Circumferential actomyosin bundles anchored by CCM1 drive endothelial cell contraction and vessel constriction

Domain: physics
Published: 2025-12-27
Editorial outcome: major_revision

Abstract
Blood vessels undergo extensive remodelling to acquire appropriate diameters, yet how endothelial cells coordinate changes in their number and shape to achieve this remains unclear. Here we show that endothelial cell contraction and rearrangements underlie the inverse relationship between cell number and vessel diameter during development. Using high-resolution imaging and manipulation of actin cytoskeleton organisation, in vivo laser ablation experiments and mathematical simulations, we reveal that tension-bearing, circumferential actomyosin bundles form in the endothelial cortex to drive endothelial cell contraction and vessel constriction. The anchorage of circumferential actin bundles to cell-cell junctions is mediated by Ccm1/Krit1 protein. Importantly, the loss of circumferential actin bundles in ccm1-deficient endothelial cells causes cell enlargement and impaired vessel constriction, culminating in vessel dilation characteristic of cerebral cavernous malformations. Our multiscale study demonstrates how circumferential actomyosin-driven endothelial cell contractions regulate vessel diameter and provides insights into mechanisms of both normal vascular development and disease pathogenesis.

Introduction
The tight control of blood vessel diameter is crucial for the efficient distribution of blood flow and tissue perfusion. During development, vessel networks are refined through vascular remodelling, a process that integrates elongation, pruning, and diameter regulation to establish a hierarchical network of arteries, veins, and capillaries1,2. The mis-regulation of vessel size can culminate in vascular malformations such as Hereditary Haemorrhagic Telangiectasia (HHT), which is characterised by enlarged arteries and veins that connect with each other to create shunts, and Cerebral Cavernous Malformation (CCM), which presents as clusters of abnormally dilated blood vessels in the brain3–5. In many vascular malformations, blood flow distribution through the vascular network is impaired and can result in bleeding and chronic pain6. Understanding how vessel diameter is defined at the cellular and molecular levels is therefore fundamental to vascular biology and disease.

Endothelial cells (ECs) are the primary building blocks of vessels, and their number and size have been recognised as critical determinants of vessel diameter. Increased EC numbers through vessel fusion7, directed migration8–10, or excess proliferation can enlarge vessels1. In addition to changes in cell number, alterations in individual EC size represent another key mechanism for remodelling vessel diameter11,12. PlexinD1-KLF213, Bone Morphogenetic Protein (BMP) and transforming growth factor beta (TGFb) signalling regulate multiple aspects of EC behaviour, including cell size. Disruption of TGFb signalling, including mutations in Alk1 and its coreceptor Endoglin11,14, or the downstream mediator, SMAD415, results in enlarged ECs that lead to aberrations in vessel diameter as observed in HHT. Similarly, EC enlargement occurs in CCM models with KRIT1/CCM1 mutation16–18. While these findings highlight EC size control as an important factor in the regulation of vascular morphology, the underlying biophysical mechanisms remain poorly understood. As recent studies have implicated the role of actin cytoskeletal remodelling in controlling EC size and vessel diameter12,19,20, we speculate that alterations in EC mechanics may underlie the pathogenesis of vascular malformations.

Here, using zebrafish intersegmental vessels (ISVs) as a model, we investigated the self-organising principles of blood vessel remodelling across subcellular, cellular and tissue scales. ISVs undergo the seemingly paradoxical behaviour of radial constriction despite an overall increase in EC number from 2 to 4 days post fertilisation (dpf). By combining live imaging with quantitative strain analysis, we resolved this paradox: vessel elongation is accommodated by additional ECs, while vessel constriction is achieved through active EC contraction and rearrangement. At the subcellular level, we identify dynamic cortical actin organisations and demonstrate that circumferential actin bundles, reinforced by non-muscle myosin II and anchored by Krit1 at junction-cortex interfaces, generate tensile forces that progressively contract ECs to narrow vessel diameter. When this cytoskeletal architecture is disrupted, either by altering cortical actin organisation, inhibiting myosin II or loss of Krit1, ECs fail to contract, rearrange abnormally, and vessels dilate. Conversely, the overexpression of Krit1 enhances circumferential actin assembly and vessel constriction, underscoring its function as a key organiser of the cortical contractile machinery. This work therefore not only provides mechanistic insight into how actin cytoskeleton organisation controls vessel diameter during normal blood vessel development but also explains how its disruption leads to pathological dilation in CCM. Overall, our study establishes a framework for understanding how EC mechanics integrate with cell number and genetic regulators to generate vessels of appropriate size.

Results
Dynamic endothelial cell behaviours underly vessel constriction and elongation
To investigate temporal changes in vessel morphometrics during vascular remodelling, we examined zebrafish arterial and venous intersegmental vessels (aISVs and vISVs) from 2 to 4 dpf (Fig. 1a). aISVs displayed a gradual, significant reduction in diameter over this period, whereas vISVs showed similar diameter between 2-3 dpf and constricted between 3-4 dpf (Fig. 1b). Quantification of EC nuclei revealed a substantial increase in cell number within both aISVs and vISVs (Fig. 1c). Although EC number has been reported to drive vessel size enlargement7,8, we observed an inverse relationship between increasing cell number and vessel diameter. Vessel length increased (Fig. 1d), and the average EC number per unit length (100 μm) showed a similar upward trend in both vessel types (Supplementary Fig. 1a), indicating a positive correlation between EC number and vessel elongation. Thus, elongation accommodates additional cells, while circumferential space becomes limited as diameter decreases. Consistently, average vessel area changed only modestly (Supplementary Fig. 1b), suggesting that ECs must deform to fit within the remodelling vessel.

To elucidate the source of EC number increase, we performed time-lapse imaging in Tg(fli1:Lifeact-mCherry)ncv7; Tg(fli1a:H2B-EGFP)ncv69 embryos from 2 to 4 dpf. In aISVs, most (>50%) underwent only positional rearrangements within the vessel. From 2-4 dpf, ~30% of vessels exhibited EC exchange with the dorsal longitudinal anastomotic vessel (DLAV) while ~10% displayed both exchange and cell division (Fig. 1e), with most exchange events involving DLAV-to-aISV immigration (Supplementary Fig. 1c, d). Thus, aISV cell number arises primarily from DLAV-derived cell immigration. In vISVs, ~80% of vessels displayed both EC division and exchange between 2-3 dpf, and ~30% still showed both behaviours between 3-4 dpf (Fig. 1f). Cell exchanges mainly involved vISV-to-DLAV emigration, with minimal contribution from the PCV (Supplementary Fig. 1e, f). Nearly all ECs of vISVs underwent mitosis, with most dividing two or more times, indicating that cell division is the main driver of venous EC number increase (Supplementary Fig. 1g). Thus, vISVs grows mainly through cell division while also serving as a cell source for the DLAV, whereas aISVs grows primarily through DLAV-derived exchange (Fig. 1g).

Endothelial cell shrinkage and rearrangement drive vessel constriction and elongation
To assess whether EC deformation contributes to ISV remodelling, we unwrapped individual ECs using a custom ImageJ script and quantified their 2-dimensional (2D) area and aspect ratio (Fig. 2a). EC significantly reduced in size in both aISVs and vISVs between 2 and 4 dpf (Fig. 2b), with similar cell aspect ratio (Supplementary Fig. 1h), suggesting that ECs shrink in both radial and axial directions. Because vessels also undergo changes in both diameter and length, we next assessed cell behaviours along the radial (circumferential) and axial (longitudinal) axes separately.

To distinguish whether vessel constriction arises from ECs becoming narrower or from cells rearranging away from the vessel circumferential surface, we applied a strain analysis. Three parameters were calculated: (i) cell number strain, reflecting expected changes in how many cells are required to span the vessel axis independent of cell shape; (ii) cell strain (in aEC or vEC), reporting whether individual ECs shrink or expand; and (iii) vessel strain (in aISV or vISV), the net vessel-level change. Together, these measures allow us to distinguish whether remodelling is driven by cell deformation, cell rearrangement, or both.

In aISVs along the radial axis (Fig. 2c), positive cell number strain between 2 and 3 dpf reflected the addition of DLAV-derived cells. Despite this, vessel strain was negative because ECs narrowed (negative cell strain), counteracting circumferential widening due to increased cell number. By 3-4 dpf, negative cell number strain showed that fewer cells encircled the circumference as they rearranged axially. Combined with continued cell narrowing, this led to a clear negative vessel strain and net constriction. Along the axial axis (Fig. 2d), cell addition and axial rearrangements supported vessel elongation (positive cell number strain, positive vessel strain) despite cell shortening from 2 to 4 dpf. Thus, aISV constriction is driven by persistent EC shrinkage and reinforced by axial rearrangement.

In vISVs, high cell division between 2 and 3 dpf generated positive radial cell number strain, yet cell narrowing (negative cell strain) offset vessel widening, keeping diameter almost unchanged. By 3-4 dpf, axial rearrangement (negative cell number strain) rather than cell narrowing produced vessel constriction (Fig. 2e). Axial elongation was supported by persistent cell division and axial rearrangement (positive cell number strain) between 2-4 dpf, overcoming cell shortening (negative cell strain) (Fig. 2f). Thus, the mechanism of vISV constriction shifts from cell shrinkage at 2-3 dpf to cell rearrangement at 3-4 dpf (Fig. 2g).

Time-lapse imaging reveals distinct cellular strategies for vessel constriction in aISVs and vISVs
To validate our static analyses with dynamic observations, we performed time-lapse imaging of ISVs from 2 to 3 dpf and 3 to 4 dpf. Cell boundaries at the front and back sides (Supplementary Fig. 2a) of the vessel were visualised with an actin reporter, (Tg(fli1:GAL4FF)ubs3; Tg(UAS:EGFP-UCHD)ubs18, hereafter referred as EGFP-UCHD, enabling direct observation of dynamic cell movements.

In aISVs, cells frequently rearranged their positions while changing shape and size (Supplementary Movie 1). New cells entered from the DLAV and migrated ventrally against flow, displacing neighbouring cells as they occupied the vessel (Fig. 3a, bi, cell labelled in blue). During these rearrangements, a transient self-seam junction formed, in which a single EC (Fig. 3a, bii, cell labelled in pink) wrapped around the entire vessel circumference (59 and 63 hpf) to form a unicellular vessel segment before unwrapping to allow rearrangement of neighbouring cells (67 hpf). Coronal views showed that such cells underwent repeated inward-outward oscillations (Fig. 3a, bii). Self-seam junctions and the accompanying rearrangements were consistently observed in all examined aISVs (n=22/22). In addition, cells rearranged axially, allowing redistribution of cellular occupancy along the vessel circumference (Fig. 3a, bii, Supplementary Fig. 3a, b and Supplementary Movie 2). In contrast, vISVs remained multicellular, with frequent cell divisions (marked by the F-actin accumulation in rounded cells, Fig. 3c, Supplementary Fig. 3c and Supplementary Movies 3 and 4). At the same time, cells decreased in size and migrated dorsally toward the DLAV, often contributing to it. Thus, vISVs accommodated cell division through cell shrinkage and axial rearrangement.

Together, these analyses confirm that aISVs constrict through persistent cell shrinkage, combined with axial rearrangement and junction remodelling, whereas vISVs constrict under conditions of high cell division, where EC shrinkage and axial rearrangement accommodate additional cells. Interestingly, cortical actin stripes were occasionally observed (Fig. 3b, black arrows), prompting us to investigate cortical actin organisation in greater detail.

Circumferential actin information is associated with ISV diameter reduction
To better understand cortical actin organisation in vessel constriction, we imaged ISVs at higher resolution using the EGFP-UCHD line and identified three major cortical actin organisations: circumferential (C), mesh (M), and longitudinal (L) along with mixed states (C-M and M-L) (Fig. 4a). At 2 dpf, both aISVs and vISVs displayed a heterogeneous mixture of these patterns, with ~50% of the aISVs exhibiting C/C-M actin and ~20% of vISVs displaying C-M actin. From 3 dpf onward, the proportion of vessels with C/C-M actin decreased, whereas the proportion of vessels with M-L/L actin became increasingly common and by 4 dpf predominated in both aISVs and vISVs (>90% of vessels) (Fig. 4b). Thus, circumferential actin is most prominent at early stages, coinciding with the period of marked EC deformation (Fig. 2c, e).

Closer inspection of circumferential actin dynamics revealed that the bundles could appear in multiple configurations: anchored to the plasma membrane (Fig. 4c and Supplementary Movie 5); connected to two cell-cell junctions (Fig. 4d and Supplementary Movie 6); or linking a junction and a membrane on the opposing side of the cell (Fig. 4e and Supplementary Movie 7). In each case, the formation of circumferential bundles coincided with inward deformation of the cell boundary. To test whether circumferential actin correlates with vessel constriction on short timescales, we performed fast time-lapse imaging at 1 min intervals. Regions enriched in circumferential actin (Fig. 4f, grey rectangle) consistently exhibited inward cell deformation and local decreases in vessel diameter (Fig. 4e, f). When circumferential actin dissipated and transitioned to mesh actin, vessel diameter gradually increased, indicating that mesh actin correlates with vessel widening (Fig. 4f and Supplementary Fig. 4). Notably, intermediate states in which circumferential and mesh actin interchanged confirmed that both C and C-M patterns (whenever C bundles were present) correlate with vessel deformation, and that their dynamic alternation produces diameter oscillations consistent with those observed in earlier time-lapse analyses (Fig. 3b). Longitudinal actin was associated with only mild decreases in diameter (Supplementary Fig. 4).

Together, these results demonstrate that cortical actin remodels dynamically during ISV remodelling and that circumferential actin correlates most strongly with vessel constriction, while mesh and longitudinal patterns do not coincide with marked narrowing.

