PROJECT SUMMARY The most common congenital heart disease (CHD) in children involves incomplete formation of the interventricular septum (IVS), which occurs in isolation or as part of more complex CHD lesions. Myocardial fiber orientation, lineage tracing and asymmetric gene expression together suggest distinct L-R patterning of the IVS. However, there exists a gap in the understanding of the developmental mechanisms of IVS patterning and VSD etiologies. This hinders improvements to CHD prenatal screening, prognosis and evaluation of extra-cardiac anomalies, as well as efforts to engineer tissue patches for heart repair. The overall objective of this proposal is to delineate how L-R IVS patterning is controlled, and how perturbations lead to VSDs. We have preliminary data that suggests the secreted guidance cues Ntn1 and Slit2 control boundary regulation for L-R patterning of the IVS. Both genes were dysregulated in a genetic pathway dependent on the CHD-linked transcription factor, Tbx5. We found that homozygous loss of function (LOF) of Ntn1 or Slit2 resulted in membranous or muscular VSDs and a trabecular-like IVS. Ntn1 LOF led to displacement of the IVS boundary toward the LV, suggesting that Ntn1 may be a key regulator of boundary positioning. Conversely, Slit2 LOF disrupted boundary integrity, leading to mixing of RV and LV lineages, suggesting Slit2 regulation of boundary maintenance. Based on these preliminary data, we hypothesize that NTN1 and SLIT2 control complementary aspects of boundary regulation to segregate LV and RV lineages at the IVS for L-R patterning and ventricular septation. To discover how these developmental cues direct ventricular septation, we propose the following three Specific Aims: (1) test the hypothesis that NTN1 from the left side of the IVS determines boundary positioning for L-R IVS patterning by chemorepulsion, (2) test the hypothesis that a SLIT2 signal from the RV and LV maintains boundary integrity to preserve cell segregation for L-R IVS patterning by chemorepulsion of IVS cells, and (3) test the hypothesis that boundary perturbations of L-R IVS patterning cause cell signature disturbances of misplaced cells and adjacent tissue microenvironments. Successful completion of this project will provide insights into the regulation of L-R IVS patterning and the origins of human CHDs. By expanding our comprehension of the basis of CHDs, we will take an important step towards our long-term goal to improve the natural history of CHDs.