Lymphatic valves are essential for efficient lymphatic transport, but the mechanisms of early lymphatic-valve morphogenesis and the role of biomechanical forces are not well understood. We found that the transcription factors PROX1 and FOXC2, highly expressed from the onset of valve formation, mediate segregation of lymphatic-valve-forming cells and cell mechanosensory responses to shear stress in vitro. Mechanistically, PROX1, FOXC2, and flow coordinately control expression of the gap junction protein connexin37 and activation of calcineurin/NFAT signaling. Connexin37 and calcineurin are required for the assembly and delimitation of lymphatic valve territory during development and for its postnatal maintenance. We propose a model in which regionally increased levels/activation states of transcription factors cooperate with mechanotransduction to induce a discrete cell-signaling pattern and morphogenetic event, such as formation of lymphatic valves. Our results also provide molecular insights into the role of endothelial cell identity in the regulation of vascular mechanotransduction.

Lymphatic vessels are lined by lymphatic endothelial cells (LECs), and are critical for health. However, the role of metabolism in lymphatic development has not yet been elucidated. Here we report that in transgenic mouse models, LEC-specific loss of CPT1A, a rate-controlling enzyme in fatty acid β-oxidation, impairs lymphatic development. LECs use fatty acid β-oxidation to proliferate and for epigenetic regulation of lymphatic marker expression during LEC differentiation. Mechanistically, the transcription factor PROX1 upregulates CPT1A expression, which increases acetyl coenzyme A production dependent on fatty acid β-oxidation. Acetyl coenzyme A is used by the histone acetyltransferase p300 to acetylate histones at lymphangiogenic genes. PROX1-p300 interaction facilitates preferential histone acetylation at PROX1-target genes. Through this metabolism-dependent mechanism, PROX1 mediates epigenetic changes that promote lymphangiogenesis. Notably, blockade of CPT1 enzymes inhibits injury-induced lymphangiogenesis, and replenishing acetyl coenzyme A by supplementing acetate rescues this process in vivo.

The cytoplasmic protein tyrosine kinase Syk has two amino-terminal SH2 domains that engage phosphorylated immunoreceptor tyrosine-based activation motifs in the signaling subunits of immunoreceptors. Syk, in conjunction with Src family kinases, has been implicated in immunoreceptor signaling in both lymphoid and myeloid cells. We have investigated the role of Syk in Fc␥ receptor (Fc␥R)-dependent and -independent responses in bone marrow-derived macrophages and neutrophils by using mouse radiation chimeras reconstituted with fetal liver cells from Syk ؊/؊ embryos. Chimeric mice developed an abdominal hemorrhage starting 2 to 3 months after transplantation that was ultimately lethal. Syk-deficient neutrophils derived from the bone marrow were incapable of generating reactive oxygen intermediates in response to Fc␥R engagement but responded normally to tetradecanoyl phorbol acetate stimulation. Syk-deficient macrophages were defective in phagocytosis induced by Fc␥R but showed normal phagocytosis in response to complement. The tyrosine phosphorylation of multiple cellular polypeptides, including the Fc␥R ␥ chain, as well as Erk2 activation, was compromised in Syk ؊/؊ macrophages after Fc␥R stimulation. In contrast, the induction of nitric oxide synthase in macrophages stimulated with lipopolysaccharide and gamma interferon was not dependent on Syk. Surprisingly, Syk-deficient macrophages were impaired in the ability to survive or proliferate on plastic petri dishes. Taken together, these results suggest that Syk has specific physiological roles in signaling from Fc␥Rs in neutrophils and macrophages and raise the possibility that in vivo, Syk is involved in signaling events other than those mediated by immunoreceptors.The cytoplasmic tyrosine kinase Syk has been implicated in a variety of hematopoietic cell responses, including immunoreceptor (1,3,4,12,22,29,44) and integrin signaling (7, 15). Syk possesses two N-terminal SH2 domains that bind in tandem to closely spaced pTyr sites located within the immunoreceptor tyrosine-based activation motifs (ITAMs) of antigen receptor subunits, such as the ␣ and ␤ chains associated with surface immunoglobulin M (IgM) in B cells (30) or the ␥ and ␤ subunits of FcεRI in mast cells (27,42).Engagement of the B-cell antigen receptor (BCR) has been suggested to result in the activation of Src family kinases, followed by phosphorylation of ITAMs and, consequently, recruitment of the Syk SH2 domains. Syk is activated, either as a direct result of SH2 binding to the phospho-ITAM or through transphosphorylation by a Src family kinase (6,31,40,43,56). Activated Syk can phosphorylate downstream targets (38) and recruits additional SH2-containing proteins that bind to pTyr sites in its SH2-kinase linker region (33). The related tyrosine kinase ZAP-70 appears to play a similar role in signaling from the T-cell receptor (TCR) (2, 53).Targeted mutagenesis of the Syk gene (5, 50) has revealed important functions for this kinase in B-and T-cell development, in BCR signaling, in macrophages, in...

During mammalian development, a subpopulation of endothelial cells in the cardinal vein (CV) expresses lymphatic-specific genes and subsequently develops into the first lymphatic structures, collectively termed as lymph sacs. Budding, sprouting and ballooning of lymphatic endothelial cells (LECs) have been proposed to underlie the emergence of LECs from the CV, but the exact mechanisms of lymph vessel formation remain poorly understood. Applying selective plane illumination-based ultramicroscopy to entire wholemount-immunostained mouse embryos, we visualized the complete developing vascular system with cellular resolution. Here, we report emergence of the earliest detectable LECs as strings of loosely connected cells between the CV and superficial venous plexus. Subsequent aggregation of LECs resulted in formation of two distinct, previously unidentified lymphatic structures, the dorsal peripheral longitudinal lymphatic vessel (PLLV) and the ventral primordial thoracic duct (pTD), which at later stages formed a direct contact with the CV. Providing new insights into their function, we found vascular endothelial growth factor C (VEGF-C) and the matrix component CCBE1 indispensable for LEC budding and migration. Altogether, we present a significantly more detailed view and novel model of early lymphatic development.

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