Introduction
The lymphatic system is a vascular network of thin‐walled capillaries and larger vessels lined by a continuous layer of endothelial cells that drain lymph from the tissue spaces of most organs and return it to the venous system for recirculation. Although much information has been gained regarding the normal and pathological growth of the vascular system (
Gale and Yancopoulos, 1999), the lack of specific lymphatic markers has made it difficult to elucidate the development of the lymphatic system. Consequently, the study of the formation of the lymphatic vasculature and its possible role in tumor metastasis has been neglected in the past, and the understanding of the precise manner by which the lymphatic system develops is still rudimentary.
Several recent reports (
Banerji et al., 1999;
Wigle and Oliver, 1999;
Karkkainen et al., 2001;
Nakano and Gunn, 2001) have described the identification of novel lymphatic markers. Furthermore, other studies have provided evidence that both VEGF‐C and VEGF‐D, which are ligands for the vascular endothelial growth factor receptor 3 (VEGFR‐3), can enhance tumor lymphangiogenesis and lymphatic metastasis (
Makinen et al., 2001;
Mandriota et al., 2001;
Skobe et al., 2001;
Stacker et al., 2001). However, a detailed comparison of the expression patterns of these recently identified lymphatic markers during early stages of lymphatic development is not yet available.
Our previous work (
Wigle and Oliver, 1999) validated Sabin's original proposal of the venous origin of the primary lymph sacs (
Sabin, 1902). Our results also indicated that the expression of Prox1 in a restricted subpopulation of endothelial cells in the embryonic veins was required to promote lymphangiogenesis and that the initial localization and subsequent migration of the lymphatic endothelial cells from the cardinal vein were polarized (the endothelial cells appear to stream together along a defined pathway). We also showed that in
Prox1‐null mice, budding and sprouting of lymphatic endothelial cells from the veins appear unaffected at embryonic day (E)10.5. However, both processes are arrested prematurely at around E11.5–E12.0 and, as a result of this arrest,
Prox1‐null mice are devoid of lymphatic vasculature (
Wigle and Oliver, 1999). Ongoing studies in our laboratory had suggested that the budding and sprouting of endothelial cells from the cardinal vein which are still detectable in
Prox1‐null embryos at E11.5–E12.0, were no longer polarized (endothelial cells followed a random path in different directions around the cardinal vein); however, the subsequent fate of those endothelial cells was undetermined.
Is it possible that in addition to its importance in the budding and sprouting of lymphatic vessels during lymphangiogenesis, Prox1 might also play a key role in the differentiation and maturation of lymphatic endothelial cells? To address this important question, we first performed a detailed, comparative analysis of heterozygous and null Prox1 embryos between E10.5 and E14.5 using antibodies to lymphatic and blood vascular markers. Here we show that in contrast to the heterozygous Prox1 embryos, in E11.5–E12.0 mutant littermates the budding and migration of the endothelial cells from the cardinal vein are no longer polarized (follows a random path). Surprisingly, we also found that these budding endothelial cells have switched their differentiation program. In contrast to the heterozygous situation in which the budding Prox1 (β‐galactosidase; β‐gal)‐positive cells corresponded to lymphatic endothelial cells, in Prox1‐null embryos these budding β‐gal‐positive endothelial cells now display markers of blood vascular endothelial cells. These observations were supported by the finding that the mutant endothelial cells did not express any of the available lymphatic markers used in this study but instead they expressed blood vascular markers. Taken together, these results indicate that Prox1 activity in a restricted subpopulation of endothelial cells in the embryonic veins is required not only to promote lymphangiogenesis but also to determine a lymphatic fate.
In addition, we have also determined that Prox1, VEGFR‐3, LYVE‐1 and SLC are expressed similarly in lymphatic endothelial cells of normal adult and tumor tissues.
Discussion
The lack of specific markers has hampered the understanding of the mechanisms controlling the development of the lymphatic vascular system. We have shown previously that
Prox1 plays a key role in lymphangiogenesis (
Wigle and Oliver, 1999). We found that
Prox1 activity is not required to initiate budding of endothelial cells from the cardinal vein, but rather to maintain the budding and sprouting of a restricted subpopulation of endothelial cells that give rise to the lymphatic vasculature (
Wigle and Oliver, 1999). By comparing the expression of lymphatic‐ and blood vascular‐specific markers in
Prox1 heterozygous and nullizygous embryos and in normal adult tissues and tumors, we have elucidated further the role of
Prox1 in the development and maintenance of the lymphatic system.
