ABSTRACT
Formation of a hierarchically organized blood vessel network by sprouting angiogenesis is critical for tissue growth, homeostasis and regeneration. How in this process endothelial cells arise in adequate numbers and arrange suitably to shape a functional vascular network is poorly understood. Here we show that YAP and TAZ promote stretch-induced proliferation and rearrangements of endothelial cells whilst preventing bleeding in developing vessels. Mechanistically, YAP and TAZ increase VE-cadherin turnover at junctions and suppress endothelial Notch and BMP signaling, two key pathways that limit sprouting and endothelial dynamics. Consequently, the loss of YAP and TAZ leads to stunted sprouting with local aggregation as well as scarcity of endothelial cells, branching irregularities and junction defects. Forced nuclear activity of TAZ instead drives hypersprouting and vascular hyperplasia. We propose a new model in which YAP and TAZ integrate mechanical signals with Notch and BMP signaling to balance endothelial cell distribution in angiogenic vessels.
INTRODUCTION
A long-standing question in developmental and cell biology relates to how cells integrate mechanical and chemical signals to orchestrate the morphogenic behaviours that ensure adequate tissue patterning. During sprouting angiogenesis, the arrangement and distribution of cells rather than their numbers appear to drive morphogenesis of the vascular tree. Recent data showing unaltered remodelling in the absence of endothelial cell apoptosis and normal branching frequency across a range of endothelial cell densities support this idea (1). In the extreme, however, too few cells will jeopardize network formation and stability (2), whereas too many cells might compromise vessel calibre control (1). Functional network formation therefore needs to establish the right number of cells in the right place, and distribute them such that the hierarchical branching pattern is supported. What establishes such a balance has remained unclear. Here we provide evidence for the yes-associated protein 1 (YAP) and its paralog WW domain containing transcription regulator 1 (TAZ) to be critically involved as endothelial cell autonomous regulators in this process.
YAP and TAZ, two transcriptional co-activators initially discovered as effectors of the Hippo signalling pathway, play a central role in organ size control via regulation of proliferation and apoptosis (3-5). In confluent cells, YAP and TAZ are phosphorylated by the Hippo kinase cascade, and retained in the cytoplasm. In sparse cells, YAP and TAZ remain unphosphorylated and can translocate to the nucleus, where they bind transcription factors inducing the expression of pro-proliferative and anti-apoptotic genes. Other stimuli have been found to regulate YAP and TAZ nuclear translocation and activity – these include, among others, G-protein coupled receptors (GPCRs) (6), junctional proteins (7, 8), and mechanical stimuli (9, 10). Furthermore, besides cell proliferation and apoptosis, YAP and TAZ also regulate cell differentiation (11), migration (12) and actomyosin contraction (13). In vascular development, the roles of YAP and TAZ are not fully understood. Yap null mutant zebrafish develop an initially normal vasculature but display increased vessel collapse and regression. Yap/Taz double mutant zebrafish die before the onset of circulation with severe developmental defects, precluding analysis of vascular development in this context (14). Endothelial-specific deletion of Yap in mice using the Tie2-Cre transgenic line is embryonically lethal due to heart valve defects caused by failed endothelial-to-mesenchymal transition (15). During post-natal development of the mouse retina, YAP was shown to regulate vascular density and branching by promoting the transcription of Angiopoetin-2 (16). While these studies point towards an important role for YAP in regulating blood vessel formation and maintenance, none of them addressed the endothelial cell autonomous requirement for YAP during sprouting angiogenesis. In addition, no study has addressed a potential requirement for TAZ during angiogenesis, and the possible redundancy between both proteins in this context. Finally, the possible interplay between YAP/TAZ and the major signalling pathways regulating angiogenesis has not been assessed.
Here, we show that YAP and TAZ are both expressed and active in sprouting ECs and critical for sprouting angiogenesis. The inducible, endothelial-specific combined deletion of YAP and TAZ leads to severe morphogenic defects consistent with impaired junctional remodelling in vivo. Furthermore, we found that the loss of YAP and TAZ decreased VE-Cadherin turnover and increased cell-cell coupling. We also discovered that endothelial YAP and TAZ strongly regulate endothelial Notch and BMP signalling in vitro and in vivo, together suggesting that YAP and TAZ integrate mechanical stimuli with key transcriptional regulators of endothelial sprouting and cell rearrangements during angiogenesis.
RESULTS
YAP and TAZ have distinct expression patterns in endothelial cells of developing vessels and localise to the nucleus at the sprouting front
Immunofluorescence staining in the postnatal mouse retina showed that YAP and TAZ are distinctly expressed in the ECs of the developing vasculature (Figure 1). While YAP is evenly expressed throughout the vasculature (Figure 1 A-D), the expression of TAZ is especially prominent at the sprouting front (Figure 1 – E-H). Furthermore, YAP is exclusively cytoplasmic in all areas of the retinal vasculature, with the exception of the sprouting front where some ECs express nuclear YAP, although at lower levels than in the cytoplasm. (Figure 1A’-D’). TAZ staining signal is very low in the remodelling plexus, arteries and veins (Figure1 E’-H’); at the sprouting front, TAZ is strongly nuclear in numerous ECs (Figure 1 E, green arrowheads and E’), and both nuclear and cytoplasmic in others (Figure 1E, red arrowheads). The nuclear signal of YAP and TAZ did not correlate with a tip or stalk cell phenotype; nuclear YAP and TAZ are rather present in a subset of tip and stalk ECs at the sprouting front. YAP and TAZ were also found at endothelial adherens junctions in veins and in the remodelling plexus, (yellow arrowheads in Fig. 1D’ and F’), as revealed by co-staining for VE-Cadherin (Figure 1 – Figure supplement 1). Together, these observations suggest that YAP/TAZ are abundant transcriptional co-activators in the endothelium, and dynamically regulated during the angiogenic process.
YAP and TAZ are required for vessel growth, branching and regularity of the vasculature
To examine the cell-autonomous role of endothelial YAP and TAZ during angiogenesis we crossed mice bearing floxed alleles of Yap or Taz (17) with mice expressing a tamoxifen-inducible Cre recombinase driven by the endothelial-restricted Pdgfb promoter (Pdgfb-iCreERT2) (18). Injection of the offspring with tamoxifen induced loss of YAP and TAZ protein in ECs during post-natal vascular development, as evidenced by immunofluorescence staining (Figure 2 – Figure supplement 1). Cre negative littermate mice were used as controls.
