Regulation of gut motility is a very complex process and is thought to require proper functioning and coordination of three major cell types: the smooth muscle cells, the enteric nervous system and a specialized group of cells called the interstitial cells of Cajal (ICC). The ICC, also commonly called the pacemaker cells of the gut have been implicated in proper gut motility. ICC reduction or loss has been linked GI motility disorders such as Hirschsprung’s disease, slow transit constipation, gastroparesis, achalasia, and intestinal pseudo-obstruction. Although the role of ICC in GI motility has been widely examined at the phenotypic level, the underlying molecular mechanisms that drive development and function remain elusive.
In this dissertation research, we generated two ICC biomarker red fluorescent protein (RFP) reporter lines, kitais32gal4 and ano1is33gal4 using the CRISPR-Cas9 mediated knock-in of the Gal4 into the zebrafish genome by homology directed repair mechanism. We further characterized their FFP expression using both confocal laser scanning microscopy and light/fluorescent microscopy. The generated reporter lines have a bright RFP expression and putative homozygous null mutants are viable and fertile. This is the first study to successfully generate ano1 reporter line in zebrafish.
Finally, we utilized both qualitative and quantitative approaches to examine the gut motility phenotypes of kita and ano1 putative null mutants at 9 dpf. Our results indicated that zebrafish intestinal motility pattern was disrupted by knock-out of kita or ano1. There was a significant reduction in the average number of contractions, average distance traveled by individual contractions, frequency of the contractions, average time taken by individual contractions. The average interval between contractions increased. Our results suggest that both kita and ano1 are critical for generation and maintenance of coordinated, steady and complete contraction in the zebrafish intestine. We provide the first evidence for the role of ano1 in zebrafish gut motility using in vivo approaches and this these finding will improve our present understanding of GI motility disorders
