ARTICLES
74 VOLUME 5 NUMBER 1 JANUARY 2004 NATURE IMMUNOLOGY
The cardiovascular circulatory system consists of a treelike hierarchy of
vessels formed from a primitive vascular network
1
. The lymphatic sys-
tem comprises a separate vascular system that also permeates most
organs of the body. The lymphatic vessels drain extracellular fluid and
dendritic cell–captured antigens through the secondary lymphoid
organs, where lymphocyte activation occurs. The sequential formation
of lymph nodes and Peyer’s patches requires lymphotoxin α
1
β
2
signal-
ing through the lymphotoxin β -receptor
2–4
. However, the lymphatic
vasculature seems to be intact in lymphotoxin β -receptor–deficient
mice, indicating that the development of secondary lymphoid organs
and the lymphatic vasculature are regulated by a distinct set of genes
5
.
The development of the lymphatic vessels in embryos starts
when a subset of endothelial cells in the cardinal vein commit to the
lymphatic lineage and sprout to form the primary lymph sacs
6–8
. In
mice, the lymphatic vasculature starts to develop at embryonic day
10.5 (E10.5), when the cardiovascular system is already function-
ing. After the formation of the initial lymph sacs, the peripheral
lymphatics are generated by centrifugal sprouting. In mice defi-
cient in the Prox1 homeobox transcription factor gene, the
endothelial cells in the cardinal vein fail to induce lymphatic mark-
ers such as VEGF receptor 3 (VEGFR-3) or lymphatic vessel
endothelial hyaluronan receptor 1 (LYVE-1), and do not commit to
the lymphatic lineage
9,10
. Accordingly, ectopic expression of Prox-1
in cultured primary blood vascular endothelial cells induces several
lymphatic endothelial cell–specific genes and downregulates blood
vessel–specific genes
11,12
.
The gene encoding the receptor tyrosine kinase VEGFR-3 is one of
the genes upregulated by Prox-1 in the lymphatic endothelial cells
10,11
.
VEGFR-3 is required for remodeling of the blood vascular network at
midgestation, but becomes downregulated in the blood vessel
endothelia after the emergence of the lymphatic vessels
13,14
. VEGFR-3
activation by its ligands VEGF-C and VEGF-D leads to proliferation,
migration and survival of cultured human adult microvascular lym-
phatic endothelial cells
15
. VEGFR-3 activation also induces lymphan-
giogenesis in adult tissues
16,17
. Furthermore, missense mutations in
the gene encoding VEGFR-3 lead to insufficient signaling through this
receptor and hypoplasia of the cutaneous lymphatic network in a sub-
set of families suffering from congenital human lymphedema (Milroy
disease; Online Mendelian Inheritance in Man, 153100)
18
.
The two known VEGFR-3 ligands, VEGF-C and VEGF-D, belong to
the larger VEGF family of growth factors that also includes VEGF, pla-
centa growth factor and VEGF-B. In addition to activating VEGFR-3,
VEGF-C and VEGF-D also activate VEGFR-2, which is expressed in
both blood and lymphatic vessel endothelia
19–21
. Proteolytic cleavage
is an important regulator of the receptor binding and thus the biologi-
cal activity of VEGF-C and VEGF-D
19,22
. Partially processed forms of
VEGF-C and VEGF-D activate VEGFR-3, whereas the fully processed
short forms are also potent stimulators of VEGFR-2.
1
Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Haartman Institute and Helsinki University Hospital, Biomedicum Helsinki,
University of Helsinki, PO Box 63, 00014 Helsinki, Finland.
2
Institute of Biomedicine, Developmental Biology, Biomedicum Helsinki and HUCH-diagnostics, PO Box
400, 00029 Helsinki, Finland.
3
Institute of Biotechnology, University of Helsinki, PO Box 56, 00014 Helsinki, Finland.
4
MRC Human Immunology Unit, Weatherall
Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, UK.
5
Neuroscience Center, University of Helsinki, PO Box 56, 00014 Helsinki,
Finland.
6
Department of Medical Biochemistry, University of Gothenburg, Box 440, 405 30 Gothenburg, Sweden. Correspondence should be addressed to K.A.
(kari.alitalo@helsinki.fi).
Published online 23 November 2003; doi:10.1038/ni1013
Vascular endothelial growth factor C is required
for sprouting of the first lymphatic vessels from
embryonic veins
Marika J Karkkainen
1
, Paula Haiko
1
, Kirsi Sainio
2
, Juha Partanen
3
, Jussi Taipale
1
, Tatiana V Petrova
1
,
Michael Jeltsch
1
, David G Jackson
4
, Marja Talikka
5
, Heikki Rauvala
5
, Christer Betsholtz
6
& Kari Alitalo
1
Lymphatic vessels are essential for immune surveillance, tissue fluid homeostasis and fat absorption. Defects in lymphatic vessel
formation or function cause lymphedema. Here we show that the vascular endothelial growth factor C (VEGF-C) is required for the
initial steps in lymphatic development. In Vegfc
–/–
mice, endothelial cells commit to the lymphatic lineage but do not sprout to
form lymph vessels. Sprouting was rescued by VEGF-C and VEGF-D but not by VEGF, indicating VEGF receptor 3 specificity. The
lack of lymphatic vessels resulted in prenatal death due to fluid accumulation in tissues, and Vegfc
+/–
mice developed cutaneous
lymphatic hypoplasia and lymphedema. Our results indicate that VEGF-C is the paracrine factor essential for lymphangiogenesis,
and show that both Vegfc alleles are required for normal lymphatic development.
