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First published online December 7, 2008
doi: 10.1242/10.1242/dev.028456
Research Report |
1 Department of Molecular Cell Biology and Immunology, VU University Medical
Center, PO box 7057, 1007 MB Amsterdam, The Netherlands.
2 Department of Genetics and Tumor Cell Biology, St Jude Children's Research
Hospital, Memphis, TN 38105, USA.
Authors for correspondence (e-mails:
guillermo.oliver{at}stjude.org;
r.mebius{at}vumc.nl)
Accepted 28 October 2008
SUMMARY
The lymphatic vasculature drains lymph fluid from the tissue spaces of most organs and returns it to the blood vasculature for recirculation. Before reaching the circulatory system, antigens and pathogens transported by the lymph are trapped by the lymph nodes. As proposed by Florence Sabin more than a century ago and recently validated, the mammalian lymphatic vasculature has a venous origin and is derived from primitive lymph sacs scattered along the embryonic body axis. Also as proposed by Sabin, it has been generally accepted that lymph nodes originate from those embryonic primitive lymph sacs. However, we now demonstrate that the initiation of lymph node development does not require lymph sacs. We show that lymph node formation is initiated normally in E14.5 Prox1-null mouse embryos devoid of lymph sacs and lymphatic vasculature, and in E17.5 Prox1 conditional mutant embryos, which have defective lymph sacs. However, subsequent clustering of hematopoietic cells within these developing lymph nodes is less efficient.
Key words: PROX1, Lymphatic endothelial cells, Lymphoid tissue inducer cell, Lymph nodes, Lymph sacs
INTRODUCTION
More than a century ago, Florence Sabin proposed a model for the
development of the mammalian lymphatic vasculature
(Sabin, 1902
). According to
this model, endothelial cells bud from the veins to form primitive lymph sacs.
From these sacs, lymphatic endothelial cells (LECs) sprout and form the entire
lymphatic vasculature network. Initial support for the venous origin suggested
by Sabin's pioneering work was provided by analysis of the expression of
Vegfr3 (Flt4), Vegfc, Prox1 and other genes, the
activity of which is crucial for the formation of the entire lymphatic
vasculature in mammalian embryos
(Kaipainen et al., 1995;
Kukk et al., 1996;
Wigle et al., 2002;
Wigle and Oliver, 1999).
Expression of Prox1 is necessary and sufficient for the specification
of the LEC phenotype in venous endothelial cells in vivo and in vitro
(Hong et al., 2002;
Petrova et al., 2002;
Wigle et al., 2002). Sabin's
original venous model was recently validated using a genetic lineage-tracing
approach (Srinivasan et al.,
2007).
In addition to proposing a venous origin for the mammalian lymphatic
vasculature, Sabin proposed that lymph nodes (LNs) originate from the
embryonic primitive lymph sacs (Sabin,
1909). A century later, this dogma has remained unchallenged
despite the recent availability of appropriate molecular markers and mouse
models, and it is still generally accepted that LNs require lymph sacs for
their formation.
In this paper, we address this important question by taking advantage of mouse models in which Prox1 functional activity is either homozygous null, hemizygous or conditionally removed from venous LEC progenitors. In these mutant mice, lymph sacs are either absent or defective, providing an ideal system in which to evaluate whether lymph sacs are required for LN formation.
We conclude that primitive lymph sacs are not necessary for the initial formation of the mammalian LN anlagen. However, their further progression into tight clusters of hematopoietic cells that interact with stromal cells appears to be sensitive to the presence of LECs and/or relatively normal lymph sacs.
MATERIALS AND METHODS
Mice
C57BL/6 mice were purchased from Harlan (Horst, The Netherlands) and
lymphotoxin-alpha-deficient (Lta-/-) mice were purchased
from Charles River (Maastricht, The Netherlands). The generation of
Prox1+/-, Prox1-/-, Tie2-Cre and
Prox1flox/flox mice was reported previously
(Wigle and Oliver, 1999;
Kisanuki et al., 2001;
Harvey et al., 2005). All
animal experiments were approved by the local animal experimentation
committee.
Mice were mated overnight, and the day of vaginal plug detection was noted as embryonic day (E) 0.5. Pregnant females were sacrificed at different time points, and embryos harvested and prepared for sectioning by embedding and freezing in OCT (Sakura Finetek Europe, Zoeterwoude, The Netherlands).
