The proepicardium delivers hemangioblasts but not lymphangioblasts to the developing heart

The mass of the myocardium and endocardium of the vertebrate heart derive from the heart-forming fields of the lateral plate mesoderm. Further components of the mature heart such as the epicardium, cardiac interstitium and coronary blood vessels originate from a primarily extracardiac progenitor cell population: the proepicardium (PE). The coronary blood vessels are accompanied by lymph vessels, suggesting a common origin of the two vessel types. However, the origin of cardiac lymphatics has not been studied yet. We have grafted PE of HH-stage 17 (day 3) quail embryos hetero- and homotopically into chick embryos, which were re-incubated until day 15. Double staining with the quail endothelial cell (EC) marker QH1 and the lymphendothelial marker Prox1 shows that the PE of avian embryos delivers hemangioblasts but not lymphangioblasts. We have never observed quail ECs in lymphatics of the chick host. However, one exception was a large lymphatic trunk at the base of the chick heart, indicating a lympho-venous anastomosis and a 'homing' mechanism of venous ECs into the lymphatic trunk. Cardiac lymphatics grow from the base toward the apex of the heart. In murine embryos, we observed a basal to apical gradient of scattered Lyve-1+/CD31+/CD45+ cells in the subepicardium at embryonic day 12.5, indicating a contribution of immigrating lymphangioblasts to the cardiac lymphatic system. Our studies show that coronary blood and lymph vessels are derived from different sources, but grow in close association with each other.     

Proliferating mesodermal cells in murine embryos exhibiting macrophage and lymphendothelial characteristics

Background  

The data on the embryonic origin of lymphatic endothelial cells (LECs) from either deep embryonic veins or mesenchymal (or circulating) lymphangioblasts presently available remain inconsistent. In various vertebrates, markers for LECs are first expressed in specific segments of embryonic veins arguing for a venous origin of lymph vessels. Very recently, studies on the mouse have strongly supported this view. However, in the chick, we have observed a dual origin of LECs from veins and from mesodermal lymphangioblasts. Additionally, in murine embryos we have detected mesenchymal cells that co-express LEC markers and the pan-leukocyte marker CD45. Here, we have characterized the mesoderm of murine embryos with LEC markers Prox1, Lyve-1 and LA102 in combination with macrophage markers CD11b and F4/80.     

Segmental territories along the cardinal veins generate lymph sacs via a ballooning mechanism during embryonic lymphangiogenesis in mice

During lymphangiogenesis in the mammalian embryo, a subset of vascular endothelial cells in the cardinal veins is reprogrammed to adopt a lymphatic endothelial fate. The prevailing model of lymphangiogenesis contends that these lymphatic precursor cells migrate away from the cardinal veins and reassemble peripherally as lymph sacs from which a lymphatic vasculature is generated. However, this model fails to account for a number of observations that, as a result, have remained anecdotal. Here, we use optical projection tomography, confocal microscopy and in vivo live imaging to uncover three key stages of lymphatic vascular morphogenesis in the mouse embryo at high resolution. First, we define territories or "pre-lymphatic clusters" of Prox1-positive lymphatic endothelial progenitor cells along the antero-posterior axis of the cardinal veins. Second, these pre-lymphatic clusters undergo progressive extrusion ("ballooning") to generate primitive lymph sacs. Third, lymphatic vessels emerge by a combination of mechanisms including sprouting from the lymph sacs and direct delamination of streams of cells from the cardinal veins. Our data support a new model for lymphatic vascular patterning and morphogenesis, as a basis for identifying the molecular cues governing these processes.     

Growth of the jugular lymph sacs and establishment of the topography of the cervical portion of the thoracic duct during early human ontogenesis

In 196 human embryos, prefetuses, fetuses and newborns, by means of a complex of morphological methods, development of the jugular lymphatic sacs and the process of settling of the thoracic duct cervical part topography have been studied. The jugular lymphatic sac anlages take place on the 6th week of the development. From the lymphatic cleft, situating in the mesenchyme near the anterior cardinal veins, multichambered cavities laid with endotheliocytes are forming,--the jugular lymphatic sacs. Connection of the initially close lymphatic sacs with the venous system takes place secondarily by the end of the embryonic period of development. In the area of the sac ostia a valve is formed, that makes morphological premises for unidirected lymph flow into the venous system. The lymph nodes developing at the place of the reducing jugular lymphatic sacs, ensure formation: from the left jugular lymphatic sac--the cervical part of the thoracic duct, from the right jugular lymphatic sac--the right lymphatic duct and the jugular and the subclavicular lymphatic trunks. Variability in the form and topography of these structures are determined both by the form and construction of the jugular lymphatic sacs and by developmental peculiarities of the lymph nodes at their place. The process of settling of the thoracic duct cervical part topography depends on age changes of its size and form, as well as on development of structures situating nearby, and by the time of birth it is not completed.     

