Partial rescue of the Tbx1 mutant heart phenotype by Fgf8: genetic evidence of impaired tissue response to Fgf8

Abstract

Tbx1 is the candidate gene of DiGeorge syndrome and is required in humans and mice for the development of the cardiac outflow tract (OFT) and aortic arch arteries. Loss of function mutants present with reduced cell proliferation and premature differentiation of cardiac progenitor cells of the second heart field (SHF). Tbx1 regulates Fgf8 expression hence the hypothesis that the proliferation impairment may contribute to the heart phenotype of mutants. Here we show that forced Fgf8 expression modifies and partially rescues the OFT septation defects of Tbx1 mutants but only if there is some residual expression of Tbx1. This genetic experiment suggests that Tbx1, directly or indirectly, affects tissue response to Fgf8. Indeed, Tbx1(-/-) mouse embryonic fibroblasts were unable to respond to Fgf8 added to the culture media and showed defective response of Erk1/2 and Rsk1. Our data suggest a coordinated pathway modulating Fgf8 ligand expression and tissue response to it in the SHF.

Figure 1. Loss of Fgf8 in Tbx1-expressing cells causes Truncus arteriosus Communis

A–B: Fgf8 in situ hybridization in control and conditional mutants (Tbx1^cre/+;Fgf8^fl/−) at E9.5 shows reduced expression in the pharyngeal region (arrow). O, otocyst; I, 1st pharyngeal arch; fl, forelimb bud. C–J: OFT and intracardiac phenotype in near term control (C,G) and Tbx1^cre/+; Fgf8^fl/− (D–F; H–J) embryos. Note the interventricular septal defect (*) and truncus arteriosus communis (TAC). K: Histogram of qRT-PCR data showing the relative expression of Tbx1 E9.5 embryos (3 embryos per genotype).

Figure 2. Forced Fgf8 expression in the Tbx1 domain partially rescues cardiovascular defects of Tbx1-hypomorphs

A–D: Isolated hearts from near-term control Tbx1^dp1/neo2 (A) and Tbx1^fgf8/neo2 (B–D) embryos show a range of phenotypes, including alignment and OFT defects. Sections of the corresponding hearts show DORV (B–B”), TAC type A4 (C'–C”, distal septation of aorta and pulmonary trunk with unseptated truncal valve overriding the interventricular septum) or TAC type A2 (D'–D”). Note the continuity between truncal and mitral valves in C” which is lost in D” (asterisk in D”). Ao, aorta; PT, pulmonary trunk; DORV, double outlet right ventricle; TAC, truncus; LA, left atrium; A2, A4 according to van Praagh's classification. Note VSD in C” and D” (arrowheads). E. qRT-PCR of Fgf8 target genes Etv4 and Etv5 expression in mouse embryonic fibroblasts (MEFs) from Tbx1^+/+ and Tbx1^−/− embryos (two independent lines per genotype) exposed to recombinant FGF8 in culture. Mutant cells do not respond properly to FGF8 stimulation. F–G. Western blots of proteins extracted from the same cells. Mutant cells express very little or undetectable level of Rsk1 and Erk1/2 phosphorylation. H: Western blot of proteins extracted from wild type and Tbx1^neo2/− embryos. Low levels of Tbx1 are sufficient to rescue Rsk1 phosphorylation response to FGF8.