RNA seq analysis for impact of high glucose and salt on Zebrafish Embryo development
Introduction Zebrafish (Danio rerio) is an important model organism extensively used for studying developmental biology and genetics. The Wnt pathway plays a crucial role in embryonic development in vertebrates. High glucose and salinity can disrupt the normal Wnt signaling pathway and cause abnormal embryonic development. In this study, we aimed to investigate the effect of high glucose and salinity on embryological development of zebrafish through the Wnt pathway. Methods embryos were treated with normal media and high glucose for 5 days from 3 hours post-fertilization(hpf) to 96hpf separately as control and experimental groups. RNA is extracted from control and experimental group for replicates for RNAseq. Day 5 Larva is fixed in 10% formalin for H&E staining to detect organogenesis changes under high glucose and Saline conditions. Zebrafish eggs were treated with normal media and high salt (2%) for 3 days from 3 hpf to 72hpf separately as control and experimental groups. Day 1 and Day 2 larvae are fixed in 10% formalin for H&E staining.  We also analyzed the Wnt signaling pathway in these fish under different glucose and salinity conditions. Results Our results showed that exposure to high glucose and salinity caused abnormalities in embryological development of the fish. We observed poor hatching rate, reduced body length, deformities in various body parts, and mortality of the fish. Glucose delayed the zebrafish embryo development by slowing the hatch rate about 24 hours. The brain, heart, and tail started showing morphology smaller in the glucose group compared to the control group at 24 hpf. Heartbeat is slower in the glucose group compared to the control group on day 2 and 3 with a statistically significant difference. The brain, heart, and tail started showing morphology smaller in the salt group compared to the control group at 48 hpf. These conditions contribute to significant physiological differences that may provide insight on functionality post-embryological development. Testing to find consistent trends in morphology and genetic differences in high salt conditions, and genetic alterations for high glucose conditions are being further researched in this experiment. 1674 genes are regulated in high glucose treatment and 4323 genes changed expression in high saline condition. We also found that high glucose and salinity disrupted the normal Wnt signaling pathway by downregulating the expression of Wnt pathway genes and proteins. The expression of Wnt pathway inhibitors, such as Axin2 and DKK1, was upregulated, while the expression of Wnt pathway activators, such as ß-catenin and Wnt3a, was downregulated in fish exposed to high glucose and salinity. Conclusion High glucose and salinity can have a detrimental effect on the embryological development of zebrafish through the Wnt signaling pathway. Our results suggest that exposure to high glucose and salinity can disrupt the normal development of zebrafish and lead to developmental abnormalities. Further studies are needed to investigate the underlying molecular mechanisms that regulate the Wnt pathway in response to high glucose and salinity in zebrafish. Overall design: Zebrafish embryos were cultured at control, high glucose and high salt conditions for 72 hours. RNA was extracted for RNA seq. Control groups vs high glucose vs high salt groups.