Mechanotransductive feedback control of endothelial cell motility and vascular morphogenesis

Reviewer #1 (Public Review):

This manuscript puts forward the concept that there is a specific time window during which YAP/TAZ driven transcription provides feedback for optimal endothelial cell adhesion, cytoskeletal organization and migration. The study follows up on previous elegant findings from this group and others which established the importance of YAP/TAZ-mediated transcription for persistent endothelial cell migration. The data presented here extends the concept at two levels: first, the data may explain why there are differences between experimental setups where YAP/TAZ activity are inhibited for prolonged times (e.g. cultures of YAP knockdown cells), versus experiments in which the transient inhibition of YAP/TAZ and (global) transcription affects endothelial cell dynamics prior to their equilibrium.

All experiments are convincing, clearly visualized and quantified.

The strength of the paper is that it clearly indicates that there are temporal controlled feedback systems which which is important for endothelial collective cell behavior.

A limitation of the study is that the inhibitory studies in vivo may include off-target effects as well. Future endeavors, including specific knockout models, optogenetics and/or transgenic zebrafish lines that visualize endothelial cell properties (proliferation and migration) will be informative to track individual endothelial cell responses upon feedback signals.

Reviewer #2 (Public Review):

Summary:

Here the effect of overall transcription blockade, and then specifically depletion of YAP/TAZ transcription factors was tested on cytoskeletal responses, starting from a previous paper showing YAP/TAZ-mediated effects on the cytoskeleton and cell behaviors. Here, primary endothelial cells were assessed on substrates of different stiffness and parameters such as migration, cell spreading, and focal adhesion number/length were tested upon transcriptional manipulation. Zebrafish subjected to similar manipulations were also assessed during the phase of intersegmental vessel elongation. The conclusion was that there is a feedback loop of 4 hours that is important for the effects of mechanical changes to be translated into transcriptional changes that then permanently affect the cytoskeleton.

The idea is intriguing and a previous paper contains data supporting the overall model. The fish washout data is quite interesting and supports the kinetics conclusions. New transcriptional profiling in this version supports that cytoskeletal genes are differentially regulated with YAP/TAZ manipulations.

Major strengths: The combination of in vitro and in vivo assessment provides evidence for timing in physiologically relevant contexts, and rigorous quantification of outputs is provided. The idea of defining temporal aspects of the system is quite interesting. New RNA profiling supports the model.

Weaknesses:

Actinomycin D blocks most transcription so exposure for hours likely leads to secondary and tertiary effects and perhaps effects on viability.

Reviewer #4 (Public Review):

Summary:

Mason DE et al. have extended their previous study on continuous migration of cells regulated by a feedback loop that controls gene expression by YAP and TAZ. Time scale of the negative feedback loop is derived from the authors' adhesion-spreading-polarization-migration (ASPM) assay. Involvement of transcription-translation in the negative feedback loop is evidenced by the experiments using Actinomycin D. The time scale of mechanotransduction-dependent feedback demonstrated by cytoskeletal alteration in the actinomycin D-treated endothelial colony forming cells (ECFCs) and that shown in the ECFCs depleted of YAP/TAZ by siRNA. The authors examine the time scale when ECFCs are attached to MeHA matrics (soft, moderate, and stiff substrate) and show the conserved time scale among the conditions they use, although instantaneous migration, cell area, and circularity vary. Finally, they tried to confirm that the time scale of the feedback loop-dependent endothelial migration by the effect of washout of Actinomycin D (inhibition of gene transcription), Puromycin (translational inhibition), and Verteporfin (YAP/TAZ inhibitor) on ISV extension during sprouting angiogenesis. They conclude that endothelial motility required for vascular morphogenesis is regulated by mechanotransduction-mediated feedback loop that is dependent on YAP/TAZ-dependent transcriptional regulation.

Strengths:

The authors conduct ASPM assay to find the time scale of feedback when ECFCs attach to three different matrics. They observe the common time scale of feedback. Thus, under very specific conditions they use, the reproducibility is validated by their ASPM assay. The feedback loop mediated by inhibition of gene expression by Actinomycin D is similar to that obtained from YAP/TAZ-depleted cells, suggesting the mechanotranduction might be involved in the feedback loop. The time scale representing infection point might be interesting when considering the continuous motility in cultured endothelial cells, although it might not account for the migration of endothelial cells that is controlled by a wide variety of extracellular cues. In vivo, stiffness of extracellular matrix is merely one of the cues.