Circumferential actomyosin bundles generate tensile forces that drive vessel constriction
To test whether circumferential actin mediates contractility, potentially generated by non-muscle myosin II (hereafter, referred to as myosin II), to drive vessel constriction, we used the transgenic lines EGFP-UCHD and Tg(6xUAS:myl9b-mCherry)rk32 (hereafter referred as myl9b-mCherry) to visualise actin and myosin II, respectively. Static imaging revealed myosin II puncta colocalised with actin across circumferential, mesh, and longitudinal organisations. Linear alignment of myosin II along actin bundles was observed in both circumferential and longitudinal organisations (Supplementary Fig. 5a, red arrowheads), but was more frequent and continuous in circumferential bundles. By contrast, mesh actin was mainly associated with punctate myosin II, which was most often positioned between the gaps of the mesh network (Supplementary Fig. 5a, yellow arrowheads).

Time-lapse imaging confirmed these spatial associations. Linear colocalisation of myosin II with circumferential bundles coincided with local vessel constriction, and 3D rendering further verified their spatial overlap (Fig. 5a–c, grey region in kymograph; Supplementary Movie 8, 9). By contrast, lower intensity punctate distributions of myosin II were accompanied by little diameter change or local widening (Fig. 5c, white region in kymograph). Analyses of additional regions demonstrated that circumferential actomyosin consistently correlated with stronger diameter reduction than other actomyosin patterns (Supplementary Fig. 5b).

To further evaluate the mechanism underlying vessel constriction, simulations were performed using a two-dimensional coarse-grained molecular dynamics model of an actomyosin system, including actin filaments, myosin motors, and passive crosslinkers, enclosed within a deformable cell membrane. In the model, dynamic processes such as actin polymerisation, depolymerisation, and active force generation by motors are present. To promote bundle formation in the circumferential direction, few (less than 1% of the total filaments) actin filaments (2 μm in length, approximately one-fourth of the vessel width) were anchored along the longitudinal membrane. Under suitable parameter conditions, circumferential actomyosin bundles were observed to form, bridging the anchored filaments on opposite sides (Supplementary Fig. 5c and Supplementary Movie 10). The circumferential stress (σxx) was found to exceed the longitudinal component (σyy) indicating that circumferential bundles promoted the contractile activity in the circumferential direction (Supplementary Fig. 5d). In contrast, when the anchored filaments were introduced along the midline, longitudinal bundles were formed (Supplementary Fig. 5e and Supplementary Movie 11). In this situation, the longitudinal stress (σyy) exceeded the circumferential stress (σxx) (Supplementary Fig. 5f). Thus, anchoring filaments at different positions can control bundle orientation and consequently the direction of stress.

Upon the introduction of membrane deformability, the circumferential contractile stress indeed reduced the vessel width near the region where bundles were formed. The bundle formation was seen from 16 to 23 mins (Fig. 5d, grey rectangle, and Supplementary Movie 12), which can be identified by the increase in circumferential alignment N2x at the central region (CR) (Fig. 5e top, see method for the definition of circumferential alignment). The bundle formation led to the generation of contractile stress in the circumferential direction (Fig. 5e middle), which in turn resulted in a clear reduction in the vessel width (Fig. 5e bottom). Similar behaviour was repeatedly observed over time (Fig. 5e), which was confirmed by the negative correlation between vessel width and circumferential alignment N2x at CR with a time lag of about 2.5 mins (Supplementary Fig. 5g). These results indicate a causal relationship between circumferential bundle formation and vessel constriction.

Guided by this simulation, we directly tested whether the different actin organisations bear tensile forces by performing laser ablation using a multiphoton microscope. Ablation on circumferential bundles resulted in the strongest recoil, rapidly producing gaps in the cortical network, whereas longitudinal and mesh bundles showed weaker and comparable recoil responses (Fig. 5f and Supplementary Movie 13–15).

Collectively, these findings demonstrate that circumferential actin, reinforced by myosin II alignment, bears the strongest tensile forces, thereby driving EC contraction and vessel constriction during the early phase of vessel remodelling (2-3 dpf). At later stages, when circumferential bundles are less frequent, mesh and longitudinal actin contribute to cortical tension that helps maintain the reduced vessel diameter.

Skewing actin organisation in endothelial cells modulates vessel constriction
Since circumferential actin correlated strongly with vessel constriction, we next asked whether altering the balance of actin organisations would impact vessel diameter. We overexpressed Fascin1a, an actin-bundling protein that promotes parallel filament assembly. A plasmid encoding 6xUAS:fascin1a-T2A-mKate2CAAX was selectively expressed in ECs of EGFP-UCHD embryos (Fig. 6a). Compared to adjacent mKate2CAAX-negative internal controls, Fascin1a overexpression (OE) significantly increased the frequency of aISVs exhibiting circumferential or C-M actin at 2 dpf (C/C-M vs other AOs, Fisher’s exact test, p=0.04), with a trend toward more C-M at 3 and 4 dpf (Fig. 6a, b). A similar trend was observed in vISVs, although the increase at 2 dpf was not significant (p=0.27). This enrichment of C/C-M patterns translated into greater constrictive capacity: between 2-3 dpf, Fascin1a-OE vessels constricted more strongly than controls (~21% aISV, ~22% vISV vs ~11% and ~9% in controls, respectively), and by 3-4 dpf both vessel types were significantly smaller than controls (Fig. 6c, d) indicating that increasing the proportion of vessels with circumferential actin enhances constriction. Notably, circumferential actin enrichment was highest at 2 dpf, whereas statistically significant diameter constriction was detected only at 3 and 4 dpf.

Conversely, to test the effect of enhancing mesh actin, we overexpressed Wasb (human homologue, Wasp), a key regulator of actin nucleation that facilitates the formation of branched actin networks9,21. Similarly, we injected a plasmid encoding 6xUAS:wasb-mCherry into EGFP-UCHD embryos (Supplementary Fig. 6a, c). Wasb-OE increased the frequency of ISVs exhibiting mesh actin, interestingly, at the expense of longitudinal rather than circumferential actin (Supplementary Fig. 6a, b). In both aISVs and vISVs, this shift was most apparent at later developmental stages (at 4 dpf, M vs other AOs, aISV, Fisher’s test aISV, p=0.04; vISV, p=0.02). Correspondingly, Wasb-OE vessels were dilated compared to controls (Supplementary Fig. 6c, d), yet continued to constrict over time to a degree comparable to controls, indicating that increasing mesh actin preserved relative narrowing despite larger absolute diameters.

Myosin II inhibition blocks endothelial cell contraction and reduces vessel constriction
To investigate the contribution of myosin II activity on EC contraction and vessel constriction, we overexpressed a plasmid encoding dominant-negative myosin light chain 9b (6xUAS:myl9bA2A3-EGFP) in Tg(fli1:GAL4FF)ubs3; Tg(fli1:myr-mCherry)ncv1 and compared mosaic overexpression cells with adjacent GFP-negative internal controls (Fig. 6e). aISVs with Myl9bA2A3-OE ECs, and therefore reduced myosin II activity, were consistently wider compared to controls from 2 to 4 dpf, whereas vISVs showed increased diameters at 2 and 4 dpf but not at 3 dpf (Fig. 6f).

In Myl9bA2A3-OE cells, cell area remained unchanged from 2 to 4 dpf (Supplementary Fig. 6f), unlike the reduction observed in controls (Fig. 2b), indicating impaired contraction. Normally, aECs narrowed, and aISVs constricted (Fig. 2c), but with myosin II inhibition, aECs enlarged along both radial and axial axes at 2-3 dpf. Radially, vessel constriction was partly preserved by enhanced radial-to-axial rearrangement (negative cell number strain), even as cells widened. Axially, elongation arose primarily from cell lengthening (positive axial cell strain) with little cell addition. Consistent with this, in Myl9bA2A3-OE vessels, total EC number increased between 2 to 4 dpf, but lacked the stepwise rise seen in controls, which showed significant growth between 2-3 and 3-4 dpf (Supplementary Fig. 6f), suggesting delayed or uncoordinated cell addition during vessel remodelling. By 3-4 dpf, continued cell expansion in the radial axis overwhelmed axial cell rearrangement, resulting in slight vessel widening, while elongation was recovered through cell addition (Fig. 6h).

In control vISVs, high proliferation at 2-3 dpf increased circumferential coverage, but cell narrowing offset vessel widening; at 3-4 dpf, axial rearrangement (negative radial cell number strain) drove constriction (Fig. 2e). Under myosin II inhibition, vISVs exhibited less radial EC narrowing compared to control, and along with enhanced radial-to-axial cell rearrangement (negative radial cell number strain) at 2-3 dpf, leading to modest constriction (negative radial vessel strain) (Fig. 6i), in contrast to the slight widening observed in controls (Fig. 2e). By 3-4 dpf, ECs reversed to radial expansion, offset the effects of axial rearrangement, leading to modest constriction, which resulted in wider vessels than controls (Fig. 6f). Axial elongation during 2-3 dpf was driven primarily by cell addition (positive axial cell number strain) despite cell shortening (Supplementary Fig. 6g), whereas at 3-4 dpf elongation was modest and supported by cell lengthening (Fig. 6i).

Despite these changes, overall vessel length remained comparable to controls (Supplementary Fig. 6h).

Together, these findings demonstrate that myosin II activity is important for coupling EC contraction with rearrangement, and that its inhibition leads to enlarged ECs, disrupted rearrangement and exchange, and ultimately impaired vessel constriction.

Krit1 promotes circumferential actin assembly, cell contraction and vessel constriction
Since our results showed that circumferential actomyosin contractility are essential for EC contraction and vessel constriction, we next asked whether defects in these processes underlie CCM. Loss-of-function mutation in ccm1/krit1 is known to cause enlarged, poorly remodelled vessels in zebrafish and mammals16,17, but the underlying cellular mechanisms remain unclear. Krit1 has been reported to localise at endothelial junctions, where it interacts with junctional complexes and cytoskeletal elements, suggesting it may scaffold cortical actin22. We confirmed in zebrafish ISVs that Krit1 is enriched at junction-cortex interfaces and colocalised with circumferential actin, bridging cell-cell junctions and cortical bundles (Fig. 7a). This observation suggests that Krit1 may anchor actin bundles at cell-cell junctions to organise cortical actin. To test this, we performed mosaic endothelial overexpression of Krit1 (mStayGold-Krit1) and compared vessels comprising Krit1-OE cells with adjacent mStayGold (mSG) -negative internal controls (Fig. 7b). Krit1-OE increased the proportion of ISVs displaying circumferential and C-M actin at 2 and 3 dpf (Fig. 7c, C/C-M vs other AOs at 2 dpf, aISV, Fisher’s exact test p=0.04; vISV, p=0.02) and enhanced vessel constriction during this period (aISV ~12% vs ~0.5%; vISV ~9% vs ~7%), resulting in reduced aISV diameter at 3 and 4 dpf and vISV diameter at 2 and 3 dpf (Fig. 7d).

We next examined a zebrafish model of CCM containing a krit1 loss-of-function mutation (krit1t26458, referred to as krit1-/- hereafter). By transplanting cells from embryos obtained from in-crosses of krit1+/-; Tg(fli1:GAL4FF)ubs3; Tg(UAS:EGFP-UCHD)ubs18 zebrafish (yielding krit1+/+, krit1+/-, and krit1-/- genotypes) into wild-type recipients, we generated mosaic ISVs that experienced normal blood flow. This allowed us to investigate cell autonomous role of Krit1 on actin organisation, cell deformation and vessel constriction. Clear differences were observed between genotypes. In wild-type and heterozygous grafts, circumferential and C-M actin were present from 2 to 3 dpf, supporting balanced cell shape control. In krit1-/- grafts, circumferential actin was strongly reduced with a shift towards longitudinal arrangements in both aISVs and vISVs (Fig. 7e, f, ML/L vs other AOs at 2 dpf, aISV, Fisher’s exact test p=0.0007; vISV, p=0.02). Consistently, vessel diameters were larger in krit1-/- aISVs at 2, 3 and 4 dpf and vISV at 3 and 4 dpf compared to wildtypes, suggesting defects in vessel constriction (Supplementary Fig. 7a, b). Vessel lengths were comparable across groups except for vISVs at 2 dpf, indicating that the primary defect lay in radial rather than axial growth (Supplementary Fig. 7c).

Analysis of cell size and strain revealed the cellular basis of this defect. Cell area in homozygous mutants remained similar to controls at 2 dpf but increased significantly at 3 dpf in vECs and in both cell types by 4 dpf, whereas control cells progressively contracted (Supplementary Fig. 7d). In control aISVs, EC contraction along radial axis promoted constriction despite cell influx between 2 and 3 dpf, and combined with axial rearrangement of cells between 3 and 4 dpf, maintained reduced diameter (Fig. 7g). By contrast, krit1-/- cells expanded rather than contracted between 2 and 4 dpf, leading to vessel dilation even as some cells left the circumference. krit1-/- cells also lengthened axially between 3 and 4 dpf, rather than axial shortening, reinforcing overall enlargement (Supplementary Fig. 7e). In control vISVs, vessel constriction between 2 and 3 dpf was achieved by counterbalancing the addition of cells with cell contraction, and between 3 and 4 dpf was maintained primarily through axial rearrangement (wild type) or rearrangement plus contraction (heterozygotes) (Fig. 7h). In krit1-/- vISVs, however, cells failed to contract and instead expanded radially, so vessel strain shifted toward reduced or even positive values. At the same time, additional cells were accommodated axially, reflected by elevated axial strain (Supplementary Fig. 7f). Thus, without Krit1, ECs lose the coordinated contraction and rearrangement that normally drive vISV constriction.

To investigate whether these defects also involve impaired actomyosin coupling, we analysed embryos derived from in-crosses of krit1+/-; Tg(fli1:GAL4FF)ubs3; Tg(UAS:EGFP-UCHD)ubs18; Tg(6xUAS:myl9b-mCherry)rk32 which generate homozygous mutants lacking blood flow, and quantified junctional and cortical intensities of actin and myosin II. Compared with transplanted krit1-/- cells that experience normal circulation, flow-deficient mutants exhibited even larger cell size and prominent cortical actin bundles that followed a diagonal-to-longitudinal direction (Supplementary Fig. 7g). In wild-type embryos, junctional actin was enriched over cortical actin (ratio > 0), whereas this enrichment was significantly reduced in krit1-/- cells, consistent with impaired junctional actin assembly (Supplementary Fig. 7h). For myosin II, mean junction/cortex ratios were similar across genotypes (ANOVA, P=0.23), but variance was reduced in mutants (Brown-Forsythe test, P=0.037). Wild-type cells showed heterogeneous distribution, while mutants displayed more uniform patterns, indicating a loss of dynamic heterogeneity that may constrain contractile responses. Thus, Krit1 is required both for junctional actin enrichment and for myosin II distribution.

