Performing in situ hybridization and quantitative PCR to assess eya2 localization and expression in zebrafish

Abstract

Rhabdomyosarcoma (RMS) is the most common soft‐tissue sarcoma found in children (Skapek, 2019) and is classified as a skeletal muscle cancer (Hsu, 2021). A common transcription factor that increases cell proliferation (how cells grow and divide) in RMS is called SIX1 (Hsu, 2021). The SIX1 protein has coregulators, such as EYA2, that regulate the increase or decrease of SIX1’s function (Sousounis, 2020). Because the coregulator EYA2 can play a critical role in the increase of SIX1's function (Blevins, 2015), we hypothesize that an increase in EYA2 will lead to uncontrolled cellular proliferation. This uncontrolled cellular proliferation could lead to the development of tumors, specifically RMS. In order to understand EYA2’s role in tumor growth and cancer, it is important to study the coregulator under normal homeostatic conditions. Specifically, understanding how muscle tissue normally utilizes eya2 during embryonic development, when cells are proliferating, can provide insight into eya2’s potential roles in muscle tumor development. Zebrafish are ideal research specimens for this research because of their easy to visualize muscular system. A technique used to visualize mRNA expression and localization in zebrafish tissues, is in situ hybridization (ISH) (Chu, 2019). Our first objective was to confirm that we can perform ISH in our laboratory. We used ISH to observe that Eya2 mRNA was generally located in the head and upper torso regions of the zebrafish at both 24 hours post fertilization (hpf) and 48 hpf, which supports previously published results (Thisse, 2004; Thisse, 2004). Additionally, we observed an increase in eya2 mRNA levels by ISH from 24 hpf to 48 hpf. We utilized quantitative PCR (qPCR) (Heid, 1996) to quantify these changes in eya2 mRNA levels. Our qPCR results indicate there was an increase in the coregulator eya2 mRNA expression from 24 hpf to 48 hpf. Overall, both research techniques, ISH and qPCR, demonstrated an increase in eya2 transcript levels from 24 hpf to 48 hpf, suggesting that this time frame would be appropriate for future studies of eya2 function in zebrafish development.