Lymph heart musculature is under distinct developmental control from lymphatic endothelium

Lymph hearts are pulsatile organs, present in lower vertebrates, that function to propel lymph into the venous system. Although they are absent in mammals, the initial veno-lymphatic plexus that forms during mammalian jugular lymph sac development has been described as the vestigial homologue of the nascent stage of ancestral anterior lymph hearts. Despite the widespread presence of lymph hearts among vertebrate species and their unique function, extremely little is known about lymph heart development. We show that Xenopus anterior lymph heart muscle expresses skeletal muscle markers such as myoD and 12/101, rather than cardiac markers. The onset of lymph heart myoblast induction can be visualized by engrailed-1 (en1) staining in anterior trunk somites, which is dependent on Hedgehog (Hh) signaling. In the absence of Hh signaling and upon en1 knockdown, lymph heart muscle fails to develop, despite the normal development of the lymphatic endothelium of the lymph heart, and embryos develop edema. These results suggest a mechanism for the evolutionary transition from anterior lymph hearts to jugular lymph sacs in mammals.     

An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype

The process of angiogenesis has been well documented, but little is known about the biology of lymphatic endothelial cells and the molecular mechanisms controlling lymphangiogenesis. The homeobox gene Prox1 is expressed in a subpopulation of endothelial cells that, after budding from veins, gives rise to the mammalian lymphatic system. In Prox1(-)(/-) embryos, this budding becomes arrested at around embryonic day (E)11.5, resulting in embryos without lymphatic vasculature. Unlike the endothelial cells that bud off in E11.5 wild-type embryos, those of Prox1-null embryos did not co-express any lymphatic markers such as VEGFR-3, LYVE-1 or SLC. Instead, the mutant cells appeared to have a blood vascular phenotype, as determined by their expression of laminin and CD34. These results suggest that Prox1 activity is required for both maintenance of the budding of the venous endothelial cells and differentiation toward the lymphatic phenotype. On the basis of our findings, we propose that a blood vascular phenotype is the default fate of budding embryonic venous endothelial cells; upon expression of Prox1, these budding cells adopt a lymphatic vasculature phenotype.     

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

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.     

Vasculogenesis and angiogenesis in the mouse embryo studied using quail/mouse chimeras

Using quail/chick chimeras, we have previously shown that different embryonic territories are vascularized through two distinct mecanisms, angiogenesis and vasculogenesis. Angiogenesis occurs in tissues of somatopleural origin, vasculogenesis occurs in territories of splanchnopleural origin. The aim of this work was to establish if these modes of vascularization were conserved in the mammalian embryo. Since in vivo manipulations with mammalian embryos are difficult to perform, we used a quail/mouse chimera approach. Mouse limb buds of somatopleural origin, and visceral organ rudiments of splanchnopleural origin, were grafted into the coelomic cavity of 2.5 day-old quail embryos. After four to seven days, the hosts were killed and the origin of the endothelial cells in the mouse tissues was determined by double staining with the quail endothelial and hematopoietic cell-specific marker, QH1 and mouse-specific VEGFR2 and VEGFR3 probes. Our findings show that the great majority of vessels which developed in the mouse limbs was QH1+, indicating that these tissues were vascularized by angiogenesis. Conversely, visceral organs were vascularized through the vasculogenesis process by mouse endothelial cells which differentiated in situ. These results demonstrate for the first time that in the mouse embryo, as previously shown in avian species, the tissues from somatopleural origin are vascularized by angiogenesis, while rudiments of a splanchnopleural origin are vascularized by vasculogenesis, both at vascular and lymphatic levels.     