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ARTICLES
NATURE IMMUNOLOGY VOLUME 5 NUMBER 1 JANUARY 2004 75
Because of the early death of Veg f r 3-deficient mice, the function of
VEGFR-3 in the initial development of the lymphatic vessels has
remained unknown
13
. In addition, the functions of VEGF-C and
VEGF-D have not been analyzed in this process. For these reasons we
targeted the Veg f c locus in mouse chromosome 8 and analyzed the
lymphatic vascular development in embryos deficient in VEGF-C.
RESULTS
Lymphatic vascular formation fails in Vegfc-deficient embryos
We gene r a t ed mice in which Ve g f c was replaced by the lacZ marker
gene (Fig. 1). Ve g f c
–/–
embryos seemed edematous from E12.5 onward
(Fig. 2a). Approximately half of the mutant embryos died between
E15.5 and E17.5 in the mixed (ICR/129Sv) background, and there were
no live-born Ve g f c
–/–
pups. Using β -galactosidase as a marker for
endogenous Ve g f c expression, we found that Ve g f c was strongly
expressed from E8.5 onward in the jugular region where the first lymph
sacs form
23
(Fig. 2b). Accordingly, staining for β -galactosidase activity
and VEGFR-3 immunoreactivity in sections of E10.5 Ve g f c
+/–
embryos
indicated that Ve g f c was abundantly expressed in the mesenchyme dor-
solateral to the VEGFR-3-positive jugular veins, which give rise to the
lymphatic endothelium (Fig. 2c). Ve g f c was also expressed in the
smooth muscle cells surrounding large arteries (Fig. 2c).
The localization and timing of Ve g f c expression indicated that VEGF-
C is needed for the development of the lymphatic vasculature. Indeed,
staining of sections from the jugular region for the lymphatic markers
VEGFR-3, LYVE-1 (ref. 24) and podoplanin
25,26
showed that lymph sacs
did not form in Ve g f c
–/–
embryos, whereas they were clearly visible in the
Ve g f c
+/–
and Ve g f c
+/+
littermates (Fig. 3a and data not shown). All lym-
phatic vessels, including the thoracic duct, were also absent at later
developmental stages (data not shown). VEGFR-3 expression persisted
in some erythrocyte-containing capillaries of Ve g f c
–/–
embryos, whereas
it was downregulated in the Ve g f c
+/–
and Ve g f c
+/+
littermates (Fig. 3a).
However, the total amount of VEGFR-3 mRNA was reduced in the
Ve g f c
–/–
embryos, whereas the amount of VEGF-D, angiopoietin-2 or
VEGFR-2 mRNA was not altered (data not shown). The veins and arter-
ies seemed normal in sections from Ve g f c
–/–
embryos stained for platelet
endothelial cell adhesion molecule 1 (PECAM-1, also known as CD31)
and smooth muscle actin (Fig. 3b and data not shown).
VEGF-C is required for lymphatic endothelial cell migration
Because embryos deficient in Prox1 also fail to form the primitive
lymph sacs
9,10
, we studied Prox-1 expression in Veg f c
–/–
embryos by
immunofluorescence. We isolated whole-mount explants of the
axial vascular system from E10.5–E13 embryos. At E10.5, we
ab
c
Figure 2 Vegfc expression is associated with lymphatic vascular development. (a) Severe edema (arrow) in an E15.5 Vegfc
–/–
embryo. (b) Left, location
of the jugular (ju) and retroperitoneal (re) lymph sacs in a 42-day-old human fetus, corresponding to the lymph sacs of E13–E14 mouse embryos. From
ref. 44; reproduced with permission. Right, β -galactosidase staining of an E10.5 Vegfc
+/–
embryo. There is Vegfc expression in regions corresponding to
lymphatic vascular formation indicated in the left panel. (c) Transverse section of an E10.5 Vegfc
+/–
embryo (at dashed line in b). There is Vegfc expression
in the mesenchymal cells (blue arrows), toward which the endothelial cells from the jugular vein (jv; stained red for VEGFR-3) eventually sprout to form the
jugular lymph sacs. da, dorsal aorta; nt, neural tube. Scale bars: a, 2 mm; b, right, 500 µm; c, 250 µm.
b
Figure 1 Genetic targeting of the Vegfc locus. (a) The homologous recombination event deletes sequences encoding the translation initiation site and
the signal sequence of Vegfc, and places lacZ under the control of the Vegfc regulatory region. H, HindIII; N, NcoI; P, PstI; Bs, BsmBI; neo
r
, neomycin
resistance; HSV-TK, herpes simplex virus thymidine kinase; WT, wild-type. (b) Southern blot of amniotic DNA after NcoI digestion and hybridization
with the 5′ external probe indicated by the red bar in a. Right margin, molecular sizes of the digested fragments. (c) Vegfc and lacZ expression
analyzed by RT-PCR from E11.5 embryos.
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