RESEARCH REPORT
191
Development 136, 191-195 (2009) doi:10.1242/dev.025353
Phospholipase Cγ2 is necessary for separation of blood and
lymphatic vasculature in mice
Hirotake Ichise1,*,†, Taeko Ichise1,*, Osamu Ohtani2 and Nobuaki Yoshida1
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 Cγ2 (PLCγ2), by positional candidate cloning. The bloodlymph shunt observed in PLCγ2-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 PLCγ2-null bone marrow cells. These findings indicate that PLCγ2
plays an essential role in initiating and maintaining the separation of the blood and lymphatic vasculature.
KEY WORDS: Mouse, PLCγ2, Lymphangiogenesis, Vascular separation, Bone marrow-derived cells, Endothelial cell
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 FLPdeleter 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 sequencelength 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 SSLPPCR 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⬘.
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.
*These authors contributed equally to this work
Author for correspondence (e-mail: [email protected])
†
Accepted 15 November 2008
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 PGKgb2-neo cassette (Gene Bridges GmbH, Dresden, Germany) by homologous
recombination in E. coli. A DNA fragment containing the modified exon 2 was
DEVELOPMENT
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 Cγ2 (PLCγ2). We also
demonstrate that the blood-filled lymphatic vessels in PLCγ2-null
mice were caused by the aberrant separation of blood and lymphatic
RESEARCH REPORT
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 antigenretrieved for PLCγ2 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-PLCγ2 (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 antirabbit 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-PLCγ2 were
assayed using anti-phosphotyrosine (4G10), according to a previously
described method (Yamaguchi et al., 2008). Antibodies used were as
follows: rabbit polyclonal anti-PLCγ1, PLCγ2 and VEGFR2 (Santa Cruz
Development 136 (2)
Biotechnology); anti-phosphorylated PLCγ1 (Tyr 783), PLCγ2 (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 2.5-5.0⫻106 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 posttransplantation, and more than 10 wild-type mice receiving Plcg2+/+ or
Plcg2+/EGFP BM cells, 12 wild-type mice receiving PLCγ2-null BM cells and
five PLCγ2-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-offunction 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 +/al⫻CAST) 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 PLCγ2. 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 bloodfilled lymphatic vascular phenotype. We therefore renamed this allele
Plcg2al. A previous study reported that PLCγ2-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.
DEVELOPMENT
192
Role of PLCγ2 in lymphangiogenesis
RESEARCH REPORT
193
Fig. 1. A spontaneous mutant mouse strain,
abnormal lymphatics (al), and positional
candidate cloning. Chylous ascites (A) and bloodfilled vessels (B) in mutant newborns. Blood-filled
vessels were Lyve1+ lymphatic vessels (C, arrows).
Arrowheads indiacte blood vessels. Scale bar: 100 μm.
(D) Genotyping of the 139 affected mice
(n=2,4,5,128) using SSLP markers mapped the al locus
to a B6-derived region between D8Ims10 and
D8Ims18. (E) Sequencing of PCR-amplified genomic
DNA showed that a G-to-A transitional mutation (an
asterisk in D) in the Plcg2 gene occurred in the
affected mice. (F) Western blot analysis of newborn
heart tissue lysates showed that PLCγ2 was not
expressed in al/al mice. PLCγ1, loading control.
Fig. 2. Aberrant lymph sacs in PLCγ2-null embryos. (A-F) Transverse
sections of E13.5 embryos immunostained for Lyve1 (green) and CD31
(red). D-F are higher magnifications of A-C, respectively. (C,F) Aberrant
separation of a lymph sac from a cardinal vein. Blue, DAPI-stained
nuclei; A, carotid artery; V, cardinal vein; L, jugular lymph sac. Arrow
indicates a site at which ECs of the two vessels are in contact.
Arrowheads indicate connections between BECs and LECs. Scale bars:
200 μm in A-C; 50 μm in D-F.
First, we characterized the blood-lymph shunt phenotype in
detail. Developing lymphatic vessels of PLCγ2-null embryos
became blood filled and dispersed peripherally during development
(see Fig. S2D in the supplementary material). At E13.5, bloodfilled 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 PLCγ2-null embryos,
but not in wild-type embryos (six embryos for each genotype; Fig.
2A,D). In PLCγ2-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.
PLCγ2 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 PLCγ2 was expressed in both BECs and
LECs in vitro (see Fig. S3A in the supplementary material).
PLCγ1, a highly conserved homologue of PLCγ2, 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.
DEVELOPMENT
Blood-filled lymphatic vessels in PLCγ2-null mice
were aberrantly formed during development and
consisted of BECs and LECs
To identify PLCγ2-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 PLCγ2 (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 PLCγ2-null
mice for the analysis described below.
RESEARCH REPORT
Fig. 3. Double immunostaining for endogenous Plcg2 genedriven EGFP and cell markers. EGFP (green), cell markers (red) and
DAPI (blue) staining are shown. (A) Transverse sections of Plcg2EGFP/EGFP
E14.5 embryos. Arrows indicate EGFP+ F4/80+ monocytes.
