Thesis Format
Monograph
Degree
Master of Science
Program
Physiology and Pharmacology
Collaborative Specialization
Developmental Biology
Supervisor
Drysdale, Thomas A.
Abstract
VACTERL association is diagnosed in infants with multiple congenital malformations in Vertebral, Anal, Cardiac, Tracheal, Esophageal, Renal and Limb (VACTERL) tissues. Recently, mutations in KYNU, HAAO or NADSYN1 genes of the nicotinamide adenine dinucleotide (NAD+) synthesis pathway have been identified in patients with VACTERL association. Similar defects were observed in offspring of mice undersupplied with NAD+ biosynthesis precursors, tryptophan, or vitamin B3. VACTERL phenotypes have also been recapitulated in Kynu and Haao knockout mice and rescued by vitamin B3 supplementation. These findings strongly support the concept that NAD+ synthesis, previously considered a universal requirement in all cells, is especially necessary for the development of several, primarily mesodermal, organ systems. We hypothesize that nadsyn1.L is differentially regulated in early development with higher expression or activity in mesodermal derivatives. To test this, we first examined the expression of nadsyn1.L in early Xenopus laevis frog embryos. nadsyn1.L transcripts were detectable by in situ hybridization in the hatching gland and developing pronephric kidney in Xenopus. Given kidney defects are a key feature of VACTERL association, we are using CRISPR/Cas9 gene editing to test whether mutations in nadsyn1.L result in developmental defects in the kidney. Preliminary evidence suggests that nadsyn1.L mutations in Xenopus embryos cause diminished kidney pronephros complexity. These experiments will provide groundwork for understanding how compromised NAD+ synthesis results in the specific developmental defects that lead to VACTERL association.
Summary for Lay Audience
A disease called VACTERL association is diagnosed in newborns with birth defects in several of the tissues making up the VACTERL acronym: Vertebrae, Anus, Cardiac (heart), Esophagus, Renal (kidney) and Limb (bone) tissues. Recently, it was established that the gene NADSYN1, which produces NAD+, a key molecule for cell health, energy and repair, is necessary in pregnant mothers to prevent VACTERL defects in their kids. However, the mechanism by which the gene NADSYN1 is required to prevent these birth defects is still unknown. This project first aimed to explore the role of the equivalent gene in the African clawed frog species Xenopus laevis, nadsyn1.L, in causing similar birth defects to those observed in limited examples in humans. The organs implicated in VACTERL-type birth defects share a common developmental origin referred to as the mesoderm (middle body layer) tissue. This considered, we hypothesized that nadsyn1.L is required for proper development of mesoderm origin organs in Xenopus frogs, especially in those tissues implicated in VACTERL association. We found that nadsyn1.L is not uniformly expressed in frog embryos, despite the previous consensus that nadsyn1.L should be required equally in every cell. Furthermore, we found that nadsyn1.L is especially expressed in the developing kidney, one of the key VACTERL tissues. We then carried out experiments to mutate the nadsyn1.L gene and found that this caused defects in the same area of the kidney. This suggests that nadsyn1.L is required for kidney development in the frogs, and partially supports our hypothesis that nadsyn1.L may be required for normal development of VACTERL tissues. Next, we sought to model this disease in a more human context by designing a new human stem cell line that has the same genetic mutation in the NADSYN1 gene as has been described in human patients with the VACTERL disease. We preliminarily found that the NADSYN1 mutation lowered the expression of several hundred genes, including key genes for development of mesoderm and other tissues. Altogether, we report here meaningful progress toward understanding the mechanistic connection between the NADSYN1 gene, NAD+ synthesis and VACTERL association in humans.
Recommended Citation
Bell, Andrew J., "Investigating the Role of NADSYN1 and NAD+ Synthesis in Experimental Models of VACTERL Association" (2024). Electronic Thesis and Dissertation Repository. 10568.
https://ir.lib.uwo.ca/etd/10568