Immunohistochemical demonstration of Angiopoietin-2 in lymphatic vascular development

The cellular expression of Angiopoietin-2 (Ang2) was studied during lymphatic development in mouse by immunohistochemistry and compared to that of lymphatic endothelial markers. At the earliest stage of lymphvasculogenesis, Prox1-identified lymphatic precursor cells of the cardinal vein displayed an intense immunoreaction for Ang2 in their cytoplasm, implying that Ang2 may adjust lymphatic specification and sprouting from the veins under the control of Prox1. Thereafter, Ang2 was constantly expressed in Prox1 and/or LYVE-1-immunopositive endothelial cells of lymphatic sacs and vessels, ranging from lymphatic capillaries to collectors, throughout embryonic and neonatal development, and the lymphatic endothelial cells simultaneously exhibited immunoreactivity to Tie2, a primary receptor for angiopoietins. These results suggest that lymphatic endothelial cells may regulate lymphatic development via their own Ang2-Tie2 signaling. Ang2 is further immunolocalized in the developing blood vessels including hepatic sinusoids, adrenal medullary vasculature and postnatal pulmonary vessels, thereby indicating that the blood vessels, which undergo vascular remodeling and sudden alteration of blood flow during the development, are also likely to express Ang2. The present study is first to demonstrate Ang2 expression in the lymphatic endothelial cells during development, and consequently Ang2 is regarded as a molecular profile of the developing lymphatic endothelial cells required for lymphatic vascular organization.     

Prox1, master regulator of the lymphatic vasculature phenotype

In contrast to the extensive molecular and functional characterization of blood vascular endothelium, little is known about the mechanisms that control the formation and lineage-specific differentiation and function of lymphatic vessels. The homeobox gene Prox1, the vertebrate homologue of the Drosophila prospero gene, has been recently identified to be required for the induction of lymphatic vascular development from preexisting embryonic veins, and studies in Prox1-deficient mice have confirmed Florence Sabin's original hypothesis about the origin of the lymphatic vascular system from embryonic veins. The recent establishment of cell culture models for the selective propagation of blood vascular and lymphatic endothelial cells, together with the findings that these cells maintain their lineage-specific differentiation in vitro, has led to the discovery that Prox1 expression is sufficient to induce a lymphatic phenotype in blood vascular endothelium. Ectopic expression of Prox1 downregulated blood vascular-associated genes and also upregulated some of the known lymphatic endothelial cell markers. Together, these studies suggest that the blood vascular phenotype represents the default endothelial differentiation and they identify an essential role of Prox1 in the program specifying lymphatic endothelial cell fate.     

Maldevelopment of dermal lymphatics in Wnt5a-knockout-mice

Maintenance of tissue homeostasis and immune surveillance are important functions of the lymphatic vascular system. Lymphatic vessels are lined by lymphatic endothelial cells (LECs). By gene micro-array expression studies we recently compared human lymphangioma-derived LECs with umbilical vein endothelial cells (HUVECs). Here, we followed up on these studies. Besides well-known LEC markers, we observed regulation of molecules involved in immune regulation, acetylcholine degradation and platelet regulation. Moreover we identified differentially expressed WNT pathway components, which play important roles in the morphogenesis of various organs, including the blood vascular system. WNT signaling has not yet been addressed in lymphangiogenesis. We found high expression of FZD3, FZD5 and DKK2 mRNA in HUVECs, and WNT5A in LECs. The latter was verified in normal skin-derived LECs. With immunohistological methods we detected WNT5A in LECs, as well as ROR1, ROR2 and RYK in both LECs and HUVECs. In the human, mutations of WNT5A or its receptor ROR2 cause the Robinow syndrome. These patients show multiple developmental defects including the cardio-vascular system. We studied Wnt5a-knockout (ko) mouse embryos at day 18.5. We show that the number of dermal lymphatic capillaries is significantly lower in Wnt5a-null-mice. However, the mean size of individual lymphatics and the LEC number per vessel are greater. In sum, the total area covered by lymphatics and the total number of LECs are not significantly altered. The reduced number of lymphatic capillaries indicates a sprouting defect rather than a proliferation defect in the dermis of Wnt5a-ko-mice, and identifies Wnt5a as a regulator of lymphangiogenesis.     

