BACKGROUND
the formation of the cardiovascular and lymphatic network is crucial for development and physiology.
the cardiovascular system fuels nearly every tissue with oxygen and nutrients and removes waste products, while the lymphatic system is important for the drainage of extravasated fluid, the uptake of fat and is a vital part of the immune system  <cit> .
blood vessel development by sprouting from pre-existing vessels is called sprouting angiogenesis.
in hypoxic environments angiogenic sprouts with tip and stalk cells emerge.
sprouts anastomose to form new functional vessels that supply oxygen to the initially hypoxic environment  <cit> .
from the cardinal vein some venous endothelial cells  differentiate into lymphatic ecs , that migrate to form lymphatic sacs which in turn sprout to form a lymphatic network similar to angiogenesis events  <cit> .
failure to establish a  vascular network leads to severe embryonic defects at mid-to late gestation, whereas misregulation after birth can lead to diseases such as cancer, chronic and inflammatory disorders and oedema .
ecs form the inner cellular lining of blood and lymphatic vessels and the heart, and differ in protein expression, morphology and function depending on the vascular bed.
exposure to external and internal cues as well as epigenetic programming results in ec macro-heterogeneity and micro-heterogeneity .
this means that the endothelium acquires site- and organ-specific structural and functional properties, which are extensively reviewed in aird et al.
.
bmp signalling has emerged as a fundamental pathway of ec identity by regulating cardiovascular and lymphatic development  <cit> .
bmps are members of the transforming growth factor beta  family with more than  <dig> bmp members identified.
bmp ligands reported to function in ecs are bmp2/4/6/7/9/ <dig>  <cit> .
bmps bind to heteromeric transmembrane receptor complexes that consist of type i  and type ii receptors  and often also a co-receptor .
ligand binding and phosphorylation of the gs-domain of the type i receptor by the type ii receptor leads to recruitment and phosphorylation of the intracellular effectors smad <dig>  smad <dig> and smad <dig>   <cit> .
smad <dig> is also known as smad <dig>  activated psmads form a complex with the common smad, smad <dig>  and translocate to the nucleus where they stimulate transcription of specific bmp target genes such as the inhibitors of differentiation , hey <dig> and smad6/7; and repress e.g.
apelin  <cit> .
bmps can also regulate other  pathways that do not involve smad proteins  <cit> .
bmp signalling is highly tuned by extracellular and intracellular modulators, but also by signalling interplay with other signalling pathways.
furthermore, bmps are known to trigger expression of different target genes in a dose-dependent manner  <cit> , a landmark of morphogens.
in addition, hemodynamic changes can induce bmp signalling and activate smad proteins in ecs  <cit> .
recently, excessive bmp <dig> has been implicated in cerebral cavernous malformation  <cit> .
moreover, other regionalized vascular disorders such as hereditary hemorrhagic telangiectasia  and pulmonary arterial hypertension  are mainly caused by mutations in the bmp receptors acvrl <dig>  or eng  and bmpr <dig> respectively .
the question remains how mutations in components of the same bmp pathway can cause such organ-specific diseases.
a better understanding of the heterogeneity in bmp signalling output in different vascular beds may provide this insight and perhaps even the opportunity for disease-specific therapy.
phosphorylated smad1/5/ <dig> are routinely used to monitor bmp transcriptional activity, however this may confound interpretation, because psmads also play a role in chromatin remodelling and mirna biogenesis  <cit> .
to investigate the transcriptional activity of bmp-smad signalling many bmp reporter mice have been generated .
in this study we examined the bre::gfp reporter mouse in which bmp response elements , derived from the id1-promotor, drive the expression of enhanced green fluorescent protein   <cit> .
the substantial decrease in gfp levels observed in smad5-deficient bre::gfp embryos corroborate the bmp-smad sensitivity of this reporter  <cit> .
a commonality between all bre-based reporters is that bre activity does not completely overlap with psmad1/5/ <dig> signalling domains  <cit>  because the onset of reporter activity first requires de novo mrna and protein synthesis and gfp maturation, and the half-life of the reporter protein may deviate from psmad1/5/ <dig>  <cit> .
moreover, psmad1/5/ <dig> can also bind with different affinities and regulate other dna-sequences like e.g.
meme <dig>  <cit> ; psmad1/5/ <dig> also has non-transcriptional functions  <cit> .
additionally, the bre::gfp reporter is heterozygous, and it is becoming apparent that gene expression in general occurs with bursts of monoallelic expression instead of constant biallelic expression  <cit> .
nonetheless, the relevance of the bre::gfp reporter mouse became apparent in our previous study.
discrete gfp localisation patterns in angiogenic endothelium of bre::gfp embryos, with an otherwise widespread psmad1/5/ <dig> localisation, singled out those cells that underwent id-mediated bmp-smad and notch co-signalling essential for robust stalk cell fate  <cit> .
in this study we aimed to further document regional differences in bmp-smad dependent transcriptional activity in murine endothelium of blood vessels, lymphatic vessels and the heart at embryonic and postnatal stages.
we defined regions with stereotypic mosaic and continuous bre::gfp localisation patterns, yet also gfp-negative regions were found in areas where bmp-smad signalling has been reported, compatible with the morphogen functions of bmp ligands.
our data support that bmp-smad signalling can play a role in phenotype switching and endothelial cell heterogeneity.
methods
mice and tissue collection
bre::gfp transgenic mice and endothelium-specific smad1;smad <dig> knockout  mice were used.
genotyping of transgenic mice was done as described  <cit> .
all embryos and postnatal organs were dissected in ice-cold diethylpyrocarbonate -treated phosphate buffered saline  and fixed overnight  in 4 % paraformaldehyde  in pbs at 4 °c.
afterwards they were rinsed with pbs and saline and stored in 70 % ethanol until processing.
fixed embryos of embryonic day   <dig> – <dig> , e <dig>  and e <dig>  and p <dig> intestines were processed for paraffin sectioning.
skin tissue from e <dig>  and e <dig>  bre::gfp embryos was dissected after fixation.
layers of muscle and tissue were carefully removed from the skin, leaving the superficial lymphatic network intact.
from each embryo two skin biopsies were harvested.
retinas were collected from fixed eyes by removing the cornea and carefully lifting the retina from the remaining eyeball.
ears were collected from postnatal pups and separated into a ventral and dorsal side of which the latter was analysed.
for each analysis a minimum of three animals was examined.
immunofluorescence
whole mount procedure
embryos, skin biopsies, retinas, mesentery and ear skins were rehydrated and blocked in 2 % bovine serum albumin  in tris buffered saline  for 3 h at room temperature .
tissues were incubated on with primary antibodies in 2 % bsa in tbs at 4 °c, except for the embryos which were kept at rt.
this was followed by blocking for 3 h in 2 % bsa in tbs and incubation with the secondary antibody on .
the list of primary antibodies and the used dilutions are provided in supplementary material .
after whole mount immunostaining of e <dig>   and e <dig>  embryos the forebrain and the abdomen caudally from the forelimb bud were transversally removed.
all ventral tissues including the heart were removed and the neural tube was then cut open at the ventral side.
the hindbrain was mounted on a glass slide with the ventral side facing up.
the mesentery was excised from the intestines after whole mount immunostaining, and the retina was cut into a four-leaf clover before mounting on a glass slide.
paraffin sections
transversal and sagittal sections  of paraffin embedded tissues were processed for immunodetection using an automated platform .
immunofluorescent triple detection of psmad1/5/ <dig>  gfp and mf <dig> was done manually.
the list of primary antibodies, as well as the conditions used, are provided in supplementary material.
antigen retrieval was done by submerging the slides in tris-edta buffer  for 30 min at 96 °c.
for psmad1/5/ <dig>  endogenous peroxidases were inactivated in 3%h2o <dig> in methanol for 30 min and the antibody signal was amplified using the perkin elmer tsa biotin system kit .
in situ hybridisation
embryos were dissected in depc-treated pbs and fixed on in 4 % pfa in pbs at 4 °c.
afterwards they were washed three times 30 min in depc-treated pbs, immersed in 15 % sucrose and snap frozen in optimal cutting temperature  compound  with liquid nitrogen.
the gfp fluorescent in situ hybridisation  probe was custom designed with the probe designer tool from stellaris .
the coding sequence of the pegfp-n <dig> plasmid  was used for probe design.
fluorescent ish  was performed according to the manufacturer’s protocol  with the addition of a permeabilisation step with 1 % tritonx <dig>  in pbs.
images were acquired using a nikon a1r eclipse ti confocal microscope.
RESULTS
co-localisation of bre::gfp transcriptional activity and gfp in endothelium
to study bmp transcriptional activity in vessel development, we used the previously generated bre::gfp reporter strain  <cit> .
we took advantage of the unstable nature of gfp mrna, the sensitivity and single cell resolution of in situ hybridisation  on the one hand and the direct co-observation of gfp fluorescence on the other hand to validate whether gfp protein localisation reflects well the bre::gfp transcriptional activity in endothelium.
we show that there is a near to absolute correlation between the gfp mrna expression and direct gfp fluorescence localisation in vascular beds like e.g.
the cardinal vein .
in other endothelial linings such as in the heart ventricles there is a good overlap between mrna and protein, yet a fraction of the cells express only gfp mrna or only gfp protein.
this is indicative of onset of transcriptional activity while gfp protein translation and maturation is still taking off in the former cells, while transcriptional activity has already terminated but gfp protein is still present in the latter cells .
this pattern suggests the dynamic turning “on” and “off” of transcriptional activity.
overall, we conclude that gfp protein patterns report with fidelity bre::gfp transcriptional activity patterns.fig.
