Generation of umami taste knockout zebrafish to study the loss-of function effects on diet intake preference, amino acid sensing and appetite control

The selection of sustainable protein sources that provide optimal nutrition continues to be a major challenge in the aquaculture industry. Studying the physiological effects of ingredients, particularly protein and amino acids composition, may provide crucial insights to improve fish diets in terms of gut health, feed intake, metabolic effects and fish growth. The mammalian umami taste receptor (T1R1-T1R3) senses amino acids, which are key nutrients in vertebrate’s diet and is not only expressed in taste buds but also in the gastrointestinal tract. However its specific functions in the gastrointestinal tract and their relevance for the regulation of food intake and metabolism is largely unknown in mammals and remains unexplored in non-mammalian species. Also, it is not yet understood how the T1R1-T1R3 “taste” information is conveyed to the central nervous system (CNS) to promote an appropriated functional response. A better understanding of how the gut senses amino acids through T1R1-T1R3 will provide new insights that will help to define novel feed ingredients and drug targets. Specifically, we aim to provide a method to study the physiological consequences of T1R1-T1R3 disruption on gut function, such as peristaltic reflexes (motility), hormone/digestive enzymes release, and on the feed intake control. We aim to understand how different amino acids are sensed in the gut and how this information is conveyed to the CNS, and therefore it is a complex multi-organ process that requires the use of functional in vivo studies. Specifically, we will disrupt the T1R1, T1R3, and α-transducin genes. To achieve this, we will make use of the clustered regularly interspaced palindromic repeats (CRISPR) associated Cas9 system, which is the fastest and most convenient approach to induce mutations in zebrafish. This approach will provide a detailed understanding of how the T1R1-T1R3 senses amino acids and which signalling pathways are activated. To identify the mutant zebrafish in which the gene of interest is disrupted, we will extract DNA from the tail fin and analyse using high resolution melt analysis (HRMA). Phenotypic characterization of the transgenic fish will include behavioural swimming activity analysis towards an attractant, and gross growth measurements. In addition, sampling (post-mortem) of the intestine for motility patterns and pellet evacuation studies and release of appetite regulating hormones analysis will be performed; gene expression profile of the umami taste receptor and signalling molecules will also be studied in various tissues.