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First published online 4 December 2008
doi: 10.1242/dev.025353
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Research Report |
2 is necessary for separation of blood and lymphatic vasculature in mice
1 Laboratory of Gene Expression and Regulation, Center for Experimental
Medicine, Institute of Medical Science, University of Tokyo, Tokyo 108-8639,
Japan.
2 Department of Anatomy, Graduate School of Medicine and Pharmaceutical Sciences
for Research, University of Toyama, Toyama 930-0194, Japan.
Author for correspondence (e-mail:
h-ichise{at}ims.u-tokyo.ac.jp)
Accepted 15 November 2008
SUMMARY
The lymphatic vasculature originates from the blood vasculature through a
mechanism relying on Prox1 expression and VEGFC signalling, and is separated
and kept separate from the blood vasculature in a Syk- and SLP76-dependent
manner. However, the mechanism by which lymphatic vessels are separated from
blood vessels is not known. To gain an understanding of the vascular
partitioning, we searched for the affected gene in a spontaneous mouse mutant
exhibiting blood-filled lymphatic vessels, and identified a null mutation of
the Plcg2 gene, which encodes phospholipase C
2 (PLC2),
by positional candidate cloning. The blood-lymph shunt observed in
PLC2-null mice was due to aberrant separation of blood and lymphatic
vessels. A similar phenotype was observed in lethally irradiated wild-type
mice reconstituted with PLC2-null bone marrow cells. These findings
indicate that PLC2 plays an essential role in initiating and
maintaining the separation of the blood and lymphatic vasculature.
Key words: Mouse, PLC2, Lymphangiogenesis, Vascular separation, Bone marrow-derived cells, Endothelial cell
INTRODUCTION
The lymphatic vasculature originates from the blood vasculature during
development. Previous studies have demonstrated that the differentiation of
lymphatic endothelial cells (LECs) is initiated by expression of the
transcription factor prospero-related homeobox 1 (Prox1), in a subpopulation
of venous endothelial cells (Wigle and
Oliver, 1999
). Vascular endothelial growth factor (VEGF) C, which
is a ligand for vascular endothelial growth factor receptor (VEGFR) 2 and 3
(Joukov et al., 1996), is not
required for Prox1-induced LEC specification, but is necessary for lymphatic
vessel formation (Karkkainen et al.,
2004).
During later development, the lymphatic vasculature separates from the
blood vasculature and acquires specialized structures. Previous studies have
shown that mice lacking either the spleen tyrosine kinase (Syk) or
Src-homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP76)
exhibited blood-lymph shunts and their lymphatic vessels contained lymphatic
vessel endothelial hyaluronan receptor 1-positive (Lyve1+)
(Prevo et al., 2001) LECs and
blood endothelial cells (BECs) (Abtahian et
al., 2003). The blood-lymph separation may also be regulated by
hematopoietic cell-derived circulating lymphatic endothelial progenitor cells
(Sebzda et al., 2006).
However, the precise roles of Syk and SLP76 in the separation process remain
unknown.
Here, we have employed a genetic approach to facilitate further
understanding of the vascular separation. Positional candidate cloning
revealed that a spontaneous mutant mouse line exhibiting blood-filled
lymphatic vessels carries a null mutation of the Plcg2 gene, which
encodes phospholipase C2 (PLC2). We also demonstrate that the
blood-filled lymphatic vessels in PLC2-null mice were caused by the
aberrant separation of blood and lymphatic vasculature during development.
Analysis of the expression pattern of a Plcg2 reporter-knock-in and
bone marrow reconstitution studies were performed to evaluate the separation
process.
MATERIALS AND METHODS
Mice
All mice were housed under pathogen-free conditions. C57BL/6J and CAST/Ei
were purchased from CLEA Japan (Tokyo, Japan) and the Jackson Laboratory (Bar
Harbor, ME, USA), respectively. The mutant mouse strain and 129/SvEv mice were
kind gifts from Dr Motoya Katsuki (National Institute for Basic Science,
Japan). The mutant mice were backcrossed eight times to 129/SvEv mice for
genetic analysis. An FLP-deleter strain, FLP66
(Takeuchi et al., 2002), was
provided by RIKEN BRC (Tsukuba, Japan). All of the work with mice conformed to
the guidelines approved by the Institutional Animal Care and Use Committee of
the University of Tokyo.
