PROJECT SUMMARY 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. Specific types of CHD developed by DS patients affect posterior cardiac structures more severely and frequently than anterior structures. A mechanistic understanding of DS-specific organogenesis defects is lacking in most cases, although a rich descriptive literature sets the stage for concerted mechanistic studies. Specifically, how Trisomy 21 (T21) causes heart morphogenesis defects, causing posterior vs anterior CHD, is poorly understood. The molecular networks identified by the proposed work, implicated in DS organ- specific cardiac progenitors, will serve as a template for understanding the molecular ontogeny of heart defects. We have defined Hedgehog (Hh) signaling as an explicit development timer during mammalian development, required for maintaining organ-specific progenitor cells and dictating their differentiation in time and space, independent from developmental patterning or proliferation. Additionally, during cardiac development retinoic acid is crucial for establishing anteroposterior polarity and specifically promoting the posterior limits of the SHF. These signaling pathways together control the balance between organ-specific progenitors and their differentiated counterparts that is fundamental to complex organogenesis of the heart. The Moskowitz lab found that when Hh signaling is abrogated in cardiac progenitors, they underwent precocious differentiation, resulting in morphogenesis failure. Aligned with the parent award, we propose the transformative hypothesis that a unifying cause of birth defects in DS is failure of heterochronic timing control of organ-specific progenitor differentiation, resulting in precocious differentiation, a reduction of organ-specific progenitors, morphogenesis failure and birth defects in multiple organs.