Disruption ofpdgfraalters endocardial and myocardial fusion during zebrafish cardiac assembly

Abstract

Cardiac development in vertebrates is a finely tuned process regulated by a set of conserved signaling pathways. Perturbations of these processes are often associated with congenital cardiac malformations. Platelet-derived growth factor receptor α (PDGFRα) is a highly conserved tyrosine kinase receptor, which is essential for development and organogenesis. Disruption of Pdgfrα function in murine models is embryonic lethal due to severe cardiovascular defects, suggesting a role in cardiac development, thus necessitating the use of alternative models to explore its precise function. In this study, we generated a zebrafish pdgfra mutant line by gene trapping, in which the Pdgfra protein is truncated and fused with mRFP (Pdgfra-mRFP). Our results demonstrate that pdgfra mutants have defects in cardiac morphology as a result of abnormal fusion of myocardial precursors. Expression analysis of the developing heart at later stages suggested that Pdgfra-mRFP is expressed in the endocardium. Further examination of the endocardium in pdgfra mutants revealed defective endocardial migration to the midline, where cardiac fusion eventually occurs. Together, our data suggests that pdgfra is required for proper medial migration of both endocardial and myocardial precursors, an essential step required for cardiac assembly and development.

Retinoid X receptor alpha is a spatiotemporally-specific therapeutic target for doxorubicin-induced cardiomyopathy in adult zebrafish

“…The aforementioned "gain-of function" outcome in GBT0419 appears to be different from what have be previously described in other GBT lines, as all have been to date demonstrably loss-of-function mutants (17,20,40,41,69). However, it is still possible that GBT0419 is a loss-of function allele at mRNA or even protein level, given that this mutant has a blunted rxraa response to 9-cis RA (Supplemental Figure 6B, 6C), and the activated RA signaling in GBT0419 could be the consequence of certain 'feed-forward' mechanism that has been previously recognized (47).…”

“…In GBT mutants, a monomeric red fluorescent protein (mRFP) gene in the RP2 vector is fused in-frame with the N-terminal part of the affected gene (20), which reports the expression pattern of the trapped gene as a chimeric protein (17,40,41). Because GBT0419 harbors the RP2 element in the 1 st intron of rxraa ( Figure 1A), we documented mRFP expression pattern in GBT0419, and crossed it with Tg(fli1a:EGFP) (42) to label endothelial cells and Tg(ttn:actn-EGFP) (17) to label cardiomyocytes, both with enhanced green fluorescent protein (EGFP), respectively.…”

Building the vertebrate codex using the gene breaking protein trap library

“…At the end of this pipeline, 204 GBT-candidate lines met the highest stringency of confirmed expression linkage and were classified as ‘GBT-confirmed lines’ ( Figure 2A ). While 57 of these GBT-confirmed lines have been previously published ( Clark et al, 2011a ; Ding et al, 2013 ; Ding et al, 2017 ; El-Rass et al, 2017 ; Ma et al, 2020 ; Westcot et al, 2015 ), 147 of these GBT-confirmed lines are newly characterized in this manuscript and were selected for confirmation based upon their expression pattern and/or homozygous phenotype ( Supplementary file 1 ). A small subset of these GBT-confirmed lines mapped to areas in the genome without annotated transcripts.…”

“…All plots show median. The data of previous protein trap systems were converted from the data in the original articles, R14-R15, our initial R-series protein trap vectors (n = 6), ( Clark et al, 2011a ; FlipTrap, FlipTrap vectors (n = 6), Trinh et al, 2011 ; FT1, FT1 vector (n = 4), Ni et al, 2012 ; RP2.1, RP2.1 vector (n = 26), Clark et al, 2011a ; Ding et al, 2013 ; Ding et al, 2017 ; El-Rass et al, 2017 ; Westcot et al, 2015 and unpublished data) ( Figure 3—source data 1 ). The graph was made in JMP14 (SAS, Cary, NC).…”

Protocol development for discovery of angiogenesis inhibitors via automated methods using zebrafish

“…The Tg(Flk1:EGFP) line was maintained and crossed using standard techniques [19]. All zebrafish experiments were approved by the St. Michael’s Hospital Animal Care Committee (Toronto, Ontario, Canada) under protocol ACC867.…”