(B) Transverse sections of Plcg2EGFP/EGFP E13.5 embryos. V, cardinal vein;
L, lymph sac. Arrows indicate EGFP+ platelets lack nuclei. Arrowhead
indicate venous EGFP+ ECs. (C) Transverse sections of Plcg2EGFP/EGFP
E14.5 embryos. Arrows indicate a subpopulation of EGFP+ ECs.
(D) Transverse sections of thoracic walls of E15.5 embryos. Arrows
indicate lymphatic vessels; arrowhead indicates Lyve-1+ EGFP+
monocytes. EGFP+ ECs were not LECs. Scale bars: 50 μm in A-D.
(E) Transverse sections of E13.5 embryos. A, carotid artery; V, cardinal
vein; L, jugular lymph sac. Panels c and d are higher magnification
images for a and b, respectively. Arrows indicate EGFP+ non-ECs.
Arrowhead indicates a venous EC. Scale bars: 200 μm in a,b; 50 μm in
c,d.
S3B in the supplementary material). By contrast, PLCγ2 in ECs was
not phosphorylated by VEGFs (see Fig. S3B,C in the supplementary
material). It remains to be elucidated which growth factors activate
PLCγ2 and how PLCγ2 functions in ECs. We examined the
discrepancy of PLCγ2 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).
Development 136 (2)
Fig. 4. A BM reconstitution study using PLCγ2-null mice.
(A) Appearance of small intestines (a,b,d,e,g,i) and Lyve1+ intestinal
lymphatic vessels (brown; c,f,h,j). a-c, PLCγ2-null BM-reconstituted
wild-type mice; d-f, wild-type BM-reconstituted PLCγ2-null mice. b is a
higher magnification of a. (d,e) Two representative segments from one
mouse. Plcg2+/EGFP (g,h) and Plcg2EGFP/EGFP (i,j) mice not subjected to BM
transplantation are shown for comparison. Scale bars: 1 mm.
(B) Sections of small intestines of BM-reconstituted mice
immunostained for Lyve1 (green) and CD31 (red). The type of
transplantation is indicated at the top of each panel. Merged images
are shown. Arrows indicate connections between CD31+, Lyve1- blood
vessels (red) and CD31- or low, Lyve1+ lymphatic vessels (green/yellow).
Scale bar: 100 μm.
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 PLCγ2 may not function in differentiated
LECs that have become separated from blood vessels; however, we
cannot exclude the possibility that transient expression of PLCγ2 in
EC-lineage cells in a temporal and spatial manner is involved in the
separation process.
BM reconstitution using Plcg2/EGFP knock-in mice
revealed that BM-derived cells contribute to
vascular separation in a PLCγ2-dependent manner
As BM-derived cell participation in vascular separation has been
suggested previously (Sebzda et al., 2006), we evaluated the BMcell contribution to the phenotype of PLCγ2-null mice. PLCγ2-null
DEVELOPMENT
194
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 fluoresceinconjugated 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
PLCγ2-null mice from the blood-lymph shunt phenotype. One to 2
months after wild-type BM transplantation (three mice), bloodfilled 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, bloodfilled 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/PLCγ2 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 PLCγ2-expressing LECs in
vivo (Fig. 3D), but found that PLCγ2 expression was induced in
LECs in vitro (see Fig. S3D in the supplementary material).
Additionally, a few PLCγ2-null, EGFP-expressing ECs were
found in PLCγ2-null BM-reconstituted mice (see Fig. S4C in the
supplementary material). BM- or hematopoietic cell-derived
LECs transiently expressing PLCγ2 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 PLCγ2 is involved in
the intracellular signalling which mediates the separation.
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.).
RESEARCH REPORT
195
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/2/191/DC1
References
Abtahian, F., Guerriero, A., Sebzda, E., Lu, M. M., Zhou, R., Mocsai, A.,
Myers, E. E., Huang, B., Jackson, D. G., Ferrari, V. A. et al. (2003).
Regulation of blood and lymphatic vascular separation by signaling proteins SLP76 and Syk. Science 299, 247-251.
Amatschek, S., Kriehuber, E., Bauer, W., Reininger, B., Meraner, P., Wolpl, A.,
Schweifer, N., Haslinger, C., Stingl, G. and Maurer, D. (2007). Blood and
lymphatic endothelial cell-specific differentiation programs are stringently
controlled by the tissue environment. Blood 109, 4777-4785.
Cursiefen, C., Chen, L., Borges, L. P., Jackson, D., Cao, J., Radziejewski, C.,
D’Amore, P. A., Dana, M. R., Wiegand, S. J. and Streilein, J. W. (2004).
VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory
neovascularization via macrophage recruitment. J. Clin. Invest. 113, 1040-1050.