In sum, we demonstrate that Krit1 promotes the assembly and stabilisation of circumferential actin at junctional sites, enabling EC contraction and vessel constriction. Uncoupling of Krit1 from the actin cytoskeleton results in the impairment of vessel remodelling and culminates in enlarged vessels that are characteristic of CCMs.

Methods
Zebrafish husbandry and manipulation
All animal experiments were approved by the Institutional Animal Care and Use Committee at RIKEN Kobe Branch (IACUC). Zebrafish (Danio rerio) were maintained and raised following standard protocols. The transgenic lines used in this study included Tg(fli1:MYR-EGFP)ncv245, Tg(fli1:h2bc1-mCherry)ncv3146, Tg(fli1a:H2B-EGFP)ncv6947, Tg(fli1:Lifeact-mCherry)ncv745, Tg(fli1:myr-mCherry)ncv148, Tg(fli1:GAL4FF)ubs349, Tg(UAS:EGFP-UCHD)ubs1850, Tg(fli1ep:Lifeact-EGFP)zf49551, Tg(6xUAS:myl9b-mCherry)rk32 (this study), while the mutant lines used were krit1t2645816 and pdgfrbSA1638937. Fish embryos were collected within 30 minutes of divider removal and were allowed to develop at 28.5°C to the appropriate stage. For genotyping of the krit1t26458 mutant, genomic DNA was extracted using the HotSHOT method, and PCR amplification was performed with specific primers (Forward 5’-CCACAAGCGTAACGTAAATG; Reverse 5’-ATCTATGGACGCAATGCAG). The resulting PCR products were purified and sequenced using the forward primer. Genotyping of the pdgfrbSA16389 mutant was performed as previously described37.

Plasmid production and injection
Plasmids used in this study were constructed using either NEBuilder® HiFi DNA Assembly or In-Fusion® HD Cloning kits, following the respective manufacturer protocols. The wasb-mCherry vector9 (a gift from Holger Gerhardt, Max Delbrück Centre for Molecular Medicine, Germany) and the Fscn1a-T2A-mKate2CAAX vector12 were amplified by PCR and incorporated into Ac/Ds transposon-based constructs. The plasmid encoding Krit117 (a gift from Salim Abdelilah-Seyfried, Potsdam University, Germany) was amplified and fused with mStayGold and cloned into Ac/Ds constructs. The myl9bA2A3-eGFP vector52 (a gift from Anne Schmidt, Institute Pasteur, France) was amplified by PCR and assembled into Tol2 transposon-based constructs. Assembly reactions employed the appropriate backbone vectors and primers as specified by the cloning protocols. For details on vectors, refer to Supplementary Data 1. For plasmid overexpression experiments, embryos were injected at the one-cell stage with 2nl of a mixture containing 50-100ng of plasmid DNA, 100-200ng Tol2 or Ac transposase mRNA, phenol red for tracking, and Milli-Q water to a final volume.

Imaging
Embryos were immobilised in 0.8% low-melt agarose (Bio-Rad) within E3 medium supplemented with 0.16mg/mL Tricaine and 0.003% phenylthiourea to reduce movement and pigmentation, respectively. Confocal imaging was performed using an inverted Olympus IX83 microscope, equipped with a Yokogawa CSU-W1 spinning disk confocal unit. Imaging was carried out with an Olympus UPLSAPOx60/NA 1.2 water immersion objective, ×40/NA 1.25 or ×30/NA 1.05 silicone oil immersion objectives and a Zyla 4.2 CMOS camera (Andor). Image acquisition was controlled using Andor iQ3 software, and z-stack images were captured for 3D reconstructions. Actin organisation was imaged using two advanced microscope setups. The first system was an Andor/Olympus Dragonfly 200 inverted microscope equipped with a 60x water immersion objective lens (Olympus UPLSAPO, NA 1.2) and a Zyla 4.2 PLUS sCMOS camera (2.0x magnification). Imaging utilised a 40µm pinhole, and z-stack were acquired at Nyquist intervals. Imaging parameters were controlled via Fusion Control software (2.3.0.50). The second system was a customised spinning disk super-resolution microscope (SDSRM)53, implemented based on the commercial IXplore IX83 SpinSR system (Evident), equipped with a 60x silicon immersion objective lens (Evident UPLSAPO 60XS, NA 1.3). z-stack interval was 0.15um.

Microangiography
Microangiography was performed for single-cell analysis. Embryos at 2, 3, and 4 dpf were injected with 1-2 nL of dextran tetramethylrhodamine (molecular weight 2000 kDa, Invitrogen) at a concentration of 10mg/mL. The dextran solution was introduced through the duct of Cuvier of the zebrafish. Imaging was performed immediately following the injection. Confocal z-stacks were acquired using an Olympus UPLSAPO 60x/1.2 NA water immersion objective.

Cell transplantation
Donor cells from embryos derived from krit1+/- t26458 in-crossed zebrafish in Tg(fli1:GAL4FF)ubs3; Tg(UAS:EGFP-UCHD)ubs18 background were collected from random locations in the blastoderm. These cells were transplanted into the lateral marginal zone of the blastoderm in wild-type recipient embryos at 4.5–6 hpf. The procedure was carried out using a CellTram® 4r Oil microinjector (Eppendorf) with a borosilicate glass capillary GC100-15 (Harvard Apparatus Ltd). The capillary tips were shaped into a smooth spoon using a horizontal micropipette puller P-87 (Sutter Instrument Co.), and an MF-900 microforge (Narishige) for precision. During transplantation, embryos were maintained in agarose-coated Petri dishes containing 0.5x E2 buffer composed of 7.5mM NaCl, 0.25mM KCl, 0.5mM MgSO4, 75µM KH2PO4, 0.5mM CaCl2, 0.35mM NaHCO3, and 50µ/mL penicillin-streptomycin. Recipient embryos were grown to 2, 3, and 4 dpf, and those exhibiting EGFP-positive ISVs were selected for microangiography and imaging. Genotyping was performed on the corresponding donor embryos. A total of 24 independent transplantation experiments was conducted.

Image processing
Raw images of actin organisation captured using the Dragonfly 200 microscopy were deconvoluted with ™Huygens Spinning Disk Deconvolution, following the standard protocol established in the Assembly unit. The deconvoluted images were subsequently processed using Fiji software (NIH). To separate the front and back sides of the vessel, the ISV was first straightened using Fiji’s Straighten tool and then divided with the Split tool. Z-stacks for each half were projected into single images using maximum intensity projection. To enhance consistency and reproducibility, a custom macro was developed in Fiji to semi-automate the straightening and splitting process. The spline of each ISV was manually traced for straightening, and the central plane of the z-stack was manually selected for splitting (see Supplementary Fig. 2).

Quantification of blood vessel diameter and length and EC number
To quantify vessel diameter and length, live Tg(fli1:MYR-EGFP)ncv2, Tg(fli1:myr-mCherry)ncv1, or Tg(fli1:GAL4FF)ubs3; Tg(UAS:EGFP-UCHD)ubs18 transgenic embryos were imaged at 2, 3, and 4 dpf. For EC number, live Tg(fli1:h2bc1-mCherry)ncv31 or Tg(fli1a:H2B-EGFP)ncv69 embryos were imaged at the same stages. Confocal z-stacks were acquired using an Olympus UPLSAPO ×40/NA 1.25 or ×30/NA 1.05 silicone oil immersion objectives. To determine the ISV diameter, a line was drawn perpendicularly across the ISV in Fiji (NIH). The intensity profile along this line was used to identify two peaks corresponding to the vessel walls. A horizontal line was manually drawn at approximately half the maximum intensity of the peaks to measure the diameter. Over three measurements were averaged for each ISV (No. 5-18). For overexpression experiments, vessel diameters were measured specifically in regions containing GFP/mCherry-positive overexpressed cells. These regions were often part of mosaic vessels comprising both OE and non-OE cells. Control measurements were taken from either non-mosaic vessels or mosaic vessels lacking GFP/mCherry expression. The length of each ISV was measured by manually tracing a polyline along the vessel’s spine in Fiji. The tracing began at the point where the ISV connected to the DLAV and extended down to its connection with the DA or PCV. To count ECs, the nuclei within ISVs No. 5-18 were manually counted in Fiji.

Analysis of EC exchange, division, and rearrangement in time-lapses
Confocal z-stacks were acquired using an Olympus UPLSAPO ×40/NA 1.25 silicone oil immersion objectives. Time-lapse imaging was performed in Tg(fli1a:H2B-EGFP)ncv69; Tg(fli1:Lifeact-mCherry)ncv7 or Tg(fli1:GAL4FF)ubs3; Tg(UAS:EGFP-UCHD)ubs18 embryos from 2-3 dpf and 3-4 dpf. Endothelial cell exchange was tracked by following nuclear movements in 2D maximum-projected time-lapses. To visualise cell rearrangements, imaged vessels were straightened and divided into front and back views, enabling the tracking of individual ECs based on junctional actin. Cell division was identified by the transient accumulation of F-actin and the subsequent appearance of daughter nuclei in the time-lapse series.

In vivo cell shape and strain analysis
Single-cell labelling of ECs within ISVs was achieved through mosaic expression of different constructs: fli1ep:lynEGFP plasmid in wild-type embryos, 6xUAS:myl9bA2A3 for overexpression experiments, or transplanted krit1 mutant cells in Tg(fli1:GAL4FF)ubs3, Tg(UAS:EGFP-UCHD)ubs18 transgenic background. At 2, 3, and 4 dpf, microangiography was performed, and vessels were imaged using an Olympus UPLSAPO 60×/NA 1.2 water immersion objective with an optical z-plane interval of 0.25µm. EC shape analysis was conducted semi-automatically using a custom-written Fiji script developed in Python, adapting methods from a previously described approach12. The analysis provided measures of cell area (a) and aspect ratio (r), which were subsequently utilised for strain analysis. Cell width (w) and cell length (l) were calculated as w = sqrt(a/r); l = sqrt(a*r). Effective cell number was calculated as Neff(radial) = πD/w at the radial axis, and Neff(axial) = L/l at the axial axis, where D is vessel diameter, and L is vessel length. Cell number strain was calculated from the effective cell number (Neff). For a quantity X (e.g., vessel diameter D, vessel length L, cell width w, cell length l, effective cell number Neff), strain (ε) between two time points (t) was calculated as: ε(t1->t2) = (X2 - X1) / X1. For example, vessel strain at the radial axis was calculated from vessel diameter, as: ε(t1->t2) = (D2 - D1) / D1, and analogous formulations were used for axial vessel strain (from vessel length), radial and axial cell strain (from cell width and cell length), and radial and axial cell-number strain (from Neff(radial) and Neff(axial)).

Analysis of actin organisation
Processed images were analysed by dividing each ISV into front and back views. Since lateral mounting causes the dorsal side to face the objective while the ventral side is obscured by the yolk, only the dorsal portion of ISVs (at positions 10 to 18) was evaluated, where fine actin structures could be reliably resolved. Actin organisation was classified manually as circumferential (C), mesh-like (M), or longitudinal (L). Both dorsal views (front and back) of an ISV were examined; if patterns differed (e.g., front=M, back=C), the ISV was assigned a combined category (CM). Percentages were calculated as the number of ISVs in each category divided by the total number of analysable ISVs across embryos. These values therefore represent the fraction of ISVs exhibiting a given AO, not the proportion of actin structures within a vessel.

Analysis of actomyosin colocalisation and dynamics
Processed images were analysed by dividing each ISV into front and back views. Multichannel z-stacks were maximally projected to 2D, and a region of interest (ROI) covering actin-myosin II colocalisation was manually drawn. For time-lapse experiments, intensity profiles for actin and myosin II were extracted over time in ImageJ, background-subtracted using the mean of a rectangular region, and converted to log10 values. To compare actin and myosin II on the same scale, log10 values across time for each vessel were further normalised by max-min scaling. Vessel diameter was measured from kymographs by tracing vessel edges and plotted together with normalised actin and myosin II intensities to assess temporal correlations. For static images of actomyosin colocalisation in wild-type and krit1 mutant embryos (obtained from in-crosses of heterozygous parents, resulting in homozygous embryos lacking blood circulation), 6 regions were manually drawn along junctions and 10 along cortical actin per vessel. Junctional actin was measured at cell–cell contacts, corresponding to the bright, continuous EGFP-UCHD signal. Cortical actin was measured within the cell interior but not contiguous with junctional pixels, usually with a lower EGFP-UCHD signal. Because endothelial cell membrane in vivo is extremely thin (~1µm), and z resolution is limited, apical and basal membranes cannot be reliably distinguished; thus, the cortical region integrates both surfaces. Actin and myosin II intensities were measured in both channels, background-subtracted using the mean of two regions outside the vessel, and averaged to yield mean junctional and cortical values. Ratios of junctional to cortical intensity were then calculated for actin and myosin II, converted to log10 values, and used to generate one averaged ratio per vessel for plotting.

Laser ablation and analysis of mean velocity of recoiled actin
Laser ablations were performed using a Zeiss LSM980 confocal microscope equipped with adjustable laser line MP-NDD (69–1300nm) and a 63x Oil immersion objective (Plan-Apochromat, NA 1.2). Ablation parameters were controlled via ZEN (Blue 3.8) software. Each ablation consisted of a single laser pulse at 920nm with 80% laser power. Ablation was applied at the 5th frame of a time-lapse sequence comprising 50 frames captured at intervals of 250–300 ms (scan speed 13, and pixel time 0.75µs), depending on the region of interest size. The location of ablation was manually drawn with a 0.3µm × 6µm box.

