Use of the Zebrafish to Explore the Connection of Pituitary Development with Congenital Craniofacial Syndromes

Abstract

This thesis project analyzes pituitary development using the zebrafish study model. Our lab focuses on studying craniofacial malformations using complementary models that include human and other mammalian cells, mice and zebrafish. Investigation in the Esrp1/2 mouse model revealed affected pituitary development. Esrp1/2 are known genetic causes of craniofacial developmental issues such as cleft lip palate. Visualization of pituitary development may aid in future research implicating ESRP1 and ESRP2 as being causative of growth hormone deficiency diseases such as congenital hypopituitarism. The effect of esrp1 and esrp2 in zebrafish on both midfacial morphogenesis and its effect on multiple germ layers have implications around there being multiple germ layer origins of the pituitary which is a currently debated topic in the field of embryology and whether the same pathways in zebrafish are evolutionarily conserved in enough ways to serve as a model for this possible research. We hypothesized that it is possible to visualize the development of the pituitary that models what is currently known about pituitary development in zebrafish. Using a number of methodologies such as whole-mount in situ hybridization (WISH), multiplexed in situ hybridization (RNAscope), and time-lapsed imaging under fluorescent imaging microscopes and light sheet microscope, we were able to visualize the pituitary at various stages of development reflecting what is currently theorized around how the pituitary develops. Additionally, we present efforts around the creation of a LIM homeobox protein (lhx3) transgenic reporter line which could be used for future research implicating mutations in ESRP1/2 as being causative for both cleft lip palate disorders and congenital hypopituitarism. As the author of this thesis paper, Lisa Tsay carried out all the experiments outlined and presented in the methods, results, and discussion sections under the guidance of Drs. Eric Liao and Shannon Carroll.