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Intravital two-photon microscopy of lymphatic
vessel development and function using a
transgenic Prox1 promoter-directed mOrange2
reporter mouse
René Hägerling*1 , Cathrin Pollmann*1 , Ludmila Kremer†, Volker Andresen‡ and Friedemann Kiefer*
*Cell Signalling Laboratory, Department of Vascular Cell Biology, Max-Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, D-48149 Münster,
Germany, †Central Transgene Facility of Max-Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, D-48149 Münster, Germany, and ‡LaVision
BioTec GmbH, Astastrasse 14, D-33617 Bielefeld, Germany
Abstract
Lymphatic vessels, the second vascular system of higher vertebrates, are indispensable for fluid tissue
homoeostasis, dietary fat resorption and immune surveillance. Not only are lymphatic vessels formed during
fetal development, when the lymphatic endothelium differentiates and separates from blood endothelial
cells, but also lymphangiogenesis occurs during adult life under conditions of inflammation, wound healing
and tumour formation. Under all of these conditions, haemopoietic cells can exert instructive influences on
lymph vessel growth and are essential for the vital separation of blood and lymphatic vessels. LECs (lymphatic
endothelial cells) are characterized by expression of a number of unique genes that distinguish them from
blood endothelium and can be utilized to drive reporter genes in a lymph endothelial-specific fashion.
In the present paper, we describe the Prox1 (prospero homeobox protein 1) promoter-driven expression of
the fluorescent protein mOrange2, which allows the specific intravital visualization of lymph vessel growth
and behaviour during mouse fetal development and in adult mice.
Structure of the lymphatic vessel system
Higher vertebrates essentially depend on the presence of
two vascular networks: the circulatory blood vascular system
and the dendriform, unidirectionally functioning, lymphatic
vessel system [1]. Lymphatic vessels collect extravasated
fluid, macromolecules and cells from the interstitium and
return them to the venous arm of the circulation. Besides
its important function for tissues fluid maintenance and fat
resorption, the lymphatic system is indispensable for immune
surveillance and immune response generation. Lymph vessels
guide extravasated innate immune cells and soluble antigen
to the lymph nodes where both are tested by, and interact
with, the cells of the adaptive immunity, most notably Band T-lymphocytes [2]. Lymphatic vessels are found in
almost all tissues, with notable exceptions being avascular
tissues, such as epidermis, hair, nails, cartilage and cornea,
Key words: fluorescent protein, lymphangiogenesis, lymphatic system, prospero homeobox
protein 1 (Prox1), two-photon microscopy.
Abbreviations used: Ang, angiopoietin; BAC, bacterial artificial chromosome; CLEC-2,
C-type lectin-like receptor 2; Cx, connexin; E, embryonic day; FoxC2, forkhead box C2; GFP,
green fluorescent protein; LEC, lymphatic endothelial cell; Lyve1, lymphatic vessel endothelial
hyaluronan receptor 1; mRFP, monomeric red fluorescent protein; NIR, near-IR; NRP, neuropilin;
OPO, optical parametric oscillator; 2P-LSM, two-photon laser-scanning microscopy; Pdpn,
podoplanin; Prox1, prospero homeobox protein 1; SLP-76, SH2 (Src homology 2)-domaincontaining leucocyte protein of 76 kDa; Syk, spleen tyrosine kinase; Tie, tyrosine kinase
with Ig-like and EGF (epidermal growth factor)-like domains; TiSa, titanium–sapphire; VEcadherin, vascular endothelial cadherin; VEGF, vascular endothelial growth factor; VEGFR3, VEGF
receptor 3.
1
2
These authors have contributed equally to this paper.
To whom correspondence should be addressed (email [email protected]).
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Authors Journal compilation and some vascularized, but immuneprivileged, tissues that
include brain, retina and bone marrow. The lymphoid system
also comprises the secondary lymphoid organs: lymph nodes,
spleen, thymus, tonsils and Peyer’s patches [3].
