Embryo implantation remains challenging to study because of its inaccessibility in situ despite its essentiality and clinical significance. Although recent studies on long-term culture of authentic and model embryos have provided significant advances in elucidating embryogenesis in vitro, they, without the uterus, cannot genuinely replicate implantation. Here, we have recapitulated bona fide implantation ex vivo at more than 90% efficiency followed by embryogenesis and trophoblast invasion using authentic mouse embryos and uterine tissue. We utilized air-liquid interface culture method with originally developed devices manufactured with polydimethylsiloxane. Notably, the system replicated the robust induction of a maternal implantation regulator COX-2 at the attachment interface, which was accompanied by trophoblastic AKT activation, suggesting a possible signaling that mediates maternal COX-2 and embryonic AKT1 that accelerates implantation. By expanding the ex vivo findings, embryonic AKT1 transduction ameliorated defective implantation of uterine origin by a COX-2 inhibitor in vivo. The system, proposing a potentially standard platform of embryogenesis, offers a concise, reproducible, and scalable screening system, suggesting significant implications for developmental biology and therapeutic strategies for recurrent implantation failure in assisted reproductive technology.
© 2025. The Author(s).

During embryogenesis, endothelial cells (ECs) are generally described to arise from a common pool of progenitors termed angioblasts, which diversify through iterative steps of differentiation to form functionally distinct subtypes of ECs. A key example is the formation of lymphatic ECs (LECs), which are thought to arise largely through transdifferentiation from venous endothelium. Opposing this model, here we show that the initial expansion of mammalian LECs is primarily driven by the in situ differentiation of mesenchymal progenitors and does not require transition through an intermediate venous state. Single-cell genomics and lineage-tracing experiments revealed a population of paraxial mesoderm-derived Etv2+Prox1+ progenitors that directly give rise to LECs. Morphometric analyses of early LEC proliferation and migration, and mutants that disrupt lymphatic development supported these findings. Collectively, this work establishes a cellular blueprint for LEC specification and indicates that discrete pools of mesenchymal progenitors can give rise to specialized subtypes of ECs.
© 2025. The Author(s).

The loss of a single copy of <i>TBX1</i> accounts for most of the clinical signs and symptoms of 22q11.2 deletion syndrome, a common genetic disorder that is characterized by multiple congenital anomalies and brain-related clinical problems, some of which likely have vascular origins. <i>Tbx1</i> mutant mice have brain vascular anomalies, thus making them a useful model to gain insights into the human disease. Here, we found that the main morphogenetic function of TBX1 in the mouse brain is to suppress vessel branching morphogenesis through regulation of <i>Vegfr3</i> We demonstrate that inactivating <i>Vegfr3</i> in the <i>Tbx1</i> expression domain on a <i>Tbx1</i> mutant background enhances brain vessel branching and filopodia formation, whereas increasing <i>Vegfr3</i> expression in this domain fully rescued these phenotypes. Similar results were obtained using an in vitro model of endothelial tubulogenesis. Overall, the results of this study provide genetic evidence that <i>VEGFR3</i> is a regulator of early vessel branching and filopodia formation in the mouse brain and is a likely mediator of the brain vascular phenotype caused by <i>Tbx1</i> loss of function.
© 2022 Cioffi et al.

The lymphatic vasculature is essential for tissue fluid homeostasis, immune cell surveillance and dietary lipid absorption, and has emerged as a key regulator of organ growth and repair 1 . Despite significant advances in our understanding of lymphatic function, the precise developmental origin of lymphatic endothelial cells (LECs) has remained a point of debate for over a century 2-5 . It is currently widely accepted that most LECs are derived from venous endothelium 4,6 , although other sources have been described, including mesenchymal cells 3 , hemogenic endothelium 7 and musculoendothelial progenitors 8,9 . Here we show that the initial expansion of mammalian LECs is driven primarily by the in situ differentiation of specialized angioblasts and not migration from venous endothelium. Single-cell RNA sequencing and genetic lineage tracing experiments in mouse revealed a population of Etv2 + Prox1 + lymphangioblasts that arise directly from paraxial mesoderm-derived progenitors. Conditional lineage labelling and morphological analyses showed that these specialized angioblasts emerge within a tight spatiotemporal window, and give rise to LECs in numerous tissues. Analysis of early LEC proliferation and migration supported these findings, suggesting that emergence of LECs from venous endothelium is limited. Collectively, our data reconcile discrepancies between previous studies and indicate that LECs form through both de novo specification from lymphangioblasts and transdifferentiation from venous endothelium.

h4>ABSTRACT/h4> The loss of a single copy of TBX1 accounts for most of the clinical signs and symptoms of 22q11.2 deletion syndrome (22q11.2DS), a common genetic disorder that is characterized by multiple congenital anomalies and brain-related clinical problems, some of which likely have vascular origins. Tbx1 mutant mice have brain vascular anomalies, thus making them a useful model to gain insights into the brain disorders associated with the human disease. Here, we found that Tbx1 has a dynamic expression pattern in brain endothelial cells (ECs), including tip cells, during early vascularization, but it is not expressed in EC progenitors. Its main morphogenetic function in the brain is to regulate negatively filopodia biogenesis and vessel branching. Because of similar phenotypes reported for Vegfr3 loss of function, we pursued a mouse genetic approach to test TBX1-VEGFR3 interaction through gain and loss of function experiments. Vegfr3 is expressed in brain ECs with extensive overlap with Tbx1 expression. We demonstrate that inactivating Vegfr3 in the Tbx1 expression domain in a Tbx1 mutant background enhances vessel branching and filopodia formation to a greater extent than that observed in the individual mutants. Furthermore, using a mouse transgenic line, we show that increasing Vegfr3 expression in the Tbx1 expression domain fully rescued the vessel branching and filopodia phenotypes caused by Tbx1 loss of function. Similar results were obtained using an in vitro model of endothelial tubulogenesis. Overall, these results provide genetic evidence that Vegfr3 is a regulator of early vessel branching and filopodia formation in the brain, and is a likely critical effector of the brain vascular phenotype caused by Tbx1 loss of function.