Novel Biochemical And Phenotypical Findings On The Nodal Signaling Components Lefty And Cripto

ABSTRACT NOVEL BIOCHEMICAL AND PHENOTYPICAL FINDINGS ON THE NODAL SIGNALING COMPONENTS LEFTY AND CRIPTO by Amapola Balancio December 2014 Advisor: Dr. William Branford, Jr. Major: Biological Sciences (Molecular and Developmental Biology) Degree: Master of Science Nodal, a Transforming Growth Factor-β (TGF-B), is a key regulator of proper development along with its atypical TGF-B inhibitor Lefty and its EGF-CFC co-receptor Cripto. Studies have shown that Nodal is unable to signal in the absence of a functional Cripto, indicating that it is a Nodal obligate co-receptor. In fact, one of the ways that Lefty prevents Nodal is by binding to Cripto and another way is through directly interacting with Nodal. To characterize the regions of Xenopus Lefty (Xlefty) that molecularly interact with Nodal and XCr1, we created truncated, as well as uncleavable, Xlefty mutants and utilize them in phenotypic and biochemical experiments. We found that the M1 domain of Xlefty is not only the region that interacts with XCr1, but it is also sufficient to inhibit Nodal signaling. Furthermore, previous literature suggested that the mature form of Xlefty blocks Nodal. That means that the cleavage of Xlefty is necessary for Nodal inhibition. We believe our results are the first to show that only mature Xlefty interacts with Cripto. Processing of Xlefty at its first cleavage site is a requirement for Nodal inhibition, yet it has been implied that the second cleavage site is unimportant to its role in embryogenesis. Our results show otherwise. In fact, it may play a crucial role in the folding and processing of Xlefty as its mutation led to a mature form that is unable to bind XCr1. Finally, we found that overexpression of XCr1 and one of the Xlefty truncations, Xlefty∆M2/M3, led to a phenotype in which embryos were unable to complete their blastopore closure. Perhaps the most affected structure was the notochord. While it can still form as long as germ layer cells differentiate into dorsal mesoderm, proper "zipping" of the notochord requires dorsal closure by convergent extension, which is one of the main cellular movements that occur during gastrulation. To our knowledge, this is a novel phenotype of XCr1 overexpression. This phenotype implies that XCr1 plays a crucial role in mesoderm differentiation and convergent extension during early development.