<dig> the gfp reporter protein faithfully recapitulates the transcriptional activation of the bre::gfp transgene.
in situ hybridisation for gfp mrna  and direct gfp fluorescence  on cryosections through the cardinal vein  and the ventricular trabeculae  at e <dig> .
dapi is used to stain nuclei.
the boxed area in  is enlarged in panels .
the corresponding low magnification view of the trabeculae in the ventricle is not provided because of insufficient intensity of the ish signal.
white arrowheads indicate ecs double positive for gfp mrna and direct gfp fluorescence.
the red and green
arrows depict ecs only positive for gfp mrna or gfp fluorescence respectively.
scale bars: 100 μm ; 50 μm ; 10 μm
the gfp localisation pattern is mostly a subdomain of the psmad1/5/ <dig> pattern  as previously reported  <cit> , which is compatible with bmp-smad non-transcriptional and morphogen functions as discussed in the background section of this paper.
the specificity of the anti-psmad1/5/ <dig> antibody was validated in endothelium-specific smad1;smad <dig> double knockout embryos .
however, in some endothelial beds like e.g.
in the cardinal vein in e <dig>  embryos, gfp localisation hardly overlapped with psmad1/5/ <dig> localisation , suggesting terminated or undetectable psmad1/5/ <dig> signalling or transcriptional activation of the bre:gfp transgene by other factors than bmp signalling.
bre::gfp transcriptional activity is present in a mosaic pattern during embryonic angiogenesis
to closely examine bre::gfp transcriptional activity in the rapidly expanding vascular plexus, we analysed embryonic day   <dig>   and e <dig>  bre::gfp mouse embryos.
tip and stalk cell formation as well as anastomosis during sprouting angiogenesis can then be investigated.
in the roof of the hindbrain sprouts are formed from the perineural vascular plexus at opposite lateral sides of the embryo that then anastomose medially in a caudal fashion from the level of the otic vesicles onwards  <cit> .
whole mount immunostainings of bre::gfp embryos showed that gfp was mainly present in and around the heart region at  <dig> somites , with little bre::gfp transcriptional activity in the endomucin-positive blood vessels .
however, in 30s embryos a scattered gfp pattern co-localised particularly within the main vessels of e.g.
the head and the intersomitic vessels .
the hindbrain roof was excised from these embryos and the dorsal vascular plexus was flat-mounted   <cit> .
in the two-dimensional vascular plexus at 22 s bre::gfp transcriptional activity occurs in a scattered or mosaic pattern throughout the plexus, which is in accordance with previous observations  <cit> .
remarkably, some tip cells were found to express low levels of gfp while the mosaic pattern in non-tip cells was more intense throughout the plexus .
this is in contrast with earlier observations where gfp was not detected in tip cells  <cit> , which likely reflects higher affinity of the current chicken polyclonal anti-gfp antibody than the previously used mouse monoclonal anti-gfp antibody.
at e <dig>  the vessels from the superficial vascular plexus start to invade the hindbrain, thus forming a multi-layered vascular network  <cit> .
depth coding of the dorsal hindbrain region at 30s revealed the superficial and deeper plexus .
the latter had slender vessels with slim tip cells, whereas the superficial plexus had thicker honeycomb-like vessels and broader tip cells, as seen at 22 s .
the gfp localisation pattern in both plexuses was mosaic and comparable to the plexus of the 22 s embryo, yet, the deeper plexus seemed to be enriched in gfp-positive ecs  compared to the superficial plexus .
additionally, a string of gfp-positive non-endothelial cells was observed in the midline at both stages.
this correlates with the presence of bmp <dig> and bmp <dig> in the dorsal midline at e <dig> -e <dig>   <cit> .
in general, the two distinct dorsal vascular plexuses demonstrate heterogeneity in bmp-smad signalling in an angiogenic vascular bed.fig.
<dig> mosaic bre::gfp transcriptional activity in midgestation mouse hindbrain vasculature.
immunodetection of endomucin and gfp in bre::gfp embryos of 22 s  and 30s .
arrowheads indicate the heart region.
boxed areas represent the head region  and the intersomitic vessels .
g overview of a 22 s dorsal vascular plexus  in a flat-mounted hindbrain roof.
arrows indicate direction of rostral  to the left, caudal  to the right.
boxed areas  show sprouts at the midline.
asterisks depict gfp-positive tip cells.
h depth coding of a 30s dorsal vascular plexus and the original picture .
blue and green colours represent more ventral and dorsal tissues respectively.
the vascular plexus at 30s consists of a superficial  and deeper plexus .
boxed areas are enlarged in the right panels.
scale bars: 500 μm ; 100 μm ; 75 μm ; 50 μm ; 25 μm
spatio-temporal changes in bre::gfp activity during retinal angiogenesis
the retina is commonly used to investigate postnatal blood vessel development.
the primary plexus develops from the optic nerve towards the peripheral margin.
around postnatal day   <dig> the primary vascular plexus invades the deeper retinal layers perpendicularly whereafter the outer plexus forms again radially.
different aspects of vessel formation can be studied because vascular sprouting happens at the periphery while remodelling occurs simultaneously in the centre  <cit> .
the gfp localisation pattern was diverse but stereotypic throughout the retinal stages investigated and the gfp levels decreased over time.
at p <dig>  the vasculature has sprouted halfway across the retina .
the sprouting front displayed relatively strong gfp signals compared to the central plexus .
many tip cells at the sprouting front as well as the arteries and veins in the centre were gfp-positive, whereas the intermediate capillary bed displayed a more mosaic gfp distribution .
moreover, arteries seemed weaker gfp-positive compared to the strong gfp-positive veins .
at p <dig> the sprouting front developed into a vascular border.
comparable to p <dig>  gfp-positive arteries and veins were observed, along with a mosaic distribution of gfp in the capillaries .
at this stage the matured vessels in the centre have sprouted into the retina to form the perpendicular vessels and the outer plexus.
approximately half of the perpendicular vessels seemed gfp-positive, though weaker than the veins of the primary plexus .
moreover, the less ramified outer plexus also displayed a mosaic gfp pattern .
overall, fewer gfp-positive cells were present in the different plexuses at p <dig>  although the mosaic distribution was maintained in the primary and outer plexus .
these data indicate a dynamic nature of bmp-smad signalling over time.fig.
<dig> different bre::gfp transcriptional activity patterns in the postnatal vasculature.
a overview of a p <dig> retina with immunodetection of endomucin and gfp.
boxed areas show the sprouting front  and the centre  of the retinal plexus.
single staining for gfp is shown in the bottom panels.
asterisks depict gfp-positive tip cells, the arrows point to the veins, while the arrowheads show the arteries.
d–g the retina at p <dig>  immunostained for endomucin and gfp, has a multi-layered vascular plexus consisting of the vascular border  and centre  of the primary plexus, the perpendicular vessels  and outer plexus .
single gfp staining is shown in the lower panels.
scale bars: 500 μm ; 75 μm
gfp localisation patterns are dynamic in the developing heart
given the well-known functions of bmps in heart development and the presence of bre::gfp activity during early heart development and valve formation  <cit> , we zoomed in deeper on the spatio-temporal patterns of bre::gfp transcriptional activity in endocardium at e <dig> -e <dig> , e <dig>  and e <dig>  and observed remarkable regional differences over time.
at e <dig>  the initial heart tube starts to loop and progressively forms the four chambered heart  <cit> .
the heart tube comprises an inner endothelial layer and an outer myocardial layer separated by extracellular matrix or cardiac jelly  <cit> .
the pro-epicardial organ progressively covers the myocardial layer with epicardium.
overall, relatively few gfp-positive cells were observed in tissue sections of the developing heart , which is consistent with former results  <cit> .
nonetheless, gfp patterns were robust and reproducible in endocardial subregions, and changed over time .fig.
<dig> dynamic bre::gfp transcriptional activity in the developing heart.
paraffin embedded hearts are sectioned transversally, unless otherwise specified, and  stained for the endothelial marker isolectinb <dig> and gfp.
dapi is used to stain nuclei.
a overview of an e <dig>  heart.
b section of the superior and inferior atrioventricular canal cushions at e <dig> .
c e <dig>  sagittal section of avc  and oft cushions .
d e <dig>  lateral cushion with gfp-positive endocardial cells  and myocardium .
e at e <dig>  the septum primum displays gfp-positive ecs on the endothelial cap .
the atrium is gfp-negative at e <dig>   while the ventricular trabeculae  have gfp-positive ecs .
h overview of an e <dig>  heart.
the tricuspid valve shows gfp-positive cells at the atrial side  at e <dig>   and e <dig>  .
gfp signals  in the intra-atrial septum at e <dig>   and e <dig>  .
some endocardial cells covering the atrial  and ventricular trabeculae  are gfp-positive  at e <dig> .
ao: aorta; avc: atrioventricular canal; iavc: inferior avc cushion; savc: superior avc cushion; bv: bicuspid valve; cv: cardinal vein; doft: distal outflow tract; ivs: inter-ventricular septum; la: left atrium; lv: left ventricle; poft: proximal outflow tract; ra: right atrium; rv: right ventricle; sp: septum primum; tv: tricuspid valve.
scale bars: 200 μm ; 100 μm ; 50 μm
cardiac cushions are the primordia of the valves and most septa in the developing heart.
they are formed in the atrioventricular canal  which separates the atria from the ventricles and the outflow tract  which bridges the ventricles with the aortic sac .
at respectively e <dig>  and e <dig>  the endocardial cells of the avc cushions and proximal oft cushions delaminate, undergo endothelial-to-mesenchymal transition  and invade the cardiac jelly .
in contrast, the distal oft cushions become mainly populated by neural crest cell derived mesenchymal cells from e <dig>  onwards  <cit> .
the endocardial cells of the superior and inferior avc cushions displayed a mosaic gfp localisation pattern until e <dig>  .
from e <dig>  onwards, patches of gfp-positive ecs were restricted to the atrial side of the superior cushion.
these streaks of gfp-positive ecs remained present at e <dig>  and e <dig>  on the medial leaflets of the tricuspid and bicuspid valve that form from these cushions .
in contrast, only few gfp-positive ecs were present in the lateral avc cushions from e <dig>  onwards .
yet, at e <dig>  the lateral leaflets of the tricuspid and bicuspid valve displayed, just like the medial leaflets, also patches of gfp-positive ecs .
patches of gfp-positive muscle cells were also observed in the avc myocardium flanking the developing cushions and valves up to e <dig> , though with varying gfp levels among neighbouring cells.
.
the ecs of the oft cushions showed a mosaic gfp localisation pattern comparable to the avc cushions at e <dig> , although the oft appeared slightly enriched in gfp-positive ecs .
remarkably, the mesenchymal cells that populate the avc and oft cushions were gfp-negative .
in contrast to the ecs of the avc and oft, the ecs of the inflow tract and the endothelial cap of the septum primum showed a ubiquitous gfp localisation pattern at e <dig>  .
as development proceeds and this septum reaches the superior cushion forming the intra-atrial septum, bre::gfp transcriptional activity decreased with only a few gfp-positive ecs still present at e <dig>  and e <dig>  .
only occasionally a gfp-positive cell was detected in atrial endothelium at e <dig>  , while the ventricles showed a mosaic gfp pattern in the ecs covering the trabeculae throughout the stages analysed .
interestingly, a mosaic pattern also emerged in the atrial endothelium coinciding with initiation of trabeculation from e <dig>  onwards .
moreover, the ecs of the aortic and pulmonary valve leaflets also displayed a mosaic gfp pattern at e <dig>  .
levels of bre::gfp activity differ in embryonic and postnatal blood and lymphatic vessels
little information is available on bmp-smad signalling in different lymphatic beds.
at e <dig>  the first lecs differentiate from venous ecs in the cardinal vein  <cit> .
these lecs bud of, migrate and assemble into lymphatic sacs by e <dig> , which will remodel into a functional lymphatic network.
we found interesting spatio-temporal differences in bre::gfp transcriptional activity during lymphangiogenesis.
in the embryo most blood and lymphatic vessels have a widespread gfp localisation pattern, while in postnatal tissues like the mesentery, intestinal villi and the ear skin many blood vessels appeared to have reduced gfp signals and the lymphatic vessels had discrete and unique gfp localisation patterns.
in general, the blood vessels, including the cardinal vein, and lymphatic vessels displayed a continuous gfp localisation pattern at e <dig> -e <dig>  .
however, the aorta showed a mosaic gfp localisation pattern .
interestingly, nearly all prox1- positive lecs budding from the cardinal vein were gfp-positive, yet still weakly positive for the blood vessel marker endomucin in e <dig>  embryos .
likewise, at e <dig>  some gfp-positive ecs were prox1- and endomucin-positive , yet in few sections at e <dig>  the gfp-positive cardinal vein adjacent to the lymphatic sac was endomucin-positive on the medial side, while the lateral side, closest to the lymphatic sac, was prox1-positive .
blood and lymphatic vessels in dorsal skin biopsies were also ubiquitously gfp-positive at e <dig>  and e <dig>  .
however, the gfp signal appeared more uniform in the blood vessels, whereas the lecs showed different levels of gfp among neighbouring cells .
other gfp-positive cord-like structures were observed at e <dig>  and e <dig>  in dorsal skin biopsies, but these were excluded as vessels because they were collagen type iv-negative .
similar structures have been described as schwann cells of sensory nerves  <cit> .fig.