To determine the phenotypic properties of Prox1‐positive cells in heterozygous and nullizygous
Prox1 embryos, we investigated the expression of three other available lymphatic markers (VEGFR‐3, LYVE‐1 and SLC) and two blood vascular markers (laminin and CD34). Functional inactivation of
VEGFR‐3 in mice disrupts the development of the cardiovascular system (
Dumont et al., 1998). VEGFR‐3 is expressed in the endothelial cells of some fenestrated blood vessels (
Partanen et al., 2000) and in angiogenic blood vessels in some tumors (
Valtola et al., 1999). However, during later embryonic development, VEGFR‐3 expression becomes largely restricted to the lymphatic vessels (
Kaipainen et al., 1995;
Wigle and Oliver, 1999). The key role that this marker plays in the lymphatic system was demonstrated by the identification of mutations in
VEGFR‐3 in several cases of congenital lymphedema (
Karkkainen et al., 2000).
LYVE‐1, a member of the Link protein superfamily, was identified recently as a lymphatic‐specific receptor for the extracellular matrix glycosaminoglycan HA (
Banerji et al., 1999). HA is thought to provide a hydrated environment that facilitates cell transformation and migration during development (
Jackson et al., 2001). LYVE‐1 may also participate in the uptake or transport of HA across the lymphatic wall (
Jackson et al., 2001;
Prevo et al., 2001). Immunohistochemical analyses have demonstrated LYVE‐1 expression on the surface of endothelial cells of lymphatic vessels (
Jackson et al., 2001;
Prevo et al., 2001).
SLC (or CCL21) is released by the lymphatic endothelium (
Gunn et al., 1998,
1999;
Zlotnik and Yoshie, 2000). The migration of leukocytes toward cells comprising the lymphatic vasculature is regulated, at least in part, by this chemokine. SLC is expressed uniformly in adult lymphatic endothelium (
Gunn et al., 1998,
1999), and is expressed in embryonic lymphatics as early as E11.5 (this work).
In the present study, we found that β‐gal‐positive endothelial cells that start to bud from the veins of Prox1‐null embryos, but are arrested at E11.5–E12.0, do not undergo lymphatic differentiation. These cells do not express any of the lymphatic markers except VEGFR‐3 (at low levels), but they do express high levels of markers such as laminin and CD34, a finding that suggests that these cells have adopted a blood vascular phenotype.
In addition to the arrest of endothelial cell budding and migration observed in Prox1‐null embryos at around E11.5, the polarity (directionality) of the budding was also defective. This finding suggests that Prox1 function is required for normal maintenance of some as yet unidentified signaling mechanism that is involved in guiding the budding and migration of the lymphatic endothelial cells. This molecule may be located in the surrounding tissue on one side of the cardinal vein, and its activity is dependent on Prox1 function, in an as yet undetermined cell‐autonomous or non‐cell‐autonomous manner.
Our findings suggest that
Prox1 activity is required not only to maintain budding and sprouting of a subpopulation of venous endothelial cells that will give rise to the lymphatic vasculature but also to determine the final fate of those budding endothelial cells. On the basis of our results, we have developed a working model of early lymphatic vascular development (
Figure 8). After the initial formation of the vascular system, the expression of LYVE‐1 and Prox1 in endothelial cells in the cardinal veins at approximately E9.5–E10.0 is probably one of the first indications that lymphangiogenesis has been initiated. All endothelial cells in the veins are probably initially bipotent and, upon the expression of at least Prox1 in a restricted subpopulation of venous endothelial cells (on only one side of the cardinal vein), those cells become committed (biased) to initiate the lymphatic differentiation program. As development proceeds, this subpopulation of LYVE‐1‐ and Prox1‐ positive endothelial cells starts to bud from the veins in an initially
Prox1‐independent manner. However, maintenance of the budding and migration requires
Prox1 activity. Normally, as the cells bud in a polarized manner, they start to express additional lymphatic endothelial markers. At this stage, SLC expression is first detected, and VEGFR‐3 expression is maintained at high levels in budding lymphatic endothelial cells, but its expression becomes weaker in blood vascular endothelial cells. The expression of these four lymphatic markers may indicate that this process becomes specified irreversibly toward the lymphatic pathway. On the basis of our results, we suggest that this step is also dependent on
Prox1 activity and on a feedback signaling mechanism required for the maintenance and polarized budding of these lymphatic endothelial cells. Endothelial cell budding and migration are arrested because of the lack of
Prox1 function, and random budding occurs because of a failure in the feedback loop signaling mechanism. As a result, neither lymphatic bias nor lymphatic specification is accomplished. Therefore,
Prox1 activity in a restricted subpopulation of endothelial cells in the embryonic veins is required not only to promote lymphangiogenesis but also to determine the lymphatic fate by the initiation of the lymphatic differentiation program of those budding venous endothelial cells. In the future, the identification of the molecules and the mechanisms involved in these developmental decisions will provide important information for our understanding of lymphangiogenesis during development and in diseases such as cancer.