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Intercellular communication orchestrates a multitude of physiologic and pathologic conditions. Algorithms to infer cell–cell communication and predict downstream signalling and regulatory networks are needed to illuminate mechanisms of stem cell differentiation and tissue development. Here, to fill this gap, we developed and applied CellComm to investigate how the aorta–gonad–mesonephros microenvironment dictates haematopoietic stem and progenitor cell emergence. We identified key microenvironmental signals and transcriptional networks that regulate haematopoietic development, including Stat3, Nr0b2, Ybx1 and App, and confirmed their roles using zebrafish, mouse and human models. Notably, CellComm revealed extensive crosstalk among signalling pathways and convergence on common transcriptional regulators, indicating a resilient developmental programme that ensures dynamic adaptation to changes in the embryonic environment. Our work provides an algorithm and data resource for the scientific community.

The hierarchical organization of properly sized blood vessels ensures the correct distribution of blood to all organs of the body, and is controlled via haemodynamic cues. In current concepts, an endothelium-dependent shear stress set point causes blood vessel enlargement in response to higher flow rates, while lower flow would lead to blood vessel narrowing, thereby establishing homeostasis. We show that during zebrafish embryonic development increases in flow, after an initial expansion of blood vessel diameters, eventually lead to vessel contraction. This is mediated via endothelial cell shape changes. We identify the transforming growth factor beta co-receptor endoglin as an important player in this process. Endoglin mutant cells and blood vessels continue to enlarge in response to flow increases, thus exacerbating pre-existing embryonic arterial-venous shunts. Together, our data suggest that cell shape changes in response to biophysical cues act as an underlying principle allowing for the ordered patterning of tubular organs.

While the generation of hematopoietic progenitor cells from pluripotent stem cells (hiPSCs) can readily be accomplished in a dish, transgene free generation of long term reconstituting hematopoietic stem cells (HSCs) remains a major challenge to the field. In order to recapitulate definitive hematopoiesis in vitro, we need a better understanding of the developmental processes occurring during HSC emergence in vivo. Despite having identified the origin of HSCs to be specialized endothelium, termed hemogenic endothelium, we have yet to fully characterize all distinct cell types which may play a role during the process of endothelial-to-hematopoietic transition. Here, we have utilized single cell RNA sequencing to profile two major hematopoietic organs in the mouse. The aorta-gonad-mesonephros (AGM) region was analyzed on embryonic day (E)10.5, just prior to the first emergence of HSCs, and the placenta on E11.5, prior to its peak of HSC potential. In addition to capturing endothelial and nascent hematopoietic stem cells, we also included cells of the surrounding embryonic tissue, allowing us to computationally predict cell-cell interactions based on ligand and receptor expression. This approach validated previously described regulators of HSC emergence, i.e. the recently published surface antigen CD44, while also predicting additional signaling pathways, which have not been implicated in HSC development so far. Using a morpholino based knockdown screening approach in zebrafish, we tested the effect of the predicted target ligand and receptor interactions on the presence of runx1+/cmyb+ cells in the aorta. Following this approach, we were able to identify novel modulators of HSC emergence in the AGM. Strikingly, direct comparison between the AGM and placental dataset revealed a population of cells within the placenta with a transcriptional signature highly similar to AGM hemogenic endothelium. Functional validation of these cells will test their ability to acquire hemogenic potential after in vitro maturation in the presence of a supportive stromal layer. Implementation of the transcriptional and cell-cell communication findings will aid enhancement of hiPSC-derived hematopoietic differentiation protocols toward the production of functionally competent HSCs.

Hematopoietic stem cells (HSCs) are born from cells in the dorsal aorta that adopt hematopoietic potential and commitment during development. Physical forces, namely wall shear stress (WSS) and cyclic stretch (CS), produced by hemodynamic blood flow are required to generate HSCs from arterial endothelium, but the mechanisms by which these forces are transduced into a “stemness” regulatory program remain unknown. Using a novel “dorsal-aorta-on-a-chip” microfluidic platform, we demonstrate that exposure to WSS and CS increases expression of the hematopoietic transcription factor RUNX1 in human CD34+ hemogenic endothelial cells derived from induced pluripotent stem cells. Notably, CS specifically induced activity of the YAP transcription factor, a mechanically-activated regulator of organ size and pluripotency. We corroborate these findings in a zebrafish model, and demonstrate that loss or gain of YAP function in vivo can blunt or augment the production of HSCs during definitive hematopoiesis. Importantly, we find that YAP is responsible for the maintenance, not initiation, of the hematopoietic program in newly specified hemogenic endothelial cells. Molecularly, we identify a stretch-induced, RhoGTPase-dependent mechanotransduction network controlling YAP nuclear availability to regulate RUNX1 expression, both in zebrafish and human cells. Moreover, we show that small molecule stimulation of RhoGTPases can rescue HSC production in zebrafish embryos with no flow, and enhance the hematopoietic potential of human iPSC-derived CD34+ endothelial cells grown in static culture. Together these findings uncover YAP as a transcriptional regulator of stem cell fate commitment during tissue morphogenesis, and suggest a pharmacologically amenable mechanotransduction pathway that could be exploited to improve the in vitro derivation of HSCs from human cells for therapeutic purposes.

Hematopoietic stem and progenitor cells (HSPCs), first specified from hemogenic endothelium (HE) in the ventral dorsal aorta (VDA), support lifelong hematopoiesis. Their de novo production promises significant therapeutic value; however, current in vitro approaches cannot efficiently generate multipotent long-lived HSPCs. Presuming this reflects a lack of extrinsic cues normally impacting the VDA, we devised a human dorsal aorta-on-a-chip platform that identified Yes-activated protein (YAP) as a cyclic stretch-induced regulator of HSPC formation. In the zebrafish VDA, inducible Yap overexpression significantly increased runx1 expression in vivo and the number of CD41+ HSPCs downstream of HE specification. Endogenous Yap activation by lats1/2 knockdown or Rho-GTPase stimulation mimicked Yap overexpression and induced HSPCs in embryos lacking blood flow. Notably, in static human induced pluripotent stem cell (iPSC)-derived HE culture, compound-mediated YAP activation enhanced RUNX1 levels and hematopoietic colony-forming potential. Together, our findings reveal a potent impact of hemodynamic Rho-YAP mechanotransduction on HE fate, relevant to de novo human HSPC production.