<dig> spatio-temporal changes in bre::gfp transcriptional activity in blood and lymphatic vessels.
a–c transverse sections at the level of the neck of e <dig>  , e <dig>   and e <dig>   bre::gfp embryos with immunodetection of gfp, the blood vessel marker endomucin and the lymphatic vessel marker prox <dig>  dapi is used to stain nuclei.
boxed areas are enlarged in the right panels, with single gfp, endomucin and prox <dig> staining next to it.
skin biopsies of e <dig>   and e <dig>   bre::gfp embryos with immunodetection of gfp, endomucin and prox <dig>  boxed areas are shown in the middle  panels with single staining for gfp in the right panel.
cv: cardinal vein; da: dorsal aorta; ls: lymphatic sac.
scale bars: 75 μm ; 50 μm ; 25 μm
since the lymphatic network is still expanding after birth, we investigated the postnatal mesentery, intestines and ear skin.
different types of lymphatic vessels occur in each of these tissues.
the mesentery contains collecting vessels, while lacteals resorb lipids from the intestines and lymphatic capillaries drain lymph from ear tissue.
interestingly, many venous ecs appeared gfp-positive in p <dig> and p <dig> mesentery , whereas only few gfp-positive arterial ecs were observed  with also some gfp-positive peri-endothelial cells covering the arteries .
the developing lymphatic vessels were predominantly gfp-negative, although bre::gfp transcriptional activity was specifically present in some valve forming regions .
at p <dig>  the arterial ecs and peri-endothelial cells appeared gfp-negative and also fewer venous ecs were gfp-positive, whereas the lymphatic vessels still showed gfp in the valve regions .
remarkably, the vessels had reduced bre::gfp transcriptional activity over time from p <dig> to p <dig> .fig.
<dig>
bre::gfp transcriptional activity in postnatal blood and lymphatic vessels.
a stitch of a p <dig> mesentery with immunodetection of gfp and the pan-endothelial marker cd <dig>  arteries, veins and collecting lymphatic vessels in a p <dig>  and p <dig>  mesentery are depicted.
arrows point to gfp-positive peri-endothelial cells.
arrowheads show gfp-positive ecs in the vicinity of a valve.
sections of p <dig> intestines with immunodetection of gfp and endomucin  or the lymphatic vessel marker lyve <dig> .
white arrows point to some gfp-positive cells in a capillary, yellow arrows show gfp-positive lecs in a lacteal.
arrowheads show some gfp-positive goblet cells.
stitch of p <dig>  and p <dig>  dorsal ear skin with immunodetection of gfp and the lymphatic marker podoplanin.
gfp-positive lecs at the branch points are depicted by arrows at p <dig>  and p <dig> .
a: artery; l: lymphatic vessel; v: vein.
scale bars: 1 mm ; 500 μm ; 75 μm
most intestinal villi comprise a lyve1-positive lymphatic vessel, called the lacteal, which is surrounded by an endomucin-positive blood capillary.
only few ecs of the blood capillaries and lacteals were gfp-positive .
other non-endothelial cells, such as goblet cells, were also gfp-positive in the villus .
in the ear skin bre::gfp transcriptional activity was absent from the lymphatic capillary bed at p <dig> and p <dig>  with the exception of a single gfp-positive lec at some branch points .
bre::gfp transcriptional activity does not correlate with proliferation
inhibitors of differentiation or ids are helix-loop-helix proteins that interact with and inhibit basic helix-loop-helix transcription factors.
through induction of these ids, the bmp pathway promotes ec migration and tube formation  <cit> .
this led to the hypothesis that gfp-positive ecs would be proliferative.
several embryonic and postnatal tissues were investigated for co-localisation of gfp and phospho histone  <dig>  in the blood and lymphatic system.
ph <dig> is only present in the m-phase of the cell cycle  <cit> .
there was rarely overlap between ph <dig> and gfp-positive blood or lymphatic ecs in the neck region at e <dig>  and e <dig>   or in dorsal skin biopsies from e <dig>  and e <dig>  embryos .
when ph <dig> seemed to be present in a gfp-positive ec, the proliferating cell was usually not in the same focal plane as the ec , or showed mainly low to no levels of gfp .
in addition, skin biopsies of e <dig>  and e <dig>  embryos were immunostained for ki <dig> which marks all active phases of the cell cycle.
proliferation decreased between e <dig>  and e <dig> , but even though many non-endothelial cells were proliferative only few gfp-positive ecs and lecs were ki67-positive .
also in the heart there was almost no co-localisation observed between ph <dig> and gfp .
furthermore, ecs of the sprouting front and the centre of the retina were not ph3-positive at p <dig>  however, at this stage non-endothelial cells were more proliferative at the sprouting front than at the centre .
overall, proliferation decreased by p <dig> but shifted towards the ecs, as now ph3-positive ecs could be observed .
some of these proliferating cells seemed more gfp-positive than others.
this is an intriguing difference in co-localisation pattern which suggests another context dependent role for bmp-smad signalling.fig.
<dig>
bre::gfp transcriptionally active ecs are rarely proliferative.
transverse sections of e <dig>   and e <dig>   bre::gfp mouse embryos with immunodetection of the proliferation marker , gfp and endomucin  or prox <dig> .
dorsal skin biopsies obtained from e <dig>   and e <dig>   bre::gfp embryos show ph <dig>  gfp and endomucin  or prox <dig>  staining.
boxed areas are enlarged in the right panels.
i overview of an e <dig>  heart with immunodetection of gfp and ph <dig>  dapi is used to stain nuclei.
the sprouting front  and centre  of p <dig>  and p <dig>  retinas with immunodetection of gfp, endomucin and ph <dig>  arrows point to ph3-positive ecs.
avc: atrioventricular canal; ivs: inter-ventricular septum; la: left atrium; lv; left ventricle; ra: right atrium; rv: right ventricle.
scale bars: 100 μm ; 75 μm ; 50 μm; 25 μm
discussion
spatio-temporal information on output of important signalling pathways in the vasculature may help to increase our understanding of how mutations in components of the same pathway can cause organ-specific vascular disorders and provide a window of opportunity for designing disease-specific therapy.
in the past decade, many bre-reporters have been generated in zebrafish and mice .
the bre::gfp reporter used in this study is not the most sensitive, as some other bmp reporters show broader patterns of transcriptional activity.
however, this precisely allows to zoom in on selective processes and dose-dependent bmp actions.
our study shows that gfp patterns in endothelium faithfully report transcriptional activation of the bre::gfp transgene, and are remarkably robust.
however the bmp-smad transcriptional output is highly dynamic in time and space, in the different cardiovascular and lymphatic beds of bre::gfp mice.
mosaic gfp localisation patterns were observed in different regions of the developing vascular tree and heart.
it was found in the dorsal vascular plexuses of midgestation mouse embryos and in the capillary bed of p4-p <dig> retinas.
in addition, at e <dig> -e <dig>  the endocardial cells of the avc and oft cushions and those lining the atrial and ventricular trabeculae also displayed a mosaic gfp pattern .
such a mosaic pattern of transcriptional activity suggests a role for bmp-smad in ec plasticity and micro-heterogeneity.
the multi-layered vascular network of the dorsal hindbrain and retina develop in a similar fashion, with the deeper plexus of the dorsal hindbrain resembling morphologically more the capillary network in the primary plexus of the retina  <cit> .
previously, we showed that the bre::gfp pattern singled out stalk cell competent cells in the dorsal vascular plexus that were undergoing bmp and notch co-signalling and that loss of bmp-smad signalling in endothelium resulted in a stalk cell defect  <cit> .
we also observed weak gfp-positive tip cells in the dorsal hindbrain and retinal plexuses.
these tip cells might have been former stalk cells that have taken over the tip cell position  <cit> , with traces of non-degraded gfp.
alternatively, bmp <dig> and bmp <dig> synthesized by cells at the midline  <cit>  may function as pro-angiogenic guidance cues that trigger an alternative bmp-smad signalling pathway in the tip cells.
circulatory bmp <dig> is likely to promote stalk cell competence through activating the mosaic transcriptional activity observed in the rest of the dorsal vascular plexus.
in the retina, bmp <dig> and bmp <dig> are important for postnatal vascular remodelling  <cit> .
remarkably, bmp <dig> was unable to induce bre activity in vitro, suggesting that the gfp signals in the centre of the retina, where vessel maturation and remodelling occurs, were the result of bmp <dig> signalling.
however, also bmp <dig>   <dig>   <dig> and  <dig> have been shown to play significant roles in retinal neurogenesis and vascularisation  <cit> .
retinal vascularisation is preceded and stimulated by the development of a vast network of neuronal cells  <cit> , the latter also depending on bmp-smad signalling  <cit> .
the retinal ecs reciprocally promote differentiation of the neuronal plexus  <cit> .
our data do not allow to distinguish the precise source and type of bmp signal, yet, bmp-smad transcriptional activity seems more imperative at the sprouting front than in the centre where the vascular plexus is maturing.
many bmps have been reported to regulate cardiac cushion development  <cit> .
in the avc cushions, bmp <dig> stimulates ecs to undergo endmt  <cit> , while bmp <dig> is important in the oft for proliferation and growth of endocardial cushions rather than endmt  <cit> .
studies with knockout mice reveal that bmp <dig>  alk <dig>  alk <dig>  bmpr <dig>  smad <dig> and smad <dig> are important for the development of the avc cushions and to a lesser extent oft cushions .
our study shows that bmp signalling induces mosaic transcriptional activity in cushion endocardium, likely to maintain an intact cushion epithelium while a few cells can undergo endmt.
whether the gfp-positive cells or rather their neighbours are subsequently triggered to undergo endmt remains to be elucidated.
