Future Physiology 2020 (Virutal) Oral Communications: A zebrafish model of embryonic growth disruption reveals an ongoing impact on metabolic and cardiac phenotype

As climate change continues to pose a global problem, the need to understand organisms immediate and ongoing responses to the changing environment is essential. Temperature modulation and altered access to nutrition have been shown to impact embryonic growth trajectory in a diverse range of animal models1-3. The ability of an organism to respond to such changes during embryogenesis is known as developmental plasticity and can heavily influence embryonic growth trajectory. There is significant that altered embryonic growth trajectory has a lasting negative impact on metabolic and cardiac health, with early life growth disruption correlating strongly with increased adiposity, cardiac dysfunction, and metabolic disorder4,5. The coordination of these diverse pathways is not fully understood. Grb10 is a negative regulator of the insulin signalling pathway, the main coordinator of embryonic growth and development. While studies have revealed that Grb10 disruption in mammals alters insulin sensitivity and body mass and size, the long-term impact has not been investigated. To address this issue, this study validated the transient disruption of grb10a expression in Danio rerio by antisense- oligonucleotide-mediated knockdown. The impact on embryonic growth was measured in terms of total body length, while the impact on metabolism was measured in terms of rate of yolk consumption and glucose uptake. Heart rate was also measured to assess cardiac health. The persistent impact on late-juvenile metabolism was measured in terms of oxygen consumption by stop-flow respirometry. An Affymetrix GeneChipTM Zebrafish Genome Array was used to monitor gene expression over the first 30 days post fertilisation. All data were compared to embryos treated with a standard control morpholino (targeting human beta globin). Phenotypic rescue and reversal were possible through injection of grb10a RNA and displayed a dose-dependent effect. Knockdown was sufficient to alter embryonic growth trajectory, respiratory rate, and cardiac function, similar to existing mammalian models. Results are quoted as the mean ± S.E.M., compared by paired t-test. Total body length (3.41 ± 0.02 mm vs 3.16 ± 0.03 mm, p<0.0001, n=42), yolk consumption (0.0848 ± 0.009 mm2 vs 0.0024 ± 0.008 mm2, p<0.0001 n=18), and glucose uptake (55701 ± 2131 AU vs 39811 ±1079 AU, p=0.0002, n=5) were significantly elevated in morphant fish, while heart rate was significantly reduced (79.1 ± 7.3 bpm vs 116.9 ± 2.3 bpm, p<0.0001, n=14). Late-juvenile morphant fish continued to present altered metabolic rate (134.40 ± 15.60 µgO2h-1g-1 vs 19.00 ± 3.28 µgO2h-1g-1, p<0.0001, n=5). Transcriptomic data analysed by Qlucore Omics Explorer 2.2 (Lund, Sweden) revealed transient modulation of grb10a expression permanently remodelled the transcriptional landscape. Multiple growth factor mediated pathways were highly impacted long after morpholino attenuation. Additionally, expression of key cardiac genes was significantly altered in adult cardiac tissue (myl7 elevated 20%, p<0.0001, n=3, nppa down 40%. p=0.0012, n=3). The enduring nature of these changes suggests the zebrafish is a suitable model for longitudinal investigation of the link between embryonic growth disruption, adult phenotype, and later life disease risk. This also provides a predictive example of the impact the rapidly changing environment can have on adult health and phenotype.