Endothelial deletion of YAP or TAZ led to mild vascular defects (Figure 2A,B,C,D). Yapfl/fl Pdgfb-iCreERT2 mice (Yap iEC-KO) presented reduced radial expansion of the vasculature (7% +/- 5.4 reduction, p=0.0123) and reduced vessel density (9% +/- 4.4 reduction, p=0.0002) (Figure 2G,H). Tazfl/fl Pdgfb-iCreERT2 mice (Taz iEC-KO) did not show altered radial expansion but displayed decreased vessel density (6% +/- 5.8 reduction, p=0.0214) (Figure 2H). Neither mutant showed a change in the branching frequency of vessels (Figure 2I). Interestingly, in Yap iEC-KO retinas the expression of TAZ was increased and TAZ more often localised to the nucleus (Figure 2 – Figure supplement 2), suggesting compensatory regulation. Taz iEC-KO retinas did not however show a clear difference in YAP expression (data not shown). Deleting both proteins in compound mutant mice (Yapfl/f Tazfl/fl Pdgfb-iCreERT2, YapTaz iEC-KO) produced a dramatic defect in blood vessel development (Figure 2E,F): the retinal vasculature showed a 21% (+/-14, p=0.0012) decrease in radial expansion (Figure 2G), a 26% (+/- 7.0, p<0.0001) decrease in capillary density (Figure 2H), and a 55% (+/- 15.4, p<0.0001) decrease in branching frequency (Figure 2I). Interestingly, the vessel loops were not only bigger in Yap/Taz iEC-KO mice (Figure 2J), but also more variable in size (Figure 2K), and shape (Figure 2L) than in control mice. These results indicate that endothelial YAP and TAZ are critical for the development of a homogeneous blood vessel network and can perform redundant functions in the endothelium.
YAP and TAZ are required for endothelial cell proliferation in response to mechanical stretch
As YAP and TAZ display pro-proliferative and anti-apoptotic roles in many cell types (3, 4), we evaluated whether the reduced vascularization of Yap/Taz iEC-KO retinas was associated with reduced cell proliferation or increased apoptosis. EC proliferation, assessed by EdU staining (Figure 3A-C), was decreased in Yap iEC-KO retinas (23% +/- 10.0, p=0.0469), whilst not affected in Taz iEC-KO. Consistent with our prior results the decrease in cell proliferation was strongest in Yap/Taz iEC-KO retinas (33% +/- 26.0, p=0.0059). Staining for cleaved caspase 3 revealed that apoptosis was unaltered by YAP/TAZ loss (Figure 3D-F).
To understand if YAP and TAZ were required for proliferation downstream of VEGF, we knocked down YAP and TAZ in human umbilical vein endothelial cells (HUVECs) using small interfering RNAs (siRNAs) (Figure 3 - Figure supplement 1) and measured the proliferation rate by flow cytometry after treatment with increasing concentrations of VEGF (Figure 3G). Interestingly, upon loss of YAP, TAZ or YAP/TAZ, ECs proliferated at similar or even increased rates compared to control cells. Furthermore, VEGF treatment did not alter the subcelular localisation of YAP and TAZ in HUVECs (Figure 3 – Figure supplement 2), suggesting that VEGF is not a primary regulator of their activity.
We next asked whether YAP and TAZ mediate endothelial proliferation in response to stretch – another crucial mitogenic stimulus for the endothelium (19). To this end, we subjected HUVECs to 24h of stretch and measured the proliferation rate in comparison to non-stretched, static cells treated with the same siRNAs, by EdU labelling (Figure 3H). Control cells responded to stretch with a 5-fold average increase in proliferation, and this effect was reduced upon knockdown of VE-Cadherin confirming previous observations (19). The knockdown of YAP and YAP/TAZ, but not TAZ alone, led to a decrease in stretch induced proliferation. Thus YAP is, similarly to VE-cadherin, required for endothelial cell proliferation in response to mechanical stimulation at cell-cell junctions.
YAP/TAZ loss leads to irregular endothelial cell distribution and haemorrhages
Further analysis of Yap/Taz iEC-KO retinas revealed severe defects at the sprouting front. Yap/Taz iEC-KO mutant retinas had 23% (+/- 12.3, p= 0.0113) fewer angiogenic sprouts than the control (Figure 4A,B yellow asterisks and Figure 4 - Figure supplement 1). Moreover, whereas control sprouts were elongated and showed long cellular protrusions towards the non vascularised front (Figure 4A’), sprouts in Yap/Taz iEC-KO retinas were rounder and lacked protrusions (Figure 4B’). The defective sprout morphology correlated with irregular spacing and frequent aggregations of ECs within the sprouts (Figure 4B’), arguing that migration and/or the rearrangement of ECs are perturbed in Yap/Taz mutant vessels. Additionally, the Yap/Taz iEC-KO vasculature displayed aberrant vessel crossings (Figure 4C,C’,C”,D,D’,D”), suggesting that vessels may frequently have failed to anastomose or stabilize connections following sprouting, and instead passed each other. Interestingly, defects in cellular rearrangements, sprouting elongation and anastomosis have previously been associated with altered stability or dynamics of endothelial cell junctions (20-24). The defects in morphology were coupled to defects in function as Yap/Taz iEC-KO retinas displayed large haemorrhages from vessel sprouts at the angiogenic front (Figure 4E,E’,F,F’), indicating loss of junctional integrity.
Together, these results argue against a cell proliferation defect being the only driver of the Yap/Taz iEC-KO phenotype and suggest that endothelial YAP/TAZ play a role in the regulation of ECs junctions.