Polymerase chain reaction (PCR) genotyping of simple sequence-length polymorphism (SSLP) markers
Genomic DNA (0.1 µg) was used for PCR and the amplification conditions
were: 94°C for 2 minutes, 30-35 cycles of 94°C, 60°C and 72°C
for 1 minute each, with a final extension at 72°C for 7 minutes. PCR
products were electrophoresed on 3-4% agarose gels in TBE buffer. Newly
designed SSLP-PCR primer pairs located in the region between D8Mit48 and 120
are as follows: D8Ims9, forward 5'-CCACAGTATACCCACATAGATT-3' and
reverse 5'-AGCGGACTGGTGACAGCACA-3'; D8Ims10, forward
5'-CTCACTGAACCATCTCACCA-3' and reverse,
5'-AGGTGCCTGTGTACAATAGA-3'; D8Ims11, forward
5'-GATCTAGTGTAGTAGCAGCA-3' and reverse
5'-TTCTGGCCTCTGTGAGAGTTTG-3'; D8Ims1, forward
5'-CCTCCATGGACACTGCACTC-3' and reverse
5'-GTGAGTTCAGTGCCAGCCAG-3'; D8Ims2, forward
5'-TCTCACATTAAGTGCGTGCC-3' and reverse
5'-AGGAGGAGTCGGATGGAAGC-3'; D8Ims18, forward
5'-ACACTACACTCAATGCACATG-3' and reverse
5'-ACAATGATGGTCTTCAGAGC-3'; and D8Ims19, forward
5'-CAAGGTGGAGACTAAGAAGC-3' and reverse
5'-CTGCTGCCACTTCATGTAAG-3'.
Generation of Plcg2/EGFP knock-in mice
A BAC clone encompassing the Plcg2 gene RPCI23-308L22 was
purchased from Invitrogen (Carlsbad, CA, USA). Part of the coding region of
exon 2 was replaced with EGFP cDNA (Clontech/TAKARA Bio, Shiga, Japan)
followed by a murine PGK polyadenylation signal sequence and an FRT-flanked
PGK-gb2-neo cassette (Gene Bridges GmbH, Dresden, Germany) by homologous
recombination in E. coli. A DNA fragment containing the modified exon
2 was subcloned into pUC-DT-A (a gift from Dr Takeshi Yagi, Osaka University,
Japan) (Yagi et al., 1993).
The linearized vector was electroporated into a Plcg2+/al
ES cell line established from Plcg2+/al blastocysts (T.I.,
unpublished). All correctly targeted G418-resistant clones possessed the
Plcg2+/EGFP genotype. B6- or 129-congenic
knock-in mice harbouring the neo cassette, except for embryos in
Fig. 3D, were used in this
study.
Immunohistochemistry
Specimens were fixed in PBS containing 4% paraformaldehyde (PFA) at 4°C
overnight and 10 µm frozen sections were prepared. For EGFP immunostaining,
trypsinization of sections was used for antigen retrieval. All sections were
incubated with 3% H2O2 in PBS prior to immunostaining.
Sections were incubated with a primary antibody, followed by incubation with
the Histofine reagent (Nichirei Biosciences, Tokyo, Japan). Prior to detection
of the primary/secondary antibody complexes, sections were incubated with a
biotinylated antibody for double immunostaining. A Streptavidin/Biotin
blocking kit (Vector laboratories, Burlingame, CA, USA) was used. Following
detection of the first antibody complexes, sections were incubated with 3%
H2O2 to quench the peroxidase activity of the Histofine
reagent, and then incubated with streptavidin-HRP (NEN/Perkin-Elmer, Waltham,
MA, USA). The TSA HRP detection system (NEN/Perkin-Elmer) and a DAB solution
were used. Frozen sections of fresh tissues were acetone fixed and are shown
in Fig. S4A (F4/80, CD11b, normal IgG) in the supplementary material. Paraffin
wax-embedded sections (7 µm) are shown in
Fig. 1 and Fig. S2C (in the
supplementary material), and were antigen-retrieved for PLC2
immunostaining by boiling in the antigen-retrieval buffer (R&D systems,
Minneapolis, MN, USA). Micrographs in figures are representative of 2-20
independent sections from two to six independent specimens. For whole-mount
immunostaining, specimens were pre-fixed in 4% PFA and then fixed in a
methanol/DMSO solution. After quenching with methanol containing 5%
H2O2 and rehydration, immunostaining was performed.
Micrographs in figures are representative of two independently stained
specimens from more than two mice.