Initial lymphatic capillaries are blind-ending thin-walled
vessels lined by a single layer of overlapping oak-leafshaped endothelial cells that lack pericyte or smooth muscle
cell coverage and a continuous basement membrane. In
the presence of low tissue fluid pressure, initial lymphatics
display little or no lumen. When the interstitial pressure
rises, specialized anchoring filaments that connect lymphatic
capillaries to the surrounding extracellular matrix exert
tension on the endothelial cells, resulting in opening of
the lumen and fluid uptake [4]. This process is largely
facilitated by the specialized discontinuous cell junctions of
lymphatic capillaries that consist of VE-cadherin (vascular
endothelial cadherin)-positive buttons, which alternate with
VE-cadherin-negative flaps at stretches where the endothelial
cells overlap [5]. These interjunctional gaps are also used by
leucocytes as sites of vessel entry [6].
The lymph, which consists of interstitial fluid, macromolecules and leucocytes, is drained into pre-collector
lymphatic vessels that combine properties of capillaries (oakleaf-shaped cells) and collecting vessels (intraluminal valves).
Transport to the venous circulation is finally accomplished
by increasingly larger collecting vessels that are lined by
a thin continuous basement membrane, show pericyte and
smooth muscle cell coverage and consist of smaller functional
Biochem. Soc. Trans. (2011) 39, 1674–1681; doi:10.1042/BST20110722
Advances in the Cellular and Molecular Biology of Angiogenesis
units called lymphangions. Each lymphangion is delimited by
intraluminal bileaflet valves that ensure directionality of flow
and are capable of active lymph transport [1]. The collecting
lymphatics drain into the thoracic and right lymphatic duct,
which are the only physiological major connections between
blood and lymph vessels.
Development of the lymphatic vessel
system
Over the last two decades, identification of a number of
molecular makers for lymphatic endothelial cells that include
the transcription factor Prox1 (prospero homeobox protein
1), the receptor tyrosine kinase or VEGFR3 [VEGF (vascular
endothelial growth factor) receptor 3], Lyve1 (lymphatic
vessel endothelial hyaluronan receptor 1) and glycoprotein
Pdpn (podoplanin) has significantly facilitated research into
lymphatic biology [7]. In mice and humans, development
of the lymphatic system starts after the primordial blood
vascular system has formed [8]. As early as 1902, Sabin [9]
postulated that LECs (lymphatic endothelial cells) derive
from venous endothelium, a hypothesis that was largely
confirmed by the analysis of genetically modified mice over
the last decade.
The first molecular marker indicating lymphatic differentiation is Lyve1, which is first expressed in the cardinal vein
from E (embryonic day) 9 onwards and indicates lymphatic
competence [10]. Then, 1 day later, around E10, polarized
expression of Prox1 can be detected, initially in a subset of
Lyve1-positive endothelial cells of the cardinal vein [11,12].
Genetic deletion has demonstrated that Prox1 is a master
control gene for the determination of LEC fate [13]. Prox1deficient endothelial cells fail to acquire the LEC phenotype
and arrest at E11.5, being incapable of budding and migrating
from the cardinal vein [14]. The signals regulating Prox1
expression are not fully understood; however, the homeobox
transcription factor Sox18 [SRY (sex-determining region on
the Y chromosome)-related HMG (high-mobility group)box 18], which is also expressed in Lyve1-positive endothelial
cells of the cardinal vein before Prox1 expression, appears
to act upstream of Prox1 [4,15]. Early during development,
VEGFR3 is expressed in all endothelial cells; however, after
development of the lymphatic system, expression becomes
restricted to LECs. One ligand for VEGFR3 is VEGFC, which is required for the migration and sprouting of
Prox1-positive LECs and for proliferation and survival of
LECs until postnatal maturation; VEGF-C-deficient mice
die prenatally because they fail to establish a functional
lymphatic system [16]. In contrast, the other VEGFR3 ligand,
VEGF-D, is dispensable for lymphangiogenesis, as VEGFD-deficient mice do not exhibit lymphatic abnormalities
[17]. Experiments in Xenopus laevis tadpoles identified a
modulatory role for VEGF-D in lymphatic development
[18]. The co-receptor NRP (neuropilin) 2 modulates
VEGFR3/VEGF-C signalling, which influences the survival
and migration of endothelial cells and is necessary for proper
lymphatic vessel sprouting in response to VEGF-C [19,20].
