28Establishment of separated pulmonary and systemic circulations in vertebrates relies on the key 29 role of neural crest cells (NCC) for the septation of the embryonic cardiac outflow tract (OFT). 30Absence of NCCs induces OFT septation defects, analogous to a loss of Bone Morphogenetic 31Proteins (BMPs) activity, though it remains unclear how BMPs control cardiac NCC differentiation 32 and behaviour. To address this question, we monitored cardiac NCC state upon gain in BMP 33 signaling, caused by the deletion of Dullard, using 3D-imaging and single cell transcriptomics. 34 Specific loss of Dullard in the NCC results in premature OFT septation, pulmonary artery 35 obstruction and embryonic death. This is caused by uncontrolled NCC convergence towards the 36 endocardium and asymmetrical myocardial differentiation, promoted by elevated levels of the 37 guiding cue Sema3c and decreased levels in mesenchymal trait markers. Furthermore, we 38 unraveled the molecular basis of the zipper-like OFT septation where graded Sema3c expression 39 follow a gradient of BMP activation in NCC along the OFT length. 40 41 Keywords 42 43 Neural crest cells, Outflow tract, BMP signaling, Sema3c, Dullard 44 45 46 47The embryonic OFT is a solitary tube called truncus arteriosus, which is remodelled into the aortic 48 (Ao) and pulmonary (Pa) arteries (1) (Fig. 1A). This morphogenesis is orchestrated in time and 49 space by several cross-interacting cell-types including the myocardial progenitors of the second 50 heart field (SHF), the endocardial cells (EC) delineating the OFT lumen, and the cardiac neural 51 crest cells (cNCC) (2,3) ( Fig. 1A). Various genetic manipulations or ablations have highlighted the 52 predominant role of cNCC in initiating and controlling OFT septation (4,5). Originally, cNCC 53 delaminate from the dorsal neural tube and migrate through the pharyngeal mesoderm to reach 54 the developing OFT ( Fig. 1A). They invade the two cardiac cushions, condense towards the 55 endocardium and trigger its rupture, thereby creating the two great arteries (6,7). The rupture of 56 the endocardium is first detected at the distal levels of the OFT, at E11.5 in mouse, and gets 57 progressively established at more proximal levels. 58
59The intense investigations to identify the molecular cues controlling cNCC stereotyped behaviour 60 and differentiation in the OFT mesenchyme have established the importance of the Bone 61 Morphogenic Proteins (BMP), secreted by the outlying myocardium cells (8-10). Indeed, BMP 62 signaling loss-of-function within the NCC lineage leads to the formation of hypoplastic cushions, 63 a shorter and non-septated OFT, phenocopying the absence of cNCC (11-13). However, little is 64 known on the molecular cascades triggered by BMP signalling responsible for the cNCC mediated 65 OFT septation. 66 OFT axis set the tempo of OFT septation from its distal to its proximal regions. Hence, our findings 80 reveal that fine tuning of BMP signaling levels in cNCC orchestrate OFT septation in time and 81 space. 82 83 Res...