To quantify actin recoil velocity, the ablated region (a gap) in the actin network was analysed by drawing a line region of interest (ROI) across the gap to generate a kymograph. The edges of the gap were manually traced on the kymograph, and the distance between these edges was measured for each time frame to track the gap distance over time. Velocity was calculated by dividing the change in distance by the frame rate. The average velocity was calculated from measurements taken during the first 10 s of the time lapse. For each ablation, three separate line ROIs were drawn across the gap, generating three average velocity measurements. The final mean velocity for each ISV was determined by averaging these three values. Each data point in the velocity bar chart represents the mean recoil velocity of an individual ablated ISV.

Model of cytoskeletal dynamics in endothelial cells in 2D
Since ECs lining blood vessels are flat, the system was approximated by a 2D model in a rectangular area of 8µm (horizontal, circumferential) × 30µm (vertical, longitudinal). The cytoskeletal network comprised filaments, motors, crosslinkers, and cell membranes, incorporating processes such as filament polymerisation/depolymerisation, motor-generated active forces, and turnover of filaments and crosslinkers to preserve network dynamics. To promote bundle formation, anchored filaments were introduced at the vertical (longitudinal) boundary or along the vertical midline. The simulation was first performed in a fixed boundary to see the bundle formation. Deformable membrane was then introduced along the vertical boundary to see the cell deformation induced by the bundle formation. This model represents an extension of our previously described framework54, and full details are provided in the Supplementary Note 1 and Supplementary Table 1.

Stress calculation
The stress tensor in the simulation was computed according to the following formula: σ αβ = 1/A * Σi>j f ij,α r ij,β, where A is the area of the system, fij is the force acting on bead j by bead i, rij = ri - rj, fij,α is the α component of fij and rij,β is the β component of rij.

Quantification of filament orientation
The degree of filament orientation in the circumferential direction (x direction) was quantified by circumferential alignment N2x = Σi (n2i,x) / Σi Ri, where ni,x = cosθi is the x component of the orientation θi of segment i. Here, each filament consists of multiple segments (See supplemental Method). This quantity was measured in the blue region in Fig. 5d in both peripheral and central (red region, between 2 and 5 μm) regions.

