Cardiovascular phenotyping of fetal mice by noninvasive high-frequency ultrasound facilitates recovery of ENU-induced mutations causing congenital cardiac and extracardiac defects.

As part of a large-scale noninvasive fetal ultrasound screen to recover ethylnitrosourea (ENU)-induced mutations causing congenital heart defects in mice, we established a high-throughput ultrasound scanning strategy for interrogating fetal mice in utero utilizing three orthogonal imaging planes defined by the fetus' vertebral column and body axes, structures readily seen by ultrasound. This contrasts with the difficulty of acquiring clinical ultrasound imaging planes which are defined by the fetal heart. By use of the three orthogonal imaging planes for two-dimensional (2D) imaging together with color flow, spectral Doppler, and M-mode imaging, all of the major elements of the heart can be evaluated. In this manner, 10,091 ENU-mutagenized mouse fetuses were ultrasound scanned between embryonic days 12.5 and 19.5, with 324 fetuses found to die prenatally and 425 exhibiting cardiovascular defects. Further analysis by necropsy and histology showed heart defects that included conotruncal anomalies, obstructive lesions, and shunt lesions as well as other complex heart diseases. Ultrasound imaging also identified craniofacial/head defects and body wall closure defects, which necropsy revealed as encephalocele, holoprosencephaly, omphalocele, or gastroschisis. Genome scanning mapped one ENU-induced mutation associated with persistence truncus arteriosus and holoprosencephaly to mouse chromosome 2, while another mutation associated with cardiac defects and omphalocele was mapped to mouse chromosome 17. These studies show the efficacy of this novel ultrasound scanning strategy for noninvasive ultrasound phenotyping to facilitate the recovery of ENU-induced mutations causing congenital heart defects and other extracardiac anomalies.