The establishment of separated pulmonary and systemic circulation in vertebrates, via cardiac outflow tract (OFT) septation, is a sensitive developmental process accounting for 10% of all congenital anomalies. Neural Crest Cells (NCC) colonising the heart condensate along the primitive endocardial tube and force its scission into two tubes. Here, we show that NCC aggregation progressively decreases along the OFT distal-proximal axis following a BMP signalling gradient. Dullard, a nuclear phosphatase, tunes the BMP gradient amplitude and prevents NCC premature condensation. Dullard maintains transcriptional programs providing NCC with mesenchymal traits. It attenuates the expression of the aggregation factor Sema3c and conversely promotes that of the epithelial-mesenchymal transition driver Twist1. Altogether, Dullard-mediated fine-tuning of BMP signalling ensures the timed and progressive zipper-like closure of the OFT by the NCC and prevents the formation of a heart carrying the congenital abnormalities defining the tetralogy of Fallot.

Generating comprehensive image maps, while preserving spatial 3D context, is essential to quantitatively assess and locate specific cellular features and cell-cell interactions during organ development. Despite the recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on 2D histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in 3D and map tissue interactions at key time points in the mouse embryo. We demonstrate the utility of the approach by providing volumetric data, 3D distribution of three main cellular components (epithelial, mesenchymal, endothelial) within the developing pancreas, and quantification of their relative cellular abundance within the tissue. Interestingly, our 3D images show that endocrine cells are constantly and increasingly in contact with endothelial cells forming small vessels, while the interactions with mesenchymal cells decrease over time. These findings suggest distinct cell-cell interaction requirement for early endocrine cell specification and late differentiation. Lastly, we combine our image data in an open-source online repository (referred to as Pancreas Embryonic Cell Atlas).

During organ formation, progenitor cells need to acquire the diversity of cell identities found in the organ as well as organize themselves into distinct structural units. How these processes are coordinated, and how tissue architecture(s) are preserved despite the dramatic cell rearrangements occurring in developing organs remain unclear. Here, we identified cellular rearrangements between acinar and ductal progenitors as a mechanism to drive branching morphogenesis in the pancreas while preserving the integrity of the acinar-ductal functional unit. Using ex vivo and in vivo mouse models, we found that pancreatic ductal cells form clefts by protruding and pulling on the acinar basement membrane, which lead to acini splitting. Newly formed acini remain connected to bifurcated branches generated by ductal cell rearrangement. IGF/PI3K pathway regulates this process by controlling ductal cell fluidity. If components of the pathway are genetically or chemically dysregulated, ductal cell fluidity prevents branching and affects pancreatic cell fates. Hence, our results explain how acinar multiplication and branch bifurcation are synchronized during pancreas organogenesis.

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...

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Generating comprehensive image maps, while preserving spatial 3D context, is essential to quantitatively assess and locate specific cellular features and cell-cell interactions during organ development. Despite the recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on 2D histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in 3D and map tissue interactions at key development time points in the mouse embryo. We used transgenic mouse models and antibodies to visualize the three main cellular components within the developing pancreas, including epithelial, mesenchymal and endothelial cell populations. We demonstrated the utility of the approach by providing volumetric data, 3D distribution of distinct progenitor populations and quantification of relative cellular abundance within the tissue. Lastly, our image data were combined in an open source online repository (referred to as Pancreas Embryonic Cell Atlas). This image dataset will serve the scientific community by enabling further investigation on pancreas organogenesis but also for devising strategies for the in vitro generation of transplantable pancreatic tissue for regenerative therapies.

Our understanding of how human skin cells differ according to anatomical site and tumour formation is limited. To address this we have created a multi-scale spatial atlas of healthy skin and basal cell carcinoma (BCC), incorporating in vivo optical coherence tomography, single cell RNA sequencing, spatial global transcriptional profiling and in situ sequencing. Computational spatial deconvolution and projection revealed the localisation of distinct cell populations to specific tissue contexts. Although cell populations were conserved between healthy anatomical sites and in BCC, mesenchymal cell populations including fibroblasts and pericytes retained signatures of developmental origin. Spatial profiling and in silico lineage tracing support a hair follicle origin for BCC and demonstrate that cancer-associated fibroblasts are an expansion of a POSTN+ subpopulation associated with hair follicles in healthy skin. RGS5+ pericytes are also expanded in BCC suggesting a role in vascular remodelling. We propose that the identity of mesenchymal cell populations is regulated by signals emanating from adjacent structures and that these signals are repurposed to promote the expansion of skin cancer stroma. The resource we have created is publicly available in an interactive format for the research community.