Investigating a Tbx1 and Pax9 genetic interaction during cardiovascular development

Abstract

Congenital cardiovascular malformations (CCVM) are the most common type of birth defect in humans and can be life threatening for the newborn. 22q11 deletion syndrome (22q11DS) is one of the most common CCVM in humans, with patients presenting a wide variety of abnormalities including craniofacial dysmorphology, immune deficiency, mental retardation and cardiovascular defects, including ventricular septal defects, abnormal right subclavian artery and interrupted aortic arch type B. TBX1 is considered the main gene underlying the cardiovascular phenotype in 22q11DS patients, however, the great phenotypic variability among 22q11DS patients suggests genetic modifiers define the presentation of the phenotype. The transcription factor Pax9, was found significantly down-regulated in Tbx1-null embryos (a mouse model of 22q11DS). The aim of this project was to determine whether Pax9 is involved in cardiovascular development and to study a potential genetic interaction between Pax9 and Tbx1 during cardiovascular development. The results show that Pax9 is required for cardiovascular development as all Pax9-null embryos have severe cardiovascular abnormalities including IAA, VSD, BAV, DORV, and abnormal or completely absent common carotids. Furthermore, a strong genetic interaction between Pax9 and Tbx1 was found, since double heterozygosity leads to lack of formation of the 4th pharyngeal arch arteries consequently leading to a significant increase in the incidence of IAA. The molecular mechanism of this interaction between Pax9 and Tbx1 was investigated. The results show that Tbx1 does not bind to any region within the Pax9 locus in vitro and in vivo. A physical interaction between Pax9 and Tbx1 proteins was also ruled out by co-immunoprecipitation. qPCR analysis revealed a significant downregulation of Gbx2 in double heterozygous embryos, and luciferase experiments revealed Pax9 is able to promote luciferase expression of a conserved regulatory region within the Gbx2 locus, whereas Tbx1 repressed luciferase expression of this Gbx2 cloned regions. The results in this dissertation suggest Pax9 and Tbx1 regulate cardiovascular development, at least in part, through regulating Gbx2.