taken together, in cushion endocardium and in angiogenic endothelium, the mosaic-perhaps stochastic-transcriptional bmp-smad activity seems to serve as a source of phenotypic diversity.
the exquisite fine-tuning of the bmp pathway, which also involves negative feedback mechanisms, may also generate switch modes of activation states.
whether the mosaicism in bmp-smad transcriptional activity is static or dynamic, with bre::gfp activity switching between ‘on’ and ‘off’ states, cannot be addressed directly in our model due to limitations in the resolution of real-time intravital microscopy, combined with the need for potentially long windows of observation.
dynamic mosaicism in expression has recently been demonstrated for von willebrand factor , and also in vitro for esm <dig> and ephrin-b <dig>  in some but not all vascular beds.
this appears to be a phenotype switching strategy for adaptive homeostasis  <cit> .
remarkably, the endocardial cells of the atria turned on mosaic bre::gfp activity several days later than the ventricular endocardial cells.
interestingly, this delayed activation correlated with the delayed onset of bmp <dig> expression  <cit>  and initiation of trabeculation in atrial myocardium at e <dig>  compared to the onset of the same process in ventricular myocardium already at e <dig> .
bmp <dig> is a well-known regulator of cardiac trabeculation and/or compaction  <cit> .
trabeculation defects are also observed in endothelium-specific smad <dig> ko and smad1/smad <dig> double ko embryos  <cit> .
our data suggest that not all ecs are equally involved in this process.
it would be interesting to evaluate whether and how expansion from a mosaic to a continuous bmp-smad transcriptional activity pattern in ventricular and atrial endocardium would impact trabeculation or provide  advantages.
remarkably, during embryonic development the lymphatic vessels showed widespread bre::gfp transcriptional activity, yet in pups gfp-positive ecs were restricted to the valve forming regions of collecting lymphatic vessels in the mesentery.
this is in agreement with the role of bmp <dig> in lymphatic valve development  <cit> .
in the lymphatic capillary bed of the ear skin, an occasional gfp-positive cell would localise at branch points.
furthermore, gfp-positive endocardial cells were observed on the atrial side of the tricuspid and bicuspid heart valve leaflets, but also in the inflow tract.
all these patterns correspond with endothelium undergoing fluid shear stress, which can induce bmp-smad signalling.
hemodynamic alterations have been reported to induce bmp <dig> and activate smad1/ <dig> in the aorta  <cit> , and to mediate arteriogenesis  <cit> .
mature ecs are characterized by a slow proliferation rate.
for example, in adult ear skin only  <dig>  % of the lecs are reported to be ki67-positive, whereas approximately 30 % of lecs are ki67-positive in embryonic skin at e <dig> -e <dig>   <cit> .
the role of bmp signalling in ec proliferation is thought to be highly context dependent  <cit> .
because bre::gfp signals peaked around midgestation and progressively decreased in postnatal stages in the vascular tree, we reasoned that correlations between bmp-smad transcriptional output and proliferation may become apparent in specific vascular beds.
remarkably, we found that proliferating ph3-positive ecs were almost invariably gfp-negative in the different vascular beds analysed, except in the p <dig> retina where more often double positive cells were observed.
a recurrent theme was - like in zebrafish embryos  <cit>  - that the abundance of gfp-positive ecs was higher in veins than in arteries in several embryonic and postnatal tissues.
for instance, the ecs in the cardinal vein showed a continuous gfp localisation pattern, whereas the aorta had a mosaic gfp pattern.
in the mesentery almost no gfp-positive ecs were observed in the arteries, yet some of its peri-endothelial cells were gfp-positive.
in contrast, many ecs of the veins were gfp-positive.
alk <dig>  endoglin and bmpr <dig> play a role in the establishment and maintenance of mural cell coverage on mature vessels  <cit> , primarily in arteries.
mutations in bmpr <dig> lead to pah which is characterized by abnormal proliferation of ecs and smooth muscle cells  in arterioles  <cit> , whereas a deletion of bmpr <dig> leads to insufficient recruitment and decreases pdgfrβ expression in mural cells  <cit> .
bmp2/bmpr <dig> signalling negatively regulates pdgfbb induced proliferation of pulmonary arterial smcs in a psmad1/5/ <dig> independent manner  <cit> .
it is likely that veins express more bmp <dig> and hence limit the number of smc coverage, but also the differences in shear stress in both vessel types may underlie the above differences.
CONCLUSIONS
we experienced that the bre::gfp reporter is an exquisitely useful tool to get grip on the complex bmp-smad transcriptional signalling contexts in vivo.
the gfp signals are robust, reproducible and highly regionalised; they correlate well with known areas of bmp signalling in the endothelium and reveal new microdomains of bmp signalling.
our study underscores the regionalised and heterogeneous nature of bmp signalling in the circulatory and lymphatic vasculature of embryos and pups, with striking shifts in transcriptional output over time in different endothelium types.
this study highlights that extrapolation of results obtained in one vascular bed to another, or generalisation, should be done with extreme care.
examining other bmp signalling reporters and intercrossing them can likely shed light on yet other facets of the complex bmp-smad signalling output.
knowledge on differential signalling output is highly valuable to better understand the ontogeny of bmp-linked diseases and may lead to improved disease-tailored therapies.
additional files
additional file 1: table s <dig>  list of primary antibodies and glycoprotein stainings.
biotin amplification was only required for prox <dig> detection.
the same antibody concentration was used for whole mount analysis, paraffin sections and cryosections, unless otherwise indicated.
additional file 2: figure s <dig>  the bre::gfp reporter is transcriptionally active in a subdomain of the psmad1/5/9-positive endothelial cells.
immunodetection of smad1/5/ <dig> and gfp in the avc and oft at e <dig>  , the ventricular trabeculae  and the cardinal vein  at e <dig> .
dapi is used to stain nuclei.
specificity of the psmad1/5/ <dig> staining was confirmed in control embryos  and endothelium-specific smad1; smad <dig> double knockout embryos  .
embryos were analyzed at e <dig>  to circumvent the embryonic lethality of mutant embryos at e <dig> .
the psmad1/5/ <dig> levels were specifically reduced in endothelium of the ec-specific smad1; smad <dig> double knockouts, while levels remained unchanged in the non-endothelial cells.
the myocardium was visualized by an anti-mf <dig> staining.
arrows indicate psmad1/5/ <dig> deficient endothelium .
at: atrium; avc: atrioventricular canal; cv: cardinal vein; oft: outflow tract.
scale bars: 100 μm ; 50 μm .
additional file 3: figure s <dig>
bre::gfp localisation patterns in the p <dig> retina.
immunodetection of endomucin and gfp in the retina at p <dig>  the retina has a multi-layered vasculature that consist of the vascular border  and centre  of the primary plexus, the perpendicular vessels  and outer plexus .
single staining for gfp is shown in the lower panels.
scale bars: 75 μm.
additional file 4: table s <dig>  gfp localisation patterns in embryonic hearts with emphasis on bre::gfp activity in endocardial cells.
the number of hearts analysed is indicated in the top row.
results are consistent for all hearts analysed, unless specifically indicated.
continuous ;  ubiquitous ;  mosaic ;  occasional cell ;  patches;  undetectable; n/a not applicable
additional file 5: figure s <dig>
bre::gfp transcriptional activity in the outflow tract and aortic and pulmonary valves.
transverse sections of e <dig>  embryos with  detection of isolectin b <dig> and gfp.
dapi is used to stain nuclei.
ubiquitous gfp signal in the inflow tract valve, and mosaic gfp localisation in the aortic  and pulmonary  valve.
scale bars: 50 μm
additional file 6: figure s <dig>
bre::gfp transcriptional activity is similar in blood and lymphatic vessels.
transverse sections through the neck of e <dig>   and e <dig>   bre::gfp embryos immunostained for gfp, endomucin, prox <dig> and dapi.
boxed areas are enlarged in the right panels with the single gfp staining next to it.
schematic overview shows the dorsal skin area taken from an e <dig>  embryo.
scale bars: 50 μm ; 25 μm .
additional file 7: figure s <dig>
bre::gfp transcriptional activity is not restricted to vessels.
e <dig>  skin biopsies with immunodetection of gfp, prox <dig> and the extracellular matrix marker collagen type iv.
boxed areas are enlarged in the right panels.
arrows show non-vessel structures.
scale bars: 75 μm ; 25 μm .
additional file 8: figure s <dig>
bre::gfp transcriptional activity does not co-localise with ki <dig> in ecs.
skin biopsy of an e <dig>  bre::gfp mouse with immunodetection of gfp, ph <dig> and prox <dig>  the merged stack in the left panel is split up in some single z-stacks to show ph3-positive cells beneath or above vessel structures .
e <dig>   and e <dig>   skin biopsies with immunodetection of a different proliferation marker , gfp and prox <dig>  boxed areas are enlarged in the right panels.
ki67-positive lecs  and blood ecs  can be observed.
scale bars: 75 μm ; 25 μm .
abbreviations
gfp green fluorescent protein
aartery
aoaorta
atatrium
avcatrioventricular canal
bmpbone morphogenetic protein
brebmp response element
bsabovine serum albumin
bvbicuspid valve
cvcardinal vein
dadorsal aorta
depcdiethylpyrocarbonate
doftdistal outflow tract
eembryonic day
ecendothelial cell
endmtendothelial-to-mesenchymal transition
fishfluorescent in situ hybridisation
hhthereditary hemorrhagic telangiectasia
iavcinferior atrioventricular canal cushion
idinhibitor of differentiation
ish
in situ hybridisation
ivsinter-ventricular septum
llymphatic vessel
laleft atrium
leclymphatic endothelial cell
lslymphatic sac
lvleft ventricle
octoptimal cutting temperature
oftoutflow tract
onovernight
ppostnatal day
pahpulmonary arterial hypertension
pbsphosphate buffered saline
pfaparaformaldehyde
ph3phospho histone 3
poftproximal outflow tract
psmadphosphorylated smad
raright atrium
rtroom temperature
rvright ventricle
ssomites
savcsuperior atrioventricular canal cushion
smcsmooth muscle cell
spseptum primum
tbstris buffered saline
tgfβtransforming growth factor beta
tvtricuspid valve
vvein
vwfvon willebrand factor
we thank past and present members of the zwijsen team, m.j. goumans, b.p.t kruithof, d. huylebroeck, r. dries, and a. benn for stimulating discussions; j. vanderlinden, a. francis and e. radaelli for immunodetection support and c. adriaens for in situ hybridisation support.
funding
kb and nc have respectively iwt and fwo fellowships.
this work is supported by vib, ku leuven, the interuniversity attraction poles program iuap vii/ <dig>  grants from the research council of the ku leuven , fwo g0542n <dig> and the hercules foundation .
availability of data and materials
all data are presented in the main paper or additional supporting files.
authors’ contributions
kb, mws, nc, lu and az conceived and designed the study; kb, mws and nc generated all the presented data; ns and lu contributed with some of the immunofluorescence analyses at an early stage of the project; bre::gfp mice were provided by smcsl and em provided technical and mouse husbandry support; kb drafted the manuscript, with help of mws, nc, smcsl and az.
all authors discussed the work, read and approved the final manuscript.
competing interests
the authors declare that they have no competing interests.
consent for publication
not applicable.
ethics approval
all animal procedures were approved by the ethical committee and performed according to the guidelines of the animal welfare committee of ku leuven, belgium .

