Cathy Pichol-Thievend

Pkd1 Regulates Lymphatic Vascular Morphogenesis during Development

Lymphatic vessels arise during development through sprouting of precursor cells from veins, which is regulated by known signaling and transcriptional mechanisms. The ongoing elaboration of vessels to form a network is less well understood. This involves cell polarization, coordinated migration, adhesion, mixing, regression, and shape rearrangements. We identified a zebrafish mutant, lymphatic and cardiac defects 1 (lyc1), with reduced lymphatic vessel development. A mutation in polycystic kidney disease 1a was responsible for the phenotype. PKD1 is the most frequently mutated gene in autosomal dominant polycystic kidney disease (ADPKD). Initial lymphatic precursor sprouting is normal in lyc1 mutants, but ongoing migration fails. Loss of Pkd1 in mice has no effect on precursor sprouting but leads to failed morphogenesis of the subcutaneous lymphatic network. Individual lymphatic endothelial cells display defective polarity, elongation, and adherens junctions. This work identifies a highly selective and unexpected role for Pkd1 in lymphatic vessel morphogenesis during development.

VEGFD regulates blood vascular development by modulating SOX18 activity.

Vascular endothelial growth factor-D (VEGFD) is a potent pro-lymphangiogenic molecule during tumor growth and is considered a key therapeutic target to modulate metastasis. Despite roles in pathological neo-lymphangiogenesis, the characterization of an endogenous role for VEGFD in vascular development has remained elusive. Here, we used zebrafish to assay for genetic interactions between the Vegf/Vegf-receptor pathway and SoxF transcription factors and identified a specific interaction between Vegfd and Sox18. Double knockdown zebrafish embryos for Sox18/Vegfd and Sox7/Vegfd exhibit defects in arteriovenous differentiation. Supporting this observation, we found that Sox18/Vegfd double but not single knockout mice displayed dramatic vascular development defects. We find that VEGFD-mitogen-activated protein kinase kinase-extracellular signal-regulated kinase signaling modulates SOX18-mediated transcription, functioning at least in part by enhancing nuclear concentration and transcriptional activity in vascular endothelial cells. This work suggests that VEGFD-mediated pathologies include or involve an underlying dysregulation of SOXF-mediated transcriptional networks.

Vegfc Regulates Bipotential Precursor Division and Prox1 Expression to Promote Lymphatic Identity in Zebrafish

Lymphatic vessels arise chiefly from preexisting embryonic veins. Genetic regulators of lymphatic fate are known, but how dynamic cellular changes contribute during the acquisition of lymphatic identity is not understood. We report the visualization of zebrafish lymphatic precursor cell dynamics during fate restriction. In the cardinal vein, cellular commitment is linked with the division of bipotential Prox1-positive precursor cells, which occurs immediately prior to sprouting angiogenesis. Following precursor division, identities are established asymmetrically in daughter cells; one daughter cell becomes lymphatic and progressively upregulates Prox1, and the other downregulates Prox1 and remains in the vein. Vegfc drives cell division and Prox1 expression in lymphatic daughter cells, coupling signaling dynamics with daughter cell fate restriction and precursor division.

Mural lymphatic endothelial cells regulate meningeal angiogenesis in the zebrafish

Mural cells of the vertebrate brain maintain vascular integrity and function, play roles in stroke and are involved in maintenance of neural stem cells. However, the origins, diversity and roles of mural cells remain to be fully understood. Using transgenic zebrafish, we identified a population of isolated mural lymphatic endothelial cells surrounding meningeal blood vessels. These meningeal mural lymphatic endothelial cells (muLECs) express lymphatic endothelial cell markers and form by sprouting from blood vessels. In larvae, muLECs develop from a lymphatic endothelial loop in the midbrain into a dispersed, nonlumenized mural lineage. muLEC development requires normal signaling through the Vegfc–Vegfd–Ccbe1–Vegfr3 pathway. Mature muLECs produce vascular growth factors and accumulate low-density lipoproteins from the bloodstream. We find that muLECs are essential for normal meningeal vascularization. Together, these data identify an unexpected lymphatic lineage and developmental mechanism necessary for establishing normal meningeal blood vasculature.

SoxF factors induce Notch1 expression via direct transcriptional regulation during early arterial development

Arterial specification and differentiation are influenced by a number of regulatory pathways. While it is known that the Vegfa-Notch cascade plays a central role, the transcriptional hierarchy controlling arterial specification has not been fully delineated. To elucidate the direct transcriptional regulators of Notch receptor expression in arterial endothelial cells, we used histone signatures, DNaseI hypersensitivity and ChIP-seq data to identify enhancers for the human NOTCH1 and zebrafish notch1b genes. These enhancers were able to direct arterial endothelial cell-restricted expression in transgenic models. Genetic disruption of SoxF binding sites established a clear requirement for members of this group of transcription factors (SOX7, SOX17 and SOX18) to drive the activity of these enhancers in vivo. Endogenous deletion of the notch1b enhancer led to a significant loss of arterial connections to the dorsal aorta in Notch pathway-deficient zebrafish. Loss of SoxF function revealed that these factors are necessary for NOTCH1 and notch1b enhancer activity and for correct endogenous transcription of these genes. These findings position SoxF transcription factors directly upstream of Notch receptor expression during the acquisition of arterial identity in vertebrates. Summary: Identification of novel enhancers in vertebrate Notch1 genes places SoxF transcription factors directly upstream of Notch signalling in controlling arterial specification and differentiation.