YAP/TAZ regulate adherens junction morphology and stability
Staining for VE-Cadherin revealed several junctional alterations in the vessels of Yap/Taz iEC-KO mice (Figure 4G-H). In control retinas, cell junctions were thin and mostly linear (Figure 4G’), while in Yap/Taz iEC-KO retinas ECs displayed tortuous junctions (Figure 4H’). VE-Cadherin staining also unveiled profound differences in the arrangement of ECs within vessels. In control retinas, ECs were arranged into multicellular tubes, highlighted by the presence of two or more VE-Cadherin junctions running longitudinally along the axis of the vessels (Figure 4G,G”). Some unicellular segments lacking VE-Cadherin staining could also be found and always correlated with decreasing calibre, indicative of regressing vessels (Figure 4G red arrowheads) (25). In contrast, in Yap/Taz iEC-KO retinas we observed many unicellular vessel segments lacking longitudinal VE-Cadherin junctions, but in vessels of normal calibre (Figure 4H, red arrowheads and H”). As junctional remodelling has been shown to be required for the cellular rearrangements that establish multicellular tubes (20), these results suggest that YAP and TAZ regulate junctional remodelling.
VE-Cadherin staining in HUVECs after YAP, TAZ and YAP/TAZ knockdown revealed altered junctional morphology. Interestingly, previous studies have correlated junctional morphology with cellular activities. In vivo, straight or linear junctions were associated with high Notch activity and stalk cell behaviour, while serrated junctions (also referred to as VE-Cadherin fingers) were found in tip cells or actively rearranging cells (21). In vitro, VE-Cadherin fingers were shown to steer migrating ECs and couple leader and follower cells (26), and have also been correlated with increased permeability in cell monolayers. More recently, junction associated intermediate lamellipodia have been associated with decreased permeability in cultured ECs (27).
To more accurately describe the differences in junctional morphology after YAP/TAZ knockdown, we defined five junctional categories (straight junctions, thick junctions, thick to reticular junctions, reticular junctions and fingers) (Figure 5E). Control cells showed mostly reticular junctions (Figure 5A,F). The knockdown of YAP and TAZ led to an increase in straight junctions and fingers, respectively (Figure 5B,C,F), whereas the combined knockdown of YAP/TAZ led to an increase in both straight junctions and fingers and to a loss of reticular junctions (Figure 5D,F). In addition, the knockdown of YAP/TAZ led to junctional breaks in the monolayer, as seen by the presence of gaps in VE-Cadherin stainings (Figure 5D, red arrowheads). Together, these observations demonstrate that YAP and TAZ together are required for the formation of reticular junctions and inhibit the formation of straight junctions and fingers. Interestingly, however, they individually have distinct effects on adherens junction morphology.
To understand whether the shift in morphology translated into a functional defect we investigated the permeability of the monolayer to 250kDa dextran molecules. Only the combined knockdown of YAP/TAZ led to a significant increase in permeability in comparison to the control situation (Figure 5G), suggesting that YAP/TAZ are both required for the barrier function of the endothelium and can compensate for each other in this particular role.
The dynamic rearrangements of ECs during sprouting require that cell-cell junctions are constantly assembled, rearranged and disassembled. To understand whether YAP and TAZ regulate the turnover of cell junctions, we pulse-labeled VE-Cadherin molecules at cell junctions using an antibody directly coupled to a fluorescent dye for 30 minutes (Figure 5H-I) (28). The antibody was subsequently washed out and cells cultured for two more hours in normal conditions, before being fixed and stained for surface VE-Cadherin using a second fluorescent label. Comparing the two sequential VE-cadherin labels allowed us to distinguish junctions with high, intermediate and low turnover rates (Figure 5J). In control cells, 44% of patches were of high turnover junctions, 24% of intermediate turnover junctions and 32% of low turnover junctions (Figure 5K). The knockdown of YAP/TAZ significantly decreased the percentage of high turnover junctions to 14% (p=0.0387) and increased the percentage of low turnover junctions to 58%. Interestingly, we found a correlation between the morphology of junctions and VE-Cadherin turnover rates (Figure 5L): straight junctions and fingers showed the lowest turnover rate, while reticular junctions showed the highest. To understand if the different VE-Cadherin turnover observed after knockdown of YAP/TAZ was caused by a shift in morphology, we compared the turnover of VE-Cadherin within the same morphological categories. Knockdown of YAP/TAZ decreased the percentage of high turnover junctions within all morphological categories, confirming a specific defect in VE-Cadherin turnover.
To test whether YAP and TAZ regulate the endocytosis of VE-Cadherin, we imaged intracellular VE-Cadherin vesicles after pulse labeling the molecule at the surface and allowing it to be endocytosed (Figure 5M,M’,N,N’). However, the detectable amount of VE-cadherin vesicles after YAP/TAZ knockdown was unaffected. In order to challenge the stability of the junctions we triggered cell junction disruption by chelating extracellular calcium with EGTA (29) (Figure 5O,O’,P,P’). Whereas VE-cadherin accumulated in intracellular vesicles in control cells, after YAP/TAZ knockdown substantial amounts of VE-cadherin antibody remained at the cell junction, signifying reduced endocytosis. Thus, YAP/TAZ promote VE-Cadherin turnover and facilitate its dynamic recycling.
As cell junctions are essential for ECs to rearrange and migrate collectively (30), and Yap/Taz iEC-KO retinas presented less elongated sprouts suggestive of a migration defect, we asked whether cell migration was also regulated by YAP/TAZ.
YAP/ TAZ are required for individual endothelial cell migration
To address the requirement of YAP and TAZ for endothelial cell migration we performed a scratch-wound assay (Figure 6A-H). While in the control situation the wound was completely closed at 16h (Figure 6A,B,I), after knockdown of YAP less than 50% of the wound area was closed at the same time point (p=0.0067) (Figure 6C,D,I). A stronger effect on endothelial cell migration was observed after the knockdown of TAZ and YAP/TAZ, with less than 20% of the wound area being closed at 16h (p=0.0006 for siTAZ vs siCTR and p=0.0013 for siYAP+siTAZ vs siCTR) (Figure 6E,F,G,H,I).