 the formation of the cardiovascular and lymphatic network is crucial for development and physiology. the cardiovascular system fuels nearly every tissue with oxygen and nutrients and removes waste products, while the lymphatic system is important for the drainage of extravasated fluid, the uptake of fat and is a vital part of the immune system. blood vessel development by sprouting from pre-existing vessels is called sprouting angiogenesis. in hypoxic environments angiogenic sprouts with tip and stalk cells emerge. sprouts anastomose to form new functional vessels that supply oxygen to the initially hypoxic environment. from the cardinal vein some venous endothelial cells differentiate into lymphatic ecs, that migrate to form lymphatic sacs which in turn sprout to form a lymphatic network similar to angiogenesis events. failure to establish a vascular network leads to severe embryonic defects at mid-to late gestation, whereas misregulation after birth can lead to diseases such as cancer, chronic and inflammatory disorders and oedema. ecs form the inner cellular lining of blood and lymphatic vessels and the heart, and differ in protein expression, morphology and function depending on the vascular bed. exposure to external and internal cues as well as epigenetic programming results in ec macro-heterogeneity and micro-heterogeneity. this means that the endothelium acquires site- and organ-specific structural and functional properties, which are extensively reviewed in aird et al.. bmp signalling has emerged as a fundamental pathway of ec identity by regulating cardiovascular and lymphatic development. bmps are members of the transforming growth factor beta family with more than bmp members identified. bmp ligands reported to function in ecs are bmp2/4/6/7/9/. bmps bind to heteromeric transmembrane receptor complexes that consist of type i and type ii receptors and often also a co-receptor. ligand binding and phosphorylation of the gs-domain of the type i receptor by the type ii receptor leads to recruitment and phosphorylation of the intracellular effectors smad smad and smad. smad is also known as smad activated psmads form a complex with the common smad, smad and translocate to the nucleus where they stimulate transcription of specific bmp target genes such as the inhibitors of differentiation, hey and smad6/7; and repress e.g. apelin. bmps can also regulate other pathways that do not involve smad proteins. bmp signalling is highly tuned by extracellular and intracellular modulators, but also by signalling interplay with other signalling pathways. furthermore, bmps are known to trigger expression of different target genes in a dose-dependent manner, a landmark of morphogens. in addition, hemodynamic changes can induce bmp signalling and activate smad proteins in ecs. recently, excessive bmp has been implicated in cerebral cavernous malformation. moreover, other regionalized vascular disorders such as hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension are mainly caused by mutations in the bmp receptors acvrl or eng and bmpr respectively. the question remains how mutations in components of the same bmp pathway can cause such organ-specific diseases. a better understanding of the heterogeneity in bmp signalling output in different vascular beds may provide this insight and perhaps even the opportunity for disease-specific therapy. phosphorylated smad1/5/ are routinely used to monitor bmp transcriptional activity, however this may confound interpretation, because psmads also play a role in chromatin remodelling and mirna biogenesis. to investigate the transcriptional activity of bmp-smad signalling many bmp reporter mice have been generated. in this study we examined the bre::gfp reporter mouse in which bmp response elements, derived from the id1-promotor, drive the expression of enhanced green fluorescent protein. the substantial decrease in gfp levels observed in smad5-deficient bre::gfp embryos corroborate the bmp-smad sensitivity of this reporter. a commonality between all bre-based reporters is that bre activity does not completely overlap with psmad1/5/ signalling domains because the onset of reporter activity first requires de novo mrna and protein synthesis and gfp maturation, and the half-life of the reporter protein may deviate from psmad1/5/. moreover, psmad1/5/ can also bind with different affinities and regulate other dna-sequences like e.g. meme; psmad1/5/ also has non-transcriptional functions. additionally, the bre::gfp reporter is heterozygous, and it is becoming apparent that gene expression in general occurs with bursts of monoallelic expression instead of constant biallelic expression. nonetheless, the relevance of the bre::gfp reporter mouse became apparent in our previous study. discrete gfp localisation patterns in angiogenic endothelium of bre::gfp embryos, with an otherwise widespread psmad1/5/ localisation, singled out those cells that underwent id-mediated bmp-smad and notch co-signalling essential for robust stalk cell fate. in this study we aimed to further document regional differences in bmp-smad dependent transcriptional activity in murine endothelium of blood vessels, lymphatic vessels and the heart at embryonic and postnatal stages. we defined regions with stereotypic mosaic and continuous bre::gfp localisation patterns, yet also gfp-negative regions were found in areas where bmp-smad signalling has been reported, compatible with the morphogen functions of bmp ligands. our data support that bmp-smad signalling can play a role in phenotype switching and endothelial cell heterogeneity. methods mice and tissue collection bre::gfp transgenic mice and endothelium-specific smad1;smad knockout mice were used. genotyping of transgenic mice was done as described. all embryos and postnatal organs were dissected in ice-cold diethylpyrocarbonate -treated phosphate buffered saline and fixed overnight in 4 % paraformaldehyde in pbs at 4 °c. afterwards they were rinsed with pbs and saline and stored in 70 % ethanol until processing. fixed embryos of embryonic day –, e and e and p intestines were processed for paraffin sectioning. skin tissue from e and e bre::gfp embryos was dissected after fixation. layers of muscle and tissue were carefully removed from the skin, leaving the superficial lymphatic network intact. from each embryo two skin biopsies were harvested. retinas were collected from fixed eyes by removing the cornea and carefully lifting the retina from the remaining eyeball. ears were collected from postnatal pups and separated into a ventral and dorsal side of which the latter was analysed. for each analysis a minimum of three animals was examined. immunofluorescence whole mount procedure embryos, skin biopsies, retinas, mesentery and ear skins were rehydrated and blocked in 2 % bovine serum albumin in tris buffered saline for 3 h at room temperature. tissues were incubated on with primary antibodies in 2 % bsa in tbs at 4 °c, except for the embryos which were kept at rt. this was followed by blocking for 3 h in 2 % bsa in tbs and incubation with the secondary antibody on. the list of primary antibodies and the used dilutions are provided in supplementary material. after whole mount immunostaining of e and e embryos the forebrain and the abdomen caudally from the forelimb bud were transversally removed. all ventral tissues including the heart were removed and the neural tube was then cut open at the ventral side. the hindbrain was mounted on a glass slide with the ventral side facing up. the mesentery was excised from the intestines after whole mount immunostaining, and the retina was cut into a four-leaf clover before mounting on a glass slide. paraffin sections transversal and sagittal sections of paraffin embedded tissues were processed for immunodetection using an automated platform. immunofluorescent triple detection of psmad1/5/ gfp and mf was done manually. the list of primary antibodies, as well as the conditions used, are provided in supplementary material. antigen retrieval was done by submerging the slides in tris-edta buffer for 30 min at 96 °c. for psmad1/5/ endogenous peroxidases were inactivated in 3%h2o in methanol for 30 min and the antibody signal was amplified using the perkin elmer tsa biotin system kit. in situ hybridisation embryos were dissected in depc-treated pbs and fixed on in 4 % pfa in pbs at 4 °c. afterwards they were washed three times 30 min in depc-treated pbs, immersed in 15 % sucrose and snap frozen in optimal cutting temperature compound with liquid nitrogen. the gfp fluorescent in situ hybridisation probe was custom designed with the probe designer tool from stellaris. the coding sequence of the pegfp-n plasmid was used for probe design. fluorescent ish was performed according to the manufacturer’s protocol with the addition of a permeabilisation step with 1 % tritonx in pbs. images were acquired using a nikon a1r eclipse ti confocal microscope.  co-localisation of bre::gfp transcriptional activity and gfp in endothelium to study bmp transcriptional activity in vessel development, we used the previously generated bre::gfp reporter strain. we took advantage of the unstable nature of gfp mrna, the sensitivity and single cell resolution of in situ hybridisation on the one hand and the direct co-observation of gfp fluorescence on the other hand to validate whether gfp protein localisation reflects well the bre::gfp transcriptional activity in endothelium. we show that there is a near to absolute correlation between the gfp mrna expression and direct gfp fluorescence localisation in vascular beds like e.g. the cardinal vein. in other endothelial linings such as in the heart ventricles there is a good overlap between mrna and protein, yet a fraction of the cells express only gfp mrna or only gfp protein. this is indicative of onset of transcriptional activity while gfp protein translation and maturation is still taking off in the former cells, while transcriptional activity has already terminated but gfp protein is still present in the latter cells. this pattern suggests the dynamic turning “on” and “off” of transcriptional activity. overall, we conclude that gfp protein patterns report with fidelity bre::gfp transcriptional activity patterns.fig. the gfp reporter protein faithfully recapitulates the transcriptional activation of the bre::gfp transgene. in situ hybridisation for gfp mrna and direct gfp fluorescence on cryosections through the cardinal vein and the ventricular trabeculae at e. dapi is used to stain nuclei. the boxed area in is enlarged in panels. the corresponding low magnification view of the trabeculae in the ventricle is not provided because of insufficient intensity of the ish signal. white arrowheads indicate ecs double positive for gfp mrna and direct gfp fluorescence. the red and green arrows depict ecs only positive for gfp mrna or gfp fluorescence respectively. scale bars: 100 μm; 50 μm; 10 μm the gfp localisation pattern is mostly a subdomain of the psmad1/5/ pattern as previously reported, which is compatible with bmp-smad non-transcriptional and morphogen functions as discussed in the background section of this paper. the specificity of the anti-psmad1/5/ antibody was validated in endothelium-specific smad1;smad double knockout embryos. however, in some endothelial beds like e.g. in the cardinal vein in e embryos, gfp localisation hardly overlapped with psmad1/5/ localisation, suggesting terminated or undetectable psmad1/5/ signalling or transcriptional activation of the bre:gfp transgene by other factors than bmp signalling. bre::gfp transcriptional activity is present in a mosaic pattern during embryonic angiogenesis to closely examine bre::gfp transcriptional activity in the rapidly expanding vascular plexus, we analysed embryonic day and e bre::gfp mouse embryos. tip and stalk cell formation as well as anastomosis during sprouting angiogenesis can then be investigated. in the roof of the hindbrain sprouts are formed from the perineural vascular plexus at opposite lateral sides of the embryo that then anastomose medially in a caudal fashion from the level of the otic vesicles onwards. whole mount immunostainings of bre::gfp embryos showed that gfp was mainly present in and around the heart region at somites, with little bre::gfp transcriptional activity in the endomucin-positive