Zebrafish core

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Project summaries and research accomplishments for the zebrafish Core in 2021/2022 are listed below: 1. The facility is collaborating with Dr. Christopher Westlake's group (LCDS, NCI-CCR) on several projects utilizing the zebrafish model system to study primary cilia formation and ciliary signaling. These studies involve the use of morpholino, CRISPR, and protein overexpression approaches combined with the biochemical analysis of protein expression and imaging of developmental structures by immunofluorescent staining and confocal or transmission electron microscopy. Using these approaches, we completed a project in FY2022 that was initiated by Dr. Huiji Zhao in Dr. Westlake's group. A gene product that was identified by Dr. Zhao in a cellular screen for regulators of mammalian multiciliogenesis was characterized, and its functional requirements compared to that of homologues in human, mouse, zebrafish and frog cells, the latter of which was conducted in collaboration with Dr. Ira Daar's group (Cancer and Developmental Biology Laboratory, NCI-CCR). This work was completed and published at EMBO Reports (Zhao H., Sun J., et al., 2022). In a second project being conducted with Dr. Saurabh Shakya, we are studying the effects of novel mutations in a signaling protein known to play an important role in ciliogenesis. Dr. Shakya will characterize the effect and/or function of the mutant proteins in fibroblasts derived from human patients, while we will compare their function in the zebrafish model system. Using a combination of morpholino and overexpression/rescue approaches, we will determine whether embryos expressing the mutant proteins present any cilia formation defects and associated ciliopathy phenotypes. This work will be included in a manuscript that is currently under preparation. In a third project, the facility is collaborating with Dr. Lu to study the function of a group of proteins involved in membrane fusion and determine whether they function in ciliogenesis. Finally, in conjunction with Dr. Westlake, the facility collaborated with two groups at Ohio State University (OSU). Working with Dr. Monica Venere's lab, we investigated the function of a kinesin motor protein associated with familial diseases that exhibit overlapping clinical features with ciliopathies. Using a CRISPR/Cas9 approach developed by the OSU Zebrafish Facility, embryos lacking the kinesin gene were generated, following which the embryos were assessed for potential cell cycle aberrations and for ciliary defects that phenocopied the characteristics of the human disease. This work will be included in a manuscript that is currently in preparation. In collaboration with Dr. Matthew Summers at OSU, the facility used a morpholino approach to study the apoptotic function of USP37, a deubiquitinating enzyme that controls responses to replication stress. This work was completed and published at J. Biol. Chem. (Stromberg BR., et al., 2021). 2. In collaboration with Dr. Russell Smith in Dr. Deborah Morrison's group (LCDS and the NCI-RASopathy Initiative, NCI-CCR), we explored the function and localization of novel B-Raf mutants associated with the cardiofaciocutaneous syndrome (CFC), one of a group of developmental disorders known collectively as the RASopathies. In this study, we determined whether early cell migrations and phenotypical defects induced by overexpression of the B-Raf mutants in zebrafish correlated with the corresponding disease presentation in humans and whether the observed phenotypes were specific to the causative mutation. This work is currently under review at Molecular Cell. 3. To support a future collaboration with Dr. Marielle Yohe (Pediatric Oncology and the NCI-RASopathy Initiative, NCI-CCR), the facility has developed assays using several fluorescent reporter transgenic lines (Tg) to assist in the analysis of RASopathy-associated mutant proteins. In particular, Tg(Col2a1:GFP) and Tg (Islet1:GFP) are used to characterize abnormalities in cartilage/skeletal and cranial motor neuron organization during development, and Tg(Dusp6:GFP) will be used as a reporter line for RAS/MAPK signaling in studies evaluating the effects of specific drug treatments. Work with the Tg(Col2a1:GFP) line was used in the manuscript from Dr. Morrison's lab currently under review. Collaborative studies between the Morrison and Yohe groups and the NCI-RASopathy Initiative will also take advantage of the zebrafish convergent-extension cell movements assay to analyze any previously uncharacterized RASopathy mutants identified through the NCI-RASopathies Initiative. This assay will be an important tool to obtain critical information regarding the severity of the mutation and the effectiveness of various drug treatments. Additionally, a novel assay to identify the presence of hypertrophic cardiomyopathy (HCM) in mutant zebrafish embryos is currently under development. Two transgenic lines, Tg(Myl7:GFP) and Tg(cmlc2:GFP) were recently acquired to characterize heart looping defects. The goal of this collaboration will be to use the zebrafish system to accelerate the development of patient-specific therapeutic approaches. 4. In collaboration with Dr. Orri Gudmundsson in Dr. Jonathan Keller (Mouse Cancer Genetics Program, NCI-CCR), the facility is continuing a project started in FY2020 aimed at analyzing the functional requirement of the zinc finger protein Pogz in zebrafish hematopoietic stem cells (HSCs). Embryos lacking pogz were generated using CRISPR/Cas9 and validation of the knockout was confirmed. Mutant embryos will be analyzed at different developmental stages by in situ hybridization in order to monitor the expression of genes specific for hematopoietic cells and to confirm the reduction or loss of specific blood lineages in the knockouts. In addition, the pogz knockout strain will be subject to FACS analysis. If defects are detected in HSCs and/or hematopoietic lineages, we will determine downstream gene targets that may be critical for Pogz function by conventional or single cell RNA-sequencing. 5. In collaboration with Dr. Esta Sterneck (LCDS, NCI-CCR), the facility is working on the development of a xenograft assay to study the metastatic behavior of human tumor cells in zebrafish, when embryos are treated with drugs that target various cancer-associated signaling pathways. The goal of this research is to identify classes of drugs that will enhance or reduce tumor cell survival. In the future, the behavior of tumor cells will also be studied using a modified xenograft approach to observe whether cells leave the circulatory system and extravasate into surrounding tissues using a transgenic line marking the vascular system.