A blood capillary plexus-derived population of progenitor cells contributes to genesis of the dermal lymphatic vasculature during embryonic development

ABSTRACT Despite the essential role of the lymphatic vasculature in tissue homeostasis and disease, knowledge of the organ-specific origins of lymphatic endothelial progenitor cells remains limited. The assumption that most murine embryonic lymphatic endothelial cells (LECs) are venous derived has recently been challenged. Here, we show that the embryonic dermal blood capillary plexus constitutes an additional, local source of LECs that contributes to the formation of the dermal lymphatic vascular network. We describe a novel mechanism whereby rare PROX1-positive endothelial cells exit the capillary plexus in a Ccbe1-dependent manner to establish discrete LEC clusters. As development proceeds, these clusters expand and further contribute to the growing lymphatic system. Lineage tracing and analyses of Gata2-deficient mice confirmed that these clusters are endothelial in origin. Furthermore, ectopic expression of Vegfc in the vasculature increased the number of PROX1-positive progenitors within the capillary bed. Our work reveals a novel source of lymphatic endothelial progenitors employed during construction of the dermal lymphatic vasculature and demonstrates that the blood vasculature is likely to remain an ongoing source of LECs during organogenesis, raising the question of whether a similar mechanism operates during pathological lymphangiogenesis. Highlighted Article: Genetic lineage tracing and targeted gene disruption and gain of function identify a novel source of lymphatic endothelial progenitors that contribute to the formation of lymphatic vasculature in mouse skin.

Dominant-negative Sox18 function inhibits dermal papilla maturation and differentiation in all murine hair types

SOX family proteins SOX2 and SOX18 have been reported as being essential in determining hair follicle type; however, the role they play during development remains unclear. Here, we demonstrate that Sox18 regulates the normal differentiation of the dermal papilla of all hair types. In guard (primary) hair dermal condensate (DC) cells, we identified transient Sox18 in addition to SOX2 expression at E14.5, which allowed fate tracing of primary DC cells until birth. Similarly, expression of Sox18 was detected in the DC cells of secondary hairs at E16.5 and in tertiary hair at E18.5. Dominant-negative Sox18 mutation (opposum) did not prevent DC formation in any hair type. However, it affected dermal papilla differentiation, restricting hair formation especially in secondary and tertiary hairs. This Sox18 mutation also prevented neonatal dermal cells or dermal papilla spheres from inducing hair in regeneration assays. Microarray expression studies identified WNT5A and TNC as potential downstream effectors of SOX18 that are important for epidermal WNT signalling. In conclusion, SOX18 acts as a mesenchymal molecular switch necessary for the formation and function of the dermal papilla in all hair types. Summary: The Sox18Op/+ mutation in mice, which mimics a hair loss condition in humans, leads to decreasing Wnt signal interactions with the hair follicle epidermis and abnormal hair follicle morphology.

Lymphatic vascular specification and its modulation during embryonic development

Despite its essential roles in development and disease, the lymphatic vascular system has been less studied than the blood vascular network. In recent years, significant advances have been made in understanding the mechanisms that regulate lymphatic vessel formation, both during development and in pathological conditions. Remarkably, lymphatic endothelial cells are specified as a subpopulation of pre-existing venous endothelial cells. Here, we summarize the current knowledge of the transcription factor pathways responsible for lymphatic specification and we also focus on the factors that promote or restrict this event.

AB0095 Forelimb Development in the Mouse: an Original Model of Organo-Dependent Lymphangiogenesis

Background Quantitative and qualitative changes affecting the lymphatic network are associated with limb and joint diseases including rheumatoid arthritis and osteoarthritis at the adult age. However how lymphatics develop during embryogenesis in the limbs remain largely unexplored. Objectives To describe for the first time lymphangiogenesis in mouse forelimbs and joints during early embryogenesis. Methods Prox1 is a key transcription factor involved in lymphatic specification and remodelling. Taking advantage of the Prox1-GFP reporter transgenic mouse line, we described lymphangiogenesis in the forelimbs and in the joints during early embryogenesis from 9.5 (E9.5) to 14.5 (E14.5) days post-coitum using confocal imaging on fixed samples stained with specific markers for lymphatic and vascular endothelia, as well as for surrounding specifying tissues including cartilage, muscles and peripheral nerves. Timelapse imaging was also performed on specimens collected at E11.5 and E12.5 to address lymphangiogenesis ex vivo. Results From E9.5 to E10.5, Prox1 positive cells emerge directly from the cardinal vein and are located at the limb bud, following an antero-posterior gradient. Elongated shape suggests migrating cells. Other markers for lymphatics including neuropillin-2 and endothelium including endomucin are expressed in a dense unpatterned vascular network involving the future forelimb suggesting an undifferentiated primary capillary plexus. From E11.5 to E12.5, this unpatterned network undergoes an extensive remodelling with the formation of an avascular zone as the skeleton mesenchyme is condensing. More strikingly, Prox1 positive cells emerge directly from this capillary plexus, suggesting an original mechanism of local lymphangiogenesis. In addition to this process, ex vivo imaging at E11.5 and E12.5 reveals that sprouting, branching, cell division and networking from the forelimb marginal vein also occur, still following an antero-posterior gradient. Later on, vascular network patterning is coordinated to other tissues specification including peripheral nerves, joints and muscles. Conclusions Altogether, our data suggest that the development of lymphatics in the forelimb represents an original model of organo-dependent lymphangiogenesis associating migration from the cardinal vein and local lymphangiogenesis from local precursors. Acknowledgements Dr Christelle Nguyen received fundings from the French Society of Rheumatology (SFR), the French Society of Rehabilitation Medicine (SOFMER) and the Bettencourt-Schueller Foundation. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3954