A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy

Abstract

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.

Pdgfrb‐Cre targets lymphatic endothelial cells of both venous and non‐venous origins

“…To assess the specificity of Pdgfrb‐Cre mediated targeting of LECs of different origins, we analysed Cre mediated recombination in the cardinal vein that provides a source of the first venous derived LECs, the peripheral longitudinal lymphatic vessel and the primordial thoracic duct, that are also referred to as the jugular lymph sacs (JLS) (Hagerling et al, 2013; Srinivasan et al, 2007; Yang et al, 2012). Immunofluorescence analysis of E13.5 Pdgfrb‐Cre ; R26‐mTmG embryos showed the expected GFP + perivascular cells (Foo et al, 2006) (Fig.…”

Cdk5 controls lymphatic vessel development and function by phosphorylation of Foxc2

“…Therefore, our further studies focused on analysis of the lymphatic valve phenotype. We analysed whether the role of Cdk5 in lymphatic valve formation also encompasses lymphovenous valves, which separate the primordial thoracic duct (pTD) and adjacent cardinal vein (CV) 3,4,25,26 . Three-dimensional reconstructions of image stacks obtained by optical sectioning of entire whole-mount immunostained E12.5 embryos showed lymphovenous valves, that is, two contact sites of double layer of endothelial cells with high Prox1 expression, in control littermates and demonstrated frequent failure of lymphovenous valve formation in Cdk5 fl/fl Tie2Cre embryos (Fig.…”

“…Whole-mount staining of E12.5 embryos was performed as previously described 3 . In brief, embryos were fixed (2 h RT), permeabilized (0.5% TX in PBS, 4°C, 48 h), blocked (1% BSA, 0.1% Tween20 in PBS, 4°C, 48 h), incubated with primary antibodies (anti-Prox1, 102-PA32 Relia Tech; anti-CD31 553370 BD Biosciences; anti-VEGFR3, AF743 R&D Systems) in blocking solution (1:200, 4°C, 1 week), washed (0.1% Tween in PBS, 2 days), incubated with secondary antibodies (AlexaFlour, Life Technologies, 1:1,000, 4°C, 1 week) and washed (0.1% Tween in PBS, 2d) before clearing with BABB.…”

“…Lymphatic endothelial cells emerge from the cardinal veins and migrate away to form the primary lymphatic vessels, that is, the lymph sacs, which get separated from the blood vasculature by lymphovenous valves [2][3][4] . Peripheral lymphatic vessels remodel into lymphatic capillaries that take up interstitial fluid and collecting lymphatic vessels that drain the lymph into the venous system.…”

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

“…CD31 is a 130 kDa transmembrane glycoprotein also known as platelet endothelial cell adhesion molecule 1 (PECAM-1). It is considered to be a pan-endothelial cell marker since it is expressed on all types of blood and lymphatic vessels 21,24,25 . CD34 is 110-kDa transmembrane glycoprotein present on most hematopoietic progenitor and stem cells, vascular endothelial cells and some lymphatic vessels 26 .…”