Immunofluorescence
Following cryosectioning (7 µm) of the embryos, sections were fixed in
dehydrated acetone for 2 minutes and then air dried for 15 minutes. Endogenous
avidin was blocked with an avidin-biotin block (Vector Laboratories,
Burlingame, CA). Sections were then preincubated in PBS supplemented with 5%
(v/v) mouse serum for 10 minutes. Incubation with the primary antibody for 45
minutes was followed by incubation with Alexa-Fluor-labeled conjugate
(Invitrogen, Breda, The Netherlands) for 30 minutes. All incubations were
carried out at room temperature. Sections were counterstained with Hoechst
33342 (Invitrogen) for 10 minutes and analyzed on a Leica TCS SP2 confocal
laser-scanning microscope (Leica Microsystems, Rijswijk, The Netherlands).
Antibodies
The antibodies GK1.5 (anti-CD4), MECA-367 [anti-mucosal vascular addressin
cell adhesion molecule 1 (MAdCAM1)], MP33 (anti-CD45), 8.1.1 (anti-podoplanin)
and ERTR7 (which recognizes extracellular matrix component secreted by
fibroblastic reticular cells) were affinity purified from the supernatants of
hybridoma cell cultures using protein G-Sepharose (Pharmacia, Uppsala,
Sweden). The antibodies were biotinylated or labeled with Alexa-Fluor 488, 546
or 633 (Invitrogen). The antibodies A7R34 (anti-IL7R
; eBioscience, San
Diego, CA), 429 (anti-VCAM1; eBioscience), Avas12a1 (anti-VEGFR2;
eBioscience), anti-MECA32 (pan-endothelial cell marker; BD Biosciences,
Erembodegem, Belgium), anti-LYVE1 (Millipore, Billerica, MA), 11D4.1
[anti-vascular endothelial (VE)-cadherin; BD Biosciences], anti-VEGFR1
(anti-FLT1; Neomarkers, Fremont, CA), AFL4 (anti-VEGFR3; eBioscience),
anti-PROX1 (ReliaTech, Braunschweig, Germany), anti-ROR
t (kindly
provided by D. Littman) (Sun et al.,
2000) and anti-β-galactosidase (MP Biomedicals, Aurora, OH)
were used biotinylated or unconjugated and visualized with Alexa-Fluor 488,
546 or 633-conjugated streptavidin, anti-rat or anti-rabbit IgG, or
anti-Armenian hamster-Cy3, as appropriate.
|
Identification of LECs in the LN anlagen
To determine the origin of mammalian LNs, we first assessed the
contribution of LECs to the developing LNs of E14.5 and E16.5 wild-type mouse
embryos. It is well established that early during LN organogenesis and upon
their interaction with lymphotoxin-expressing hematopoietic lymphoid tissue
inducer (LTi) cells, lymphotoxin-beta receptor (LTβR)-expressing
mesenchymal cells differentiate into specialized stromal organizer cells
(Mebius, 2003;
Vondenhoff et al., 2007).
However, before this LTβR-dependent process initiates, some type of
signal induces the accumulation of LTi cells and stromal cells
(Eberl et al., 2004;
White et al., 2007;
Yoshida et al., 2002). Until
now, the inductive signals from the primitive lymph sacs or differentiating
LECs were considered the primary candidates in the process that initiates the
clustering of LTi cells and stromal cells.
To characterize the presence of LECs at the site of LTi cell clusters, we
immunostained wild-type LN anlagen with antibodies against CD4, which is
expressed by LTi cells (Mebius et al.,
1996), IL7R, which is expressed by LTi cells and their
precursors (Cupedo et al.,
2004; Yoshida et al.,
2002) and CD45 (PTPRC - Mouse Genome Informatics), which is
expressed by all hematopoietic cells. This analysis revealed that 
50% of
the CD45+ cells in the anlagen corresponded to LTi cells, as
indicated by their expression of CD4 and IL7R at E14.5
(Fig. 1A). Cluster size
increased between E14.5 and E16.5 (Fig.
1B). At E14.5, all of the LN anlagen were present in the embryo;
however, the inguinal and popliteal LN anlagen consisted of very small
clusters of LTi cells (data not shown). Therefore, the anlagen containing
larger clusters of LTi cells (i.e. axillary, brachial, renal, cervical,
mesenteric, thymic and aortic) were used for detailed analysis.
