this mechanism, we have been contracted to create knockouts for 15 distinct genes for three labs: seven for the Blackshear lab (NIEHS), six for the Stratakis lab and two for the Porter lab. We generated carriers of at least two distinct frame-shifting alleles for 10 of these and one frame-shifting allele for another. We identified gene-specific CRISPR/Cas9 components (i.e., gRNAs) that mutagenize with sufficient efficiency for four of the five remaining genes, and we used these to produce likely carriers of multiple alleles that are currently being raised. We are still performing tests to identify a suitable gRNA for the fifteenth gene.

Precise genome editing

We have also continued to troubleshoot methods for precise genome editing. Our approach, which combines co-injection of “rescue” templates along with the CRISPR/Cas9 components, is discussed above in the context of our support for the Kaler lab. Furthermore, to help ensure that our precise genome editing efforts reflect state-of-the-art practices and insights, we have established or reinforced contact and dialogue with the laboratories of Raman Sood (NHGRI Zebrafish Core), Shawn Burgess (NHGRI), David Grunwald (University of Utah), and Darius Balciunas (Temple University).

Steady source of zebrafish embryos

As part of the Central Aquatic Facility, NICHD has two large and two small mass embryo production systems (MEPS) distributed between two of our procedure rooms. Initial optimization by the Research Animal Management Branch (RAMB) and Charles River staff already had one of the two larger MEPS running on a continuous basis, with one embryo collection per week. However, NICHD research through the NICHD Zebrafish Core and through other facilities requires embryos more frequently. For small-molecule screens, at least 1500 embryos per experimental day are required. For microinjection experiments, two morning waves of at least 500 synchronously fertilized embryos are required. To achieve these goals, working together with RAMB and Charles River, we successfully adjusted husbandry, feeding and embryo collection methods and timing to enable the collection of two synchronous waves of a sufficient quantity of freshly fertilized embryos on three days per week. More recently, we reduced the workload for staff and the production burden for the fish by having only one collection day per week for each of two MEPS. Over time, we will assess yields and fecundity associated with this reduced collection schedule in 2017–2018. In addition to the EK strain we have been using, we will introduce a second wild-type zebrafish strain, TAB5, that is better suited for precise genome editing projects, so that one MEPS will house each strain.

Robust software and hardware for longitudinal growth studies

An increasingly common phenotypic characterization of genetic variants in zebrafish is the measurement of their sizes and weights and how certain genetic conditions can alter these parameters over time. i.e., change their growth. Indeed, the use of the Core’s macro-photography lens and milligram balance to measure and weigh various genetic models has risen strikingly since the Core’s inception in 2012. Of relevance to the NICHD Zebrafish Core’s projects, growth defects have been found in some of the Stratakis and Porter lab’s genetic zebrafish models. Traditional approaches to size measurement and zebrafish husbandry present three challenges that we are trying to overcome: (1) they are labor-intensive; (2) they require life-threatening immobilization of the fish; and (3) there is no practical strategy for re-identifying the individual fish for longitudinal measurements over time. In 2014, the Core contracted Viewpoint Life Sciences to write software for their behavior tracking systems designed to measure birds-eye view lengths and surface areas of free-swimming zebrafish embryos, larvae, juveniles, and adults. A promising package was installed on the three Viewpoint behavioral tracking units owned by NHGRI and shared by NICHD. Last year, the software was delivered and tested, and we found that it can recapitulate manual measurements with an overall accuracy of about 90%. We will work with Viewpoint to reformulate the software for greater accuracy. Last year, we also worked with R&D Aquatics to develop a system for rearing individual zebrafish for the entirety of their approximately two months of larval and juvenile growth. We confirmed 100% survival for these first two months of life in an initial trial of wild-type fish. We plan to combine this hardware and software to enable the generation and comparison of growth curves for individual wild-type and mutant zebrafish using a minimum of investigator effort.

Additional Funding

• NICHD Customers: $14,250 in fee-for-use charges
• Non-NICHD Customers: $17,280 in fee-for-use charges

Publications

1. Gore A, Athans B, Iben J, Johnson K, Russanova V, Castranova D, Pham V, Butler M, Williams-Simons L, Nichols J, Bresciani E, Feldman B, Kimmel C, Liu P, Weinstein B. Epigenetic regulation of hematopoiesis by DNA methylation. eLife 2016;5:e11813.
2. Trivellin G, Bjelobaba I, Daly AF, Larco DO, Palmeira L, Faucz FR, Thiry A, Leal LF, Rostomyan L, Quezado M, Schernthaner-Reiter MH, Janjic MM, Villa C, Wu TJ, Stojilkovic SS, Beckers A, Feldman B, Stratakis CA. Characterization of GPR101 transcript structure and expression patterns. J Mol Endocrinol 2016;57:97-111.

Collaborators

• Perry Blackshear, PhD, Signal Transduction Laboratory, NIEHS, Research Triangle Park, NC
• Harold Burgess, PhD, Section on Behavioral Neurogenetics, NICHD, Bethesda, MD
• Stephen Kaler, MD, Section on Translational Neuroscience, 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
• Mendel Tuchman, MD, Children's National Medical Center, Washington, DC
• 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.

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