Given that cells aggregated at the sprouting front of Yap/Taz iEC-KO retinas, we wondered whether in addition to defective directional cell migration they also lacked the ability to shuffle with the neighbouring cells. Recent data illustrated that collectively migrating ECs in vitro move in streams and swirls and display straight junctions along the lateral boundaries and fingers along the front and rear (26). To investigate collective cell migration we therefore analysed the arrangement of cells in a confluent monolayer (Figure 6 J-O). Control cells displayed a cobblestone appearance without identifiable subgroups of cells (Figure 6J). In contrast, after knockdown of YAP/TAZ cells adopted elongated shapes and arranged into streams and swirls (Figure 6K). To quantity this effect we used the longest axis of the EC nucleus as a proxy for the orientation of each cell and developed a measure of monolayer coordination based on the alignment of cells with their neighbours (Figure 6L,L’,M,M’, N). A score of 1 would signify parallel alignment between all cells, and a score of 0 random alignment of the population. Control cells displayed higher than random alignment with their closest neighbours, but cells beyond 300μm from each other were arranged at random (Figure 6I). While the knockdown of YAP did not affect the alignment score of cells, the knockdown of TAZ led to increased alignment. The combined knockdown of YAP/TAZ led to an even higher degree of coordination, with higher alignment scores across all distances between cells. These results suggest that YAP/TAZ promote the ability of cells to distribute individually within monolayers.
Nuclear YAP and TAZ inhibit Notch and BMP signalling in endothelial cells
To gain insight into the nuclear function of YAP and TAZ, we generated a Pdgfb-iCreERT2 -inducible TAZ gain-of-function mouse allele, in which a mutated version of TAZ (TAZ S89A) is introduced in the Rosa26R locus and expressed by a CAG promoter following Cre-mediated excision of an upstream stop codon (Figure 7 – Figure supplement 1). The TAZ S89A mutation results in enhanced nuclear localization of TAZ as it escapes phosphorylation by the upstream Hippo kinase LATS (31). The allele also expresses nuclear EGFP by means of an IRES sequence, allowing the identification of recombined cells expressing the TAZ mutant protein. Taz iEC-GOF retinas exhibited 25% increased sprouting (+/- 12.2, p=0.0074) (Figure 7A,B yellow asterisks and Figure 7C) and 19% increased branching (+/- 8.3, p=0.0012) (Figure 7D). Thus, driving nuclear TAZ expression, leads in many aspects to the opposite phenotype of Yap/Taz iEC-KO retinas, suggesting that the loss of the nuclear function of YAP/TAZ plays a key role in the development of the observed vascular loss-of-function phenotypes.
To elucidate the transcriptional targets of YAP and TAZ, we performed unbiased transcriptome analysis on HUVECs transduced with adenoviruses encoding for YAP and TAZ gain-of-function mutants or GFP as a control (AdYAPS127A, AdTAZS89A, AdGFP) (Figure 7 – Figure supplement 2). Forced activation of YAP and TAZ led to congruent gene expression changes including the canonical YAP/TAZ target genes CYR61, ANKRD1, and CTGF, as expected. Interestingly, YAP and TAZ also suppressed numerous Notch and BMP target genes. During sprouting angiogenesis, Notch and BMP9/10 signalling restrict the acquisition of a tip cell phenotype by activated ECs (32-38). These results were confirmed by qRT-PCR analysis (Figure 7E): AdYAPS127A and AdTAZS89A cells expressed significantly less LFNG, DLL4 and HES1 (Notch target genes), SMAD6, UNC5B and ID1 (BMP target genes) and HEY1 (a common Notch and BMP target gene) than control cells. Consistent with these findings, knockdown of YAP or TAZ lead to a substantial increase in Notch reporter activity (Figure 7 – Figure supplement 3A) and target gene expression (Figure 7F). Similar effects were observed for the BMP pathway (Figure 7 – Figure supplement 3B and Figure 7F), while TEAD-driven reporter activity and YAP/TAZ target genes were repressed (Figure 7 – Figure supplement 3C and Figure 7F).
To understand if Notch and BMP signalling were also affected in vivo, we stained Yap/Taz iEC-KO retinas for DLL4 and phospho-SMAD1/5/8. In control retinas, DLL4 expression was highest at the leading edge, decreasing over the first 100μm from the sprouting front, beyond which the expression was evenly low throughout the vessels in the plexus (Figure 7G,H and Figure 7 – Figure supplement 4). In Yap/Taz iEC-KO retinas the expression of DLL4 at the sprouting front was higher; additionally, the area of high DLL4 was broader, decreasing for up to 200μm from the sprouting front before flattening to the lower levels of the plexus. Moreover, staining Yap/Taz iEC-KO retinas for pSMAD1/5/8 showed a ∼10 fold increase in the number of ECs positive for pSMAD1/5/8 at the sprouting front (p<0.0001) (Figure 7,I’, J, J’,K).
Together, these results identify that endothelial YAP and TAZ repress Notch and BMP signalling during angiogenesis and retinal vascular expansion.