Extraembryonic origin of circulating endothelial cells

Circulating endothelial cells (CEC) are contained in the bone marrow and peripheral blood of adult humans and participate to the revascularization of ischemic tissues. These cells represent attractive targets for cell or gene therapy aimed at improving ischemic revascularization or inhibition of tumor angiogenesis. The embryonic origin of CEC has not been addressed previously. Here we use quail-chick chimeras to study CEC origin and participation to the developing vasculature. CEC are traced with different markers, in particular the QH1 antibody recognizing only quail endothelial cells. Using yolk-sac chimeras, where quail embryos are grafted onto chick yolk sacs and vice-versa, we show that CEC are generated in the yolk sac. These cells are mobilized during wound healing, demonstrating their participation to angiogenic repair processes. Furthermore, we found that the allantois is also able to give rise to CEC in situ. In contrast to the yolk sac and allantois, the embryo proper does not produce CEC. Our results show that CEC exclusively originate from extra-embryonic territories made with splanchnopleural mesoderm and endoderm, while definitive hematopoietic stem cells and endothelial cells are of intra-embryonic origin.     

Intravital two-photon microscopy of lymphatic vessel development and function using a transgenic Prox1 promoter-directed mOrange2 reporter mouse

Lymphatic vessels, the second vascular system of higher vertebrates, are indispensable for fluid tissue homoeostasis, dietary fat resorption and immune surveillance. Not only are lymphatic vessels formed during fetal development, when the lymphatic endothelium differentiates and separates from blood endothelial cells, but also lymphangiogenesis occurs during adult life under conditions of inflammation, wound healing and tumour formation. Under all of these conditions, haemopoietic cells can exert instructive influences on lymph vessel growth and are essential for the vital separation of blood and lymphatic vessels. LECs (lymphatic endothelial cells) are characterized by expression of a number of unique genes that distinguish them from blood endothelium and can be utilized to drive reporter genes in a lymph endothelial-specific fashion. In the present paper, we describe the Prox1 (prospero homeobox protein 1) promoter-driven expression of the fluorescent protein mOrange2, which allows the specific intravital visualization of lymph vessel growth and behaviour during mouse fetal development and in adult mice.     

A hypothesis on the role of primitive macrophages in initial embryonic lymphatic development

Recent evidences have shown that macrophages are tightly related to pathological lymphangiogenesis. However, the effects which primitive macrophages take in embryonic lymphatic development remains unclear. Here, we postulate that the primitive macrophages may play an important role in initial embryonic lymphatic development. The possible mechanism: primitive macrophages induce BECs to transdifferentiate into LECs and initiate the budding, moreover, the lymph sacs are not only formed by LECs but also some scattered cells with macrophage characteristics preferentially located in the loose mesenchyme.     

Expression of lymphatic endothelium-specific hyaluronan receptor LYVE-1 in the developing mouse kidney

Our knowledge of the embryonic development of the lymphatic vessels within the kidney is limited. The aim of this study was to establish the time of appearance and the distribution of intra-renal lymphatic vessels in the developing mouse kidney by using the lymphatic marker, LYVE-1. Kidneys from embryonic day 12 (E12) to E18, from neonates at post-natal day 1 (P1) to P21, and from adults were studied. In the adult mouse kidney, LYVE-1 was expressed mainly in the lymphatic endothelial cells (LECs) and in a subset of endothelial cells in the glomerular capillaries. However, in the developing mouse kidney, LYVE-1 was also expressed transiently in F4/80⁺/CD11b^– immature macrophages/dendritic cells and in the developing renal vein. LYVE-1⁺ lymphatic vessels connected with extra-renal lymphatics were detected in the kidney at E13. F4/80⁺/CD11b^–/LYVE-1⁺ immature macrophages/dendritic cells appeared prior to the appearance of LYVE-1⁺ renal lymphatic vessels and were closely intermingled or even formed part of the lymphatic vascular wall. Prox1 was expressed only in the LYVE-1⁺ LECs from fetus to adult-hood, but not in LYVE-1⁺ endothelial cells of the developing renal vein and macrophages/dendritic cells. Thus, lymphatic vessels of the kidney might originate by extension of extra-renal lymphatics through an active branching process possibly associated with F4/80⁺/CD11b^–/LYVE-1⁺ macrophages/dendritic cells.