Statistics and reproducibility
All statistical analyses were performed using GraphPad Prism 10. The specific tests used for each dataset are indicated in the corresponding figure legends. Comparisons between two groups were evaluated using unpaired two-tailed Student’s t tests, while comparisons among more than two groups were assessed using one-way ANOVA followed by Tukey’s post hoc test. Categorical data were analysed with Fisher’s exact test. Exact p-values are reported in the figures. Statistical significance was defined as p<0.05. Additional details are provided in the figure legends. The sample size (number of cells or vessels) was chosen based on the number of embryos expressing the transgene of interest (e.g., transient overexpression of plasmids) or of the desired genotype obtained per experiment. The number of embryos used for time-lapse imaging per experiment ranged from 3 to 4 per experiment for overnight imaging, 6 to 8 for short (e.g., 20−30 min) but high temporal resolution imaging, since time was a limiting factor. Data exclusions: (1) Cell area measurements: endothelial cells located in the ventral region of ISVs (connected to the dorsal aorta) were excluded as these cells are significantly smaller that cells located in the middle and dorsal regions of the ISVs. In addition, analysis of actin organisation was only performed in middle-dorsally located cells. 2) Analysis of actin organisation: ISVs with weak reporter expression, poor image quality, or undergoing remodelling without flow were excluded. Images or schematics in figures were organised or manually drawn using Adobe Illustrator 2022.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a nicely written paper in which the authors use transgenic zebrafish lines and confocal imaging to measure vessel and endothelial cell morphometrics in arterial (a) and venous (v) intersegmental vessels (ISVs) from 2-4 days post-fertilization. They find that aISVs contain fewer, larger ECs and vISVs contain more, smaller ECs, with increased EC number arising largely from cell divisions after sprouting and anastomosis are complete. These findings confirm and modestly extend (to later timepoints) previously published work (e.g. Rosa et al. Development 2022). Beyond these morphometric analyses, the authors focus on the role of different types of actin in controlling EC size and vessel diameters. They suggest that circumferential actomyosin is the key driver of constriction in aISVs and vISVs between 2-4 dpf. While this is a logical idea, more rigor is required to establish causation or strong correlation. The imaging is well-presented and morphometric measurements seem sound. The work is mostly descriptive and provides modest new insight into the mechanism of endothelial cell shape changes and vessel diameter changes.\n\nMajor concerns\n1. The primary novel conclusion from this work is that circumferential actomyosin is responsible for EC contraction and decreased ISV radius between 2 and 4 days post-fertilization (dpf). However, data are largely observational, and temporal changes in actin organization and cell size and vessel diameter do not always correlate. For example, the authors do not address how circumferential actin, present primarily at and analyzed only at 2 dpf, accounts for diameter decreases in aISV and vISV, which are only measurable at 4 dpf (Fig. 1b, and presumably Fig. 5e,f; 6g,h, although stats not shown). How is tension maintained and diameter restricted in the absence of these structures at 4 dpf?\n2. Similarly, at 2 dpf, even on short time scales, the apparent loss of circumferential actin over time correlates with constriction and maintenance of smaller vessel diameter (Fig. 3e,f). Can the authors show, in wild type ISVs, that regions that have circumferential actin contract more than regions without it, on this same time scale?\n3. Actomyosin/diameter correlation (Fig. 4a-c) was assayed only at a region of circumferential actomyosin. Additional correlations between actin/myosin/diameter should be shown, both for regions with high and low colocalization, to strengthen conclusions.\n4. In Fig. 5, while there is an apparent decrease in pure circumferential actin at 2 dpf in aISV and vISV with wasb-OE, as expected, there is an apparent increase in mixed (mesh + circumferential) actin with wasb-OE at 3 dpf in aISV and vISV, and at 4 dpf in aISV. Accordingly, these data do not strongly support a role for circumferential actin in constriction.\n5. The authors do not directly consider a role for pericytes in narrowing of ISV diameter, Pericytes are present on ISVs as early at 2 dpf (Ando Development 2016; ref. 29; Colijn et al. Dev Dyn 2024).\n6. The percent of each actin type is not consistent in control in Figs. 3, 5, 6, suggesting high variability from clutch to clutch, inconsistent timing, and/or high interoperator variability/subjectivity from observer to observer. Can the authors please address this?\n7. In Fig. 6k,l, myosin disruption seems to increase aEC size and decrease radial strain, diameter. By contrast, the same treatment increases vEC size but has no effect on radial strain. Why are these parameters uncoupled in vISVs and coupled in aISVs?\n8. The authors nicely detail morphometric differences in arterial versus venous ISVs, but do not explore the mechanisms that underlie these differences. Do the authors think that the differences simply reflect increased proliferative potential of vECs?\n\nMinor concerns\n1. All movies would benefit from annotation to draw the eye to events of interest.\n2. Figure 1b shows a significant increase in aISV diameter and non-significant decrease in vISV between 2 and 3 dpf, but this is buried by a general statement that “both vessel types exhibit a significant reduction in diameter…” over this time (2-4 dpf).\n3. The methods state that ISVs 10-18 (so 9 per embryo) were analyzed, but figures and legends say that N = total number of ISVs. So for 3b, for example, n= 42 could mean a few as 42/9 = 4-5 embryos. For statistical purposes, intra-embryo measurements should be averaged, and each N should be a single embryo, not a single ISV. Regardless, number of embryos analyzed should be clearly stated.\n4. Is there any correlation between actin type and position within ISV? For example, the circumferential actin shown in Figure 3a is at a bend.\n5. Line 240: vISVs (9 ECs at 3 pdf) are longer than aISVs (5 ECs at 3 dpf), so cell numbers could be normalized to length for fairer comparisons.\n6. In the myl9bA2A3-EGFP overexpression experiments, no gal4 driver is mentioned in the text, Fig. 5 legend, or methods. The only other transgene described is Tg(fli1:myr-mCherry).\n7. Figure 4a-c: Data are from an aISV at 2 dpf. Were the additional data (from total of 20 movies, not shown) all aISV, or also vISV?\n8. Figure 5b, c: statistical analysis required to prove that wasb-OE alters actin organization\n9. Line 316-317, Fig. 5g-i: Results statement needs to be clarified. There is no increase in diameter in vISV with myl9bA2A3-OE at 3 dpf. Also, the authors should clarify whether vessel diameters were measured in mosaic WT and OE ISVs (as shown in g), non-mosaic vessels, or a mixture of both. Particularly interesting would be to measure diameters in WT and OE regions of mosaic vessel.\n10. Line 319, Fig. S4: The only significant cell area increases with myl9bA2A3-OE are at 4 dpf in both aISV and vISV; it should be stated in the text that there are no increases at 2 and 3 dpf.\n11. Line 360, Fig. S5: Text says the area of krit1-/- ECs is increased, but this is not true for aECs at 2 dpf and not significant for vECs at 2 dpf.\n12. Line 362, Fig 6g,h: Text states that both ISV types have “significantly enlarged lumens at 2, 3, and 4 dpf compared to wild-type and krit1+/- EC-containing vessels.” This is untrue according to statistical analysis.\n13. The DA and PCV also get smaller in diameter over early developmental time. Can you speculate on whether your findings are likely applicable to larger multicellular tubes?\n14. Reference 15 is now published, please update.", "reviewer_2": "This manuscript by Chen et al. addresses the mechanisms that lead to zebrafish intersegmental vessels remodeling during development. Whether and how acto-myosin contraction allows vessel constriction during vessel remodeling is the driving question. A recent paper in Angiogenesis by Yin et al. 2024, has investigated the role of acto-myosin contraction at cell-cell junctions for lumen formation in zebrafish without addressing the question of vessel caliber. Therefore, the questions here have yet not been addressed directly.\n\nHowever, the general claim that circumferential actomyosin bundles and not the other actin organizations drive endothelial cell deformations is not enough demonstrated. Moreover, the contribution of other described phenomena such as cell exchange and cell division to vessel constriction is not well taken into account.\n\nMajor comments:\n1- First, it is difficult to follow the terminology describing the different events. The exact meaning of terms like cell rearrangement, cell exchange, cell shape deformation, cell size and their relationship is not clear and makes figure 1 difficult to follow. Terms should be more clearly explained and maintained consistent between methods and the main results section. The results discuss the tissue strain while the methods describe the calculations for a vessel strain which can be confusing.\n2- The method description needs to be much more clear and a schematic should be added to figure 1 to explain how the geometry of the cell is measured with an ellipse depicted with the major and minor axis which then leads to the various calculations described. Cv and Cr should be more clearly described as the length of the radial and vertical axes of the ECs assuming an ellipse.\n3- In figure 2b, it is quite difficult to follow the variation in diameter of the vessels at the front and back sides. A curve representing actin intensity and vessel diameter over time as in figure 4c would help. The respective contribution of actin rings and cell rearrangement (with new cells entering the zone) is not clear.\n4- The following part of the manuscript describes the role of actin remodeling on vessel constriction. However, this relies on a manual annotation of three types of actin which is a highly biased method. For example in fig3c, d, fig 5, fig6, the manual annotation of the actin fibers does not seem very close to the reality. Actin segmentation and categorization should be automated to make it more reliable. In Fig 3, the percentage of the various actin organizations is quantified. But then the spatiotemporal relationship between actin organization and vessel/cell shape changes is only described for circumferential actin. Are the other actin types also involved ? if not, this needs to be shown to claim that it is primarily circumferential actin that drives vessel constriction.\n5- This brings up the numbers of technical compared to biological replicates which are not clear. Indeed, there is no clear reporting throughout the entire paper of independent trials/embryos relative to the vessels or cells/vessel quantified. The “n” is reported above several bar plots but very unclear what this n includes. Moreover, there is no appropriate statistical reporting section in the methods where this should also be elaborated upon. On first glance, since source data files are missing, the p values look very low for the distribution that can be seen in the bar plots.\n6- Lines 308-309 conclude that circumferential actin is essential for vessel constriction but the OE-vessels still constrict and the difference between control and OE is 17 versus 14% which seems hardly enough to support this conclusion.\n7- One interesting point raised is the contribution of flow to vessel dilation in Krit-/- embryos. This still open question in the field needs more investigation on how no flow exacerbates CCM phenotype, and in what ways – actin/cell shape/proliferation/migration. Therefore, the effect of flow on the Krit phenotype should be more thoroughly explored and quantified. A movie of krit1-/- ISV with flow or no flow should be shown.\n\nMinor comments:\n8- Fig. 4d: which dpf is this comparison performed on? For vISVs this difference between recoil velocity of circumferential and longitudinal actin is really not significant and the sample size is also small. Are the vISVs from different embryos or within one independent trial?\n9- Lines 302-305- claim that there is a reduction in circumferential actin in both aISVs and vISVs at 2/3/4dpf in wasb OE ECs. This seems to not correlate with the box plots (eg. 3 and 4dpf for aISVs show higher C-M in the wasb-OE).\n10- Lines 313-331: the section discusses the effects on cell and vessel strains from Myosin II inhibition but does not discuss the effects on cell number changes which are identified as an important factor in Fig. 1. A quantification of ES/number of cells could help clarify this.", "reviewer_3": "Summary of the study: Using the zebrafish model, the authors intent to address how transitions in endothelial cell (EC) number and shape are coordinated to define vessel diameter during development remains. Their excellent data revealed that EC deformations, rearrangements and transient formation of self-seam junctions play key roles in regulating cell number and vessel diameter. By performing high-resolution analysis of actin cytoskeleton organization disclosed the generation of tension bearing, circumferential actomyosin bundles in the endothelial cortex that drive EC deformation and vessel constriction. In a vascular disease model of CCM, they showed that the loss of circumferential actin bundles in krit1/ccm1-deficient ECs causes cell enlargement and impaired vessel constriction that culminate in dilated vessels. Their strong data support that circumferential actomyosin-driven EC deformations in controlling vessel size and in the prevention of vascular malformations.\nThis is a very novel and well performed study with high quality data. I only have a minor issue regarding the myosin II activity in CCM disease model. They showed inhibition of myosin II activity leads to disrupted cell deformation and increased vessel diameter. Yet, CCM samples exhibited increased pospho-MLC2 thus increased myosin activity. How these can be reconciled?", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to Reviewers’ Comments\nWe thank the reviewers for their valuable feedback. Below, we provide point-by-point\nresponses (in blue) to all comments and have made corresponding revisions to address\nthese concerns.\nReviewers’ Comments:\nReviewer #1 (Remarks to the Author)\nThis is a nicely written paper in which the authors use transgenic zebrafish lines and\nconfocal imaging to measure vessel and endothelial cell morphometrics in arterial (a)\nand venous (v) intersegmental vessels (ISVs) from 2-4 days post-fertilization. They find\nthat aISVs contain fewer, larger ECs and vISVs contain more, smaller ECs, with\nincreased EC number arising largely from cell divisions after sprouting and anastomosis are complete. These findings confirm and modestly extend (to later timepoints) previously published work (e.g. Rosa et al. Development 2022). Beyond these morphometric analyses, the authors focus on the role of diQerent types of actin\nin controlling EC size and vessel diameters. They suggest that circumferential\nactomyosin is the key driver of constriction in aISVs and vISVs between 2-4 dpf. While\nthis is a logical idea, more rigor is required to establish causation or strong correlation.\nThe imaging is well-presented and morphometric measurements seem sound. The\nwork is mostly descriptive and provides modest new insight into the mechanism of\nendothelial cell shape changes and vessel diameter changes.\nMajor concerns:\n1. The primary novel conclusion from this work is that circumferential actomyosin is\nresponsible for EC contraction and decreased ISV radius between 2 and 4 days post-\nfertilization (dpf). However, data are largely observational, and temporal changes in\nactin organization and cell size and vessel diameter do not always correlate. For\nexample, the authors do not address how circumferential actin, present primarily at\nand analyzed only at 2 dpf, accounts for diameter decreases in aISV and vISV, which are\nonly measurable at 4 dpf (Fig. 1b, and presumably Fig. 5e,f; 6g,h, although stats not\nshown).\nWe thank the reviewer for pointing out the discrepancy between the stage of circumferential\nactin analysis and the stage at which vessels become significantly narrower. We revisited\nthe data that we had used for the first submission and realized that each data point\nrepresented one diameter measure, not the average diameter per vessel. We apologize for\nthis mistake and have corrected it so that each data point now represents the average of a\nvessel. Additionally, we have included average diameter measurements from at least two\nadditional independent experiments. With these corrections and updates, we observe that\ndiameter decreases from 2 dpf for aISVs (Fig. 1b), when circumferential actin is most\nfrequent. This continuous vessel narrowing from 2 dpf is also observed in control or wildtype\nvessels in Fig. 6d and f, Supplementary Fig.6d and Supplementary Fig.7b. Time-lapse\nimaging provide further evidence of diameter fluctuations with transient narrowing events at\n2 dpf (Fig. 4E, Supplementary Movie 7), demonstrating that vessel constriction occurs as\nearly as 2 dpf (Fig. 1b). Together, these data demonstrate that EC constriction begins at 2 dpf,\nbut significant reduction in vessel diameter becomes apparent by 4 dpf.\nWe also repeated the strain analyses at two separate intervals, 2-3 dpf and 3-4 dpf, instead\nof a single 2-4 dpf interval, to gain a more detailed understanding of endothelial cell (EC)\nbehaviours during vessel remodelling (Fig. 2c – g, lines 135 - 162). Specifically, we calculated\n(i) cell number strain, indicating how many cells span the vessel axis; (ii) cell strain,\nreflecting EC shrinkage or expansion; and (iii) vessel strain, representing the net tissue-level\nchange. These parameters clarify how cell rearrangement and size changes contribute to\nvessel constriction. Strain analysis revealed that aISV constriction begins at 2 dpf, when\ncell addition coincides with EC narrowing, and is later reinforced by axial rearrangement. In\nvISVs, early proliferation increases cell number but is oVset by EC narrowing, with later\nconstriction driven primarily by axial cell rearrangement. Together, these findings show that\ncircumferential actin enrichment at 2 dpf marks the onset of endothelial contraction\nand vessel constriction, while the measurable diameter reduction by 4 dpf reflects the\ncumulative outcome of these early contractile events.\nHow is tension maintained and diameter restricted in the absence of these structures\nat 4 dpf?