Weaknesses:

ASPM assay is based on attachment-dependent phenomenon. The time scale including the inflection point determined by ASPM experiments using cultured cells and the mechanotransduction-based theory do not seem to fit in vivo ISV elongation. Although it is challenging to find the conserved theory of continuous cell motility of endothelial cells, the data is preliminary and does not support the authors' claim. There is no evidence that mechanotransduction solely determines the feedback loop during elongation of ISVs. The points to be addressed are listed in recommendations for the authors.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1:

(1) Point for more elaborate discussion: Apparently the timescale of negative feedback signals is conserved between endothelial cell migration in vitro (with human cells) and endothelial migration during the formation of ISVs in zebrafish. What do you think might be an explanation for such conserved timescales? Are there certain processes within cytoskeletal tension build up that require this quantity of time to establish? Or does it relate to the time that is needed to begin to express the YAP/TAZ target genes that mediate feedback?

The underlying mechanisms responsible for the conserved timescale is a major direction that we continue to explore. Localization of YAP/TAZ to the nucleus is likely not rate-limiting. We showed previously that acute RhoA activation produced significant YAP/TAZ nuclear localization within minutes, while subsequent co-transcriptional activity aligned with the gene expression dynamics observed here (Berlew et al., 2021). We hypothesize that the dynamics of YAP/TAZdependent transcription and the translation of those target genes are rate-limiting for initial feedback loop completion (tic = 4 hours). This is supported by work from us and others in a variety of cell lines showing YAP/TAZ transcriptional responses take place during the first few hours after activation. (Franklin et al., 2020; Mason et al., 2019; Plouffe et al., 2018) While our data identify mediators of initial feedback loop completion, the molecular effectors that determine the timescale of new cytoskeletal equilibrium establishment (teq = 8 hours) remain unclear.

(2) Do you expect different timescales for slower endothelial migratory processes (e.g. for instance during fin vascular regeneration which takes days)?

We selected the ISV development model because it exhibits similar migratory kinetics to our previously-explored human ECFC migration in vitro. The comparable kinetics allowed us to study dynamics of the feedback loop in vivo on similar time scales, but we have not explored models featuring either slower or faster dynamics.

It would be interesting to test how feedback dynamics are impacted in distinct endothelial migratory processes. Our data suggest that the feedback loop is necessary for persistent migration; however, YAP and TAZ respond to a diversity of upstream regulators in addition to mechanical signals, which might depend on the process of vascular morphogenesis. For example, after fin amputation, inflammation and tissue regeneration may impact the biochemical and mechanical environment experienced by the endothelium. Additionally, cells display different migratory behaviors in ISV morphogenesis compared to fin regeneration. During ISV formation, sprouting tip cells migrate dorsally through avascular tissue, followed by stalk cells. (Ellertsdóttir et al., 2010) In contrast, the fin vasculature regenerates by forming an intermediate vascular plexus, where some venous-derived endothelial cells migrate towards the sprouting front, while others migrate against it. (Xu et al., 2014) We are excited to study the role of this feedback loop in these different modes of neovessel formation in future studies.

(3) Is the ~4hrs and 8hrs feedback time window a general property or does it differ between specific endothelial cell types? In the veins the endothelial cells generate less stress fibers and adhesions compared to in the arteries. Does this mean that there might be a difference in the feedback time window, or does that mean that certain endothelial cell types may not have such YAP/TAZcontrolled feedback system?

Recent studies suggest that venous endothelial cells are the primary endothelial subtype responsible for blood vessel morphogenesis. (Lee et al., 2022, 2021; Xu et al., 2014) They are highly motile and mechanosensitive, migrating against blood flow. (Lee et al., 2022) The Huveneers group has shown that the actin cytoskeleton is differently organized in adult arteries and veins in response to biomechanical properties of its extracellular matrix, rather than intrinsic differences between arterial and venous cells. (van Geemen et al., 2014) This suggests that arterial and venous cells have distinct cytoskeletal setpoints due to mechanical cues in their environment (Price et al., 2021). We expect this to impact the degree of cytoskeletal remodeling and cell migration at equilibrium, rather than the kinetics of the feedback loop per se, though we have not yet tested this hypothesis. Testing these predictions on cytoskeletal setpoint stability and adaptation is a major direction that we continue to explore.

