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The NICHD Zebrafish Core; Primary Germ Layer Formation

Benjamin Feldman, PhD, Staff Scientist and Director of the NICHD Zebrafish Core

The NICHD Zebrafish Core was established in May 2012. The research on primary germ layer formation is also making contributions to the field of Developmental Biology. The goal of the NICHD Zebrafish Core is to provide 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 NICHD Zebrafish Core comprises Thomas Sargent (Chair), Harold Burgess, Ajay Chitnis, Igor Dawid, and Brant Weinstein.

The Zebrafish Core

The Core's activities consist of (i) oversight and support of client-specific projects, (ii) maintenance and improvement of equipment and infrastructure, (iii) improvement of operational procedures, and (iv) educational outreach. During 2013–14, Feldman engaged in research projects with five labs: three from NICHD, one from NCI, and one from the Children’s National Medical Center.

Zebrafish model of Smith-Lemli-Opitz syndrome (SLOS)

SLOS is an autosomal recessive, multiple malformation syndrome with pediatric onset characterized by intellectual disability and aberrant behavior. In collaboration with Forbes Porter, several zebrafish lines are being investigated that carry mutant alleles of dhcr7, the zebrafish ortholog to human gene DHCR7. The lines were established in the NICHD Zebrafish Core via TALEN genome engineering during 2012–13. 2013–14 was dedicated to the search and identification of phenotypes associated with the mutant alleles affecting metabolism, morphology, viability, fertility, and behavior. The project will continue in 2014–15.

Function of zebrafish orthologs to human genes implicated in childhood gigantism

Gigantism arises due to excess growth hormone (GH) secretion during childhood, before the growth plates close. In collaboration with Constantine Stratakis, the zebrafish ortholog to a human gene implicated as a driver of gigantism when overexpressed in infants was cloned during 2012–13 and its effects (when transiently overexpressed) on morphology and growth were investigated. In 2013–14, constructs of various genes for whole-mount RNA stains marking the zebrafish pituitary and hypothalamus were assembled and probes synthesized in preparation for co-localization studies to determine where the zebrafish ortholog is expressed and how overexpression of the gene affects pituitary morphology. In anticipation of chronic longitudinal overexpression studies, the following tools were assembled: a Gal4 line that drives expression of upstream activating sequence (UAS)–bearing constructs in the pituitary; a Gal4 line that drives pan-neuronal expression; and a pan-neuronal GFP–expressing line. UAS–based transgenic constructs were built, and an alternative (cre-lox–based) transgenic design was planned and vector precursor backbones were assembled. Plans and purchases were also made for specialized holding tanks and software to facilitate longitudinal growth measurements. This project will continue into 2014–15.

Function of zebrafish orthologs to human genes implicated in familial adrenal hyperplasia

In collaboration with Constantine Stratakis, the following novel approach was initiated in 2012–13 to understand the molecular function of a particular human gene, ARMC5, which is implicated as a tumor suppressor for adrenal hyperplasia. Global RNA sequencing was performed on zebrafish embryos in which the zebrafish ortholog to the gene, armc5, was (i) downregulated by antisense morpholino oligonucleotide injection, (ii) upregulated by RNA injection, or (iii) unperturbed. The sequencing was outsourced, the data received in 2013–14, and initial analyses were performed. Identification and validation of targets and pathways affected by both up- and downregulation will continue in 2014–15. A genetic armc5 mutant generated by the Sanger TILLING project was obtained. The studies will include validation in these mutants.

RNA expression constructs were obtained and antisense probes generated for in situ RNA stains of genes whose expression marks the zebrafish inter-renal primordium, a tissue that is the functional equivalent to the human adrenal gland. In situ staining of zebrafish armc5 RNA was also performed and protocol optimization was initiated for co-localization studies to determine whether armc5 is expressed in the inter-renal primordium. The project will continue into 2014–15.

Modeling copper deficiency–associated distal motoneuropathy

The Menkes gene on Xq13.3 encodes ATP7A, a P‐type cation-transporting ATPase localized to the plasma membrane and the trans‐Golgi network (TGN) and critical for proper intracellular copper distribution. Two ATP7A missense mutations, T994I and P1386S cause a milder syndrome than Menkes that is still debilitating to children and young adults.

In 2013–14, a project was initiated in collaboration with Stephen Kaler to clarify the structure-function relationship of ATP7A and motor neuron defects. In anticipation of experiments that will look for motor neuron defects in atp7a mutants, a zebrafish line carrying mutations in the orthologous atp7a gene and a transgenic line that expresses GFP in motor neurons were obtained from outside sources and crossed. RNA expression constructs were also designed and molecular cloning initiated for future structure-function assays that will compare the ability of synthetic RNA from WT and mutant alleles to rescue putative motor neuron defects as well as other defects in atp7a null embryos. The project will continue into 2014–15.

Human metastatic cell behaviors in a whole-body (zebrafish embryo) microenvironment

The dual goals of this project, conducted with Kandice Tanner, is to (i) determine the trophic range of certain metastatic melanoma and breast carcinoma cell lines and (ii) document cellular dynamics during early tumor formation from metastatic cells that have seeded into new microenvironments. Initiated in 2013–14, we have thus far optimized delivery of cells into the anterior CNS of embryos (for tumor-formation studies) and the delivery of cells into the circulatory system (for trophism studies). We also optimized several central aspects of imaging on confocal microscopes, including multi-day time-lapse studies. The project will continue into 2014–15.