DISCUSSION
The present study aimed to provide a detailed understanding of the distribution and function of endothelial YAP and TAZ in angiogenesis. Our finding that YAP and TAZ were present in the nucleus of ECs at the sprouting front of developing vessels shows parallels with other cell types where nuclear YAP and TAZ are detected in actively proliferating areas of developing tissues. Interestingly, however, YAP and TAZ show distinct expression patterns in ECs, although these proteins show a high degree of redundancy in many other cell types. While TAZ was predominantly expressed in the sprouting front where it accumulated strongly in endothelial nuclei, YAP was mostly cytoplasmic both in the sprouting front and also in more mature, remodelling vessels. These data are in agreement with a role for YAP in vessel maintenance. Also in zebrafish, the loss of YAP did not impact vessel formation but led to vessel collapse and regression (14). Furthermore, in addition to nuclear and cytoplasmic YAP and TAZ, we also detected junctional localization of these proteins in retinal vessels. A previous study by Giampietro and colleagues (7) has shown that endothelial YAP associates with adherens junction proteins at stable junctions and that this prevents its nuclear accumulation and transcriptional activity. Whether this is also true for TAZ has previously not been addressed. A sequestration of YAP and TAZ either in the cytoplasm or bound to junctional proteins can potentially serve different and not necessarily mutually exclusive roles: preventing their nuclear activity, keeping a pool of protein ready to shuttle to the nucleus and drive gene expression, and having other cytoplasmic functions. It is not yet entirely clear what regulates the subcellular localisation of YAP and TAZ in the developing vasculature. ECs at the sprouting front and in more mature vessels have different adherens junctions, experience distinct levels of signalling from secreted angiogenic molecules and are exposed to different levels of shear stress by the blood. Adherens junctions in sprouting vessels undergo dynamic remodelling that accompanies endothelial cell shape changes and migration, in contrast to mature vessels where they appear more stable in terms of shape (21, 39). Endothelial YAP and TAZ relocate to the nucleus upon disruption of cell junctions or loss of VE-Cadherin (shown for YAP by Choi and colleagues (16) and confirmed in our analysis also for TAZ, data not shown). Interestingly, we did not find junctional localisation of YAP or TAZ at the sprouting front, supporting the idea that more dynamic junctions fail to sequester YAP and TAZ away from the nucleus. Conceptually, these data would support the hypothesis that the subcellular localisation of YAP and TAZ in the vasculature is at least in part regulated by the maturation of adherens junctions. In zebrafish, Nakajima and colleagues (14) showed that YAP nuclear relocation correlated with lumenisation of sprouting vessels, and they attributed this to the effect of shear stress on YAP. In the mouse retina, hemodynamic fluid laws predict that vessels at the sprouting front experience very low levels of shear (40), arguing against YAP and TAZ being activated by shear in this model. Additionally, we found no difference in the subcellular localisation of YAP or TAZ between arteries and veins, i.e. vessels that experience distinct shear stress levels. However, it is possible that local and fast changes in shear stress levels are more relevant to regulate YAP and TAZ than sustained shear. In support of this idea, YAP and TAZ appear not to respond to 12 or 24h of laminar shear (41), but translocate to the nucleus after only 10 minutes of laminar shear (14). Finally, although VEGF, a pro-angiogenic molecule secreted by astrocytes at the avascular front, drives endothelial proliferation and migration, we found no evidence for VEGF induced YAP and TAZ nuclear translocation. Other pro-angiogenic molecules, either locally produced or blood-borne, could regulate endothelial YAP and TAZ during development; future work will help clarify these questions and how different chemical and mechanical stimuli come together to regulate YAP and TAZ.
The role of YAP in the development of the retinal vasculature has previously been studied by Choi and colleagues using intra-ocular injection of siRNAs (16). However, we observed that YAP and TAZ are also expressed in pericytes. Thus to address the cell autonomous role of YAP and TAZ we took advantage of an endothelial specific inducible Cre to inactivate YAP and/or TAZ genetically during angiogenesis. The mild phenotype of the single mutants in comparison to the drastic phenotype of the compound mutant indicates functional redundancy in the endothelium. The compound loss of endothelial YAP and TAZ leads in the mouse retina to a decrease in the radial expansion of vessels, vascular density, branching and sprouting. This phenotype could be a consequence of a decreased number of ECs caused by a proliferation defect (42). However, our further discovery that YAP and TAZ are required to establish homogeneity in the plexus and prevent cellular aggregations suggests that endothelial YAP/TAZ signalling is not only required to provide adequate number of cells but is also critically involved in ensuring adequate EC distribution. We propose that endothelial YAP/TAZ operate in several mechanisms that jointly establish a balance of the right number of endothelial cells in the right place. First, endothelial YAP/TAZ drive proliferation in response to mechanical stimulation at the cell-cell junction, and not in response to VEGF. We propose that in this way endothelial YAP/TAZ provide a cell intrinsic mechanism of locally controlling cell densities, in contrast to growth factor mediated cell proliferation instructed by the surrounding tissue. Second, endothelial YAP/TAZ increase VE-Cadherin turnover at cell-cell junctions, which is essential for cells to rearrange. This corroborates recent findings in mouse hepatocytes where YAP antagonises adherens junction stability (43). The authors showed that YAP regulates hepatocyte adherens junctions in response to increased actomyosin contractility by increasing myosin II light chain gene expression. Accordingly, the transcriptional, nuclear role of YAP was required for junctional regulation. Together with our observations, these findings indicate the existence of a positive feedback loop where stable junctions sequester YAP and TAZ from the nucleus, therefore maintaining less junctional turnover, while remodelling junctions allow YAP and TAZ to relocate to the nucleus where they increase VE-Cadherin turnover. Our results also suggest that a high VE-Cadherin turnover at the sprouting front is required in order to maintain junctional integrity and prevent bleedings. Third, YAP/TAZ decrease cell-cell coupling and increase the ability of cells to migrate individually. Together, our results therefore identify a role for YAP/TAZ in promoting endothelial cellular rearrangements through the regulation of junctional turnover and collectiveness of cell migration. Conceptually, linking stretch induced proliferation to balance cell numbers with modulation of junctional turnover to facilitate cell rearrangements seems ideally suited to achieve the required balance of cell distribution for functional vascular patterning.
Molecularly, how YAP and TAZ affect this complex cell behavior is not clear. Our results identify that endothelial YAP/TAZ reduce the expression of Notch and BMP signaling in ECs. Interestingly, temporal fluctuations of Notch signalling in sprouting ECs are required for their shuffling behaviour in sprouting assays, and heterogeneity in the Notch-activation phase between contacting cells drives vessel branching (42). Given that YAP/TAZ dynamically shuttle between cytoplasm and nucleus, it is tempting to speculate that they may affect not only Notch-signalling levels, but also their dynamics. However, further work and new tools will be required to address these questions. On the other hand, the BMP9/10-Alk1 pathway has recently been proposed as an important driver of collective migration of ECs in particular in response to blood flow (44). Therefore, the newly identified roles of endothelial YAP/TAZ in regulating Notch and BMP signalling may prove critical for endothelial cell migration and rearrangements not only within new vascular sprouts, but also in already perfused vessels. Based on our current evidence, we propose that endothelial YAP/TAZ function as integrators of mechanical stimulation and Notch/BMP signaling to balance local cell densities and endothelial cell arrangements during sprouting angiogenesis.