(4) The experiments are based on perturbations to prove that transcriptional feedback is needed for endothelial migration. What would happen if the feedback systems is always switched on? An experiment to add might be to analyse the responsiveness of endothelial cells expressing constitutively active YAP/TAZ.

This is a problem that we are actively pursuing. Though the feedback system forms a coherent loop, we anticipate that the identity of the node of the loop selected for constitutive activation will influence the outcome, depending on whether that node is rate-limiting for feedback kinetics and the extent of intersection of that node with other signaling events in the cell. For example, we have observed that constitutive YAP activation drives profound changes to the transcriptional landscape including, but not limited to, RhoA signaling (Jones et al., 2023). We further anticipate that constitutive activation of feedback loop nodes may alter feedback dynamics, while dynamic or acute perturbation will be required to dissect these contributions in real time. For these reasons, ongoing work in the lab is pursuing these questions using optogenetic tools that enable precise spatial and temporal control (Berlew et al., 2021).

(5) To investigate the role of YAP-mediated transcription in an accurate time-dependent manner the authors may consider using the recently developed optogenetic YAP translocation tool: https://doi.org/10.15252/embr.202154401

We are enthusiastic about the power of optogenetics to interrogate the nodes and timescales of this feedback system, and we are now funded to pursue this line of research.

Reviewer #2:

The idea is intriguing, but it is not clear how the feedback actually works, so it is difficult to determine if the events needed could occur within 4 hrs. Specifically, it is not clear what gene changes initiated by YAP/TAZ translocation eventually lead to changes in Rho signaling and contractility. Much of the evidence to support the model is preliminary. Some of the data is consistent with the model, but alternative explanations of the data are not excluded. The fish washout data is quite interesting and does support the model. It is unclear how some of the in vitro data supports the model and excludes alternatives.

Major strengths:

The combination of in vitro and in vivo assessment provides evidence for timing in physiologically relevant contexts, and a rigorous quantification of outputs is provided. The idea of defining temporal aspects of the system is quite interesting.

Major weaknesses:

The evidence for a "loop" is not strong; rather, most of the data can also be interpreted as a linear increase in effect with time once a threshold is reached. Washout experiments are key to setting up a time window, yet these experiments are presented only for the fish model. A major technical challenge is that siRNA experiments take time to achieve depletion status, making precise timing of events on short time scales problematic. Also, Actinomycin D blocks most transcription so exposure for hours likely leads to secondary and tertiary effects and perhaps effects on viability. No RNA profiling is presented to validate proposed transcriptional changes.

We thank the reviewer for these helpful suggestions. We have expanded our explanation of the history and known mediators of the feedback loop in the introduction. We and, independently, the Huveneers group recently reported that human endothelial cells maintain cytoskeletal equilibrium for persistent motility through a YAP/TAZ-mediated feedback loop that modulates cytoskeletal tension. (Mason et al., 2019; van der Stoel et al., 2020) Because YAP and TAZ are activated by tension of the cytoskeleton (Dupont et al., 2011), suppression of cytoskeletal tension by YAP/TAZ transcriptional target genes constitutes a negative feedback loop (Fig. 1A). We described key components of this cell-intrinsic feedback loop, which acts as a control system to maintain cytoskeletal homeostasis for persistent motility via modulation of Rho-ROCK-myosin II activity. (Mason et al., 2019) Both we and the Huveneers group found that perturbation of genes and pathways regulated by YAP/TAZ mechanoactivation can functionally rescue motility in YAP/TAZ-depleted cells (e.g., RhoA/ROCK/myosin II, NUAK2, DLC1). (Mason et al., 2019; van der Stoel et al., 2020) We further showed previously that both YAP/TAZ depletion and acute YAP/TAZ-TEAD inhibition consistently increased stress fiber and FA maturation and arrested cell motility, accounting for these limitations of siRNA. (Mason et al., 2019)

Enduring limitations to the temporal, spatial, and cell-specific control of the genetic and pharmacologic methods have inspired us to initiate alternative approaches, which are the subject of ongoing efforts. Further research will be necessary in the zebrafish to determine the extent to which the observed migratory dynamics are driven by cytoskeletal arrest.