\nLaser ablation experiments revealed tension in circumferential, mesh, and longitudinal actin,\nwith circumferential actin generating more than 1.5 times higher tension than mesh and\nlongitudinal actin (Fig. 5f). This elevated contractile force in circumferential actin is essential\nfor cell deformation at 2-3 dpf, driving vessels toward their final diameter. Following this\ninitial cell contraction phase, mesh and longitudinal actin organizations likely function to\nmaintain the reduced cell size. This concept aligns with our previous findings (Kondrychyn\net al., 2020), which demonstrated that decreased branched actin results in enlarged ECs\nand consequently dilated vessels. We therefore propose that while circumferential actin\nbundles provide the primary contractile force for cell deformation, mesh and longitudinal\nactin bundles contribute to the cortical tension necessary to stabilize the contracted cell\nand constricted vessel. Although cell-cell junctions were not investigated during vessel\nremodelling in this study, junctional tension likely exists (Supplementary Fig. 7g and h,\nkrit1+/+) and contributes to cell size and vessel shape regulation. The coordinated action of\nthese distinct actin networks—circumferential bundles for active contraction and\nmesh/longitudinal networks for structural maintenance—ensures precise control of vessel\ndiameter during development.\n2. Similarly, at 2 dpf, even on short time scales, the apparent loss of circumferential\nactin over time correlates with constriction and maintenance of smaller vessel\ndiameter (Fig. 3e,f). Can the authors show, in wild type ISVs, that regions that have\ncircumferential actin contract more than regions without it, on this same time scale?\nWe have plotted the diameter of vessels exhibiting circumferential, mesh and longitudinal\nactin on the same time scale in Supplementary Fig. 4. These additional analyses show that\nin 11 of 12 movies, the formation of circumferential actin correlates with decrease in vessel\ndiameter. In 8/12 cases, the dissipation of circumferential actin is followed by vessel\nwidening. In contrast, the formation of mesh actin formation is correlated with vessel\nwidening (5 of 6 movies). Longitudinal actin formation is associate with varying degree of\nvessel constriction. This is mentioned in lines 217 - 224.\n3. Actomyosin/diameter correlation (Fig. 4a-c) was assayed only at a region of\ncircumferential actomyosin. Additional correlations between actin/myosin/diameter\nshould be shown, both for regions with high and low colocalization, to strengthen\nconclusions.\nWe have plotted the correlation between myl9b colocalization at circumferential actin and\ndiameter in Supplementary Fig. 5b. In the 16 vessels analyzed, periods of low actomyosin\ncolocalization is consistently correlated with wider diameter while periods of high\ncolocalization (grey box) is correlated with narrowing diameter. These observations are\nmentioned in lines 250 - 252.\n4. In Fig. 5, while there is an apparent decrease in pure circumferential actin at 2 dpf in\naISV and vISV with wasb-OE, as expected, there is an apparent increase in mixed (mesh\n+ circumferential) actin with wasb-OE at 3 dpf in aISV and vISV, and at 4 dpf in aISV.\nAccordingly, these data do not strongly support a role for circumferential actin in\nconstriction.\nWe agree with the reviewer that the wasb-OE experiment weakly supports the role of\ncircumferential actin in vessel constriction and have moved these data to Supplementary\nFig. 6a - d. We have instead worked towards strengthening the causal role of circumferential\nactin in vessel constriction by performing the following:\ni) Endothelial overexpression of Fascin 1, an actin bundling protein (Fig. 6a -d, lines 302 -\n313). These new data show that Fascin 1-OE increased the number of vessels with\ncircumferential actin formation and augmented the constriction of aISVs and vISVs.\nii) Endothelial overexpression of Krit1 (Fig. 7b-d, lines 380 - 386). Elevated Krit1 expression\nalso resulted in more vessels with circumferential actin and enhanced vessel\nconstriction.\niii) Mathematical modelling of F-actin, myosin and crosslinkers enclosed by a membrane\nrevealed that the stress generated in the circumferential direction was larger than that\nin the longitudinal direction, and that the generated stress can deform the membrane\n(Fig. 5d-e, lines 254 - 284).\n5. The authors do not directly consider a role for pericytes in narrowing of ISV diameter,\nPericytes are present on ISVs as early at 2 dpf (Ando Development 2016; ref. 29; Colijn\net al. Dev Dyn 2024).\nThank you for raising the question of whether pericytes have a role in ISV constriction.\nAlthough pericytes are present on ISVs as early as 2 dpf, they do not completely cover ISVs\nsuch that by 120 hpf (5 dpf), 40% of ISVs remained uncovered by pericytes (Ando et al., 2016,\nDevelopment). Furthermore, there is preferential coverage of aISVs than vISVs, with only\n~50% covered by pericytes (Ando et al., 2016, Development). Given the scarce coverage,\nespecially around vISVs, we hypothesized that pericytes do not play a significant role in\ndevelopmental vessel constriction. Nevertheless, we addressed Reviewer 1’s concern by\nexamining pdgfrb-/- zebrafish, which lack functional Pdgfrβ signalling and have decreased\nnumber of mural cells around ISVs and cerebral central artery (Ando et al., 2016,\nDevelopment). By measuring aISV and vISV diameters at 2, 3 and 4 dpf in pdgfrb+/+ , pdgfrb+/-\nand pdgfrb-/- zebrafish (Supplementary Fig. 9), we found that both aISVs and ISVs in pdgfrb-/-\nzebrafish constrict to the same extent as wildtype embryos, with no statistical diVerence in\ndiameter at each developmental stage. These results show that pericytes do not regulate ISV\nconstriction during developmental vascular remodelling. This new data is discussed in lines\n503 - 507.\n6. The percent of each actin type is not consistent in control in Figs. 3, 5, 6, suggesting\nhigh variability from clutch to clutch, inconsistent timing, and/or high interoperator\nvariability/subjectivity from observer to observer. Can the authors please address this?\nThe imaging sessions for 2, 3, and 4 dpf embryos were conducted within an approximate 4-\nhour window due to imaging throughput across several independent experiments. Although\nactin remodelling is dynamic as shown in our 1-minute time-lapse recordings, we also\nnoticed that actin organizations oscillate and generally remain within the same classification\nfor extended periods. To confirm stability, we re-examined embryos imaged first after\ncompleting the session and observed that their ISV actin patterns were comparable to those\nrecorded ~4 hours earlier. Thus, the observed diVerences between developmental stages\nare unlikely to result from minor timing variation within each imaging batch.\nThe inconsistency in actin classification in control cells between experiments may be due to\ndiVerent numbers of ECs analyzed - fewer samples were analyzed in the Wasb-OE and Krit1\ntransplantation experiments compared to wildtype ECs. We have therefore performed more\nexperiments to increase the number of control ECs for Wasb-OE experiment, Krit1+/+ ECs as\nwell as WT ECs (see Table 1 below) and replotted the proportion of diVerent actin\norganizations found in control ECs from the diVerent experimental conditions (see figure\nbelow). By increasing the sample size, the proportion of actin type becomes more similar to\nwildtype, especially for vISVs at 2, 3 and 4 dpf and aISVs at 2dpf. In aISVs at 3 and 4 dpf, where\nsample size is small, there is still a slight disparity in actin type which will likely be corrected\nif sample size is increased even further. Regardless, we still observe a decrease in the\nproportion of circumferential actin and an increase in longitudinal actin from 2 to 4 dpf for\neach control conditions.\nExperiment Vessel type Developmental stage No. of vessels (first No. of vessels No. of increased\nsubmission) (resubmitted) samples\nWildtype aISV 2 dpf 42 44 2\n(Fig. 3b) 3 dpf 46 62 16\n4 dpf 36 50 14\nvISV 2 dpf 19 56 37\n3 dpf 48 82 34\n4 dpf 26 77 51\nControl ECs in aISV 2 dpf 15 26 11\nWasb OE 3 dpf 10 19 9\nexperiment (Fig. 4 dpf 7 11 4\n5b &c) vISV 2 dpf 11 16 5\n3 dpf 6 15 9\n4 dpf 7 15 8\nkrit1+/+ ECs (Fig. aISV 2 dpf 11 20 9\n6b & c) 3 dpf 10 15 5\n4 dpf 10 11 1\nvISV 2 dpf 7 12 5\n3 dpf 13 18 5\n4 dpf 6 11 5\nAnother possible explanation for the diVerence in the proportion of actin classification is\nthat the experimental condition to analyze actin organization in control ECs in the wasb-OE\nexperiment is diVerent from that used for analyzing actin organization in wildtype embryos.\nIn the wasb-OE experiment, control ECs analyzed are wildtype ECs in embryos with mosaic\nendothelial overexpression of Wasb. In this experimental setting, Wasb-OE induces regional\ndilation of blood vessels (new Supplemental Fig. 6a, c and d) that can alter wall shear stress\n(WSS) distribution in the vascular network (Maung Ye and Phng, 2023). Surrounding wildtype\nECs may respond to the altered WSS by remodelling its actin network. This experimental\ncondition diVers from the condition used to classify actin organization of wildtype ECs (Fig.\n3b) where wildtype embryos were imaged, and their direct comparison should be treated\nwith caution.\n7. In Fig. 6k,l, myosin disruption seems to increase aEC size and decrease radial strain,\ndiameter. By contrast, the same treatment increases vEC size but has no eQect on radial\nstrain. Why are these parameters uncoupled in vISVs and coupled in aISVs?\nWe appreciate the reviewer’s point and have clarified the analysis by separating intervals (2-\n3 and 3-4 dpf) and explicitly reporting cell strain, vessel radial strain, and radial cell-number\nstrain (formerly E.number). Importantly, size (absolute dimension) and strain (temporal\nchange) describe distinct aspects of vessel remodelling.\nIn aISVs, myosin II inhibition prevented normal EC narrowing, showing positive cell strain at\nboth intervals (Fig. 6h). Early constriction (2-3 dpf) was partly preserved by enhanced radial-\nto-axial rearrangement (negative cell number strain), but this compensation weakened by 3-\n4 dpf, leading to vessel widening. In vISVs, reduced EC narrowing combined with stronger\nradial-to-axial redistribution at 2-3 dpf produced modest constriction, while later cell\nexpansion oVset rearrangement, resulting in weaker constriction overall.\nAlthough vISVs exhibited similar net vessel strain to controls, their ECs possess less\ncircumferential actin and experience higher proliferative compaction, which may stabilize\ndiameter despite altered contractility. Overall, myosin II inhibition disrupts the coordination\nbetween cell contraction and rearrangement, impairing vessel constriction.\n8. The authors nicely detail morphometric diQerences in arterial versus venous ISVs,\nbut do not explore the mechanisms that underlie these diQerences. Do the authors\nthink that the diQerences simply reflect increased proliferative potential of vECs?\nAs venous ECs proliferate more frequently than arterial EC, ECs in the vISVs may be\nsubjected to more compressive forces that may promote vEC shrinkage. We attempted to\naddress this question by inhibiting cell proliferation with aphidicolin and hydroxyurea.\nHowever, this treatment resulted in the reduction of endothelial cell number in both venous\nand arterial ISVs, preventing us to investigate the proliferative capacity of vECs on vessel\nmorphometrics (Supplemental Fig. 10). This is discussed in lines 511 -517.\nMinor concerns\n1. All movies would benefit from annotation to draw the eye to events of interest.\nAll movies are now annotated.\n2. Figure 1b shows a significant increase in aISV diameter and non-significant decrease\nin vISV between 2 and 3 dpf, but this is buried by a general statement that “both vessel\ntypes exhibit a significant reduction in diameter…” over this time (2-4 dpf).\nWe revisited and corrected our data, and added additional data as discussed above. The\nrevised analysis shows that aISVs display a gradual, significant reduction in diameter\nbetween 2 and 4 dpf, whereas vISVs showed similar diameter between 2-3 dpf and\nconstricted between 3-4 dpf (lines 96-98).\n3. The methods state that ISVs 10-18 (so 9 per embryo) were analyzed, but figures and\nlegends say that N = total number of ISVs. So for 3b, for example, n= 42 could mean a\nfew as 42/9 = 4-5 embryos.\nThis is not always the case as not all ECs are bright enough to image. ISVs with weak reporter\nexpression, poor image quality, or undergoing remodelling without flow were excluded. The\nnumber of embryos and experiments are now included in the figure legend.\nFor statistical purposes, intra-embryo measurements should be averaged, and each N\nshould be a single embryo, not a single ISV. Regardless, number of embryos analyzed\nshould be clearly stated.\nWe have found that aISVs (or vISVs) do not all exhibit the same actin organisation in the same\nembryo. The reason for the heterogeneity in actin organisation is unclear but may represent\ndiVerent EC status/behaviour (cell rearrangement, proliferative) or a consequence of\ndiVerent perivascular environment (shear stress/pressure level, ECM deposition). Therefore,\nit is not possible to “average” actin organisation from one embryo.\nWe have now clearly stated that n = ISV, and the number of embryos analyzed and\nexperiments performed are now stated in figure legends.\n4. Is there any correlation between actin type and position within ISV? For example, the\ncircumferential actin shown in Figure 3a is at a bend.\nWe thank the reviewer for this observation. Because the ventral region of ISVs lies deeper in\nthe tissue and cannot be imaged at suVicient resolution, we restricted our analysis of\ncortical actin organization to the dorsal part of ISVs, where imaging quality allows reliable\nclassification (see Methods). At 2 dpf, 14% of aISVs displayed circumferential (C) and 34%\nC-M actin organizations. Within this dorsal region, circumferential actin was not restricted\nto vessel bends (in approximately 47% of ISV showing C/C-M patterns), while in the\nremaining vessels it occurred along straight segments. Thus, circumferential actin is not\npositionally confined but can arise throughout the dorsal ISV.\n5. Line 240: vISVs (9 ECs at 3 pdf) are longer than aISVs (5 ECs at 3 dpf), so cell numbers\ncould be normalized to length for fairer comparisons.\nUpon the reviewer’s suggestion, we have plotted EC number per 100 µm vessel and added\nthis graph in Supplemental Fig. 1a. We have kept the graph showing the absolute number of\nECs per aISV or vISV in Fig. 1c.\n6. In the myl9bA2A3-EGFP overexpression experiments, no gal4 driver is mentioned in\nthe text, Fig. 5 legend, or methods. The only other transgene described is Tg(fli1:myr-\nmCherry).\nWe have now included the gal4 driver line in the text (line 331).\n7. Figure 4a-c: Data are from an aISV at 2 dpf. Were the additional data (from total of 20\nmovies, not shown) all aISV, or also vISV?\nWe have now shown the analysis in Supplementary Figure 5b and the vessel type (aISV or\nvISV) is written above each plot. These data are further mentioned in main text in lines 250-\n252.\n8. Figure 5b, c: statistical analysis required to prove that wasb-OE alters actin\norganization\nWe have now performed Fisher’s exact test to analyse categorical data. The shift in AO in\nwasb-OE vessels is mentioned in lines 320-322.\n9. Line 316-317, Fig. 5g-i: Results statement needs to be clarified. There is no increase\nin diameter in vISV with myl9bA2A3-OE at 3 dpf. Also, the authors should clarify whether\nvessel diameters were measured in mosaic WT and OE ISVs (as shown in g), non-mosaic\nvessels, or a mixture of both. Particularly interesting would be to measure diameters in\nWT and OE regions of mosaic vessel.\nWe thank the reviewer for this comment and have clarified the text in lines 333-335. Vessel\ndiameters were measured specifically in regions containing GFP-positive Myl9bA2A3-OE\ncells. These regions were often part of mosaic vessels comprising both OE and non-OE cells.\nControl measurements were taken from either non-mosaic vessels or mosaic vessels\nlacking GFP expression. This is mentioned in Methods in lines 643-647.\n10. Line 319, Fig. S4: The only significant cell area increases with myl9bA2A3-OE are at\n4 dpf in both aISV and vISV; it should be stated in the text that there are no increases at\n2 and 3 dpf.\nThis is now clarified in lines 337-339.\n11. Line 360, Fig. S5: Text says the area of krit1-/- ECs is increased, but this is not true\nfor aECs at 2 dpf and not significant for vECs at 2 dpf.\nWe added more experiments (see table below) and have corrected the text to state that “Cell\narea in homozygous mutants remained similar to controls at 2 dpf but increased significantly\nat 3 dpf in vECs and in both vessel types by 4 dpf”. Lines\nGenotype Vessel type Developmental stage No. of cells (first No. of cells No. of increased\nsubmission) (resubmitted) samples\nWild type aISV 2 dpf 4 12 8\n3 dpf 11 14 3\n4 dpf 13 17 4\nvISV 2 dpf 5 7 2\n3 dpf 6 12 6\n4 dpf 8 14 6\nHeterozygous aISV 2 dpf 17 23 6\n3 dpf 12 18 6\n4 dpf 14 14 0\nvISV 2 dpf 10 25 15\n3 dpf 14 17 3\n4 dpf 20 20 0\nHomozygous aISV 2 dpf 15 20 5\n3 dpf 11 16 5\n4 dpf 5 11 6\nvISV 2 dpf 7 17 10\n3 dpf 8 19 11\n4 dpf 13 23 10\n12. Line 362, Fig 6g,h: Text states that both ISV types have “significantly enlarged\nlumens at 2, 3, and 4 dpf compared to wild-type and krit1+/- EC-containing vessels.”\nThis is untrue according to statistical analysis.\nAfter analysing more vessels from transplantation experiments (see table below), we have\ncorrected this statement to clarify that vessel diameters were larger in krit1-/- aISVs at 2, 3\nand 4 dpf and vISV at 3 and 4dpf, compared to wildtype controls (Supplementary Fig. 7b,\nlines 398-400).\nGenotype Vessel type Developmental stage No. of vessels (first No. of vessels No. of increased\nsubmission) (resubmitted) samples\nWild type aISV 2 dpf 3 8 5\n3 dpf 6 9 3\n4 dpf 9 12 3\nvISV 2 dpf 5 6 1\n3 dpf 3 6 3\n4 dpf 5 10 5\nHeterozygous aISV 2 dpf 15 21 6\n3 dpf 7 11 4\n4 dpf 7 8 1\nvISV 2 dpf 6 14 7\n3 dpf 5 7 2\n4 dpf 9 9 0\nHomozygous aISV 2 dpf 7 11 4\n3 dpf 5 8 3\n4 dpf 4 6 2\nvISV 2 dpf 5 14 9\n3 dpf 5 13 8\n4 dpf 7 13 6\n13. The DA and PCV also get smaller in diameter over early developmental time. Can\nyou speculate on whether your findings are likely applicable to larger multicellular\ntubes?\nTo address this question, we have imaged endothelial cells in both DA and PCV. We also\nfound the existence of circumferential actin bundles as well as actomyosin asters,\nsuggesting that larger vessels generate diVerent actin structures, including circumferential\nactin, for vessel remodelling (Suplementary Fig. 8, lines 459 - 465).\n14. Reference 15 is now published, please update.\nThank you for pointing out that this paper is now published. Its reference is now updated.