MATERIAL AND METHODS
Mice and treatments
For loss of function experiments the following mouse strains were used: Yap fl/fl and Taz fl/fl(17), Pdgfb-iCreERT2 (18). To generate a conditional TAZ gain-of-function mouse model, a cDNA coding for a 3xFLAG-tagged human TAZ (WWTR1) carrying an alanine substitution at serine 89 (S89A)(45) was inserted into a Rosa26 targeting vector downstream of the ubiquitous CAG promoter. The cDNA also included an internal ribosome entry sequence (IRES) and a nuclear-localized enhanced green fluorescence protein (nEGFP)(46) for monitoring transgene expression. To allow Cre-dependent expression of 3xFLAG-TAZS89A and of the EGFP reporter, a floxed (loxP-flanked) transcriptional STOP cassette was incorporated between the 3xFLAG-TAZS89A-IRES-nEGFP sequence and the CAG promoter. The linearized targeting vector was transfected into embryonic stem (ES) cells derived from C57BL/6 mice, and homologous recombinants were identified by Southern blotting analysis. Correctly targeted ES cells were implanted into foster mothers and resulting chimaeras bred to C57BL/6 mice to screen for germline transmission. The mouse model was developed together with genOway. The Rosa26-3xFLAG-TAZS89A-IRES-nEGFP allele was kept heterozygous in the experimental studies.
Mice were maintained at the London Research Institute and at the Max Delbruck Center for Molecular Medicine (loss of function mice) and at the Max Planck Institute for Heart and Lung Research (gain of function mice) under standard husbandry conditions. To induce Cre-mediated recombination 4-hydroxytamoxifen (Sigma, 7904) was injected intraperitoneally (IP) (20 μL/g of 1 mg/mL solution) at postnatal day 1 and day 3 and eyes were collected at P6. In all loss and gain of function experiments control animals were littermate animals without Cre expression. Male and female mice were used for the analysis.
For endothelial cell proliferation assessment in the retina, mouse pups were injected IP 2 hours before culling with 20 uL/g of EdU solution (0.5 mg/mL; Thermo Fischer Scientific, C10340).
Cell culture
HUVECs from pooled donors (PromoCell) were cultured in EGM2-Bulletkit without antibiotics (Lonza) and used until passage 6. For YAP and TAZ gain of function experiments HUVECs were obtained from Lonza, cultured in endothelial basal medium (Lonza) supplemented with hydrocortisone (1 μg ml−1), bovine brain extract (12 μg ml−1), gentamicin (50 μg ml-1), amphotericin B (50 ng ml−1), epidermal growth factor (10 ng ml−1) and 10% fetal bovine serum (Life Technologies) and used until passage 4.
For knockdown experiments, HUVECs were transfected with SMARTpool: siGENOME siRNAs purchased from Dharmacon (Yap #M-012200-00-0005, Taz #M-016083-00-0005, VE-Cadherin # M-003641-01-0005 and non-targeting siRNA Pool 1 #D001206-13-05). Briefly, subconfluent (70-80%) HUVECs were transfected with 25 nM siRNA using Dharmafect 1 transfection reagent following the protocol from the manufacturer; transfection media was removed after 24h and experiments were routinely performed on the third day after transfection.
To activate YAP and TAZ signalling in ECs, FLAG-YAPS127A- or 3x-FLAG-TAZS89A-encoding adenoviruses were generated in the adenoviral type 5 backbone lacking the E1/E3 genes (Vector Biolabs). GFP-encoding adenoviruses were used as a control. Infections were carried out by incubating sub-confluent HUVECs (70-80%) with starvation media (EBM containing 0.1% BSA) for 4 hours followed by the addition of adenoviral particles and polybrene (Santa Cruz). After 4 hours, HUVECs were washed with Hanks Buffer for at least five times and then cultured in complete EBM media with 10% FCS and supplements overnight. All experiments were performed 24 hours post transduction.
Immunofluorescence staining
To perform retina immunofluorescence, eyes were collected from postnatal day 6 mice and fixed in 4% PFA in PBS for 1h at 4C. Retinas were dissected in PBS and permeabilised/ blocked for 1h at room temperature in 1% BSA, 2% FBS, 0.5% Triton X100, 0.01% Na deoxycholate and 0,02% Na Azide in PBS. Primary and secondary antibodies were incubated overnight at 4C and for 2h at room temperature, respectively, both in 1:1 PBS: blocking buffer. Isolectin staining was performed overnight at 4C in Pblec after retinas were equilibrated for 1h in Pblec at room temperature. Retinas were post-stained fixed in 2% PFA in PBS for 10 minutes. To mount the samples Vectashield mounting medium. (Vector Labs, H1000) or ProLong Gold (Thermo Fisher Scientific) was used. Imaging was done by laser scanning confocal microscopy (Carl Zeiss LSM700, LSM780 and Leica TCS SP8). Processing of samples was carried out in tissues from littermates under the same conditions.
For immunofluorescence in HUVECs, cells were grown in #1.5 coverslips coated with polylysine and gelatin 0.2%. At the end of the experiment cells were fixed in 4% PFA for 10min, permeabilised in 0.3% Triton-X100 in blocking buffer for 5min and blocked in 1% BSA 20mM Glycine in PBS for 30 min. Primary and secondary antibodies were incubated for 2 and 1 hours, respectively, in blocking buffer. Nuclei labeling was performed by incubating cells with DAPI for 5 min (Life technologies, D1306).
A list of the primary antibodies used can be found in Supplementary Table 1.
