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 goal of the 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 Core comprises Thomas Sargent (Chair), Harold Burgess, Ajay Chitnis, Igor Dawid, and Brant Weinstein. The Core's activities consist of (1) oversight and support of client-specific projects, (2) maintenance and improvement of equipment and infrastructure, (3) improvement of operational procedures, and (4) service and educational outreach. Feldman's research on primary germ layer formation is also making contributions to the field of Developmental Biology.
Oversight and support of client-specific projects
In the past year, we engaged in research projects with seven labs: four from NICHD, one from NCI, one from NHLBI, and one from the Children’s National Medical Center.
Zebrafish model of the human pediatric disease Smith-Lemli-Opitz syndrome (SLOS)
SLOS is an autosomal recessive, multiple malformation syndrome with pediatric onset. It is characterized by intellectual disability and aberrant behavior. Several zebrafish lines are being investigated that carry mutant alleles of dhcr7, the zebrafish ortholog of DHCR7, the human gene. The lines were established through the Core via TALEN genome engineering two years ago. In the interim, our efforts focused on defining phenotypes associated with the mutant alleles affecting metabolism, morphology, viability, fertility, and behavior. During the past year, we conducted experiments to determine which phenotypes were modulated by environmental and metabolic interventions. This project is ongoing.
Function of zebrafish orthologs to human genes implicated in childhood gigantism
Gigantism arises as a result of excess growth hormone (GH) secretion during childhood, before the growth plates close. Two years ago, the zebrafish ortholog of a human gene implicated as a driver of gigantism was cloned and its effects (when transiently overexpressed) on zebrafish morphology and growth were investigated. We recently completed a description of this gene’s native expression in zebrafish. To understand whether chronic misexpression of this gene can drive hypertophic growth in zebrafish, we built a construct that expresses the gene in response to Gal4 and established zebrafish lines carrying the construct. We plan to cross these lines with lines that express Gal4 in the pituitary, hypothalamus, or throughout the central nervous system (CNS). We have developed novel software and zebrafish holding systems to facilitate longitudinal growth measurements. The project in ongoing.
Function of zebrafish orthologs of human genes implicated in familial adrenal hyperplasia
The following novel approach was initiated two years ago. To understand the molecular function of the human gene ARMC5, implicated as a tumor suppressor for adrenal hyperplasia, global RNA sequencing was performed on zebrafish embryos in which the zebrafish ortholog to this gene, armc5, was either downregulated by antisense morpholino oligonucleotide injection, upregulated by RNA injection, or unperturbed. The sequencing was outsourced. Identification and validation of targets and pathways affected by both up- and down-regulation are ongoing. We obtained a genetic armc5 mutant generated by the Sanger TILLING project and bred it to homozygosity. The above 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. We also performed in situ staining of zebrafish armc5 RNA and initiated protocol optimization for co-localization studies to determine whether armc5 is expressed in the inter-renal primordium. The project is ongoing.
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 disease but is still debilitating to children and young adults.
Two years ago, we began a project to clarify the structure-function relationship of ATP7A and motor neuron defects. From outside sources we obtained a zebrafish line carrying mutations in the orthologous atp7a gene and a transgenic line that expresses GFP in motor neurons and crossed them in anticipation of experiments that will search for motor neuron defects in atp7a mutants, which were recently documented. Last year, RNA expression constructs were also built in anticipation of structure-function assays that will compare the ability of synthetic RNA from wild-type and mutant alleles to rescue motor neuron defects as well as other defects in atp7a-null embryos. The project was awarded a Bench-to-Bedside grant.
Assessing human metastatic cell behaviors in a whole-body (zebrafish embryo) microenvironment
The goal of this project is twofold: to determine the trophic range of certain metastatic melanoma and breast carcinoma cell lines and to document cellular dynamics during early tumor formation from metastatic cells that have seeded into new microenvironments. Two years ago, we 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). Last year, we collected a substantial amount of trophism data. We also imported optically transparent and immuno-compromised zebrafish lines, which will permit the observation of tumor behaviors for longer periods of time. The project is ongoing.