We verified that the auto-fluorescent EGFP signal or false-positive
staining by normal IgG or isotype control antibodies did not affect the
staining results. Images were acquired with an Olympus microscope (Olympus,
Tokyo, Japan). Antibodies used were as follows: rat anti-mouse Lyve-1 (R&D
Systems); rat anti-GFP (Nacalai-Tesque, Kyoto, Japan); biotinylated rat
anti-mouse CD31 (BD-Pharmingen, Franklin Lakes, NJ, USA); biotinylated goat
anti-mouse Lyve-1 (R&D Systems); biotinylated rat anti-mouse F4/80
(eBioscience, San Diego, CA, USA); rat anti-mouse F4/80 (eBioscience); rat
anti-mouse CD11b (eBioscience); and rabbit polyclonal anti-PLC2 (Santa
Cruz Biotechnology, Santa Cruz, CA, USA).
Culture of ECs and fibroblasts
Human umbilical vein ECs (HUVECs) and human dermal microvascular LECs
(HdMLECs) from pooled donors (Lonza, Basel, Switzerland) were cultured using
an EGM-2 MV bullet kit (Lonza) according to the manufacturer's protocol. Mouse
mesenteric ECs were prepared and used for analysis as described previously
(Yamaguchi et al., 2008). ECs
and mouse primary embryonic fibroblasts were cultured in EBM-2 basal medium
(Lonza) and Dulbecco's modified Eagle's medium (DMEM), respectively,
containing 0.5% serum without supplemental growth factors for 16 hours.
Recombinant human VEGF165 (Peprotech EC, London, UK), rat VEGFC
(R&D Systems) or platelet-derived growth factor-BB (PDGF-BB) (Peprotech
EC) were added to the medium at a final concentration of 100 ng/ml. Cells were
incubated for 10 minutes and harvested for analysis.
Western blotting
Cell lysates (20 µg) were resolved by SDS-PAGE and semi-dry-blotted onto
PVDF membranes (Millipore, Billerica, MA, USA). Western blot analysis was
performed using a rabbit primary antibody and HRP-conjugated anti-rabbit IgG.
Antibody-labelled bands were visualized with an ECL detection system (GE
Healthcare Bio-Sciences, Piscataway, NJ, USA) and X-ray film (Fujifilm, Tokyo,
Japan). Proteins immunoprecipitated by anti-PLC2 were assayed using
anti-phosphotyrosine (4G10), according to a previously described method
(Yamaguchi et al., 2008).
Antibodies used were as follows: rabbit polyclonal anti-PLC1,
PLC2 and VEGFR2 (Santa Cruz Biotechnology); anti-phosphorylated
PLC1 (Tyr 783), PLC2 (Tyr 759) and VEGFR2 (Tyr 1175) (Cell
Signaling Technology, Danvers, MA, USA); and 4G10 (Upstate/Millipore,
Billerica, MA, USA). Results shown in figures are representative of duplicate
or triplicate experiments.
Bone marrow reconstitution studies
Bone marrow (BM) cells were obtained from 8- to 12-week-old
Plcg2/EGFP knock-in or wild-type female mice, and were intravenously
injected at a total number of 2.5-5.0x106 cells in DMEM into
recipient syngenic 8- to 12-week-old mice that had received a total body
irradiation of 1200 rad prior to transplantation. Mice were examined 1-6
months post-transplantation, and more than 10 wild-type mice receiving
Plcg2+/+ or Plcg2+/EGFP BM cells, 12
wild-type mice receiving PLC2-null BM cells and five PLC2-null
mice (four Plcg2EGFP/EGFP and one
Plcg2al/al mice) receiving Plcg2+/+ or
Plcg2+/EGFP BM cells were used for analysis.
RESULTS AND DISCUSSION
Positional candidate cloning of a spontaneous mutant mouse strain revealed that a loss-of-function mutation of the Plcg2 gene leads to blood-lymph shunts
We identified spontaneous mouse mutants among offspring of a sibling pair
of mice from a mixed genetic background of C57BL/6J and 129/SvEv. These
mutants exhibited chylous ascites (Fig.
1A) and blood-filled lymphatic vessels in the intestine, heart,
diaphragm and skin (Fig. 1B,C,
data not shown for other tissues). Progeny tests indicated that the mutation
is inherited in an autosomal recessive manner. We named the strain
`abnormal lymphatics (al)'. The homozygous mutants
(al/al mice) have blood-filled lymphatic vessels at mid-gestation,
and die spontaneously by bleeding during development or in adulthood (data not
shown).