blood vessels. however, in 30s embryos a scattered gfp pattern co-localised particularly within the main vessels of e.g. the head and the intersomitic vessels. the hindbrain roof was excised from these embryos and the dorsal vascular plexus was flat-mounted. in the two-dimensional vascular plexus at 22 s bre::gfp transcriptional activity occurs in a scattered or mosaic pattern throughout the plexus, which is in accordance with previous observations. remarkably, some tip cells were found to express low levels of gfp while the mosaic pattern in non-tip cells was more intense throughout the plexus. this is in contrast with earlier observations where gfp was not detected in tip cells, which likely reflects higher affinity of the current chicken polyclonal anti-gfp antibody than the previously used mouse monoclonal anti-gfp antibody. at e the vessels from the superficial vascular plexus start to invade the hindbrain, thus forming a multi-layered vascular network. depth coding of the dorsal hindbrain region at 30s revealed the superficial and deeper plexus. the latter had slender vessels with slim tip cells, whereas the superficial plexus had thicker honeycomb-like vessels and broader tip cells, as seen at 22 s. the gfp localisation pattern in both plexuses was mosaic and comparable to the plexus of the 22 s embryo, yet, the deeper plexus seemed to be enriched in gfp-positive ecs compared to the superficial plexus. additionally, a string of gfp-positive non-endothelial cells was observed in the midline at both stages. this correlates with the presence of bmp and bmp in the dorsal midline at e -e. in general, the two distinct dorsal vascular plexuses demonstrate heterogeneity in bmp-smad signalling in an angiogenic vascular bed.fig. mosaic bre::gfp transcriptional activity in midgestation mouse hindbrain vasculature. immunodetection of endomucin and gfp in bre::gfp embryos of 22 s and 30s. arrowheads indicate the heart region. boxed areas represent the head region and the intersomitic vessels. g overview of a 22 s dorsal vascular plexus in a flat-mounted hindbrain roof. arrows indicate direction of rostral  to the left, caudal  to the right. boxed areas show sprouts at the midline. asterisks depict gfp-positive tip cells. h depth coding of a 30s dorsal vascular plexus and the original picture. blue and green colours represent more ventral and dorsal tissues respectively. the vascular plexus at 30s consists of a superficial and deeper plexus. boxed areas are enlarged in the right panels. scale bars: 500 μm; 100 μm; 75 μm; 50 μm; 25 μm spatio-temporal changes in bre::gfp activity during retinal angiogenesis the retina is commonly used to investigate postnatal blood vessel development. the primary plexus develops from the optic nerve towards the peripheral margin. around postnatal day the primary vascular plexus invades the deeper retinal layers perpendicularly whereafter the outer plexus forms again radially. different aspects of vessel formation can be studied because vascular sprouting happens at the periphery while remodelling occurs simultaneously in the centre. the gfp localisation pattern was diverse but stereotypic throughout the retinal stages investigated and the gfp levels decreased over time. at p the vasculature has sprouted halfway across the retina. the sprouting front displayed relatively strong gfp signals compared to the central plexus. many tip cells at the sprouting front as well as the arteries and veins in the centre were gfp-positive, whereas the intermediate capillary bed displayed a more mosaic gfp distribution. moreover, arteries seemed weaker gfp-positive compared to the strong gfp-positive veins. at p the sprouting front developed into a vascular border. comparable to p gfp-positive arteries and veins were observed, along with a mosaic distribution of gfp in the capillaries. at this stage the matured vessels in the centre have sprouted into the retina to form the perpendicular vessels and the outer plexus. approximately half of the perpendicular vessels seemed gfp-positive, though weaker than the veins of the primary plexus. moreover, the less ramified outer plexus also displayed a mosaic gfp pattern. overall, fewer gfp-positive cells were present in the different plexuses at p although the mosaic distribution was maintained in the primary and outer plexus. these data indicate a dynamic nature of bmp-smad signalling over time.fig. different bre::gfp transcriptional activity patterns in the postnatal vasculature. a overview of a p retina with immunodetection of endomucin and gfp. boxed areas show the sprouting front and the centre of the retinal plexus. single staining for gfp is shown in the bottom panels. asterisks depict gfp-positive tip cells, the arrows point to the veins, while the arrowheads show the arteries. d–g the retina at p immunostained for endomucin and gfp, has a multi-layered vascular plexus consisting of the vascular border and centre of the primary plexus, the perpendicular vessels and outer plexus. single gfp staining is shown in the lower panels. scale bars: 500 μm; 75 μm gfp localisation patterns are dynamic in the developing heart given the well-known functions of bmps in heart development and the presence of bre::gfp activity during early heart development and valve formation, we zoomed in deeper on the spatio-temporal patterns of bre::gfp transcriptional activity in endocardium at e -e, e and e and observed remarkable regional differences over time. at e the initial heart tube starts to loop and progressively forms the four chambered heart. the heart tube comprises an inner endothelial layer and an outer myocardial layer separated by extracellular matrix or cardiac jelly. the pro-epicardial organ progressively covers the myocardial layer with epicardium. overall, relatively few gfp-positive cells were observed in tissue sections of the developing heart, which is consistent with former results. nonetheless, gfp patterns were robust and reproducible in endocardial subregions, and changed over time.fig. dynamic bre::gfp transcriptional activity in the developing heart. paraffin embedded hearts are sectioned transversally, unless otherwise specified, and stained for the endothelial marker isolectinb and gfp. dapi is used to stain nuclei. a overview of an e heart. b section of the superior and inferior atrioventricular canal cushions at e. c e sagittal section of avc and oft cushions. d e lateral cushion with gfp-positive endocardial cells and myocardium. e at e the septum primum displays gfp-positive ecs on the endothelial cap. the atrium is gfp-negative at e while the ventricular trabeculae have gfp-positive ecs. h overview of an e heart. the tricuspid valve shows gfp-positive cells at the atrial side at e and e. gfp signals in the intra-atrial septum at e and e. some endocardial cells covering the atrial and ventricular trabeculae are gfp-positive at e. ao: aorta; avc: atrioventricular canal; iavc: inferior avc cushion; savc: superior avc cushion; bv: bicuspid valve; cv: cardinal vein; doft: distal outflow tract; ivs: inter-ventricular septum; la: left atrium; lv: left ventricle; poft: proximal outflow tract; ra: right atrium; rv: right ventricle; sp: septum primum; tv: tricuspid valve. scale bars: 200 μm; 100 μm; 50 μm cardiac cushions are the primordia of the valves and most septa in the developing heart. they are formed in the atrioventricular canal which separates the atria from the ventricles and the outflow tract which bridges the ventricles with the aortic sac. at respectively e and e the endocardial cells of the avc cushions and proximal oft cushions delaminate, undergo endothelial-to-mesenchymal transition and invade the cardiac jelly. in contrast, the distal oft cushions become mainly populated by neural crest cell derived mesenchymal cells from e onwards. the endocardial cells of the superior and inferior avc cushions displayed a mosaic gfp localisation pattern until e. from e onwards, patches of gfp-positive ecs were restricted to the atrial side of the superior cushion. these streaks of gfp-positive ecs remained present at e and e on the medial leaflets of the tricuspid and bicuspid valve that form from these cushions. in contrast, only few gfp-positive ecs were present in the lateral avc cushions from e onwards. yet, at e the lateral leaflets of the tricuspid and bicuspid valve displayed, just like the medial leaflets, also patches of gfp-positive ecs. patches of gfp-positive muscle cells were also observed in the avc myocardium flanking the developing cushions and valves up to e, though with varying gfp levels among neighbouring cells.. the ecs of the oft cushions showed a mosaic gfp localisation pattern comparable to the avc cushions at e, although the oft appeared slightly enriched in gfp-positive ecs. remarkably, the mesenchymal cells that populate the avc and oft cushions were gfp-negative. in contrast to the ecs of the avc and oft, the ecs of the inflow tract and the endothelial cap of the septum primum showed a ubiquitous gfp localisation pattern at e. as development proceeds and this septum reaches the superior cushion forming the intra-atrial septum, bre::gfp transcriptional activity decreased with only a few gfp-positive ecs still present at e and e. only occasionally a gfp-positive cell was detected in atrial endothelium at e, while the ventricles showed a mosaic gfp pattern in the ecs covering the trabeculae throughout the stages analysed. interestingly, a mosaic pattern also emerged in the atrial endothelium coinciding with initiation of trabeculation from e onwards. moreover, the ecs of the aortic and pulmonary valve leaflets also displayed a mosaic gfp pattern at e. levels of bre::gfp activity differ in embryonic and postnatal blood and lymphatic vessels little information is available on bmp-smad signalling in different lymphatic beds. at e the first lecs differentiate from venous ecs in the cardinal vein. these lecs bud of, migrate and assemble into lymphatic sacs by e, which will remodel into a functional lymphatic network. we found interesting spatio-temporal differences in bre::gfp transcriptional activity during lymphangiogenesis. in the embryo most blood and lymphatic vessels have a widespread gfp localisation pattern, while in postnatal tissues like the mesentery, intestinal villi and the ear skin many blood vessels appeared to have reduced gfp signals and the lymphatic vessels had discrete and unique gfp localisation patterns. in general, the blood vessels, including the cardinal vein, and lymphatic vessels displayed a continuous gfp localisation pattern at e -e. however, the aorta showed a mosaic gfp localisation pattern. interestingly, nearly all prox1- positive lecs budding from the cardinal vein were gfp-positive, yet still weakly positive for the blood vessel marker endomucin in e embryos. likewise, at e some gfp-positive ecs were prox1- and endomucin-positive, yet in few sections at e the gfp-positive cardinal vein adjacent to the lymphatic sac was endomucin-positive on the medial side, while the lateral side, closest to the lymphatic sac, was prox1-positive. blood and lymphatic vessels in dorsal skin biopsies were also ubiquitously gfp-positive at e and e. however, the gfp signal appeared more uniform in the blood vessels, whereas the lecs showed different levels of gfp among neighbouring cells. other gfp-positive cord-like structures were observed at e and e in dorsal skin biopsies, but these were excluded as vessels because they were collagen type iv-negative. similar structures have been described as schwann cells of sensory nerves.fig. spatio-temporal changes in bre::gfp transcriptional activity in blood and lymphatic vessels. a–c transverse sections at the level of the neck of e, e and e bre::gfp embryos with immunodetection of gfp, the blood vessel marker endomucin and the lymphatic vessel marker prox dapi is used to stain nuclei. boxed areas are enlarged in the right panels, with single gfp, endomucin and prox staining next to it. skin biopsies of e and e bre::gfp embryos with immunodetection of gfp, endomucin and prox boxed areas are shown in the middle panels with single staining for gfp in the right panel. cv: cardinal vein; da: dorsal aorta; ls: lymphatic sac. scale bars: 75 μm; 50 μm; 25 μm since the lymphatic network is still expanding after birth, we investigated the