Genetic manipulations of MC4R for increased growth and feed efficiency in fish
Authors: Gothilf Yoav , Cone Roger , Levavi-Sivan Berta , Harpaz Sheenan 
Published: January 2022

The hypothalamic melanocortin system plays a central role in the regulation of food consumption and energy homeostasis in mammals. Accordingly, our working hypothesis in this project was that genetic editing of the mc4r gene, encoding Melanocortin Receptor 4 (MC4R), will enhance food consumption, feed efficiency and growth in fish. To test this hypothesis and to assess the utility of mc4r editing for the enhancement of feed efficiency and growth in fish, the following objectives were set: Test the effect of the mc4r-null allele on feeding behavior, growth, metabolism and survival in zebrafish. Generate mc4r-null alleles in tilapia and examine the consequences for growth and survival, feed efficiency and body composition. Generate and examine the effect of naturally-occurring mc4r alleles found in swordfish on feeding behavior, growth and survival in zebrafish. Define the MC4R-mediated and MC4R-independent effects of AgRP by crossing mc4r- null strains with fish lacking AgRP neurons or the agrpgene. Our results in zebrafish did not support our hypothesis. While knockout of the agrpgene or genetic ablation of hypothalamic AgRP neurons led to reduced food intake in zebrafish larvae, knockout (KO) of the mc4r gene not only did not increase the rate of food intake but even reduced it. Since Melanocortin Receptor 3 (MC3R) has also been proposed to be involved in hypothalamic control of food intake, we also tested the effectofmc3r gene KO. Again, contrary to our hypothesis, the rate of food intake decreased. The next step was to generate a double mutant lucking both functional MC3R and MC4R. Again, the double KO exhibited reduced food intake. Thus, the only manipulation within the melanocortin system that affected food intake in consistent with the expected role of the system was seen in zebrafish larvae upon agrpKO. Interestingly, despite the apparent reduced food intake in the larval stage, these fish grow to be of the same size as wildtype fish at the adult stage. Altogether, it seems that there is a compensatory mechanism that overrides the effect of genetic manipulations of the melanocortin system in zebrafish. Under Aim 3, we introduced the Xna1, XnB1l, and XnB2A mutations from the Xiphophorus MC4R alleles into the zebrafish MC4R gene. We hypothesized that these MC4R mutations would act as dominant negative alleles to increase growth by suppressing endogenous MC4R activity. When we examined the activity of the three mutant alleles, we were unable to document any inhibition of a co-transfected wild type MC4R allele, hence we did not introduce these alleles into zebrafish. Since teleost fish possess two agrpgenes we also tested the effect of KO of the agrp2 gene and ablation of the AgRP2 cells. We found that the AgRP2 system does not affect food consumption but may rather be involved in modulating the stress response. To try to apply genetic editing in farmed fish species we turned to tilapia. Injection of exogenous AgRP in adult tilapia induced significant changes in the expression of pituitary hormones. Genetic editing in tilapia is far more complicated than in zebrafish. Nevertheless, we managed to generate one mutant fish carrying a mutation in mc4r. That individual died before reaching sexual maturity. Thus, our attempt to generate an mc4r-mutant tilapia line was almost successful and indicate out non-obvious capability to generate mutant tilapia.