In order to map a candidate gene causing the phenotype, we designed a
strategy of inter-subspecific backcross mapping between the 129-congenic
mutant mice (Mus musculus domesticus) and CAST/Ei mice (Mus
musculus castaneus) (see Fig. S1 in the supplementary material). We
obtained (129 +/alxCAST) F1 mice and then backcrossed them to
129 +/al mice, because al/al mice were not useful for
backcrossing owing to reproductive abnormalities. We identified affected mice
by the presence of blood-filled intestinal lymphatic vessels
(Fig. 1B) and then performed
PCR genotyping to find genomic regions with homozygous M. m.
domesticus genotypes. Genetic mapping using SSLP markers that distinguish
the two subspecies mapped the mutation to the distal half of chromosome 8,
between D8Mit48 and the terminus (data not shown). Further mapping using SSLP
markers that distinguish three inbred strains identified a 
1 Mb
non-recombinant B6-derived region between D8Ims9 and D8Mit120. High-resolution
mapping narrowed the mutation to a 220 kb region between D8Ims10 and
D8Ims18 (Fig. 1D). This region
contains the Plcg2 gene and one uncharacterized gene homologous to
the NAD(P)H steroid dehydrogenase-like (Nsdhl) gene. Sequencing of
PCR-amplified cDNA and genomic DNA identified a single A-G substitution
located in exon 2 of the Plcg2 gene
(Fig. 1E). The mutation results
in a translational stop at amino acid 54 which is a tryptophan in the
N-terminal pleckstrin homology (PH) domain of PLC2. Western blot
analysis confirmed the translational stop mutation
(Fig. 1F). These data indicate
that the mutation in Plcg2 results in a loss-of-function and leads to
the blood-filled lymphatic vascular phenotype. We therefore renamed this
allele Plcg2al. A previous study reported that
PLC2-null mice showed haemorrhaging during development and in adulthood
(Wang et al., 2000), but did
not report any lymphatic vascular abnormalities. Haemorrhaging may be caused
by rupture of blood-filled fragile lymphatic vessels by mechanical stress or
pressure of the blood flow.
Blood-filled lymphatic vessels in PLC2-null mice were aberrantly formed during development and consisted of BECs and LECs
To identify PLC2-expressing cells responsible for the phenotype, we
generated Plcg2/EGFP knock-in mice by replacing exon 2 of
Plcg2 with EGFP cDNA (see Fig. S2A in the supplementary material).
Plcg2EGFP/EGFP mice did not express PLC2 (see Fig.
S2B,C in the supplementary material) and were phenotypically indistinguishable
from Plcg2al/al mice (see Fig. S2D,E in the supplementary
material). We used the knock-in mice as PLC2-null mice for the analysis
described below.
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2-null embryos became blood filled
and dispersed peripherally during development (see Fig. S2D in the
supplementary material). At E13.5, blood-filled lymph sacs consisted of both
Lyve1+ LECs and CD31+, Lyve1- BECs
(Fig. 2, arrowheads), and the
ECs of the lymph sacs remained close to those of the cardinal veins
(Fig. 2B,E, arrows). These
results were commonly observed in PLC2-null embryos, but not in
wild-type embryos (six embryos for each genotype;
Fig. 2A,D). In PLC2-null
embryos, blood cells may remain in developing lymph sacs during vascular
separation, or transmigrate where the endothelial sheets remain in contact
between veins and lymph sacs (Fig.
2B,E). Fused vessels would allow blood to flow directly into the
lymph sac (one out of two lymph sacs in one out of six embryos;
Fig. 2C,F). Blood accumulation
in developing lymph sacs/vessels may lead to lymph sac/vessel over-expansion
and LEC attachment to developing blood vessels, followed by occasional fusion
between blood and lymphatic vessels.
PLC2 is expressed in vivo in BECs, but not in LECs
We next performed an immunohistochemical analysis using an anti-GFP
antibody. Plcg2/EGFP was expressed in a variety of embryonic and
adult tissues. In addition to F4/80+ monocytes/macrophages
(Fig. 3A), and platelets
(Fig. 3B), Plcg2/EGFP
was expressed in a subset of ECs (Fig.
3C), but not in vascular smooth muscle cells (data not shown).
Plcg2/EGFP was expressed predominantly in arterial ECs, rarely in
venous ECs and not in LECs (Fig.
3D,E). By contrast, Western blot analysis showed that PLC2
was expressed in both BECs and LECs in vitro (see Fig. S3A in the
supplementary material).