postnatal mesentery, intestines and ear skin. different types of lymphatic vessels occur in each of these tissues. the mesentery contains collecting vessels, while lacteals resorb lipids from the intestines and lymphatic capillaries drain lymph from ear tissue. interestingly, many venous ecs appeared gfp-positive in p and p mesentery, whereas only few gfp-positive arterial ecs were observed with also some gfp-positive peri-endothelial cells covering the arteries. the developing lymphatic vessels were predominantly gfp-negative, although bre::gfp transcriptional activity was specifically present in some valve forming regions. at p the arterial ecs and peri-endothelial cells appeared gfp-negative and also fewer venous ecs were gfp-positive, whereas the lymphatic vessels still showed gfp in the valve regions. remarkably, the vessels had reduced bre::gfp transcriptional activity over time from p to p.fig. bre::gfp transcriptional activity in postnatal blood and lymphatic vessels. a stitch of a p mesentery with immunodetection of gfp and the pan-endothelial marker cd arteries, veins and collecting lymphatic vessels in a p and p mesentery are depicted. arrows point to gfp-positive peri-endothelial cells. arrowheads show gfp-positive ecs in the vicinity of a valve. sections of p intestines with immunodetection of gfp and endomucin or the lymphatic vessel marker lyve. white arrows point to some gfp-positive cells in a capillary, yellow arrows show gfp-positive lecs in a lacteal. arrowheads show some gfp-positive goblet cells. stitch of p and p dorsal ear skin with immunodetection of gfp and the lymphatic marker podoplanin. gfp-positive lecs at the branch points are depicted by arrows at p and p. a: artery; l: lymphatic vessel; v: vein. scale bars: 1 mm; 500 μm; 75 μm most intestinal villi comprise a lyve1-positive lymphatic vessel, called the lacteal, which is surrounded by an endomucin-positive blood capillary. only few ecs of the blood capillaries and lacteals were gfp-positive. other non-endothelial cells, such as goblet cells, were also gfp-positive in the villus. in the ear skin bre::gfp transcriptional activity was absent from the lymphatic capillary bed at p and p with the exception of a single gfp-positive lec at some branch points. bre::gfp transcriptional activity does not correlate with proliferation inhibitors of differentiation or ids are helix-loop-helix proteins that interact with and inhibit basic helix-loop-helix transcription factors. through induction of these ids, the bmp pathway promotes ec migration and tube formation. this led to the hypothesis that gfp-positive ecs would be proliferative. several embryonic and postnatal tissues were investigated for co-localisation of gfp and phospho histone in the blood and lymphatic system. ph is only present in the m-phase of the cell cycle. there was rarely overlap between ph and gfp-positive blood or lymphatic ecs in the neck region at e and e or in dorsal skin biopsies from e and e embryos. when ph seemed to be present in a gfp-positive ec, the proliferating cell was usually not in the same focal plane as the ec, or showed mainly low to no levels of gfp. in addition, skin biopsies of e and e embryos were immunostained for ki which marks all active phases of the cell cycle. proliferation decreased between e and e, but even though many non-endothelial cells were proliferative only few gfp-positive ecs and lecs were ki67-positive. also in the heart there was almost no co-localisation observed between ph and gfp. furthermore, ecs of the sprouting front and the centre of the retina were not ph3-positive at p however, at this stage non-endothelial cells were more proliferative at the sprouting front than at the centre. overall, proliferation decreased by p but shifted towards the ecs, as now ph3-positive ecs could be observed. some of these proliferating cells seemed more gfp-positive than others. this is an intriguing difference in co-localisation pattern which suggests another context dependent role for bmp-smad signalling.fig. bre::gfp transcriptionally active ecs are rarely proliferative. transverse sections of e and e bre::gfp mouse embryos with immunodetection of the proliferation marker, gfp and endomucin or prox. dorsal skin biopsies obtained from e and e bre::gfp embryos show ph gfp and endomucin or prox staining. boxed areas are enlarged in the right panels. i overview of an e heart with immunodetection of gfp and ph dapi is used to stain nuclei. the sprouting front and centre of p and p retinas with immunodetection of gfp, endomucin and ph arrows point to ph3-positive ecs. avc: atrioventricular canal; ivs: inter-ventricular septum; la: left atrium; lv; left ventricle; ra: right atrium; rv: right ventricle. scale bars: 100 μm; 75 μm; 50 μm; 25 μm discussion spatio-temporal information on output of important signalling pathways in the vasculature may help to increase our understanding of how mutations in components of the same pathway can cause organ-specific vascular disorders and provide a window of opportunity for designing disease-specific therapy. in the past decade, many bre-reporters have been generated in zebrafish and mice. the bre::gfp reporter used in this study is not the most sensitive, as some other bmp reporters show broader patterns of transcriptional activity. however, this precisely allows to zoom in on selective processes and dose-dependent bmp actions. our study shows that gfp patterns in endothelium faithfully report transcriptional activation of the bre::gfp transgene, and are remarkably robust. however the bmp-smad transcriptional output is highly dynamic in time and space, in the different cardiovascular and lymphatic beds of bre::gfp mice. mosaic gfp localisation patterns were observed in different regions of the developing vascular tree and heart. it was found in the dorsal vascular plexuses of midgestation mouse embryos and in the capillary bed of p4-p retinas. in addition, at e -e the endocardial cells of the avc and oft cushions and those lining the atrial and ventricular trabeculae also displayed a mosaic gfp pattern. such a mosaic pattern of transcriptional activity suggests a role for bmp-smad in ec plasticity and micro-heterogeneity. the multi-layered vascular network of the dorsal hindbrain and retina develop in a similar fashion, with the deeper plexus of the dorsal hindbrain resembling morphologically more the capillary network in the primary plexus of the retina. previously, we showed that the bre::gfp pattern singled out stalk cell competent cells in the dorsal vascular plexus that were undergoing bmp and notch co-signalling and that loss of bmp-smad signalling in endothelium resulted in a stalk cell defect. we also observed weak gfp-positive tip cells in the dorsal hindbrain and retinal plexuses. these tip cells might have been former stalk cells that have taken over the tip cell position, with traces of non-degraded gfp. alternatively, bmp and bmp synthesized by cells at the midline may function as pro-angiogenic guidance cues that trigger an alternative bmp-smad signalling pathway in the tip cells. circulatory bmp is likely to promote stalk cell competence through activating the mosaic transcriptional activity observed in the rest of the dorsal vascular plexus. in the retina, bmp and bmp are important for postnatal vascular remodelling. remarkably, bmp was unable to induce bre activity in vitro, suggesting that the gfp signals in the centre of the retina, where vessel maturation and remodelling occurs, were the result of bmp signalling. however, also bmp and have been shown to play significant roles in retinal neurogenesis and vascularisation. retinal vascularisation is preceded and stimulated by the development of a vast network of neuronal cells, the latter also depending on bmp-smad signalling. the retinal ecs reciprocally promote differentiation of the neuronal plexus. our data do not allow to distinguish the precise source and type of bmp signal, yet, bmp-smad transcriptional activity seems more imperative at the sprouting front than in the centre where the vascular plexus is maturing. many bmps have been reported to regulate cardiac cushion development. in the avc cushions, bmp stimulates ecs to undergo endmt, while bmp is important in the oft for proliferation and growth of endocardial cushions rather than endmt. studies with knockout mice reveal that bmp alk alk bmpr smad and smad are important for the development of the avc cushions and to a lesser extent oft cushions. our study shows that bmp signalling induces mosaic transcriptional activity in cushion endocardium, likely to maintain an intact cushion epithelium while a few cells can undergo endmt. whether the gfp-positive cells or rather their neighbours are subsequently triggered to undergo endmt remains to be elucidated. taken together, in cushion endocardium and in angiogenic endothelium, the mosaic-perhaps stochastic-transcriptional bmp-smad activity seems to serve as a source of phenotypic diversity. the exquisite fine-tuning of the bmp pathway, which also involves negative feedback mechanisms, may also generate switch modes of activation states. whether the mosaicism in bmp-smad transcriptional activity is static or dynamic, with bre::gfp activity switching between ‘on’ and ‘off’ states, cannot be addressed directly in our model due to limitations in the resolution of real-time intravital microscopy, combined with the need for potentially long windows of observation. dynamic mosaicism in expression has recently been demonstrated for von willebrand factor, and also in vitro for esm and ephrin-b in some but not all vascular beds. this appears to be a phenotype switching strategy for adaptive homeostasis. remarkably, the endocardial cells of the atria turned on mosaic bre::gfp activity several days later than the ventricular endocardial cells. interestingly, this delayed activation correlated with the delayed onset of bmp expression and initiation of trabeculation in atrial myocardium at e compared to the onset of the same process in ventricular myocardium already at e. bmp is a well-known regulator of cardiac trabeculation and/or compaction. trabeculation defects are also observed in endothelium-specific smad ko and smad1/smad double ko embryos. our data suggest that not all ecs are equally involved in this process. it would be interesting to evaluate whether and how expansion from a mosaic to a continuous bmp-smad transcriptional activity pattern in ventricular and atrial endocardium would impact trabeculation or provide advantages. remarkably, during embryonic development the lymphatic vessels showed widespread bre::gfp transcriptional activity, yet in pups gfp-positive ecs were restricted to the valve forming regions of collecting lymphatic vessels in the mesentery. this is in agreement with the role of bmp in lymphatic valve development. in the lymphatic capillary bed of the ear skin, an occasional gfp-positive cell would localise at branch points. furthermore, gfp-positive endocardial cells were observed on the atrial side of the tricuspid and bicuspid heart valve leaflets, but also in the inflow tract. all these patterns correspond with endothelium undergoing fluid shear stress, which can induce bmp-smad signalling. hemodynamic alterations have been reported to induce bmp and activate smad1/ in the aorta, and to mediate arteriogenesis. mature ecs are characterized by a slow proliferation rate. for example, in adult ear skin only  % of the lecs are reported to be ki67-positive, whereas approximately 30 % of lecs are ki67-positive in embryonic skin at e -e. the role of bmp signalling in ec proliferation is thought to be highly context dependent. because bre::gfp signals peaked around midgestation and progressively decreased in postnatal stages in the vascular tree, we reasoned that correlations between bmp-smad transcriptional output and proliferation may become apparent in specific vascular beds. remarkably, we found that proliferating ph3-positive ecs were almost invariably gfp-negative in the different vascular beds analysed, except in the p retina where more often double positive cells were observed. a recurrent theme was - like in zebrafish embryos  - that the abundance of gfp-positive ecs was higher in veins than in arteries in several embryonic and postnatal tissues. for instance, the ecs in the cardinal vein showed a continuous gfp localisation pattern, whereas the aorta had a mosaic gfp pattern. in the mesentery almost no gfp-positive ecs were observed in the arteries, yet some of its peri-endothelial cells were gfp-positive. in contrast, many ecs of the veins were gfp-positive. alk endoglin and bmpr play a role in the establishment and maintenance of mural cell coverage on mature vessels, primarily in arteries. mutations in bmpr lead to pah which is characterized by abnormal proliferation of ecs and smooth muscle cells in arterioles, whereas a deletion of bmpr leads to insufficient recruitment and decreases pdgfrβ expression in mural cells. bmp2/bmpr signalling negatively regulates pdgfbb induced proliferation of pulmonary arterial smcs in a psmad1/5/ independent manner. it is likely that veins express more bmp and hence limit the number of smc coverage, but also the differences in shear stress in both vessel types may underlie the above differences.  