\nReviewer #2 (Remarks to the Author):\nThis manuscript by Chen et al. addresses the mechanisms that lead to zebrafish\nintersegmental vessels remodeling during development. Whether and how acto-\nmyosin contraction allows vessel constriction during vessel remodeling is the driving\nquestion. A recent paper in Angiogenesis by Yin et al. 2024, has investigated the role of\nacto-myosin contraction at cell-cell junctions for lumen formation in zebrafish\nwithout addressing the question of vessel caliber. Therefore, the questions here have\nyet not been addressed directly.\nHowever, the general claim that circumferential actomyosin bundles and not the other\nactin organizations drive endothelial cell deformations is not enough demonstrated.\nMoreover, the contribution of other described phenomena such as cell exchange and\ncell division to vessel constriction is not well taken into account.\nMajor comments:\n1- First, it is diQicult to follow the terminology describing the diQerent events. The\nexact meaning of terms like cell rearrangement, cell exchange, cell shape\ndeformation, cell size and their relationship is not clear and makes figure 1 diQicult to\nfollow. Terms should be more clearly explained and maintained consistent between\nmethods and the main results section. The results discuss the tissue strain while the\nmethods describe the calculations for a vessel strain which can be confusing.\nWe have extensively rewritten the manuscript to improve the clarity of the diVerent cellular\nbehaviours in relation to changes in vessel morphometrics (diameter and length). We have\nalso separated the former Figure 1 into two figures:\n- The new Figure 1 focuses of vessel morphometrics and cell exchanges and division. We\nhave also included an illustration to explain what is meant by cell exchange.\n- The new Figure 2 is dedicated to describing changes in cell shape. We have included\nillustrations to summarise changes in cell area and data from strain analysis.\nWhile rewriting the manuscript, we have paid attention to being consistent in our choice of\nterms. Accordingly, tissue strain has now been changed to vessel strain.\nCurrent term Previous term\nVessel strain Tissue strain\nCell strain Cell strain\nCell number strain EVective cell strain due to number/ E.number\nRadial axis Radial axis/minor axis\nAxial axis Vertical axis/major axis\n2- The method description needs to be much more clear and a schematic should be\nadded to figure 1 to explain how the geometry of the cell is measured with an ellipse\ndepicted with the major and minor axis which then leads to the various calculations\ndescribed. Cv and Cr should be more clearly described as the length of the radial and\nvertical axes of the ECs assuming an ellipse.\nWe have now included a schematic to explain how the radial and axial axes of the cell (Fig.\n2a). In the revised manuscript, we use the term “radial axis” to refer to the “minor axis”, and\n“axial axis” to refer to the “major/vertical axis”. We have also included a more detailed\nexplanation on Methods in the section ‘In vivo cell shape analysis’. Cr is cell strain at the\nradial axis and Cv is cell strain at the vertical axis. In lines 674-680, we explained that cell at\nradial axis is cell width (w) and at axial axis is cell length (l). For a quantity X (cell width w or\ncell length l), strain (ԑ) between two time points (t) was calculated as:\n𝑋 −𝑋\n# \"\n𝜀 (𝑡 → 𝑡 ) =\n! \" # 𝑋\n\"\nFor example, cell strain at radial axis at 2-3dpf, would be calculated as:\n𝑤 −𝑤\n(%&' #%&'\n𝜀 )𝑡 → 𝑡 * =\n$ #%&' (%&' 𝑤\n#%&'\nto indicate the change of cell width at the circumference between two timepoints.\n3- In figure 2b, it is quite diQicult to follow the variation in diameter of the vessels at the\nfront and back sides. A curve representing actin intensity and vessel diameter over time\nas in figure 4c would help. The respective contribution of actin rings and cell\nrearrangement (with new cells entering the zone) is not clear.\nThe intention of this figure (new Fig. 3b) is to illustrate the dynamics of cell rearrangement\nwithin the circumference of the vessel at two regions. Region 1 (demarcated by serrate pink\nline) is a unicellular segment of the vessel while region 2 (demarcated by serrated blue line)\nis a multicellular segment of the same vessel. We showed the front and back sides of the\nvessel to showcase the junction organization so to highlight the diVerences in junctions at\nthe front and back sides. We appreciate that this may be confusing and have added a\nmaximum projection of the vessel in this figure. We have also rearranged the figure to\nhighlight the dynamics of the two cells at the cross-sections of the 2 regions. We hope that\nthese additional changes will help the reviewer understand the images better.\nAs the purpose of this figure is not to correlate the intensity of actin and vessel diameter,\nwe do not see that it is fitting to place a curve representing actin intensity and vessel diameter\nover time here. We would like to refer the reviewer to new Supplementary Figure 5b for 16\ncurves showing the relationship between circumferential actin (and myosin II) intensity and\nvessel diameter. These curves consistently show that high circumferential actin intensity is\nassociated with decreased vessel diameter and vice versa.\nWe are unsure what is meant by “actin rings”. We assume that the reviewer is referring to the\ncortical actin stripes that we investigated further by higher resolution imaging (from new\nFigure 4 onwards) and classified them as circumferential actin bundles. Although these\nactin structures are circumferential, they are not rings because they do not surround the\nentire circumference of the vessel. In this study, we did not examine the relationship\nbetween its formation and cell rearrangement but focused on the role of circumferential\nactin in driving cell contraction and consequently, vessel constriction.\n4- The following part of the manuscript describes the role of actin remodeling on vessel\nconstriction. However, this relies on a manual annotation of three types of actin which\nis a highly biased method. For example in fig3c, d, fig 5, fig6, the manual annotation of\nthe actin fibers does not seem very close to the reality. Actin segmentation and\ncategorization should be automated to make it more reliable.\nWe agree that there may be a degree of bias in characterizing the actin organization\nsubjectively, and three members of the lab have independently attempted to automate the\nanalysis of actin structures detected in endothelial cells in vivo. However, each person\nencountered the same problem. Due to a high spectrum of actin intensity, from very high\njunctional signal to weakly labelled cortical actin structures that cannot be detected by\nautomated thresholding (e.g. Otsu method), the number and type of actin filaments\ngenerated by image processing and segmentation do not represent what is detectable by eye.\nThus, although a lot of time was invested to develop an image analysis method to annotate\nactin organization objectively, it did not yield reliable result, and we concluded that the\nestablishment of a robust and reliable automated method of actin organisation requires\nimmense work and constitutes its own project. We therefore resorted to the analysis of actin\norganisation by eye, and the circumferential-to-longitudinal transition between 2 and 4 dpf\nwas observed by two other researchers independently, supporting reproducibility.\nAfter receiving reviewers’ comments, we have again tried to automate the detection of actin\norientation and established an automated nematic-orientation workflow (lines 528 – 533,\nSupplementary Fig. 11, Supplementary Method 1). Actin orientations were binned into three\nclasses (−1 to −0.33 = circumferential (C); −0.33 to +0.33 = mesh; +0.33 to 1 = longitudinal\n(L)) to generate frequency plots. While this analysis broadly supports a temporal transition\nin actin organisation, it generally reports fewer C and more (L) categories than our five-bin\nmanual scheme. This discrepancy most likely reflects method-intrinsic factors: first, the\nuniform thresholding that removes junctional signal can also exclude adjacent thick\ncircumferential bundles, reducing apparent C; second, referencing θ to a single overall\nvessel axis on curved or asymmetric segments biases near-circumferential orientations\ntowards 0 while preserving streaks near +1, right-shifting medians into the L bin; third, per-\nvessel median summarisation compresses mixed distributions, so vessels that appear\nmesh-dominant can cross the +0.33 boundary and be labelled as L; last, intermediate states\nare collapsed by design (manual C-M and M-L map to mesh and L, respectively), inflating\nthose bins at the expense of C. Despite these diVerences, both approaches support a time-\ndependent transition in actin organisation during remodelling. Future work should prioritise\nsegmentation that captures the full intensity dynamic range of cortical actin and calculation\nof orientation relative to the local centreline tangent rather than a global axis.\nWe have clarified our manual classification in lines 686-689 and included images of C-M and\nM-L actin organizations in Fig. 4a to illustrate how the two classifications were determined.\nIn Fig 3, the percentage of the various actin organizations is quantified. But then the\nspatiotemporal relationship between actin organization and vessel/cell shape changes\nis only described for circumferential actin. Are the other actin types also involved ? if\nnot, this needs to be shown to claim that it is primarily circumferential actin that drives\nvessel constriction.\nWe have plotted the diameter of vessels exhibiting circumferential, mesh and longitudinal\nactin on the same time scale in Supplementary Fig. 4. These additional analyses show that\nin 12 of 12 movies, the formation of circumferential actin correlates with decrease in vessel\ndiameter. In 8/12 cases, the dissipation of circumferential actin is followed by vessel\nwidening. In contrast, the formation of mesh actin formation is correlated with vessel\nwidening (5 of 6 movies). Longitudinal actin formation is associate with varying degree of\nvessel constriction. This is mentioned in lines 217 - 224. Based on these correlative analyses,\ncircumferential actin appears to be the primary actin that drives vessel constriction.\nTo strengthen the causal role of circumferential actin in vessel constriction, we have\nperformed additional experiments:\ni) Endothelial overexpression of Fascin 1, an actin bundling protein (Fig. 6a -d, lines\n302 - 313). These new data show that Fascin 1-OE increased the number of\nvessels with circumferential actin formation and a corresponding augmented\nconstriction of aISVs and vISVs.\nii) Endothelial overexpression of Krit1 (Fig. 7b-d, lines 380 - 386). Elevated Krit1 expression\nalso resulted in more vessels with circumferential actin and enhanced vessel\nconstriction.\niii) Mathematical simulations of F-actin, myosin and crosslinkers enclosed by a membrane\nrevealed that the stress generated in the circumferential direction was larger than that\nin the longitudinal direction, and that the generated stress can deform the membrane\n(Fig. 5d-g, lines 254 - 284).\n5- This brings up the numbers of technical compared to biological replicates which are\nnot clear. Indeed, there is no clear reporting throughout the entire paper of independent\ntrials/embryos relative to the vessels or cells/vessel quantified. The “n” is reported\nabove several bar plots but very unclear what this n includes. Moreover, there is no\nappropriate statistical reporting section in the methods where this should also be\nelaborated upon. On first glance, since source data files are missing, the p values look\nvery low for the distribution that can be seen in the bar plots.\nWe have now written the number of ISVs/cells, embryos and independent experiments, and\nstatistical analysis used in all figure legends. Statistical analysis is added in methods section\nin lines 762-768. Source data is now provided in the resubmission.\n6- Lines 308-309 conclude that circumferential actin is essential for vessel constriction\nbut the OE-vessels still constrict and the diQerence between control and OE is 17\nversus 14% which seems hardly enough to support this conclusion.\nWe agree with the reviewer that the data from wasb-OE experiment weakly supports the role\nof circumferential actin in vessel constriction and have moved these data to Supplementary\nFig. 6. We have now obtained new data (Fascin1-OE, Krit1-OE and mathematical simulation\ndescribed above) that provide stronger evidence for the role of circumferential actin in vessel\nconstriction.\n7- One interesting point raised is the contribution of flow to vessel dilation in Krit-/-\nembryos. This still open question in the field needs more investigation on how no flow\nexacerbates CCM phenotype, and in what ways - actin/cell\nshape/proliferation/migration. Therefore, the eQect of flow on the Krit phenotype\nshould be more thoroughly explored and quantified. A movie of krit1-/- ISV with flow or\nno flow should be shown.\nWe agree with the reviewer’s point that the question of how flow reduces the severity of Krit1\nLOF phenotype is an important but unresolved question. While we agree that the analyses\nof actin organization, cell shape, proliferation and migratory behaviours of krit1-/- ECs in the\npresence and absence of blood flow would contribute to the understanding of the\nrelationship between blood flow and CCM formation, it is beyond the scope of this study for\ntwo reasons. The first is that our primary goal is to understand the cellular mechanism of\nblood vessel remodelling, not the eVects of flow on krit1-/- ECs. The second is that the\nduration to complete experiments required to fully understand how flow regulates actin, cell\nshape, proliferation and migration in krit1-/- ECs far exceeds the time allocated for the\nrevision of the manuscript. We estimate that this will take 9 to 12 months given that these\nexperiments require cell transplantations (a technically diVicult experiment that only yields\nabout 5% zebrafish with grafts composed of krit1-/- ECs) and the detailed nature of the image\nanalyses. Further, if we only add a movie of krit1-/- ISV with flow or no flow (which the reviewer\nrequested), it does not add much value to the existing data.\nWe have consulted this matter with the editor, who has agreed that addressing this specific\ncomment is out of the scope of the paper. Instead, we have added a statement “Notably, cell\nenlargement was more severe in flow-deficient krit1-/- cells (from in-cross, Supplementary\nFig. 7h) than in transplanted cells exposed to flow, suggesting that blood flow may partially\nmitigate the severity of Krit1 loss-of-function, a cellular mechanism that merits further\ninvestigation” in lines 489 - 492.\nMinor comments:\n8- Fig. 4d: which dpf is this comparison performed on?\nFigures showed embryos at 2dpf. We have now specified this in Figure 5 legend.\nFor vISVs this diQerence between recoil velocity of circumferential and longitudinal\nactin is really not significant and the sample size is also small. Are the vISVs from\ndiQerent embryos or within one independent trial?\nWe have reanalysed and performed more experiments to increase sample size. The updated\nresults now show a significant diVerence in recoil velocity between circumferential and\nlongitudinal actin in vISVs (P = 0.0131).\nWe have now clarified the sample size and vessel type in the figure legend: aISV n=12/8/9\nand vISV n=13/9/10 in C/M/L, from 27 embryos in 3 experiments.\n9- Lines 302-305- claim that there is a reduction in circumferential actin in both aISVs\nand vISVs at 2/3/4dpf in wasb OE ECs. This seems to not correlate with the box plots (eg.\n3 and 4dpf for aISVs show higher C-M in the wasb-OE).\nWe have re-written the results from the wasb-OE experiments and have also placed this set\nof data in Supplementary Fig. 6. In the revised manuscript, we have shifted the focus to\nincreased mesh actin (lines 318 - 322): “Wasb-OE increased the frequency of ISVs exhibiting\nmesh actin, interestingly, at the expense of longitudinal rather than circumferential actin\n(Supplementary Fig. 6a-b). In both aISVs and vISVs, this shift was most apparent at later\ndevelopmental stages (at 4dpf, M vs other AOs, aISV, Fisher’s test aISV, p=0.04; vISV, p=0.02).\n10- Lines 313-331: the section discusses the eQects on cell and vessel strains from\nMyosin II inhibition but does not discuss the eQects on cell number changes which are\nidentified as an important factor in Fig. 1. A quantification of ES/number of cells could\nhelp clarify this.\nWe thank the reviewer for this important suggestion. To address it, we performed additional\nMyl9bA2A3-OE experiments and quantified endothelial cell number together with vessel\nlength in both aISVs and vISVs. These data allowed us to calculate cell number strain\n(previously, E.number), which reflects changes in how many cells span the vessel axis,\nproviding direct insight into cell addition by cell proliferation and rearrangement dynamics.\nThe results are now included in Fig. 6h-i and Supplementary Fig. 6e-f and discussed in lines\n343 – 349 and 359 - 363. Incorporating these analyses clarifies that myosin II inhibition alters\nthe temporal coordination between cell rearrangement, cell contraction, and vessel\nconstriction.\nReviewer #3 (Remarks to the Author):\nSummary of the study: Using the zebrafish model, the authors intent to address how\ntransitions in endothelial cell (EC) number and shape are coordinated to define vessel\ndiameter during development remains. Their excellent data revealed that EC\ndeformations, rearrangements and transient formation of self-seam junctions play key\nroles in regulating cell number and vessel diameter. By performing high-resolution\nanalysis of actin cytoskeleton organization disclosed the generation of tension bearing,\ncircumferential actomyosin bundles in the endothelial cortex that drive EC deformation\nand vessel constriction. In a vascular disease model of CCM, they showed that the loss\nof circumferential actin bundles in krit1/ccm1-deficient ECs causes cell enlargement\nand impaired vessel constriction that culminate in dilated vessels. Their strong data\nsupport that circumferential actomyosin-driven EC deformations in controlling vessel\nsize and in the prevention of vascular malformations.\nThis is a very novel and well performed study with high quality data. I only have a minor\nissue regarding the myosin II activity in CCM disease model. They showed inhibition of\nmyosin II activity leads to disrupted cell deformation and increased vessel diameter.\nYet, CCM samples exhibited increased pospho-MLC2 thus increased myosin activity.\nHow these can be reconciled?\nWe thank the reviewer for the positive evaluation of our work and for pointing out the disparity\nin myosin II activity in published works on ccm1-deficient ECs and our zebrafish model.