PLC1, a highly conserved homologue of PLC2, is required for
VEGFA/VEGFR2 signalling (Liao et al.,
2002) (Sakurai et al.,
2005), and is phosphorylated in response to VEGFA and VEGFC (see
Fig. S3B in the supplementary material). By contrast, PLC2 in ECs was
not phosphorylated by VEGFs (see Fig. S3B,C in the supplementary material). It
remains to be elucidated which growth factors activate PLC2 and how
PLC2 functions in ECs. We examined the discrepancy of PLC2
expression in LECs using primary LECs from Plcg2+/EGFP
mice, and found that Plcg2/EGFP was expressed in a subpopulation of
LECs (see Fig. S3D in the supplementary material). This expression pattern may
be due to altered gene expression caused by changes in the EC microenvironment
(Amatschek et al., 2007). These
results suggest that PLC2 may not function in differentiated LECs that
have become separated from blood vessels; however, we cannot exclude the
possibility that transient expression of PLC2 in EC-lineage cells in a
temporal and spatial manner is involved in the separation process.
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2-dependent manner), we evaluated the BM-cell contribution to the phenotype of
PLC2-null mice. PLC2-null BM cells were used to reconstitute the
BM of lethally irradiated wild-type mice, and these mice developed blood-lymph
shunts in the intestines (Fig.
4A), in which heterogeneous vessels consisting of CD31+
BECs and Lyve-1+ LECs (Fig.
4B) were found. Accumulation of blood- and chyle-containing
ascites was also observed (10 out of 12 mice), but intravenously injected
fluorescein-conjugated isolectin-B4 bound to intestinal LECs (two mice; data
not shown). Additionally, lymphatic vessels cast by intravenously injected
fluorescein-conjugated gelatin (two mice; data not shown) confirmed that the
blood-lymph shunt was due to connections between blood and lymphatic
vessels.
We next examined whether wild-type BM cells could rescue PLC2-null
mice from the blood-lymph shunt phenotype. One to 2 months after wild-type BM
transplantation (three mice), blood-filled lymphatic vessels were distributed
throughout the intestine, and one mouse had peritoneal haemorrhaging (data not
shown). However, at 6 months (two mice), blood-lymph shunts were not observed
in most of the intestine (Fig.
4A,d) and a remarkable lymphatic vascular re-organization had
occurred (Fig. 4A,f), implying
that the fused vessels could be repaired. However, blood-filled lymphatic
vessels still remained in the heart (data not shown) and in a few areas of the
intestine (Fig. 4A,e), and
connections between the two vasculatures were still found in the intestine
(Fig. 4B). These results
indicate that BM-derived cells contribute to the vascular separation process,
even though they were not able to completely rescue the phenotype.
In the intestine, BM-derived cells included many F4/80+
CD11b+ monocytes/macrophages in the mucosa and submucosa (see Fig.
S4A,B in the supplementary material) and a few ECs (see Fig. S4C in the
supplementary material). Monocytes/macrophages, via a Syk/SLP76/PLC2
signalling pathway (Wilde and Watson,
2001), may contribute to lymphangiogenesis
(Cursiefen et al., 2004;
Maruyama et al., 2005). Several
reports have shown, or suggested, the presence of BM-derived LECs
(Religa et al., 2005;
Kerjaschki et al., 2006;
Sebzda et al., 2006), although
their importance in lymphangiogenesis has been debated
(He et al., 2004). We did not
detect PLC2-expressing LECs in vivo
(Fig. 3D), but found that
PLC2 expression was induced in LECs in vitro (see Fig. S3D in the
supplementary material). Additionally, a few PLC2-null, EGFP-expressing
ECs were found in PLC2-null BM-reconstituted mice (see Fig. S4C in the
supplementary material). BM- or hematopoietic cell-derived LECs transiently
expressing PLC2 during their differentiation may be also candidates for
cells that mediate the vascular separation process.
Future studies using cell type-specific knockouts and transgenic animals
will address the issues of which types of BM-derived cells are necessary for
vascular separation, and how PLC2 is involved in the intracellular
signalling which mediates the separation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/2/191/DC1
Footnotes
We thank Motoya Katsuki and Takeshi Yagi for providing the mouse strains and the DT-A plasmid, respectively, and Kaori Yamanaka, Akiko Hori, Hiroko Nakatani and Yuko Ohtani for technical assistance. This work was supported by grants from the Japan Foundation of Cardiovascular Research (to H.I.), the Japan Society for the Promotion of Science (to T.I.), and the Ministry of Education, Culture, Sports, Science and Technology, Japan (to H.I., T.I. and N.Y.).
* These authors contributed equally to this work 
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