we experienced that the bre::gfp reporter is an exquisitely useful tool to get grip on the complex bmp-smad transcriptional signalling contexts in vivo. the gfp signals are robust, reproducible and highly regionalised; they correlate well with known areas of bmp signalling in the endothelium and reveal new microdomains of bmp signalling. our study underscores the regionalised and heterogeneous nature of bmp signalling in the circulatory and lymphatic vasculature of embryos and pups, with striking shifts in transcriptional output over time in different endothelium types. this study highlights that extrapolation of results obtained in one vascular bed to another, or generalisation, should be done with extreme care. examining other bmp signalling reporters and intercrossing them can likely shed light on yet other facets of the complex bmp-smad signalling output. knowledge on differential signalling output is highly valuable to better understand the ontogeny of bmp-linked diseases and may lead to improved disease-tailored therapies. additional files additional file 1: table s list of primary antibodies and glycoprotein stainings. biotin amplification was only required for prox detection. the same antibody concentration was used for whole mount analysis, paraffin sections and cryosections, unless otherwise indicated. additional file 2: figure s the bre::gfp reporter is transcriptionally active in a subdomain of the psmad1/5/9-positive endothelial cells. immunodetection of smad1/5/ and gfp in the avc and oft at e, the ventricular trabeculae and the cardinal vein at e. dapi is used to stain nuclei. specificity of the psmad1/5/ staining was confirmed in control embryos and endothelium-specific smad1; smad double knockout embryos. embryos were analyzed at e to circumvent the embryonic lethality of mutant embryos at e. the psmad1/5/ levels were specifically reduced in endothelium of the ec-specific smad1; smad double knockouts, while levels remained unchanged in the non-endothelial cells. the myocardium was visualized by an anti-mf staining. arrows indicate psmad1/5/ deficient endothelium. at: atrium; avc: atrioventricular canal; cv: cardinal vein; oft: outflow tract. scale bars: 100 μm; 50 μm. additional file 3: figure s bre::gfp localisation patterns in the p retina. immunodetection of endomucin and gfp in the retina at p the retina has a multi-layered vasculature that consist of the vascular border and centre of the primary plexus, the perpendicular vessels and outer plexus. single staining for gfp is shown in the lower panels. scale bars: 75 μm. additional file 4: table s gfp localisation patterns in embryonic hearts with emphasis on bre::gfp activity in endocardial cells. the number of hearts analysed is indicated in the top row. results are consistent for all hearts analysed, unless specifically indicated. continuous; ubiquitous; mosaic; occasional cell; patches; undetectable; n/a not applicable additional file 5: figure s bre::gfp transcriptional activity in the outflow tract and aortic and pulmonary valves. transverse sections of e embryos with detection of isolectin b and gfp. dapi is used to stain nuclei. ubiquitous gfp signal in the inflow tract valve, and mosaic gfp localisation in the aortic and pulmonary valve. scale bars: 50 μm additional file 6: figure s bre::gfp transcriptional activity is similar in blood and lymphatic vessels. transverse sections through the neck of e and e bre::gfp embryos immunostained for gfp, endomucin, prox and dapi. boxed areas are enlarged in the right panels with the single gfp staining next to it. schematic overview shows the dorsal skin area taken from an e embryo. scale bars: 50 μm; 25 μm. additional file 7: figure s bre::gfp transcriptional activity is not restricted to vessels. e skin biopsies with immunodetection of gfp, prox and the extracellular matrix marker collagen type iv. boxed areas are enlarged in the right panels. arrows show non-vessel structures. scale bars: 75 μm; 25 μm. additional file 8: figure s bre::gfp transcriptional activity does not co-localise with ki in ecs. skin biopsy of an e bre::gfp mouse with immunodetection of gfp, ph and prox the merged stack in the left panel is split up in some single z-stacks to show ph3-positive cells beneath or above vessel structures. e and e skin biopsies with immunodetection of a different proliferation marker, gfp and prox boxed areas are enlarged in the right panels. ki67-positive lecs and blood ecs can be observed. scale bars: 75 μm; 25 μm. abbreviations gfp green fluorescent protein aartery aoaorta atatrium avcatrioventricular canal bmpbone morphogenetic protein brebmp response element bsabovine serum albumin bvbicuspid valve cvcardinal vein dadorsal aorta depcdiethylpyrocarbonate doftdistal outflow tract eembryonic day ecendothelial cell endmtendothelial-to-mesenchymal transition fishfluorescent in situ hybridisation hhthereditary hemorrhagic telangiectasia iavcinferior atrioventricular canal cushion idinhibitor of differentiation ish in situ hybridisation ivsinter-ventricular septum llymphatic vessel laleft atrium leclymphatic endothelial cell lslymphatic sac lvleft ventricle octoptimal cutting temperature oftoutflow tract onovernight ppostnatal day pahpulmonary arterial hypertension pbsphosphate buffered saline pfaparaformaldehyde ph3phospho histone 3 poftproximal outflow tract psmadphosphorylated smad raright atrium rtroom temperature rvright ventricle ssomites savcsuperior atrioventricular canal cushion smcsmooth muscle cell spseptum primum tbstris buffered saline tgfβtransforming growth factor beta tvtricuspid valve vvein vwfvon willebrand factor we thank past and present members of the zwijsen team, m.j. goumans, b.p.t kruithof, d. huylebroeck, r. dries, and a. benn for stimulating discussions; j. vanderlinden, a. francis and e. radaelli for immunodetection support and c. adriaens for in situ hybridisation support. funding kb and nc have respectively iwt and fwo fellowships. this work is supported by vib, ku leuven, the interuniversity attraction poles program iuap vii/ grants from the research council of the ku leuven, fwo g0542n and the hercules foundation. availability of data and materials all data are presented in the main paper or additional supporting files. authors’ contributions kb, mws, nc, lu and az conceived and designed the study; kb, mws and nc generated all the presented data; ns and lu contributed with some of the immunofluorescence analyses at an early stage of the project; bre::gfp mice were provided by smcsl and em provided technical and mouse husbandry support; kb drafted the manuscript, with help of mws, nc, smcsl and az. all authors discussed the work, read and approved the final manuscript. competing interests the authors declare that they have no competing interests. consent for publication not applicable. ethics approval all animal procedures were approved by the ethical committee and performed according to the guidelines of the animal welfare committee of ku leuven, belgium.
our data reveals the highly dynamic nature of bmp-smad mediated transcriptional regulation in time and space throughout the vascular tree, supporting that bmp-smad signalling can be a source of phenotypic diversity in some, but not all, healthy endothelium. to better understand the in vivo context-dependency of bmp-smad signalling, we investigated differences in bmp-smad transcriptional activity in different vascular beds during mouse embryonic and postnatal stages. postnatal lymphatic vessels showed however only discrete micro-domains of transcriptional activity. dynamic shifts in transcriptional activity were also observed in the endocardium of the developing heart, with a general decrease in activity over time. patches of transcriptional activity seemed to correlate with vasculature undergoing hemodynamic alterations. bone morphogenetic protein signalling has emerged as a fundamental pathway in endothelial cell biology and deregulation of this pathway is implicated in several vascular disorders. this knowledge can provide insight in vascular bed or organ-specific diseases and phenotypic heterogeneity within an endothelial cell population. a mosaic pattern of gfp was present in various angiogenic sprouting plexuses and in endocardium of cardiac cushions and trabeculae in the heart. the bre::gfp mouse allows to investigate selective context-dependent aspects of bmp-smad signalling. bmp-smad signallingsprouting angiogenesisvalve developmentlymphangiogenesisendocardiumphenotype switchingheterogeneityretinastochastic expressionmorphogeniwtsb 111224beets karen fwo26334g0542n13criem nathan zwijsen an interuniversity attraction poles program vii/07zwijsen an onderzoeksraad, ku leuven goa11/012zwijsen an hercules foundationinframouse zw09-03zwijsen an http://dx.doi.org/ /501100004040ku leuvenvib issue-copyright-statement© the author 2016
 bone morphogenetic protein signalling has emerged as a fundamental pathway in endothelial cell biology and deregulation of this pathway is implicated in several vascular disorders. bmp signalling output in endothelial cells is highly context- and dose-dependent. phosphorylation of the bmp intracellular effectors, smad1/5/ is routinely used to monitor bmp signalling activity. to better understand the in vivo context-dependency of bmp-smad signalling, we investigated differences in bmp-smad transcriptional activity in different vascular beds during mouse embryonic and postnatal stages. for this, we used the bre::gfp bmp signalling reporter mouse in which the bmp response element from the id1-promotor, a smad1/5/ target gene, drives the expression of gfp.  a mosaic pattern of gfp was present in various angiogenic sprouting plexuses and in endocardium of cardiac cushions and trabeculae in the heart. high calibre veins seemed to be more bre::gfp transcriptionally active than arteries, and ubiquitous activity was present in embryonic lymphatic vasculature. postnatal lymphatic vessels showed however only discrete micro-domains of transcriptional activity. dynamic shifts in transcriptional activity were also observed in the endocardium of the developing heart, with a general decrease in activity over time. surprisingly, proliferative endothelial cells were almost never gfp-positive. patches of transcriptional activity seemed to correlate with vasculature undergoing hemodynamic alterations.  the bre::gfp mouse allows to investigate selective context-dependent aspects of bmp-smad signalling. our data reveals the highly dynamic nature of bmp-smad mediated transcriptional regulation in time and space throughout the vascular tree, supporting that bmp-smad signalling can be a source of phenotypic diversity in some, but not all, healthy endothelium. this knowledge can provide insight in vascular bed or organ-specific diseases and phenotypic heterogeneity within an endothelial cell population. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords bmp-smad signallingsprouting angiogenesisvalve developmentlymphangiogenesisendocardiumphenotype switchingheterogeneityretinastochastic expressionmorphogeniwtsb 111224beets karen fwo26334g0542n13criem nathan zwijsen an interuniversity attraction poles program vii/07zwijsen an onderzoeksraad, ku leuven goa11/012zwijsen an hercules foundationinframouse zw09-03zwijsen an http://dx.doi.org/ /501100004040ku leuvenvib issue-copyright-statement© the author 2016 