Finding neuroprotective drugs to mitigate hyperammonemia, a consequence of urea cycle defects and liver failure
Exposure of the brain to high levels of ammonia causes neuro-cognitive deficits, intellectual disabilities, coma, and death. Two years ago, the Core developed a strategy to use zebrafish embryos to identify small molecules capable of diminishing the effects of hyperammonemia. The protocol was then substantially improved with the addition of an automated system for quantifying the spontaneous movement of experimental embryos. Last year, we screened a library of hundreds of small molecules with known safety profiles for humans and identified several promising candidates for follow-up validation studies in zebrafish and other animal models. The project is ongoing.
Other projects
During the past year, the Core assisted the Lippincott-Schwartz laboratory (NICHD) with the acquisition of transgenic fish lines that label various cells of the immune system, in preparation for their own project that will examine the intersection of regeneration and immunity. Core resources were also used by the Waterman laboratory (NHLBI) to examine how altering the level of selected cytoskeletal proteins affects cellular behaviors.
Maintenance and improvement of equipment and infrastructure
All equipment has been maintained in good order. The Core also acquired the following: a state-of-art automated whole-mount in situ hybridization machine; a new camera and microscope adaptor components to establish a new photomicroscopy station; a portable UV-illumination system and microscope blackout hood to visualize fluorescent zebrafish lines in the lab and as an educational outreach activity. A new generation of wild-type (WT) zebrafish was introduced and new generations were established for other mutant and transgenic lines frequently needed for Core projects and/or educational outreach. The Core also added several new lines of fish its collection, including transparent, colored (Glo-fish), and immuno-compromised lines.
Improvement in operational procedures
The 'do-it-yourself and pay-as-you-go' model of facilitated, supervised research is working well; additional services must be limited due to the small size of the Core's staff (Feldman only). These can include monthly meetings with Feldman, the PI, and all relevant trainees; RNA syntheses upon request; zebrafish line preservation via sperm freezing and line recovery via in vitro fertilization. This past year, Feldman adapted optimized approaches for Crispr/Cas9 mutagenesis and is providing quality-controlled reagents, protocols, and training for Crispr/Cas9 mutagenesis to users of the Core. Feldman is also now offering to generate F0 carriers of mutations upon request for a limited number of genes per year.
Service and educational outreach
Service
Feldman led the team responsible for writing the new NICHD Mission Statement, as part of the major reorganization that took place last year.
Educational Outreach
Feldman helped orchestrate 2015 'Take Your Child to Work Day' events at the Central Aquatics Facility and received a group award for his previous year's similar activity. He demonstrated principles of embryology and gene expression to children and families, using zebrafish, at the Rockville Science day in 2015. He hosted a high-school student from The School Without Walls in Washington DC, together with her mentor, for a one-week 'Environmentor' project (a National Council for Science and the Environment activity), and the project was ranked in first place. Feldman also provided consultation and zebrafish embryos on several occasions to Bill Wallace and his students from Georgetown Day School in Washington DC, enabling them to complete and prepare posters summarizing several science projects.
Primary germ layer formation
Feldman's overall research goal in independent work is to elucidate molecular and cellular events that control the formation of mesoderm and/or mesendoderm in zebrafish, with an eye towards general principles that are relevant to other species, particularly humans. A year ago, Feldman completed an extensive chapter on mesoderm formation for a textbook that was published in 2015. Other manuscripts based on earlier work at NHGRI are still in preparation. Data from one of these earlier projects was included in a paper from Harold Burgess's lab (Reference 2).
Additional Funding
- $16,400 in supplemental funding was obtained in FY15 as follows: $3,900 in fee-for-use charges from NICHD laboratories, $2,500 in fee-for-use charges from other NIH laboratories, and $10,000 as the first of three installments of a Bench-to-Bedside award.
Publications
- Feldman B. Taking the middle road: vertebrate mesoderm formation and the blastula-gastrula transition. Principles of Developmental Genetics, 2nd Edition. Ed: Moore S. Academic Press 2014; 203-236.
- Horstick EJ, Jordan DC, Bergeron SA, Tabor KM, Serpe M, Feldman B, Burgess HA. Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish. Nucleic Acids Res 2015; 43(7):e48.
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, D(med)Sci, 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 http://zcore.nichd.nih.gov.