Lymphatic vessel remodelling and
maturation
After separation from the cardinal vein, the emerging
lymphatic endothelial cells organize into primary lymph sacs,
from which a first lymphatic plexus appears by centrifugal
sprouting. Through subsequent maturation, which involves
the transmembrane Eph-receptor ligand ephrinB2, this plexus
remodels into a functional lymphatic vasculature. Mice with
a mutation in the PDZ-domain-binding motif of ephrinB2
fail to remodel the lymphatic vasculature into an organized
network of capillaries and collectors and lack luminal valve
formation [21,22]. Besides ephrinB2, Ang (angiopoietin)
growth factors and their kinase receptors Tie [tyrosine
kinase with Ig-like and EGF (epidermal growth factor)like domains]-1/2 are involved in the remodelling and
stabilization of blood and lymph vasculature [23–25].
A further important factor in the maturation of lymphatic
vessels is the transcription factor FoxC2 (forkhead box C2),
which is expressed in developing lymphatic vessels and
adult lymphatic valves. Loss of FoxC2 leads to defective
lymphatic remodelling, lack of valves and increased pericyte
coverage [26]. Up-regulation of FoxC2 and subsequently
Lyve1 expression mark the maturation of collecting vessels
and are followed by the accumulation of basement membrane
proteins. Although VEGFR3, Prox1 and FoxC2 remain high
in lymphatic valves, their expression recedes in the collecting
vessel trunks. Prox1 expression is, however, retained in
the entire lymphatic vessel system. Lyve1 expression is
almost lost in collecting vessels, but stays high in lymphatic
capillaries [27]. Recently, Cx (connexin) 37 and Cx43 were
both shown to be required for the formation of lymphatic
valves. Both connexins are expressed in embryonic lymphatic
endothelium, but later become restricted to LECs upstream
and downstream of intraluminal valves. Mice deficient for
Cx37 and Cx43 develop lymphoedema, chylothorax and
show reflux of lymph [28].
Platelets and myeloid cells in
blood–lymphatic vessel separation
A crucial step during the development of the lymphatic
vasculature is its separation from the blood circulation, except
for the above-mentioned connections to the subclavian veins.
Failure of blood and lymph vessels to separate results in lethal
haemorrhages and oedema during fetal development. In the
adult, formation of illegitimate blood–lymphatic anastomosis
causes the development of chylous ascites and fatal peritoneal
bleedings [29]. Analysis of mice deficient for the non-receptor
Syk (spleen tyrosine kinase), and the adaptor SLP-76 [SH2
(Src homology 2)-domain-containing leucocyte protein of
76 kDa] suggested that blood–lymphatic separation is a lifelong active process [30]. Syk and its downstream signalling
mediators SLP-76 and PLCγ (phospholipase Cγ ) appear to
act in different cell types via distinct mechanisms.
Genetic fate mapping experiments in mouse fetuses
identified a Syk-expressing myeloid population that largely
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consists of M2-polarized monocytes. These Syk-expressing
cells produce chemokines and growth factors such as VEGFC and VEGF-D and exhibit a pro-lymphangiogenic activity.
In Syk-deficient embryos, this myeloid population massively
accumulates in the fetal skin and is characterized by a strongly
increased lymphangiogenic activity, resulting in lymphatic
hyperproliferation and ultimately the formation of blood–
lymphatic shunts [31].
A number of recent studies have demonstrated that,
in addition, platelets are crucially involved in the process
of blood–lymphatic separation. Carramolino et al. [32]
demonstrated erythrocyte-filled lymph sacs in embryos
deficient for the homeodomain transcription factor Meis1
(myeloid ecotropic viral integration site 1), which lack
megakaryocytes and consequently platelets. Furthermore,
targeted ablation of the megakaryocyte lineage reproduced
the appearance of blood-filled lymph sacs and, at the
same time, excluded defects in the haemopoietic stem cell
compartment. Anti-thrombocyte antibody staining located
platelets, which interacted with the venous vessel wall close
to the sites of nascent lymph sacs [32]. Similarly, Uhrin
et al. [33] showed that local activation of platelets in the
cardinal vein at sites of newly forming lymph sacs is
required for proper blood lymphatic separation. This study
proposed that platelets clot in response to endothelial Pdpn
and thereby prevent blood from entering the lymphatics.
