Utilizing mpx:gfp transgenic zebrafish to assess eya3's potential effects on neutrophils.

Rhabdomyosarcoma (RMS) is a malignant cancer that develops in children's muscles. Although RMS is one of the most common soft tissue sarcomas in children (NIH, 2020), there are a limited number of treatment options. As a potential new treatment target, we are investigating the roles of eya3, a transcriptional regulator, in normal muscle development. Previous research has shown a connection between eya3 and the innate immune system (Liu, 2012; Okabe, 2009), sometimes indicating eya3 is involved in innate immune responses, other times demonstrating that eya3 inhibits immune responses (Vartuli, 2018). Innate immune cells include neutrophils, which express mpx or myeloid-specific peroxidase. To investigate how eya3 affects neutrophil location and/or levels in developing embryos, we utilized mpx:gfp transgenic zebrafish to visualize green fluorescent neutrophils via fluorescent imaging. After injecting eya3 morpholinos into wildtype zebrafish embryos to decrease eya3 expression, we used quantitative PCR (qPCR) to quantify the amount of neutrophils present. While our fluorescent imaging confirmed the presence of green fluorescent neutrophils in the mpx:gfp zebrafish at 96 hours post fertilization (hpf), we have yet to image neutrophils in our experimental group at the same timepoint. Our qPCR results suggest a decrease in mpx expression at 24 hpf with eya3 knockdown. We plan to confirm the potential decrease in neutrophil levels following eya3 knockdown with visualization by fluorescent microscopy. It will be interesting to determine if neutrophil levels are decreased in all locations, or depleted only from some locations in the zebrafish. © FASEB.

Citation

Gloria Baxter, Nicole Larson, Violet Tessier, Jenean O'Brien. Utilizing mpx:gfp transgenic zebrafish to assess eya3's potential effects on neutrophils. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2022 May;36 Suppl 1

PMID: 35556657

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