\nWhile some studies report ROCK-driven hypercontractiliy, others showed reduced\njunctional myosin II activity. To elucidate the contractile state of krit1-/- ECs during vessel\nremodelling, we examined the localization of actin and myosin II using Tg(fli1:GAL4FF)ubs3;\nTg(UAS:EGFP-UCHD)ubs18; Tg(6xUAS:myl9b-mCherry)rk32 (Supplementary Figure 7g, lines 422\n- 431). We found that the loss of Krit1 alters the distribution of actin and myosin II at junctions\nversus cortex, potentially aVecting force transmission and EC transmission. The diVerences\nin myosin II activity in in vitro and in vivo studies, and the implications of our findings, are now\ndiscussed in lines 475 -495.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank all the reviewers for their insightful and important comments, which improved our\nmanuscript tremendously. Please find our point-by-point response below.\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have very nicely addressed all of my previous concerns regarding the original\nsubmission, as evidenced by numerous additional experiments presented in both the main\ntext and in the supplement. They convincingly demonstrate a role for circumferential\nactomyosin in endothelial cell contraction and vessel narrowing and new data strengthen\nsupport for a role for the CCM protein, KRIT1, in this process.\nWe thank the reviewer for the positive feedback on our revised manuscript and are delighted\nthat they are satisfied with our revision.\nReviewer #2 (Remarks to the Author):\nFirst we would like to acknowledge the authors for their efforts in thoroughly revising the\nmanuscript. The use of a consistent terminology over the manuscript helps its understanding\nlike the new explanations on the quantified parameters.\nAdditional data have been acquired for many of the figures improving the robustness of the\nstatistical analyses and numbers of studied vessels, embryos and experiments have been\nspecified as requested.\nNew experiments with overexpression of Krit and Fascin nicely complement the data.\nHowever, some parts still need attention:\nWe thank the reviewer for acknowledging our efforts during the revision process. Please find\nbelow our point-by-point response to additional concerns.\n-We noticed that in Fig1c only one experiment has been performed to quantify EC number\nper vessel at 4 dpf which is surprising as many other quantifications have been done at this\ntime point in other figures.\nWe thank the reviewer for highlighting this point. We agree that including an additional\nbiological replicate at 4 dpf would improve the robustness of the quantification. We have\nperformed an independent repeat experiment and updated the figure, figure legend (lines\n1012 – 1013), statistical analyses and source data accordingly. The latest result with\nadditional replicate is consistent with the previously observed increase in EC number\nbetween 2 and 4 dpf.\n-In figure 2g, at 3-4 dpf, it is said that aEC shrinks while vEC shortens. Please explains what\nit means either directly in the figure or in its legend.\nWe thank the reviewer for pointing out the need for clarification. We have revised the legend\nof Fig. 2g to explicitly define the terminology used:\nShorten: cell length decreases at axial axis; Narrow: cell width decreases at radial axis;\nShrink: reduces in both axial and radial axis.\nThis is revised in lines 1037- 1039.\n-In figure 4c-f, it is not said whether these are aISV or vISV.\nWe clarified the vessel type in the figure legend. Lines 1063-1064.\n-In the paragraph line 298-313, it should be mentioned that whereas circumferential actin is\nmore enriched at 2 dpf, diameter constriction is only statistically significant at 3 and 4 dpf.\nThank you for the suggestion. We have now added this clarification to the corresponding\nparagraph in the Results section “Notably, circumferential actin enrichment was highest at 2\ndpf, whereas statistically significant diameter constriction was detected only at 3 and 4 dpf.”\nLine 318-320.\n-Line 377: please cite the original paper from Glading et al., JCB 2007.\nWe apologise for the incorrect citation and have now cited the original paper from Glading et\nal., JCB 2007.\n- Paragraph 422-431 and sup fig 7h: a new Junction/Cortex Ratio is introduced in the\nquantifications but the term cortex is not clearly defined. Does this \"cortex actin\" actually\ncorresponds to the total intracellular actin content or to only a zone and which one ?\nConcerning the linear fibers that are present in krit1 -/- ECs (Fig 7g), why are they not\nconsidered as circumferential actin fibers ? They also cross the cell from one junction to\nanother. We are not necessarily asking for more experiments but for more clarity about what\nis seen here and its interpretation. Obviously, the wrong location in z of these contractile\nfibers is key for the krit-/- phenotype and could explain the paradox raised by reviewer 3\nconcerning the increased pMLC in these cells.\nThe “cortex actin” measured in these analyses corresponds to a defined intracellular zone,\nnot total actin content. Specifically, 10 cortical actin regions per vessel were manually\nselected from regions within the cell interior where EGFP-UCHD signal was present (but not\ncontiguous with junctional pixels). Junctional actin was sampled at cell–cell contacts, where\nEGFP-UCHD intensity is highest. We agree that the spatial position of contractile fibres is\ncritical, particularly in light of in vitro studies showing that CCM1/CCM2 loss promotes\nROCK1-dependent ventral stress fibres. However, as endothelial cells in zebrafish vessels\nare extremely thin (~1-2 µm) and z-resolution is limited, apical and basal membranes cannot\nbe reliably distinguished in our images; thus, the cortical region integrates both apical and\nbasal surfaces. This definition has now been clearly described in the Methods section in line\n739-752.\nIn our in vivo data, the prominent linear actin fibres observed in krit1−/− ECs (Supplementary\nFig. 7g) are clearly distinct from the circumferential bundles seen in wild-type cells. They\nexhibit a diagonal-to-longitudinal orientation along the vessel axis rather than the\nperpendicular orientation characteristic of circumferential bundles. We now specified this in\nline 437-439 and added arrows and arrowheads to indicate junctional and cortical actin,\nrespectively, in Supplementary Fig. 7g). Also, most of these longitudinal actin structures do\nnot connect to cell-cell junctions. Together with the reduced junction/cortex actin ratio and\naltered myosin II distribution, these observations indicate a redistribution of contractile\nstructures away from junctional-circumferential actin scaffolds toward a more homogeneous\npattern. This pattern is consistent with the notion that increased pMLC in krit1−/− cells does\nnot translate into effective circumferential contraction because contractility is mis-localized\nand reoriented.\n-Line 484: in Lisowska et al (ref 29), it is shown that in 2D, ROCK1 promotes ventral actin\nstress fibers connected to focal adhesions and ECM and not “cortical stress fibers” as said\nhere.\nWe thank the reviewer for catching this inaccuracy and we apologize for the inappropriate\nphrasing. The text has now been corrected to refer specifically to “ventral actin stress fibres,”\nin line 504.\n-Line 491: Please refer to fig 7e when talking about transplanted cells exposed to flow, it will\nfacilitate the comparison. In this paragraph, mosaicism should also be commented as it may\nalso impact the acto-myosin organization of the mutant cell and may contribute to the\ndifferences between pure homozygous vessels and transplanted populations.\nWe thank the reviewer for this helpful suggestion. We now explicitly refer to Fig. 7e when\ndescribing transplanted krit1−/− cells exposed to flow. We also acknowledge that mosaicism\nmay influence the actomyosin organization of transplanted mutant cells by placing them in a\nwild-type mechanical environment, and this clarification has now been incorporated into the\nrevised manuscript in line 517-520.\n-Line 539: When talking about the effect of CCM mutant cells on neighbouring ones, Shapeti\net al, Nat com 2024 should also be cited.\nThank you for pointing out the relevance of the work of Shapeti et al. on the influence of\nCCM mutant cells on neighbouring wildtype cells. We have now cited this work in the\nmanuscript (line 566).\n-Movies 8 and 18 don’t work.\nWe apologise for this technical error (they played on our computer). We have remade .avi\nfiles from .tif files for Movies 8 and 18 and hope that the reviewers can now open and view\nthese movies.\nReviewer #3 (Remarks to the Author):\nThe authors have carefully addressed my previous concerns on Rock/MLC activation in\nCCM pathogenesis.\nWe are pleased that we have addressed the reviewer’s concerns.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank all the reviewers for their insightful and important comments, which improved our\nmanuscript tremendously. Please find our point-by-point response below."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have very nicely addressed all of my previous concerns regarding the original submission, as evidenced by numerous additional experiments presented in both the main text and in the supplement. They convincingly demonstrate a role for circumferential actomyosin in endothelial cell contraction and vessel narrowing and new data strengthen support for a role for the CCM protein, KRIT1, in this process.", "reviewer_2": "First we would like to acknowledge the authors for their efforts in thoroughly revising the manuscript.The use of a consistent terminology over the manuscript helps its understanding like the new explanations on the quantified parameters.\nAdditional data have been acquired for many of the figures improving the robustness of the statistical analyses and numbers of studied vessels, embryos and experiments have been specified as requested.\nNew experiments with overexpression of Krit and Fascin nicely complement the data.\nHowever, some parts still need attention:\n-We noticed that in Fig1c only one experiment has been performed to quantify EC number per vessel at 4 dpf which is surprising as many other quantifications have been done at this time point in other figures.\n-In figure 2g, at 3-4 dpf, it is said that aEC shrinks while vEC shortens. Please explains what it means either directly in the figure or in its legend.\n-In figure 4c-f, it is not said whether these are aISV or vISV.\n-In the paragraph line 298-313, it should be mentioned that whereas circumferential actin is more enriched at 2 dpf, diameter constriction is only statistically significant at 3 and 4 dpf.\n-Line 377: please cite the original paper from Glading et al., JCB 2007.\n- Paragraph 422-431 and sup fig 7h: a new Junction/Cortex Ratio is introduced in the quantifications but the term cortex is not clearly defined. Does this \"cortex actin\" actually corresponds to the total intracellular actin content or to only a zone and which one ?\nConcerning the linear fibers that are present in krit1 -/- ECs (Fig 7g), why are they not considered as circumferential actin fibers ? They also cross the cell from one junction to another. We are not necessarily asking for more experiments but for more clarity about what is seen here and its interpretation. Obviously, the wrong location in z of these contractile fibers is key for the krit-/- phenotype and could explain the paradox raised by reviewer 3 concerning the increased pMLC in these cells.\n-Line 484: in Lisowska et al (ref 29), it is shown that in 2D, ROCK1 promotes ventral actin stress fibers connected to focal adhesions and ECM and not “cortical stress fibers” as said here.\n-Line 491: Please refer to fig 7e when talking about transplanted cells exposed to flow, it will facilitate the comparison. In this paragraph, mosaicism should also be commented as it may also impact the acto-myosin organization of the mutant cell and may contribute to the differences between pure homozygous vessels and transplanted populations.\n-Line 539: When talking about the effect of CCM mutant cells on neighbouring ones, Shapeti et al, Nat com 2024 should also be cited.\n-Movies 8 and 18 don’t work.", "reviewer_3": "The authors have carefully addressed my previous concerns on Rock/MLC activation in CCM pathogenesis.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank the reviewer for acknowledging our efforts during the revision process. Please find below our point-by-point response to additional concerns.\n-We noticed that in Fig1c only one experiment has been performed to quantify EC number per vessel at 4 dpf which is surprising as many other quantifications have been done at this time point in other figures.\nWe thank the reviewer for highlighting this point. We agree that including an additional biological replicate at 4 dpf would improve the robustness of the quantification. We have performed an independent repeat experiment and updated the figure, figure legend (lines 1012 – 1013), statistical analyses and source data accordingly. The latest result with additional replicate is consistent with the previously observed increase in EC number between 2 and 4 dpf.\n-In figure 2g, at 3-4 dpf, it is said that aEC shrinks while vEC shortens. Please explains what it means either directly in the figure or in its legend.\nWe thank the reviewer for pointing out the need for clarification. We have revised the legend of Fig. 2g to explicitly define the terminology used:\nShorten: cell length decreases at axial axis; Narrow: cell width decreases at radial axis;\nShrink: reduces in both axial and radial axis.\nThis is revised in lines 1037- 1039.\n-In figure 4c-f, it is not said whether these are aISV or vISV.\nWe clarified the vessel type in the figure legend. Lines 1063-1064.\n-In the paragraph line 298-313, it should be mentioned that whereas circumferential actin is more enriched at 2 dpf, diameter constriction is only statistically significant at 3 and 4 dpf.\nThank you for the suggestion. We have now added this clarification to the corresponding paragraph in the Results section “Notably, circumferential actin enrichment was highest at 2 dpf, whereas statistically significant diameter constriction was detected only at 3 and 4 dpf.”\nLine 318-320.\n-Line 377: please cite the original paper from Glading et al., JCB 2007.\nWe apologise for the incorrect citation and have now cited the original paper from Glading et al., JCB 2007.\n- Paragraph 422-431 and sup fig 7h: a new Junction/Cortex Ratio is introduced in the quantifications but the term cortex is not clearly defined. Does this \"cortex actin\" actually corresponds to the total intracellular actin content or to only a zone and which one ?\nConcerning the linear fibers that are present in krit1 -/- ECs (Fig 7g), why are they not considered as circumferential actin fibers ? They also cross the cell from one junction to another. We are not necessarily asking for more experiments but for more clarity about what is seen here and its interpretation. Obviously, the wrong location in z of these contractile fibers is key for the krit-/- phenotype and could explain the paradox raised by reviewer 3 concerning the increased pMLC in these cells.\nThe “cortex actin” measured in these analyses corresponds to a defined intracellular zone, not total actin content. Specifically, 10 cortical actin regions per vessel were manually selected from regions within the cell interior where EGFP-UCHD signal was present (but not contiguous with junctional pixels). Junctional actin was sampled at cell–cell contacts, where EGFP-UCHD intensity is highest. We agree that the spatial position of contractile fibres is critical, particularly in light of in vitro studies showing that CCM1/CCM2 loss promotes ROCK1-dependent ventral stress fibres. However, as endothelial cells in zebrafish vessels are extremely thin (~1-2 µm) and z-resolution is limited, apical and basal membranes cannot be reliably distinguished in our images; thus, the cortical region integrates both apical and basal surfaces. This definition has now been clearly described in the Methods section in line 739-752.\nIn our in vivo data, the prominent linear actin fibres observed in krit1−/− ECs (Supplementary Fig. 7g) are clearly distinct from the circumferential bundles seen in wild-type cells. They exhibit a diagonal-to-longitudinal orientation along the vessel axis rather than the perpendicular orientation characteristic of circumferential bundles. We now specified this in line 437-439 and added arrows and arrowheads to indicate junctional and cortical actin, respectively, in Supplementary Fig. 7g). Also, most of these longitudinal actin structures do not connect to cell-cell junctions. Together with the reduced junction/cortex actin ratio and altered myosin II distribution, these observations indicate a redistribution of contractile structures away from junctional-circumferential actin scaffolds toward a more homogeneous pattern. This pattern is consistent with the notion that increased pMLC in krit1−/− cells does not translate into effective circumferential contraction because contractility is mis-localized and reoriented.\n-Line 484: in Lisowska et al (ref 29), it is shown that in 2D, ROCK1 promotes ventral actin stress fibers connected to focal adhesions and ECM and not “cortical stress fibers” as said here.\nWe thank the reviewer for catching this inaccuracy and we apologize for the inappropriate phrasing. The text has now been corrected to refer specifically to “ventral actin stress fibres,” in line 504.\n-Line 491: Please refer to fig 7e when talking about transplanted cells exposed to flow, it will facilitate the comparison. In this paragraph, mosaicism should also be commented as it may also impact the acto-myosin organization of the mutant cell and may contribute to the differences between pure homozygous vessels and transplanted populations.\nWe thank the reviewer for this helpful suggestion. We now explicitly refer to Fig. 7e when describing transplanted krit1−/− cells exposed to flow. We also acknowledge that mosaicism may influence the actomyosin organization of transplanted mutant cells by placing them in a wild-type mechanical environment, and this clarification has now been incorporated into the revised manuscript in line 517-520.\n-Line 539: When talking about the effect of CCM mutant cells on neighbouring ones, Shapeti et al, Nat com 2024 should also be cited.\nThank you for pointing out the relevance of the work of Shapeti et al. on the influence of CCM mutant cells on neighbouring wildtype cells. We have now cited this work in the manuscript (line 566).\n-Movies 8 and 18 don’t work.\nWe apologise for this technical error (they played on our computer). We have remade .avi files from .tif files for Movies 8 and 18 and hope that the reviewers can now open and view these movies."}]

Decision letter