Poster T16 - Two-Photon Microscopy-Guided Femtosecond Laser Photoablation at the Proximal Outflow Tract Induces Tetralogy of Fallot in Avian Embryos

Introduction: Malformation of the embryonic cardiac outflow tract (OFT) causes many clinical serious congenital heart defects (CHDs). CHDs affect about 2% of live births and may cause 30% of premature fetal losses. Despite their prevalence, over 80% of CHDs lack an identifiable genetic cause. Among the most severe CHDs is Tetralogy of Fallot (ToF), which consists of four key anomalies: ventricular septal defect, overriding aorta, pulmonary stenosis, and hypertrophic right ventricle. Notably, more than 90% of ToF cases have no known genetic basis, and the genes linked to the remaining 10% do not induce ToF in animal models.

Mechanical forces play a crucial role in heart remodeling, yet their impact on CHD development remains underexplored. Previous microsurgical hemodynamic interventions on chick embryos have demonstrated that altered blood flow can lead to CHDs. However, these procedures are challenging to localize and standardize, resulting in phenotypic variations that complicate causality determination.

We hypothesize that the local mechanical environment of the cardiac cushions controls their precisely-timed remodeling, and that even minor localized changes in this environment will lead to CHDs. To test this hypothesis, we targeted the proximal outflow tract (OFT) cushions, which are implicated in the development of Tetralogy of Fallot. Using two-photon microscopy-guided deep femtosecond photoablation, we induced localized hemodynamic perturbations. We further hypothesize that ablation of specific cushions will result in clinically relevant CHDs by altering the growth and remodeling programs through hemodynamic changes.

Materials and

Methods: We performed laser photoablation on the proximal outflow tract (OFT) at the Hamburger-Hamilton stage 23 (HH23). We harvested both the ablated embryos and the sham controls at stages HH31 and HH36. We documented morphological changes in the ablated hearts using nano-CT scanning and 3D analysis. We measured the changes in mechanical properties of the proximal OFT cushions at key developmental stages using a micropipette aspiration assay. To identify the growth and maturation trajectory of the OFT, we employed immunohistochemistry, characterizing the ECM and analyzing the activity of Yap, Notch, BMP, and endocardial to mesenchymal transition.

Results, Conclusions, and Discussions: More than half of embryos survive to the Hamburger-Hamilton stage 36 (HH36). Our results indicate that ablation of the proximal outflow tract (OFT) cushion consistently generates tetralogy of Fallot (ToF) in over 30% of the surviving embryos. Closer examination reveals that the ablated embryos exhibit delayed OFT development that results in OFT misalignment and incomplete separation.
Notably, there has never been a reliable embryonic model for ToF, and our model is the first to achieve this. Our results indicate that mechanobiology is a stronger driving force in the development of ToF. This finding provides deeper insights into the causes of ToF and has the potential to inform the development of early prevention and intervention methods.

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