Image analysis
Analysis of radial expansion, capillary density, branching frequency, proliferating ECs, apoptosis and sprouting numbers was done using Fiji. Radial expansion corresponds to the mean distance from the optic nerve to the sprouting front (8 measurements in tilescans of two whole retinas per animal). Capillary density corresponds to the vessel area (measured by thresholding IB4 signal) divided by the field of view area (6–8 images of (425 μm)2 between artery and vein per animal). Branching frequency was measured by manually counting all branching points in a field of view (4-5 images of (200 μm)2 between artery and vein per animal). The plexus regularity was assessed through the standard deviation of the size and the circularity of the vascular loops in the plexus (using same images as for analysis of capillary density). Vascular loops were segmented by thresholding the IB4 signal to avoid artifacts we excluded loops with a size smaller than 86 um^2 for the analysis. Endothelial proliferation was measured by manually counting the number of EdU positive endothelial nuclei (ERG positive) and dividing by the vessel area (measured by thresholding IB4 signal) (4 images of (425 μm)2 containing the sprouting front and localized on top of arteries per animal). Apoptosis was measured manually by counting the number of cleaved caspase 3 positive figures and dividing by the vessel area (measured by thresholding IB4 signal) (tilescan of one whole retina per animal). The number of sprouts was measured manually (3 images of 425x850μm of the sprouting front per animal). To quantify DLL4 intensity the outline of the sprouting front and the position of the arteries were manually defined using IB4 staining. Vessels were segmented by thresholding the IB4 staining in Fiji. Then, DLL4 intensity inside the vasculature was normalised with the average DLL4 intensity outside of the vasculature. Subsequently, for every pixel inside the vasculature (excluding the arteries) the distance to the sprouting front was calculated. The normalised DLL4 values within each bin were averaged (15μm bins from 0 to 500 μm). For each retina quarter a curve was obtained, and the average and SEM of these curves was shown in the graph (one retina quarter was used per animal). To quantify pSMAD1/5/8 status the number of pSMAD1/5/8 positive endothelial nuclei was manually counted and dividing by the total number of endothelial nuclei (defined by being ERG positive) (3 images of (225μm)2 containing the sprouting front were used per animal).
To analyse YAP/TAZ subcellular localisation in HUVECs we adapted a previously existing cytoplasm-to-nucleus translocation assay pipeline from Cell Profiler (47). Briefly, YAP or TAZ staining intensity was measured both inside the nucleus of the cell and in a 12 pixels wide ring of cytoplasm grown radially from the nucleus. The nucleus localisation was determined using a DAPI mask. We calculated the ratio between the nucleus and cytoplasm intensity and categorised cells as having nuclear localisation, nuclear and cytoplasmic and cytoplasmic localisation when the ratio was >1.2, between 1.2 – 0.8 and <0.8 respectively.
Cell junction morphology analysis was done in confluent monolayers of HUVECs stained for VE-Cadherin. 5 morphological categories were defined: straight, thick, thick to reticular, reticular and fingers. We acquired 5 images of (160μm)2 per condition per experiment, divided each image in (16 μm)2 patches, and randomly grouped these patches. The classification into categories was done manually and blindly for the condition.
To analyse cell coordination we used confluent cells labelled for DAPI. The nuclei were automatically segmented using a customized Python algorithm relying on the Scikit Image Library. By fitting an ellipse to each nucleus we obtained its major and minor axis, and the angle of the major axis with the x-axis of the image was assigned to the nucleus as its orientation. This way each nucleus in the images was assigned a position given by its midpoint and an orientation. Next we analyzed the average alignment of the nuclei of two cells depending on their distance. As the nuclei don’t have a directionality (i.e. they are nematics as opposed to vectors), the angles between two nuclei range from 0 corresponding to the nuclei being parallel, to π/2 corresponding to them spanning a right angle. For any two cells in each image we calculated the angle and the Euclidean distance between them, and then we binned the cells depending on their distance. We introduced a parameter called ‘alignment’ which is 1 if all cells are perfectly aligned and 0 for a completely random distribution of cell orientations.
VEGF treatment and YAP/TAZ staining
Confluent HUVECs were maintained in VEGF free media for 24h. VEGF treatment was then performed for 6h with 0ng/mL, 4ng/mL, 20ng/mL or 100ng/mL of VEGF-165 (PrepoTech, 450-32). Immunofluorescence staining and analysis of YAP and TAZ subcellular localisation was performed as above described.
VEGF treatment and proliferation assessment
Knockdown HUVECs were maintained in VEGF free media for 24h. VEGF treatment was then performed for 24h with 0ng/mL, 4ng/mL, 20ng/mL or 100ng/mL of VEGF-165. Cells were pelleted, ressuspended in 90% cold Methanol and stored at -20C° before further processing. Cells were then ressuspended in Propidium Iodide/RNase staining solution (Cell signaling, 4087) for 30 minutes before cell cycle analysis by flow cytometry (LSRII, BD). Data was analysed using BD FACSDiva™ software.
Mechanical stretch application and proliferation assessment
HUVECs were plated on collagen I - 0.2% gelatine-coated Bioflex plates (BF-3001C, Flexcell International Corporation). Gene knockdown was preformed as previously described. Cells were incubated in transfection media for 24h, and allowed to recover in fresh complete media for 4h. Afterwards cells were incubated for 24h in serum starvation media (0,1%BSA in EBM2 pure media) to form a confluent, quiescent monolayer. Cyclic stretch (0.25Hz, 15% elongation) was then applied for 24h using a Flexcell® FX-5000™ Tension System. Control cells were placed in the same incubator but not on the Flexcell® device (static conditions). EdU pulsing was performed after 20h of the 24h stretch period. At the end of the experiment cells were fixed in 4% PFA and EdU staining was performed according to the manufacturer’s protocol (Click-It EdU C10337 Life Technologies). Nuclei were labelled with DAPI. Three regions of interested were acquired per sample in a Carl Zeiss LSM700 scanning confocal microscopes (Zeiss, Germany). Quantification of proliferation was done using a CellProfiler pipeline. Percentage of S phase cells was determined as percentage of EdU positive nuclei over the total number of nuclei.
Permeability assay
24h after siRNA transfection cells were re-plated into fibronectin coated Transwell® membranes (Costar 3460) at confluence and incubated for 2 more days to stabilize cell junctions. On the third day after transfection 0.5mg/mL of 250kDa FITC Dextran in cell media (Sigma FD250) was added to the top well. Fluorescence on the bottom well was measured after 6h in a Gemini XPS fluorescent plate reader. In each experiment 3 wells were measured per condition.
Pulse chase VE-Cadherin experiment for quantification of low, intermediate and high turnover junctions
Cells were labelled live with a non-blocking monoclonal antibody directed against extracellular VE-Cadherin and directly coupled with Alexa-Fluor647 (BD Pharmingen, #555661, 1:200) for 30 minutes. Cells were then washed 2x with PBS and incubated with complete media for additional 2 hours. Cells were fixed with 4% PFA and stained for VE-Cadherin (Santa Cruz Biotechnology, #6458, 1:200) with a secondary antibody coupled with Alexa-Fluor-488. 5 (160μm)2 images per condition per experiment were acquired in a Carl Zeiss LSM700 confocal laser scanning microscope using the same acquisition settings. Max projection of z stack and merging of channels was done in Fiji. Images were divided in (16 μm)2 patches and the patches were randomly grouped. Patches were classified into a morphological category and into low, intermediate or high turnover categories, manually and blindly for the condition.