To identify early YAP/TAZ-regulated transcriptional changes, we have added RNA profiling of control and YAP/TAZ depleted cells cultured on stiff matrices for four hours. Genes upregulated by YAP/TAZ depletion were enriched for Gene Ontology (GO) terms associated with Rho protein signal transduction, vascular development, cellular response to vascular endothelial growth factor (VEGF) stimulus, and endothelial cell migration (Fig. 9B). These data support a role for YAP and TAZ as negative feedback mediators that maintain cytoskeletal homeostasis for endothelial cell migration and vascular morphogenesis.

Reviewer #3:

The authors used ECFC - endothelial colony forming cells (circulating endothelial cells that activate in response to vascular injury).

Q: Did the authors characterize these cells and made sure that they are truly endothelial cells - for example examine specific endothelial markers, arterial-venous identity markers & Notch signalling status, overall morphology etc prior to the start of the experiment. How were ECFC isolated from human individuals, are these "healthy" volunteers - any underlying CVD risk factors, cells from one patient or from pooled samples, what injury where these humans exposed to trigger the release of the ECPFs into the circulation, etc. The materials & methods on ECFC should be expanded.

Human umbilical cord blood-derived ECFCs were isolated at Indiana University School of Medicine and kindly provided by Dr Mervin Yoder. Cells were cultured as described by the Yoder group (Rapp et al., 2011) and our prior paper (Mason et al., 2019). We have expanded the materials and methods section to describe the source and characterization of these cells.

The authors suggest that loss of YAP/TAZ phenocopies actinomycin-D inhibition - "both transcription inhibition and YAP/TAZ depletion impaired polarization, and induced robust ventral stress fiber formation and peripheral focal adhesion maturation". However, the cell size of actinomycin-D treated cells (Fig. 1B, top right panel), differs from the endothelial cell size upon siYAP/TAZ (Fig. 1E, top right panel) - and vinculin staining seems more pronounced in actinomycin-D treated cells (B, bottom right) when compared to siYAP/TAZ group. Cell shape is defined by acto-myosin tension.

Q: Besides Fraction of focal adhesion >1um; focal adhesion number did the authors measure additional parameters related to cytoskeleton remodelling / focal adhesions that can substantiate their statement on similarity between loss of YAP/TAZ and actinomycin-D treatment. Would it be possible to make a more specific genetic intervention (besides YAP/TAZ) interfering with the focal adhesion pathway as opposed to the broad spectrum inhibitor actinomyocin-D.

Our previous paper (Mason et al., 2019) delineated the mechanistic relationships between YAP/TAZ signaling, focal adhesion turnover, actomyosin polymerization, and the intervening mechanisms of myosin regulation. Specifically, we demonstrated that YAP/TAZ regulate the myosin phosphatase kinase, NUAK2, and ARHGAP genes to mediate this feedback. Expanding on this work, the current study aimed to define the temporal kinetics of the cytoskeletal mechanotransductive feedback in vitro and in vivo. We used actinomycin-D and YAP/TAZ depletion to interrogate the role of transcriptional regulation and YAP/TAZ signaling, respectively. In this revision, we have added RNA profiling that identifies early YAP/TAZ-regulated transcriptional changes and further points to other molecular mediators of focal adhesions (e.g. TRIO, RHOB, THBS1) that will be the subjects of future studies.

Q: Does the actinomycin-D treatment affect responsiveness to Vegf? induce apoptosis or reduce survival of the ECFC?

