Zebrafish core

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

Project summaries and research accomplishments for the zebrafish facility in 2020/2021 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 FY2021 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 is currently under review at EMBO Reports (Zhao et al., 2021). 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 has made progress towards developing xenograph assays in zebrafish using fluorescently-labeled mammalian tumor cells. The goal of this project will be to investigate cilia requirements in tumorigenesis and metastasis. 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 is currently under review at J. Biol. Chem. (Stromberg 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 will be included in a manuscript currently in preparation. 3. To support a future collaboration with Dr. Marielle Yohe (Pediatric Oncology and the NCI-RASopathy Initiative, NCI-CCR), the facility is carrying 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 develop assays for the characterization of 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. 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. The ultimate 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. 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 crossed with two transgenic reporter lines (Cd41:GFP and Myb:GFP) that will facilitate the tracking of hematopoietic stem cells for fluorescence microscopy and 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 xenograph 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 circulating tumor cells (CTC) will also be studied using a modified xenograph approach to observe cluster formation and/or their invasiveness/intravasation. Single cell suspensions or pre-formed clusters of GFP-tagged CTCs will be injected into the pericardium of embryos, following which drug treatments will be applied and the formation of stable clusters quantified. In addition, whether cells leave the circulatory system and extravasate into surrounding tissues will also be evaluated using a transgenic line marking the vascular system, which was recently acquired.