Pulse chase VE-Cadherin experiment and EGTA treatment
Cells were labelled live with a non-blocking monoclonal antibody directed against extracellular VE-Cadherin and directly coupled with Alexa-Fluor647 (BD Pharmingen, #555661, 1:200) for 30 minutes. Cells were then washed 2x with PBS and incubated with 4mM EGTA or vehicle in complete media for 30 minutes. Cells were fixed with 4% PFA and stained for VE-Cadherin (Santa Cruz Biotechnology, #6458, 1:200) with a secondary antibody coupled with Alexa-Fluor-488.
Scratch wound assay
24h after siRNA transfection cells were re-plated into a scratch wound assay device (IBIDI). On the following day a cell free gap of 500μm was created by removing the insert of the device. Images were taken immediately after removing the insert (0h) and after 16h. The cell free area was measured in Fiji and used to calculate the percentage of wound closure at 16h.
RNA extraction and quantitative real time-polymerase chain reaction
RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. For HUVECs transfected with adenoviruses carrying YAP and TAZ gain of function mutations, 2µg of total RNA were reverse transcribed to cDNA using M-MLV reverse transcriptase (Invitrogen). For HUVECs transfected with siRNAs 90ng of RNA were reverse transcribed using RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific). qRT-PCR was performed using TaqMan reagents and probes (Applied Biosystems) (listed in Supplementary Table 2). qRT-PCR reactions were run on a StepOnePlus real-time PCR instrument (ThermoFisher Scientific) or Quant Studio 6 Flex (Applied Biosystems) and expression levels were normalised to human ACTB or human HPRT1 using the 2deltaCT method.
Western blot
Protein was extracted from HUVECs using M-PER protein extraction reagent with Halt Protease and Phosphatase inhibitors (Pierce). Proteins concentration was assessed using a BCA protein assay kit (Pierce). Proteins were separated by SDS–PAGE and blotted onto nitrocellulose membranes (Bio-Rad). Membranes were probed with specific primary antibodies and then with peroxidase-conjugated secondary antibodies. The following antibodies were used: YAP 63.7 (Santa Cruz Biotechnology, sc-101199, 1:1000), GAPDH (Millipore, MAB374, 1:4000). The bands were visualized by chemiluminescence using an ECL detection kit (GE Heathcare) and a My ECL Imager (Thermo Scientific).
Dual luciferase reporter assay
Renilla-luciferase reporter assays for TEF-1 (48), RBPj (49), BRE (50, 51) and FOPflash (52)-Luciferase promoter activity were performed as follows: 48 hours after gene knockdown by siRNA HUVECs were cotransfected with 600 ng of Luciferase reporter gene construct and 300 ng of pRL-CMV (Promega) using Lipofectamine2000 and incubated for 4 hours. Cell extracts were prepared 72 hours post siRNA transfection and 24 hours post Luciferase reporter transfection, and luciferase activity was measured using a dual luciferase system as described (53). Experiments were carried out in duplicates and results were normalized to the correspondent FOPflash/Renilla measurement.
Microarray and gene set enrichment analysis
Microarray studies were performed as described(46). In brief, total RNA was extracted from HUVECs using the RNeasy kit (Qiagen) and RNA quality assessed with the 6000 nano kit and an Agilent Bioanalyser. RNA was labelled according to the Affymetrix Whole Transcript Sense Target Labeling protocol. Affymetrix GeneChip® Human Gene 2.0 ST arrays were hybridized and scanned using Affymetrix protocols. Data were analysed using the Affymetrix expression console using the RMA algorithm; statistical analysis was done using DNAStar Arraystar 11. Heat maps of gene signatures were plotted using RStudio, Inc.
Statistical Analysis
Statistical analyses were performed using GraphPad Prism software and statistical significance was determined using unpaired Student t-test. When technically possible the investigators were blinded to genotype during experiments and quantification.
Author Contribution
FN designed the study, performed experiments, analyzed the results and wrote the manuscript; AKB, YTO, ACV, AS and JRC performed experiments, analyzed the results and reviewed the manuscript; IH and EBK performed experiments; SA performed quantitative analysis and analyzed results; CAF provided critical feedback. HG and MP designed the study and wrote the manuscript.
Competing interests
None.
Acknowledgments
We thank members of the Vascular Biology (Berlin) and Vascular Patterning (VIB – Leuven) Laboratories for helpful discussions. We thank the Cancer Research UK - London Research Institute and the Max Delbrück Center for Molecular Medicine Animal Facilities for animal care and technical support. We thank Dr. Axel Behrens for kindly providing the Tazfl/fl mice. We thank Dr. Walter Birchmeier and Dr. Daniel Besser for providing the Normalizer and BRE-luc reporter, Dr. Eric Sahai and Dr. Nic Tapon for providing the TEF1-reporter, and Dr. Michael Gotthardt and Dr. Michael Radke for access to the Flexcell® Tension System and technical assistance. We specially thank Dr. Veronique Gebala, Dr. Andre Rosa and Dr. Baptiste Coxam for helpful comments on the manuscript.
FN was financially supported by the Fundação para a Ciência e a Tecnologia (FCT), CRUK-CRICK and the MDC. ACV and AKB were supported by the DZHK (German Centre for Cardiovascular Research), AS was supported by the EMBO (European Molecular Biology Organization), JRC was supported by the FCT. CAF is supported by the FCT, EC-ERC Starting Grant, Portugal2020 program. MP is supported by the Max Planck Society, the ERC Starting Grant ANGIOMET, the Deutsche Forschungsgemeinschaft, the Excellence Cluster Cardiopulmonary System, the LOEWE grant Ub-Net, the DZHK, the Stiftung Charité and the EMBO Young Investigator Program. HG is supported by the DZHK and ERC Consolidator Grant Reshape 311719.