To identify LECs within the LN anlagen, we immunostained adjacent sections
with the stromal and endothelial markers MAdCAM1, VE-cadherin (cadherin 5),
VEGFR1 (FLT1), VEGFR2 (KDR), MECA32 (PLVAP) and with the LEC markers LYVE1,
podoplanin, PROX1 and VEGFR3 (FLT4)
(Banerji et al., 1999;
Breier et al., 1996;
Breiteneder-Geleff et al.,
1999; Wigle and Oliver,
1999; Cupedo et al.,
2004; Kaipainen et al.,
1995; Mebius et al.,
1996; Hallmann et al.,
1995). At E14.5, the axillary LN anlage was surrounded by
LYVE1+ (Fig. 1C) or
PROX1+ and podoplanin+
(Fig. 1E) LECs, and, central to
the cluster of hematopoietic cells, a VEGFR1+ VEGFR2+
MECA32- blood vessel was detected
(Fig. 1G, arrowhead). At E16.5,
this blood vessel was located distal from the hematopoietic cluster and
opposed to the MAdCAM1+ LYVE1+ LECs
(Fig. 1H, arrowhead). Smaller
VEGFR1- VEGFR2+ MECA32+ blood vessels were
also located among stromal cells at both developmental stages
(Fig. 1G,H).
Lymph sacs are not required to initiate LN formation
Next, we determined whether LECs that colocalized with the earliest
clusters of LTi cells within the LN anlagen provided some type of inductive
signal that is required for the formation of these clusters. To this end, and
to conclusively address whether LECs/lymph sacs regulate the initiation of
mammalian LN development, we took advantage of available
Prox1-/- mouse embryos
(Wigle and Oliver, 1999).
Prox1 activity is necessary for the specification of the LEC
phenotype in venous endothelial cells located in the embryonic cardinal veins
(Wigle et al., 2002). Upon
specification of the LEC phenotype by PROX1 and in agreement with Sabin's
original proposal (Sabin,
1902), the LEC progenitors leave the cardinal vein, form the
primitive lymph sacs and, subsequently, the entire lymphatic network
(Wigle and Oliver, 1999;
Wigle et al., 2002). In
Prox1-null embryos, LEC specification does not take place; therefore,
these mutant embryos lack all LEC derivatives, such as lymph sacs and
lymphatic vasculature (Wigle and Oliver,
1999; Wigle et al.,
2002).
|
). This
analysis revealed that the accumulation of LTi cells and MAdCAM1-expressing
stromal cells was not affected in the LEC- and lymph sac-deficient E14.5
Prox1-null embryos (Fig.
2C,F,I). This result indicated that LECs and/or primitive lymph
sacs are not required during the initial step leading to the formation of the
LN anlagen. However, the organization of the MAdCAM1+ cells
appeared to be affected in the mutant LN anlagen. Normally, at this stage,
MAdCAM1+ cells encapsulate the hematopoietic cells
(Fig. 2G, arrow); instead, in
Prox1-null embryos, MAdCAM1+ cells were intermingled with
the hematopoietic cells and did not encapsulate the hematopoietic clusters
(Fig. 2I, arrow). Also, in
contrast to wild-type embryos, E14.5 Prox1-/- embryos did
not appear to contain the inguinal and popliteal LN anlagen. As these anlagen
normally form last, their absence is likely to be due to a developmental delay
of the Prox1-null embryos.
Next, we confirmed that the clusters of CD45+CD4+
cells detected in Prox1-null embryos correspond to LTi cells and
therefore truly represent the earliest LN anlagen. To do this, we
immunostained sections of E14.5 Prox1-null embryos for RORt
(RORC), a nuclear orphan receptor required for the generation of LTi cells
(Eberl et al., 2004). The
clustered CD45+CD4+ hematopoietic cells within the early
LN anlagen expressed RORt, thereby confirming their LTi cell identity
(see Fig. S1A-C in the supplementary material). This initial analysis
conclusively demonstrated that, contrary to the accepted dogma, the initiation
of mammalian LN formation does not require lymph sacs.
Defective lymphatic vasculature does not affect LN anlagen formation
To determine whether reduced levels of Prox1 activity resulting in
a defective lymphatic vasculature affect the normal formation of the LN
anlagen, we analyzed Prox1-heterozygous mice that exhibited
mispatterned and leaky lymphatic vasculature
(Harvey et al., 2005).
Expression of CD45, CD4 and IL7R (LTi cells and their precursors),
VEGFR1, MECA32 and VEGFR2 (blood vasculature) and of PROX1, podoplanin and
MAdCAM1 (LECs and stromal cells; our unpublished results)
(Cupedo et al., 2004;
Mebius et al., 1996) was
compared in E14.5 wild-type, Prox1+/- and
Prox1-/- embryos. No obvious changes were seen in the
morphology or size of the LTi cell clusters
(Fig. 3A,B), in the occurrence
of the larger VEGFR1+ VEGFR2+ MECA32- blood
vessels (Fig. 3D,E, arrows), in
the lining of the lymphatic endothelium
(Fig. 3G,H, arrows), or in the
occurrence of the smaller VEGFR1- VEGFR2+
MECA32+ blood vessels (Fig.
