Zebrafish core

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Project summaries and research accomplishments for the zebrafish Core in 2022/2023 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, transgenic lines 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. Dr Westlake's group is investigating patient mutations associated with regulation of Rab11 ciliogenesis function. We are using a combination of morpholino and overexpression/rescue approaches in zebrafish to perform functional studies of the human variants and determine whether we could model ciliopathy-related disease. A revised manuscript is being prepared for resubmission to Nature Communication that included several additional zebrafish experiments including the development of a CRIPSR line requested by the reviewers. In a second 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. In a third project, the facility worked with Dr. Wang and in collaboration with Dr.Daar's group to identify ciliogenesis defects associated with Rab protein functioning in multiciliogenesis. This work was interrupted due to the departure of Dr. Wang but may resume in the future. In a third project with Dr. Saha, the facility is developing tools to perform 2-3 color live imaging of membrane tubules using expression of fluorescently labeled proteins in developing zebrafish. Both transient and transgenic (Tg) expression of Rab proteins, known to be involved in membrane tubule trafficking and ciliogenesis, are monitored overtime using the spinning disk confocal from Dr. Westlake. These novel tools would allow to provide the first evidence of a rab-dependent trafficking cascade important for membrane tubule formation in vivo. This work, if successful, will be combined with Dr- Saha's work in human cells and included in a manuscript that is currently in preparation. Furthermore, the development of transgenic lines and other fluorescent markers amenable for the zebrafish system will be used in the future to perform multicolor live ciliogenesis assays. Finally, in collaboration with Dr. Kristen Johnson at UNH Manchester, the facility is studying the expression pattern of a transcription factor important for multiciliated cells formation in developing embryos. Overexpression of mutant proteins as well as 2 colors in situ hybridization chain reaction techniques will be used to assess the function of the transcription factor. 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 cardiofaciocutaneous syndrome (CFC), one of a group of developmental disorders known collectively as the RASopathies. In this study, we determined whether early cell migration 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 was completed and published at Molecular Cell (Spencer-Smith R. et al., 2022). This recent study was critical to establish standards for a RASopathy-specific phenotypical pipeline that now includes several powerful quantitative assays such as convergent-extension cell movements, heart edema formation analysis and measurements of the ceratohyal angles using the Tg(col2a1:GFP) line. This pipeline is currently employed in two separate collaborative projects with Elizabeth Terrel and Dan Ritt from Dr. Deborah Morrison's group to identify the function of other RAF variants and test novel drug treatments. 3. In collaboration with Dr. Marielle Yohe (Pediatric Oncology and the NCI-RASopathy Initiative, NCI-CCR), the facility is developing fluorescent reporter transgenic lines (Tg) to assist in the analysis of RASopathy-associated mutant proteins. Collaborative studies with the Yohe group also use the RASopathy phenotypical pipeline developed for the Morrison group and, in particular the convergence-extension assay, to analyze any previously uncharacterized RASopathy mutants identified through the NCI-RASopathies Initiative. These assays will be important to obtain critical information regarding the severity of the mutations and the effectiveness of various drug treatments. The Tg(Dusp6:GFP) will also be used as a reporter line for RAS/MAPK signaling activation in studies evaluating the effects of specific 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 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. Dr. Yohe invited Dr. Christine Kettenhofen (Insinna) to write the zebrafish models section of a chapter on non-NF1 Rasopathies for an upcoming book edited by Dr. Kate Rauen on Rasopathies. The chapter was submitted. Finally, a collaboration with Dr. Michael Sargen from the NCI Clinical Genetics Branch (DCGEG) was proposed in an application for a tenure-track position within the Lasker Clinical Research Scholars Program. A major focus of Dr. Sargens' research will be to identify germline variants contributing to melanoma development. As part of this research, Dr. Sargen would be collaborating with Dr. Yohe to perform cell-based functional studies of variants of interest and our facility would perform in vivo experiments using zebrafish models. A letter of support for the Lasker application was written and the proposal submitted. 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. Once the facility obtains the F2 generation for the double knock-out line, the project will be interrupted due to the retirement of Dr. Keller and the line will be transferred to Dr. Gudmundsson to complete the project in his new laboratory. 5. In collaboration with Dr. Esta Sterneck (LCDS, NCI-CCR), the facility performed pilot experiments to establish a xenograft assay to study the metastatic behavior of human tumor cells in zebrafish. Several drug treatments that target various cancer-associated signaling pathways were tested. The goal of this research will be 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.