PROJECT SUMMARY / ABSTRACT: Congenital or neonatal accumulation of chyle in the pleural space is the most common cause of pleural effusion affecting 1 in 10,000 births with mortality rates between 20-60%. Neonatal patients with a spontaneous accumulation of chyle frequently exhibit bilateral pleural effusion, severe respiratory distress, tachypnea, and cyanosis, suggesting the mechanical effect of compression on lung compliance and impairment of gas exchange in alveoli. Although Dr. Bartloet established accumulation of chyle in the pleural space as a lymphatic anomaly in 1633, the mechanism for their formation and treatment have not been fully defined. Recent studies reveal the requirement of prenatal lymphatic function to drain pleural fluid and promote the inflation of lungs at birth, which is required for viability. To accomplish this, an extensive network of lymphatic vessels within the pleura, the intercostal space, the perivascular spaces of arterioles and venules, and the connective tissue of the terminal and respiratory bronchioles maintain fluid homeostasis and promote effective gas exchange. Abnormal dilation of these lymphatic vessels, known as lymphangiectasia, is frequently associated with neonatal chylous effusion, immature lungs, and severe respiratory distress with mortality. Even though Dr. Rudolf Virchow described neonatal pulmonary lymphangiectasia as early as 1856, the underlying causal etiology and treatment options remain elusive. Our preliminary studies identify an unexpected causal link between pathological MAPK activation and lymphangiectasia in mice and humans. Pathological activation of lymphatic MAPK causes severe pulmonary lymphangiectasia, accumulation of chyle in the pleural space, and complete lethality. Preliminary analyses of human pathological tissue samples from patients diagnosed with lymphangiectasia revealed sustained MAPK activation within lymphatic endothelial cells and recapitulated the murine phenotype. Mechanistically, the genome-wide phosphorylated MAPK occupancy screen revealed direct regulation of an evolutionarily conserved genetic program required for lymphatic vessel structure and function. This research program aims to identify how the MAPK signaling pathway establishes and maintains a specific transcriptional program for lymphatic vessel development. In addition, proposed studies will identify the mechanisms by which specific kinases and transcription factors interact to regulate the chromatin recruitment of MAPK within developing pulmonary lymphatic vasculature. The set of proposed studies has broad significance for understanding how signaling pathways intersect with chromatin-modifying transcription factors to regulate the development of organ-specific lymphatic vasculature and could be highly applicable to the entire field of congenital vascular diseases.