The mucin-type transmembrane glycoprotein T1α/Pdpn is
expressed in the cardinal vein around E11 and later in Prox1positive LECs. Mice lacking this protein die at birth due to
respiratory failure, exhibit severe lymphoedema and show
blood-filled lymphatics [34]. Pdpn interacts with CLEC2 (C-type lectin-like receptor 2) on platelets and myeloid
cells. Interestingly, CLEC-2-dependent activation of platelets
requires the Syk/SLP-76 signalling pathway [35,36].
Regulation of lymph vessel growth by
myeloid cells
During pathological conditions, such as tumour growth
and inflammation, subsets of myeloid cells are known
to promote angiogenesis by producing pro-angiogenic
factors [37]. In particular, macrophages display VEGFdependent and VEGF-independent pro-angiogenic activities
[38]. Depending on their polarization state (M1 or M2),
macrophages can exert tumour-suppressive or tumourpromoting effects. During inflammation-induced lymphangiogenesis, VEGF-A, -C and -D derived from CD11b +
macrophages play a critical role [39,40]. M2-polarized TEMs
(Tie-2-expressing monocytes), which can be recruited by
tumour-cell-secreted Ang-2, are potent stimulators of tumour
angiogenesis [41]. Inhibition of M-CSF (macrophage colonystimulating factor), suppresses tumour angiogenesis as well as
lymphangiogenesis [42].
Besides providing pro-angiogenic factors, macrophages
may also act directly on sprouting vessels. In adipose tissue,
VEGF/VEGFR-2-dependent accumulation of Lyve1 + mac
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Authors Journal compilation rophages is crucial for the formation of a dense vessel network
[43]. Zumsteg et al. [44] showed that, in vitro, macrophages
can form and contribute to lymphatic-like structures.
During this process, macrophages appear to incorporate
preferentially at tip and branching points, indicating a role
in endothelial sprouting [44]. An as yet unidentified role
for macrophages in angiogenesis was recently described by
Fantin et al. [45], who showed that Tie-2- and NRP1expressing tissue macrophages can interact with endothelial
tip cells and thereby provide a bridge to promote vascular
anastomosis.
Imaging lymphatic vessel growth
The formation of new lymphatic vessels through sprouting
from an existing vessel bed, as well as the subsequent pruning
and maturation steps, are highly complex processes, which
involve intricate interactions of different cell and tissue
types. An in-depth understanding of the regulation of lymph
vessels indispensably relies on the ability to image vascular
behaviour under various physiological settings. Ideally,
optical sectioning is applied which allows the visualization
of the spatial arrangement of a vessel bed in a minimally
disturbed piece of tissue and is therefore far superior to the
traditional analysis of histological sections. The process of
sectioning and staining is unavoidably associated with the loss
of material and tissue distortion, making subsequent digital
reconstruction of the vascular network tedious and timeconsuming. Over the last decade, a number of monomeric
fluorescent proteins that span a large spectral range from
yellow–orange to dark red, have been derived by mutagenesis
of mRFP (monomeric red fluorescent protein) [46]. Owing
to the superior tissue penetration of red light, red-shifted
fluorescent proteins are particularly well suited as genetically
encoded markers for different molecules, cells or tissue
types [47]. In the context of visualizing vascular structures,
the use of fluorescent genetically encoded markers bears
significant benefits. Because fluorescent proteins render
immunochemical staining unnecessary, antibody penetration
no longer limits the depth of analysis, which is now
only restricted by optical limits. Ultimately, genetically
encoded markers should also allow the dynamic imaging
of live samples. In addition, the specific marking can be
used to isolate or enrich cell populations of interest using
FACS.