We have not looked specifically at the effect of actinomycin-D treatment on responsiveness to VEGF. However, actinomycin-D has been reported to reduce transcription of VEGF receptors (E et al., 2012). In contrast, we found that YAP/TAZ depletion upregulated GO terms associated with endothelial cell migration and response to VEGF stimulus (Fig. 9B), as well as receptors to angiogenic growth factors, including KDR and FLT4 (Fig. 9E). These results suggest YAP/TAZ depleted cells may be more sensitive to VEGF stimulation but remain nonmotile due to cytoskeletal arrest.

We showed previously that long-term treatment with actinomycin-D reduces ECFC survival (Mason et al., 2019).

Q: Which mechanism links ECM stiffness with endothelial surface area in the authors scenario. In zebrafish, activity of endothelial guanine exchange factor Trio specifically at endothelial cell junctions (Klems, Nat Comms, 2020) and endoglin in response to hemodynamic factors (Siekmann, Nat Cell Biol 2017) have been show to control EC shape/surface area - do these factors play a role in the scenario proposed by the authors.

Our new transcriptional profiling indicates both Trio and endoglin are regulated through YAP and TAZ in human ECFCs. We plan to follow up on these findings.

Q: The authors report that EC migrate faster on stiff substrate, and concomitantly these cells have a larger surface area. What is the physiological rationale behind these observations. Did the authors observe such behaviors in their zebrafish ISV model? How do these observations integrate with the tip - stalk cell shuffling model (Jakobsson & Gerhardt, Nat Cell Biol, 2011) and Notch activity in developing ISVs.

This question raises important distinctions between the mode of migration in ISV morphogenesis and endothelial cells adherent to substrates. Cells behave and respond to mechanical cues differently in 2D vs. 3D matrices. (LaValley and Reinhart-King, 2014) Additionally, the microenvironment in vivo is much more complex, combining numerous biochemical signals and changing mechanical properties. (Whisler et al., 2023) We are actively investigating the downstream targets of YAP/TAZ mechanotransduction and how that integrates with other pathways known to regulate vascular morphogenesis, such as Notch signaling.

The authors examined the formation of arterial intersegmental vessels in the trunk of developing zebrafish embryos in vivo. They used a variety of pharmacological inhibitors of transcription and acto-myosin remodelling and linked the observed morphological changes in ISV morphogenesis with changes in endothelial cell motility.

Q: Reduced formation and dorsal extension of ISVs may have several reasons, including reduced EC migration and proliferation. The Tg(fl i1a:EGFP) reporter however is not the most suitable line to monitor migration of individual endothelial cells. Can the authors repeat the experiments in Tg(fl i1a:nEGFP); Tg(kdrl:HRAS-mCherry) double transgenics to visualize movement-migration of the individual endothelial cells and EC proliferation events, in the different treatment regimes.

So far, we have not tracked individual endothelial cells during ISV morphogenesis. We agree this is the best approach and are pursuing a similar technique for these experiments.

ISV formation is furthermore affected by Notch signalling status and a series of (repulsive) guidance cues.

Q: Does de novo blockade of gene expression with Actinomycin D affect Notch signalling status, expression of PlexinD - sFlt1, netrin1 or arterial-venous identify genes.

While we have not performed gene expression analysis under the Actinomycin D condition, Actinomycin D functions as a broad transcription inhibitor. We are currently pursuing the downstream targets of YAP/TAZ mechanotransduction in both ECFCs and zebrafish.

Remark: The authors may want to consider using the Tg(fl i1:LIFEACT-GFP) reporter for in vivo imaging of actin remodelling events.

We thank the reviewer for their helpful suggestion.

Remark: the authors report "As with broad transcription inhibition, in situ depletion of YAP and TAZ by RNAi arrested cell motility, illustrated here by live-migration sparklines over 10 hours: siControl: , siYAP/TAZ: (25 μm scale-bar: -)". Can the authors make a separate figure panel for this, how many cells were measured?

Please refer to our previous publication for the complete details on this data (Mason et al., 2019). We have added the citation in the text.

Remark: in the wash-out experiments, exposure to the inhibitors is not the same in the different scenarios - could it be that the longer exposure time induces "toxic" side effect that cannot be "washed out" when compared to the short treatment regimes?

This is a possible limitation of the pharmacological approach and have included it in the discussion section. We are currently exploring alternative approaches to interrogate the timescale of the feedback loop more precisely.

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