The fundamental question for the field of Down Syndrome (DS) basic research is how an extra copy of human chromosome 21 (HSA21) translates into the organ-specific pathologies that are observed in the DS population. Structural pathologies in DS include congenital heart malformations, including a 2000-fold increased risk for Atrioventricular Septal Defects (AVSDs). A mechanistic understanding of DS-specific organogenesis defects is lacking, and specifically, how Trisomy 21 (T21) causes Congenital Heart Disease is poorly understood. In the parent award, we have defined Hedgehog (Hh) signaling as an explicit development timer of cardiac differentiation during mammalian development. Furthermore, there is plentiful evidence that suggests that T21 causes Hedgehog signaling defects. In this supplement, we propose to evaluate cardiac progenitors in Tc(HSA21q;MAC)1Yakaz ("TcMAC21"), a new mouse model of DS with a stably segregating HSA21q-MAC hybrid chromosome that is nearly complete compared to the human HSA21q chromosome. Importantly, TcMAC21 mice exhibit penetrant AVSDs. We propose to determine whether AVSDs in TcMAC21 result from failure of Hedgehog-signaling dependent timing control of SHF cardiac progenitor differentiation timing. This Supplement addresses INCLUDE Component 1: Targeted high risk - high reward basic science studies in areas highly relevant to Down syndrome. We propose the transformative hypothesis that the cause of cardiac defects in DS is failure of Hedgehog-signaling-dependent timing control of progenitor differentiation, resulting in precocious differentiation, reduction of cardiac progenitor numbers, and cardiac morphogenesis failure resulting in CHD.