Finding neuroprotective drugs to mitigate hyperammonemia

Exposure of brain to high ammonia causes neuro‐cognitive deficits, intellectual disabilities, coma and death. In 2012–13, the Core, in collaboration with Ljuba Caldovic and Mendel Tuchman, developed a strategy to use zebrafish embryos to identify small molecules capable of diminishing the effects of hyperammonemia. The protocol was substantially improved in 2013–14 with the addition of an automated system for quantifying the spontaneous movement of experimental embryos. A library of hundreds of small molecules with known safety profiles for humans was screened this year and several promising candidates identified for follow-up validation studies in zebrafish and other animal models. The project will continue into 2014–15.

Other projects

The NICHD zebrafish core assisted the Manzini lab from George Washington University in 2012–14 by in conducting a few transient gene disruption experiments while their own zebrafish facility was being constructed. The project is no longer active. Projects for 2014-15 are being planned with one lab from NHLBI and one lab from NICHD.

Maintenance and improvement of equipment and infrastructure

All equipment has been maintained in good order. The core acquired two new microscopes, free of charge, via property transfers. A large-capacity new 28° incubator with day/night light control was purchased and added to the core. A multi-component system for imaging and quantifying zebrafish behaviors was acquired, with the main components purchased by NHGRI and auxiliary components purchased by NICHD. Equipment has been placed in room 6/B241A and room 6/B140. This equipment has greatly facilitated the SLOS and hyperammonemia projects, and additional software and auxiliary equipment was recently purchased to facilitate the gigantism project. In accordance with the NIH-wide “clean sweep” for hazardous agents, a careful inventory was made of all the NICHD core space in rooms 6/B140, 6/B134 and 6/B139, including all boxes in the freezer and refrigerator. Four new computers were purchased to replace four older ones that were no longer reliable for image acquisition and bioinformatics. A smaller camera and lighter microscope base were acquired to facilitate microscope transportation for educational outreach activities. A new generation of wild-type (WT) zebrafish was introduced into room 6/B134 and new generations were established for other mutant and transgenic lines frequently needed for core projects and/or educational outreach.

Improvement of operational procedures

The Core's "do-it-yourself and pay-as-you-go" model of facilitated, supervised research is working well; additional services must be limited owing to the small size of the NICHD Zebrafish Core staff (Feldman only). However, two additional services were added in 2013–14. First, for laboratories with long-term projects, monthly meetings with Feldman and all relevant trainees were established. Second, because in vitro synthesis of RNA is challenging and has proven to be a bottleneck, Feldman is now offering to perform any requested RNA syntheses himself, and he has added Bioanalyzer-based quality assessments to the standard flow of projects involving synthetic RNA. Additional improvements include the implementation of web-based calendars for scheduling equipment, space and use of WT breeders in rooms B140, B134 and B241A, and the addition of sperm freezing, in vitro fertilization, whole-mount in situ antibody staining, and microangiography injections to the suite of procedures Feldman is able to teach and assist with.

Educational outreach

Feldman helped orchestrate and presented "Take Your Child to Work Day" events at the Central Aquatics Facility and organized a display on "RNA Expression Domains During Development" at the 3^rd USA Science & Engineering Festival in Washington DC. He also provided embryos and consultation to biology teachers at Woodrow Wilson High School, Washington, DC, and additional consultation on the use of zebrafish for K-12 education to a STEM content specialist from the Montgomery County Public School System and to a biology teacher from Georgetown Day School in Washington, DC.

Primary germ layer formation

Feldman’s overall research goal in his independent work is to elucidate molecular and cellular events that control the formation of mesoderm and/or mesendoderm in zebrafish, with a view towards general principles that are relevant to other species, particularly humans. He completed an extensive chapter on mesoderm formation for a new edition of a textbook that is currently in press. Feldman presented some of this chapter's content as a poster at the Mid-Atlantic Society for Developmental Biology Meeting at The Johns Hopkins University in May, 2014. Other manuscripts based on earlier work at NHGRI are still in preparation. Data from one of these earlier projects was included in a paper that is currently under revision.

Additional Funding

As per the core's fee-for-use system, supplemental funding from other NIH sources was obtained as follows: $2250 from NICHD laboratories and $2500 form other NIH laboratories.

Publications

Feldman B, Tuchman M, Caldovic L. A zebrafish model of hyperammonemia. Mol Genet Metab 2014;113:142-147.
Feldman B. Taking the middle road: vertebrate mesoderm formation and the blastula-gastrula transition. Principles of Developmental Genetics, 2nd Edition 2014;204-227.

Collaborators

Harold Burgess, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Ljubica Caldovic, PhD, Center for Genetic Medicine Research, Children's National Medical Center, Washington, DC
Stephen Kaler, MD, Molecular Medicine Program, NICHD, Bethesda, MD
Chiara Manzini, PhD, George Washington University, Washington, DC
Forbes D. Porter, MD, PhD, Program on Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
Thomas Sargent, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Constantine Stratakis, MD, DMedSci, Program on Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
Kandice Tanner, PhD, Laboratory of Cell Biology, NCI, Bethesda, MD
Mendel Tuchman, MD, Children's National Medical Center, Washington, DC

Contact

For more information, email bfeldman@mail.nih.gov or visit zcore.nichd.nih.gov.

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