The forming limb skeleton serves as a signaling center for limb vasculature patterning via regulation of Vegf

Idit Eshkar-Oren, Sergey V. Viukov, Sharbel Salameh, Sharon Krief, Chun-do Oh, Haruhiko Akiyama, Hans-Peter Gerber, Napoleone Ferrara and Elazar Zelzer

Limb development constitutes a central model for the study of tissue and organ patterning; yet, the mechanisms that regulate the patterning of limb vasculature have been left understudied. Vascular patterning in the forming limb is tightly regulated in order to ensure sufficient gas exchange and nutrient supply to the developing organ. Once skeletogenesis is initiated, limb vasculature undergoes two seemingly opposing processes: vessel regression from regions that undergo mesenchymal condensation; and vessel morphogenesis. During the latter, vessels that surround the condensations undergo an extensive rearrangement, forming a stereotypical enriched network that is segregated from the skeleton. In this study, we provide evidence for the centrality of the condensing mesenchyme of the forming skeleton in regulating limb vascular patterning. Both Vegf loss- and gain-of-function experiments in limb bud mesenchyme firmly established VEGF as the signal by which the condensing mesenchyme regulates the vasculature. Normal vasculature observed in limbs where VEGF receptors Flt1, Flk1, Nrp1 and Nrp2 were blocked in limb bud mesenchyme suggested that VEGF, which is secreted by the condensing mesenchyme, regulates limb vasculature via a direct long-range mechanism. Finally, we provide evidence for the involvement of SOX9 in the regulation of Vegf expression in the condensing mesenchyme. This study establishes Vegf expression in the condensing mesenchyme as the mechanism by which the skeleton patterns limb vasculature.

Prox1 physically and functionally interacts with COUP-TFII to specify lymphatic endothelial cell fate

Sunju Lee, Jinjoo Kang, Jaehyuk Yoo, Sathish K. Ganesan, Sarah C. Cook, Berenice Aguilar, Swapnika Ramu, Juneyong Lee, and Young-Kwon Hong

Specification of endothelial cell (EC) fate during vascular development is controlled by distinct key regulators. While Notch plays an essential role in induction of arterial phenotypes, COUP-TFII is required to maintain the venous EC identity. Homeodomain transcription factor Prox1 functions to reprogram venous ECs to lymphatic endothelial cells (LECs). Here, we report that the venous EC fate regulator COUP-TFII is expressed in LECs throughout development and physically interacts with Prox1 to form a stable complex in various cell types including LECs. We found that COUP-TFII functions as a coregulator of Prox1 to control several lineage-specific genes including VEGFR-3, FGFR-3, and neuropilin-1 and is required along with Prox1 to maintain LEC phenotype. Together, we propose that the physical and functional interactions of the 2 proteins constitute an essential part in the program specifying LEC fate and may provide the molecular basis for the hypothesis of venous EC identity being the prerequisite for LEC specification.

A mechanism of sialylation functionally silences the hyaluronan receptor LYVE-1 in lymphatic endothelium

Thomas D. Nightingale, Matthew E. F. Frayne, Steven Clasper, Suneale Banerji, and David G. Jackson

The major lymphatic endothelial hyaluronan receptor LYVE-1, a Link superfamily glycoprotein similar to the hyaluronan-binding/inflammatory leukocyte homing receptor CD44 was initially implicated in hyaluronan (HA) -mediated cell adhesion and lymph borne hyaluronan metabolism. However, the apparently normal phenotype of LYVE-1 knockout mice and the recent demonstration that the receptor undergoes cytokine-induced endocytosis independent of HA uptake have cast doubt on such functions. Here we present new data that reconcile these anomalies by showing that LYVE-1 is functionally "silenced" in cell specific fashion by autoinhibitory glycosylation. We demonstrate that LYVE-1 transfected in HEK 293T fibroblasts and Jurkat T cells is competent to bind HA, whereas the endogenous receptor in cultured lymphatic endothelial cells, or the receptor transfected in CHO and HeLa cells is not. Moreover, through a combination of mutagenesis and functional analysis in HEK 293T fibroblasts and glycosylation defective CHO cell lines we reveal that the inhibitory mechanism is reversible and is exerted by terminal sialylation most likely through 2-3 or 2-6 linkage to O-glycans. Finally, we provide evidence that the mechanism operates in vivo by showing that native LYVE-1 in primary lymphatic endothelial cells is extensively sialylated and that HA binding can be reactivated by neuraminidase treatment of the soluble ectodomain. These results reveal unexpected complexity in the regulation of LYVE-1 function and raise the possibility that this receptor, like CD44, may become active after appropriate unmasking in vivo.

Endothelial cell O-glycan deficiency causes blood/lymphatic misconnections and consequent fatty liver disease in mice

Jianxin Fu, Holger Gerhardt, J. Michael McDaniel, Baoyun Xia, Xiaowei Liu, Lacramioara Ivanciu, Annelii Ny, Karlien Hermans, Robert Silasi-Mansat, Samuel McGee, Emma Nye, Tongzhong Ju, Maria I. Ramirez, Peter Carmeliet, Richard D. Cummings, Florea Lupu, and Lijun Xia

Mucin-type O-glycans (O-glycans) are highly expressed in vascular ECs. However, it is not known whether they are important for vascular development. To investigate the roles of EC O-glycans, we generated mice lacking T-synthase, a glycosyltransferase encoded by the gene C1galt1 that is critical for the biosynthesis of core 1–derived O-glycans, in ECs and hematopoietic cells (termed here EHC T-syn–/– mice). EHC T-syn–/– mice exhibited embryonic and neonatal lethality associated with disorganized and blood-filled lymphatic vessels. Bone marrow transplantation and EC C1galt1 transgene rescue demonstrated that lymphangiogenesis specifically requires EC O-glycans, and intestinal lymphatic microvessels in EHC T-syn–/– mice expressed a mosaic of blood and lymphatic EC markers. The level of O-glycoprotein podoplanin was significantly reduced in EHC T-syn–/– lymphatics, and podoplanin-deficient mice developed blood-filled lymphatics resembling EHC T-syn–/– defects. In addition, postnatal inactivation of C1galt1 caused blood/lymphatic vessel misconnections that were similar to the vascular defects in the EHC T-syn–/– mice. One consequence of eliminating T-synthase in ECs and hematopoietic cells was that the EHC T-syn–/– pups developed fatty liver disease, because of direct chylomicron deposition via misconnected portal vein and intestinal lymphatic systems. Our studies therefore demonstrate that EC O-glycans control the separation of blood and lymphatic vessels during embryonic and postnatal development, in part by regulating podoplanin expression.