Imaging genetically encoded fluorescent
proteins using two-photon microscopy
A meaningful analysis of vascular structures in living tissue
must be powerful enough to obtain data from deep
tissue layers (>100 μm), which are not accessible to
traditional fluorescence or confocal microscopy. 2P-LSM
(two-photon laser-scanning microscopy) provides ideal
conditions for deep tissue imaging. Owing to the shortage
of endogenous chromophores absorbing in the NIR
Advances in the Cellular and Molecular Biology of Angiogenesis
Figure 1 Determination of two-photon biophysical properties of mRFP derivatives
(A) Schematic diagram of the 2P-LSM setup. Fluorophores are simultaneously excited by NIR-pulsed two-photon
laser lines at wavelengths of 820 nm and 1100 nm respectively. Reflected fluorescence light was filtered using
wavelength-specific bandpass filters and detected by non-descanned (ND) GaAsP (gallium arsenide phosphide) detectors.
(B) Two-photon-excitation spectrum of mOrange determined in transfected Cos-1 cells expressing different levels of protein.
(C) Two-photon-excitation spectra of various mRFP derivatives determined in transfected Cos1 cells. Maximal fluorescence
intensities of the different proteins were normalized to 1. (D) Two-photon-bleaching curves for several fluorescent proteins,
as measured by fluorescence decay after illumination with 1100 nm NIR-pulsed two-photon laser light in transfected Cos-1
cells and plotted as normalized intensities against total exposure time. Half-time was determined as 50 % of initial intensity.
mOrange2 exhibits a 4-fold greater photostability than mOrange. rel. intensity, relative intensity; a.u., arbitrary units.
(near-IR), the NIR two-photon excitation light provided by
femtosecond pulsed TiSa (titanium–sapphire) lasers is less
scattered, penetrates deeper and causes less photobleaching
and toxicity in tissue compared with the visible light used in
classical far-field fluorescence microscopy [48]
For intravital imaging, we use a custom 2P-LSM designed
by LaVision BioTec, which is equipped with two TiSa
lasers, one of which is directly routed into the scan optics.
The second TiSa laser is coupled to an OPO (optical
parametric oscillator) resulting in a wavelength extension
that provides femtosecond pulsed-IR light in the range
1070–1250 nm [49] (Figure 1A). To determine the twophoton imaging parameters of various mRFP derivatives, we
measured their two-photon excitation spectra. Surprisingly,
we found the optimal excitation wavelength for the mRFP
derivatives mStrawberry, mOrange, mCherry and mPlum,
but also for TurboFP635, to be approximately 1100 nm,
while excitation below 1050 nm was negligible (Figures 1B
and 1C). Our findings have two important implications.
First, because NIR light with wavelength greater than
1050 nm is not efficiently generated by TiSa lasers and hence
excitation efficiency for mRFP derivatives is suboptimal,
they emphasize the vital importance of OPO-based 2P-LSM.
Secondly, because of the unexpected property that all mRFP
derivates are optimally excited at 1100 nm, multicolour
imaging is immediately possible, as long as the emission
signals can be separated satisfactorily. In addition, an analysis
of the photostability of the mRFP derivatives demonstrated
significantly decreased bleaching of mOrange2, resulting in
a more than doubled fluorescence half-life compared with
its predecessor mOrange (Figure 1D). Similar results have
recently been reported using OPO-based 2P-LSM for the
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Figure 2 Confirmation of lymphatic-specific expression of the fluorescent reporter protein mOrange2 in adult and fetal
prox1-mOrange2-pA-BAC mice
(A) A cDNA encoding mOrange2–SV40pA (simian virus 40 polyadenylation site) and an ampicillin-resistance cassette for
positive selection were introduced at the Prox1 start codon into the BAC clone RP23-190F21 replacing part of exon 2
using Red/ET recombination. (B) Endogenous expression of prox1-mOrange2 in lymph vessels of the skin imaged by
fluorescence stereomicroscopy and in the ear imaged by confocal laser-scanning microscopy. (C) Skin whole mount of a
prox1-mOrange2-pA-BAC embryo (E14.5) counterstained with positively identifying markers. The vascular marker PECAM1
is expressed in blood vessels and more weakly in lymphatic vessels, whereas Lyve1 is expressed exclusively in lymphatic
vessels. fl., fluorescence.
imaging of tumour and dendritic cells in the skin and
inflammation in the brain [50,51].