He, Y., Rajantie, I., Ilmonen, M., Makinen, T., Karkkainen, M. J., Haiko, P.,
Salven, P. and Alitalo, K. (2004). Preexisting lymphatic endothelium but not
endothelial progenitor cells are essential for tumor lymphangiogenesis and
lymphatic metastasis. Cancer Res. 64, 3737-3740.
Joukov, V., Pajusola, K., Kaipainen, A., Chilov, D., Lahtinen, I., Kukk, E.,
Saksela, O., Kalkkinen, N. and Alitalo, K. (1996). A novel vascular endothelial
growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2)
receptor tyrosine kinases. EMBO J. 15, 290-298.
Karkkainen, M. J., Haiko, P., Sainio, K., Partanen, J., Taipale, J., Petrova, T. V.,
Jeltsch, M., Jackson, D. G., Talikka, M., Rauvala, H. et al. (2004). Vascular
endothelial growth factor C is required for sprouting of the first lymphatic
vessels from embryonic veins. Nat. Immunol. 5, 74-80.
Kerjaschki, D., Huttary, N., Raab, I., Regele, H., Bojarski-Nagy, K., Bartel, G.,
Krober, S. M., Greinix, H., Rosenmaier, A., Karlhofer, F. et al. (2006).
Lymphatic endothelial progenitor cells contribute to de novo lymphangiogenesis
in human renal transplants. Nat. Med. 12, 230-234.
Liao, H. J., Kume, T., McKay, C., Xu, M. J., Ihle, J. N. and Carpenter, G. (2002).
Absence of erythrogenesis and vasculogenesis in Plcg1-deficient mice. J. Biol.
Chem. 277, 9335-9341.
Maruyama, K., Ii, M., Cursiefen, C., Jackson, D. G., Keino, H., Tomita, M.,
Van Rooijen, N., Takenaka, H., D’Amore, P. A., Stein-Streilein, J. et al.
(2005). Inflammation-induced lymphangiogenesis in the cornea arises from
CD11b-positive macrophages. J. Clin. Invest. 115, 2363-2372.
Prevo, R., Banerji, S., Ferguson, D. J., Clasper, S. and Jackson, D. G. (2001).
Mouse LYVE-1 is an endocytic receptor for hyaluronan in lymphatic
endothelium. J. Biol. Chem. 276, 19420-19430.
Religa, P., Cao, R., Bjorndahl, M., Zhou, Z., Zhu, Z. and Cao, Y. (2005).
Presence of bone marrow-derived circulating progenitor endothelial cells in the
newly formed lymphatic vessels. Blood 106, 4184-4190.
Sakurai, Y., Ohgimoto, K., Kataoka, Y., Yoshida, N. and Shibuya, M. (2005).
Essential role of Flk-1 (VEGF receptor 2) tyrosine residue 1173 in vasculogenesis
in mice. Proc. Natl. Acad. Sci. USA 102, 1076-1081.
Sebzda, E., Hibbard, C., Sweeney, S., Abtahian, F., Bezman, N., Clemens, G.,
Maltzman, J. S., Cheng, L., Liu, F., Turner, M. et al. (2006). Syk and Slp-76
mutant mice reveal a cell-autonomous hematopoietic cell contribution to
vascular development. Dev. Cell 11, 349-361.
Takeuchi, T., Nomura, T., Tsujita, M., Suzuki, M., Fuse, T., Mori, H. and
Mishina, M. (2002). Flp recombinase transgenic mice of C57BL/6 strain for
conditional gene targeting. Biochem. Biophys. Res. Commun. 293, 953-957.
Wang, D., Feng, J., Wen, R., Marine, J. C., Sangster, M. Y., Parganas, E.,
Hoffmeyer, A., Jackson, C. W., Cleveland, J. L., Murray, P. J. et al. (2000).
Phospholipase Cgamma2 is essential in the functions of B cell and several Fc
receptors. Immunity 13, 25-35.
Wigle, J. T. and Oliver, G. (1999). Prox1 function is required for the development
of the murine lymphatic system. Cell 98, 769-778.
Wilde, J. I. and Watson, S. P. (2001). Regulation of phospholipase C gamma
isoforms in haematopoietic cells: why one, not the other? Cell Signal 13, 691-701.
Yagi, T., Nada, S., Watanabe, N., Tamemoto, H., Kohmura, N., Ikawa, Y. and
Aizawa, S. (1993). A novel negative selection for homologous recombinants
using diphtheria toxin A fragment gene. Anal. Biochem. 214, 77-86.
Yamaguchi, T., Ichise, T., Iwata, O., Hori, A., Adachi, T., Nakamura, M.,
Yoshida, N. and Ichise, H. (2008). Development of a new method for isolation
and long-term culture of organ-specific blood vascular and lymphatic endothelial
cells of the mouse. FEBS J. 275, 1988-1998.
DEVELOPMENT
Role of PLCγ2 in lymphangiogenesis