3D,E, arrowheads) between wild-type and
Prox1-heterozygous littermates. By contrast, the larger
VEGFR1+ VEGFR2+ MECA32- blood vessels
(Fig. 3F) and the lining of the
lymphatic endothelium (Fig. 3I)
were not detected in Prox1-null littermates.
|
Progression of LN formation is affected in Prox1 conditional mutant mice
After determining that the lack of LECs and lymph sacs in
Prox1-null embryos does not affect the initiation of LN formation,
and that despite the reduced PROX1 levels in Prox1-heterozygous
embryos, LN formation occurs normally, we addressed whether a greatly reduced
number of LECs and the defective primary lymph sacs would affect the
initiation of LN formation or the further development of the initial clusters
of LTi cells into bona fide LNs. To do this, we took advantage of available
Prox1 conditional mutant embryos
(Harvey et al., 2005) in which
Prox1 activity is removed from venous LEC progenitors. Floxed
Prox1 mice (Harvey et al.,
2005) were crossed with a Tie2-Cre transgenic line.
Around E10.5, Tie2 (Tek) is expressed in endothelial cells of the cardinal
veins (Kisanuki et al., 2001;
Sato et al., 1993;
Harvey et al., 2005;
Srinivasan et al., 2007). We
have previously shown that upon PROX1 expression, these venous endothelial
cells adopt a LEC phenotype and bud from the veins to form the primary lymph
sacs (Wigle et al., 2002).
Therefore, we used Tie2-Cre to generate Prox1
conditional-null embryos in which lymphangiogenesis is severely compromised
(Srinivasan et al., 2007).
Although variable, some of these Prox1 conditional mutant embryos
contain only a few PROX1-expressing LECs in or around the anterior cardinal
vein at E11.5. At E15.5, they exhibit only some occasional, scattered
superficial LECs (Srinivasan et al.,
2007). Importantly, some of the more severely affected mutant
embryos exhibit no deep lymphatic vasculature
(Srinivasan et al., 2007). In
summary, although standard Prox1-null embryos are completely devoid
of LECs and therefore of lymph sacs and lymphatic vasculature, severely
affected Prox1 conditional mutant embryos exhibit small and
morphologically defective lymph sacs (our unpublished observations)
(Srinivasan et al., 2007).
E17.5 Prox1 conditional mutant embryos were generated by the
intercross of Prox1+/- and
Tie2-Cre/Prox1flox/+ mice
(Srinivasan et al., 2007). As
previously indicated (Srinivasan et al.,
2007), only occasional, scattered superficial PROX1-expressing
LECs were present in some of the most severely affected
Tie2-Cre/Prox1flox/- mutant embryos. As revealed by
immunostaining with antibodies against MAdCAM1, CD4 and IL7R, all LNs
were present in E17.5 Prox1 conditional mutant embryos (data not
shown). In the most severely affected embryo (based on excision efficiency and
the limited number of PROX1-expressing LECs), the size of the CD4-expressing
LTi cell clusters was greatly reduced (data not shown). In less severely
affected embryos with more PROX1-expressing LECs, LTi cell clusters of normal
appearance and that colocalized with MAdCAM1+ LYVE1+
cells were observed (Fig. 4B).
However, further analysis of the stromal organizer cells within the developing
LNs showed reduced expression of MAdCAM1 and VCAM1 in the conditional mutant
embryos (Fig. 4C,D). These
results suggest that LECs and/or the lymphatic vasculature help to position
LTi cells in such a way that mesenchymal cells are stimulated to differentiate
toward stromal organizers. The reduced number of LECs and lymphatic vessels
present in these conditional mutants hamper normal mesenchymal cell
differentiation.
|
). Additional analyses are necessary to identify the inductive signals involved in attracting and clustering the first LTi cells at the sites where LNs will develop. We have observed that LNs often develop at locations where blood vessels bifurcate; therefore, signals that are required for blood vessel branching might stimulate the initiation of LN formation. Accordingly, the first inductive signals should lead to chemokine expression that is responsible for attracting the first LTi cells. Which chemokines are instrumental for this process and how they are induced will need further study.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/1/29/DC1
Footnotes
We thank S. Srinivasan for critical reading of the manuscript and Angela McArthur for scientific editing. This work was supported by a VICI grant (918.56.612) (R.E.M.) and a Genomics grant (050-10-120) (M.F.V.) from the Netherlands Organization for Scientific Research, and by R01-HL073402 (G.O.) from the National Institutes of Health, by Cancer Center Support CA-21765 from the National Cancer Institute, and by the American Lebanese Syrian Associated Charities (ALSAC). Deposited in PMC for release after 12 months.
* These authors contributed equally to this work 
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