Generating a reporter mouse for the
visualization of lymphatic vessels
In combination with GFP (green fluorescent protein),
regulatory elements of the Tie-2 and VE-cadherin promoter
regions have been successfully used to visualize blood vessels
in the mouse [52,53]. We wanted to generate a mouse
model for the visualization of lymphatic vessels that can
be combined with existing genetically labelled mice already
bearing either green- or red-labelled cells. We decided to use
the Prox1 promoter to drive expression of the mOrange2
protein in lymphatic endothelium. Prox1-driven expression
is particularly suitable for monitoring lymph vessels both
C The
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Authors Journal compilation in embryos and adults, because Prox1 serves as a master
regulator of lymphatic differentiation during embryonic
lymphangiogenesis and its expression is maintained also
in adult LECs [27]. The mOrange2 protein is spectrally
sufficiently distinct from green and red fluorescent proteins
to be optically separated by narrow-bandpass filters and its
increased photostability makes it a good choice for long-term
observations in vivo.
We used recombineering in bacteria to generate a
BAC (bacterial artificial chromosome) construct comprising
approximately 100 kb upstream and downstream of the
Prox1-initiating ATG, to which the mOrange2 cDNA was
fused in-frame (Figure 2A). Transgenic mice harbouring
this construct, prox1-mOrange2-pA-BAC mice, showed
faithful labelling of lymphatic capillaries in various organs,
including mesentery, skin and lymph nodes (Figures 2B
Advances in the Cellular and Molecular Biology of Angiogenesis
Figure 3 Intravital two-photon imaging of lymphatic vessels
(A) Superficial lymphatic vasculature of the fetal skin (E16.5) imaged using 2P-LSM. Superficial lymphatics (marked by
mOrange2 expression) show different developmental and remodelling stages of fetal lymphangiogenesis. (B) 2P-LSM
intravital microscopy of lymphatic valve action. A series of images is presented showing the opening of a lymphatic valve
upon lymph flow in an adult mouse. Lymphatic vessels were marked by mOrange2 expression and the surrounding tissue is
visualized through second harmonic signals from the extracellular matrix. (C) Lymphatic valve architecture and lymph flow
after FITC injection. Two-photon images visualize a lymphatic collecting vessel including a saddle-shaped lymphatic valve
(left-hand panel, red) and FITC uptake into lymphatic vessels (central panel, green content within the vessel) embedded in
abundant connective tissue (right-hand panel, merged image showing FITC-filled lymphatic vessel and surrounding tissue).
and 2C). During the course of our analysis, a Prox1promoter driven GFP reporter model was published
that showed a very similar, if not identical, expression
pattern [54].
Intravital 2P-LSM of prox1-mOrange2-pA-BAC mice
finally allows us to visualize the superficial lymphatic vessel
network in developing mouse embryos (Figure 3A), as
well as lymphatic vessels and valve movement in the skin
of adult mice (Figures 3B and 3C). This approach offers,
for the first time, the unique opportunity to visualize the
growth and formation of lymphatic vessels in a living
organism and promises for the near future exciting, so far
inaccessible, insights into the biology of the lymphatic vessel
system.
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Acknowledgments
We thank D. Vestweber for his continued support and R. Tsien,
Department of Pharmacology, Department of Chemistry and
Biochemistry, University of California San Diego, San Diego, CA,
U.S.A., for the gift of mOrange2.
Funding
The project was supported by the Deutsche Forschungsgemeinschaft
[grant numbers SFB629 and SFB656 to F.K. and R.H.] and the Max
Planck Society.
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Received 1 September 2011
doi:10.1042/BST20110722
C The
C 2011 Biochemical Society
Authors Journal compilation 1681