Zebrafish core

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Project summaries for FY 2025 are listed below: 1. The facility is collaborating with Dr. Christopher Westlake's group (LCDS, NCI-CCR) on several projects: Investigation of normal and disease function of WDR44 during zebrafish development. In 2024, our facility continued ongoing projects initiated with the identification of novel patient variants associated with WDR44, an X-linked gene involved in regulation of Rab11 ciliogenesis function and responsible for a spectrum of ciliopathy. The first part of the work was published in Nature Communication (Accogli et al., 2024) and several other pathogenic variants are currently being investigated using a combination of morpholino and overexpression/rescue approaches in zebrafish to perform functional studies and determine whether we could model ciliopathy-related disease. In additional to our over-expression studies, the facility developed a wdr44 CRISPR knockout line currently under investigation. Investigation of membrane trafficking, tubulation and fusion regulator function in ciliogenesis and ciliopathy. Abnormal expression of Rab proteins has been reported in multiple cancers. In a project with Dr. Saha, the facility developed tools to perform 2-3 color live imaging of membrane tubules using expression of fluorescently labeled Rab proteins in developing zebrafish. Both transient and transgenic (Tg) expression of these proteins, known to be involved in membrane tubule trafficking and ciliogenesis, were monitored overtime using the spinning disk confocal from Dr. Westlake. This work provided the first evidence of a Rab-dependent trafficking cascade important for membrane tubule formation in vivo and was published in Cell Reports (Saha I, Insinna C and Westlake. CJ, 2024). Furthermore, these assays could be used in the future to study newly identified proteins associated with Rab-dependent tubulation events. Finally, the facility is also collaborating with Dr. Lu to study the function of a group of proteins involved in membrane fusion and determine whether they function in ciliogenesis. This work will be combined with Dr. Lu's work in human cells and included in a manuscript that is currently in preparation. Extramural collaboration In collaboration with Dr Oueslati and Dr Teixeira (University of Laval), the facility developed an optogenetic approach to investigate links between WDR44 and neurodegenerative diseases via the formation of alpha-synuclein aggregates in vivo. This work was included in a manuscript that will be submitted to Nature early August. 2. In collaboration with Dr. Marielle Yohe (LCDS, Pediatric Oncology and the NCI-RASopathy Initiative, NCI-CCR), the facility assists in the functional analysis of newly identified variants associated with a group of developmental disorders known collectively as the RASopathies and the evaluation of RAS/MAPK targeted agents'efficacy. Our previous studies with the Morrison laboratory helped to establish standards for a RASopathy-specific phenotypical pipeline that 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. Collaborative studies with the Yohe group use the RASopathy phenotypical pipeline to analyze any previously uncharacterized RASopathy variants identified through the NCI-RASopathies Initiative. These assays are important to obtain critical information regarding the severity of the mutations and the effectiveness of various drug treatments. Our primary focus during this FY has been to utilize these assays for several HRAS variants observed in Costello syndrome. The functional analysis of these HRAS variants will be included in a manuscript currently in preparation. Efforts were invested this year in the development of an inducible transgenic line expressing a HRAS variant. Several novel assays were used to identify the presence of cardiac defects including hypertrophic cardiomyopathy (HCM), heart looping or size defects in mutant zebrafish embryos. The functional analysis of this HRAS variant and the transgenic line will be included in a manuscript currently in preparation. The goal of this collaboration will be to use the zebrafish system to accelerate the development of patient-specific therapeutic approaches. Notably, this year a zebrafish chapter on non-NF1 RASopathies was published (Insinna C and Yohe M. Chapter 40: Modeling the non-NF1 RASopathies. In The RASopathies, edited by Katherine Rauen, 2024). Finally, in 2023, a collaborative project using zebrafish with Dr. Michael Sargen from the NCI Clinical Genetics Branch (DCGEG) consists of identifying germline variants contributing to melanoma development. Dr. Sargen collaborates with Dr. Yohe to perform cell-based functional studies of variants of interest and our facility performs in vivo experiments using zebrafish. Using the convergence extension assay and drug treatments, the severity of several LZTR1 variants was assessed in vivo. This work was included in a manuscript that is currently in preparation. 3. In collaboration with Dr. Deborah Morrison's group (LCDS and the NCI-RASopathy Initiative, NCI-CCR), we explored the function and localization of novel RAS/RAF mutants associated with RASopathies. In a prior study (Spencer-Smith et al., Molecular Cell, 2022), we determined whether early cell migration and phenotypical defects induced by overexpression of the BRAF mutants in zebrafish correlated with the corresponding disease presentation in humans and whether the observed phenotypes were specific to the disease-associated mutation. The RASopathy-specific pipeline is currently employed in several collaborative projects with Elizabeth Terrel and Dan Ritt from Dr. Deborah Morrison's group to examine the biological activity of other RAF variants and to test novel drug treatments. 4. The primary goal of Dr. Sheppard's translational research group is to develop more efficacious therapies for patients with lymphatic malformations. Specifically, her research has focused on a subtype of complex lymphatic anomaly, called central conducting lymphatic anomaly (also known as channel type lymphatic malformation, CCLA), with severe morbidity and mortality, associated with RASopathies. Dr. Sheppard identifies novel pathogenic variants in the RAS/MAPK that cause CCLA and respond to MEK inhibition and uses the established RASopathy-specific phenotypical pipeline from the facility to determine the function of these variants and to test drug treatments. Our functional analysis of SHOC2 variants is included in a manuscript currently under review with the Journal of Exp. Medicine. 5. In collaboration with Dr. Lucero in Dr. Schnermann's laboratory (Chemical Biology Laboratory, NCI-CCR), the facility trained Dr. Lucero to establish in vivo xenografts models needed to address antibody drug conjugate toxicity, payload properties and labeling chemistries on tumor. In vivo imaging is conducted by Dr.Lucero and Dr. Stephen Lockett at OMAL (NCI-Frederick). The group uses zebrafish as a platform to image the uptake and activation of fluorophore-antibody conjugates in tumor, helping define off-target distribution and understand whether Antibody drug Conjugates' linkers activated by the tumor microenvironment improve tumor selectivity. 6. In 2024, the facility started a collaboration with Dr. Yohe and Dr. Turbyville from the RAS Initiative to develop the use of an ERKTR biosensor for expression in zebrafish and enable in vivo studies of RAS and MAPK signaling dynamics to facilitate the development of RAS-targeted therapies. An image analysis pipeline was developed to quantify signaling kinetics in real-time and this tool will be made accessible to several of our collaborators.