Combined forces of hydrostatic pressure and actin polymerization drive endothelial tip cell migration and sprouting angiogenesis

Cell migration is a key process in the shaping and formation of tissues. During sprouting angiogenesis, endothelial tip cells invade avascular tissues by generating actomyosin-dependent forces that drive cell migration and vascular expansion. Surprisingly, ECs can still invade if actin polymerization is inhibited. In this study, we show that endothelial tip cells employ an alternative mechanism of cell migration that is dependent on Aquaporin (Aqp)-mediated water inflow and increase in hydrostatic pressure. In the zebrafish, ECs express aqp1a.1 and aqp8a.1 in newly formed vascular sprouts in a VEGFR2-dependent manner. Aqp1a.1 and Aqp8a.1 loss-of-function studies show an impairment in intersegmental vessels formation because of a decreased capacity of tip cells to increase their cytoplasmic volume and generate membrane protrusions, leading to delayed tip cell emergence from the dorsal aorta and slower migration. Further inhibition of actin polymerization resulted in a greater decrease in sprouting angiogenesis, indicating that ECs employ two mechanisms for robust cell migration in vivo. Our study highlights an important role of hydrostatic pressure in tissue morphogenesis.


INTRODUCTION
The blood vascular system is a dynamic, multicellular tissue that adapts to the metabolic demands of development, growth and homeostasis by altering its pattern and morphology.Macroscopic transformation in the vascular network is achieved by microscopic changes in endothelial cell (EC) behaviours.For example, vascular expansion through sprouting angiogenesis requires the coordination of EC migration, proliferation, anastomosis, lumen formation and cell rearrangements, all of which require specific cell shape changes.At the initial phase of sprouting angiogenesis, endothelial tip cells generate numerous membrane protrusions such as filopodia and lamellipodia that drive migration and anastomosis (Figueiredo et al., 2021;Gerhardt et al., 2003;Phng et al., 2013;Sauteur et al., 2017).During tubulogenesis, apical membranes deform by generating inverse blebs to expand lumens so that blood can be transported efficiently through the blood vascular network (Gebala et al., 2016).However, following vessel perfusion, ECs become less dynamic and instead develop resistance to the deforming forces of blood pressure to maintain vessel morphology (Kondrychyn et al., 2020).EC shape is therefore dynamic and changes depending on the morphogenetic state of vascular development.
It is well established that localized remodelling of actin cytoskeleton and non-myosin II contractility are instrumental in driving cell shape changes during tissue morphogenesis (Clarke and Martin, 2021;Munjal and Lecuit, 2014;Murrell et al., 2015).More recently, there is an accumulating body of work implicating a role of water flow and the resulting changes in hydrostatic pressure as another mechanical mechanism of cell shape change (Choudhury et al., 2022;Chugh et al., 2022;Li et al., 2020).For example, in the osmotic engine model of cell migration, water inflow at the leading edge and outflow at the rear leads to forward translocation of tumour cells in confined microenvironment (Stroka et al., 2014).Additionally, hydrostatic pressure from the extracellular environment can drive tissue morphogenesis, as demonstrated during mouse blastocyst formation (Chan et al., 2019;Dumortier et al., 2019), development of the otic vesicle in the zebrafish (Mosaliganti et al., 2019) and blood vessel lumen expansion (Gebala et al., 2016).Hydrostatic pressure can therefore generate sufficient forces to shape tissues and organs to their proper form and size during development.
We have previously discovered that EC migration persists after the inhibition of actin polymerization and in the absence of filopodia to generate new blood vessels in the zebrafish (Phng et al., 2013).This observation suggests the existence of an alternative mechanism of migration independent of actin polymerization.In this study, we sought to determine whether hydrostatic pressure regulates EC migration during sprouting angiogenesis in the zebrafish by investigating the function of Aquaporins (Aqp), which are transmembrane water channels that increase water permeability of cell membranes to promote transcellular water flow (Day et al., 2014;Farinas et al., 1997;Kozono et al., 2002;Preston et al., 1992).We discovered that ECs of newly formed intersegmental vessels (ISV) express aqp1a.1 and aqp8a.1 mRNA, and observed the enrichment of Aqp1a.1 and Aqp8a.1 proteins in the leading edge of migrating tip cells.Detailed single cell analyses showed that endothelial tip cells lacking aqp1a.1 and aqp8a.1 expression have reduced cell volume, membrane protrusions and migration capacity.As a result, there is defective sprouting angiogenesis and ISV formation in aqp1a.1;aqp8a.1 double mutant zebrafish.Notably, when actin polymerization is inhibited in aqp1a.1;aqp8a.1 double mutant zebrafish, there is a greater impairment in EC migration and sprouting angiogenesis, demonstrating the additive function of actin polymerization and hydrostatic pressure (that is created by water inflow) in generating membrane protrusions to drive EC migration in vivo.
While aqp1a.1 is widely expressed in blood vessels of the head and trunk (dorsal aorta (DA), caudal artery (CA), ISVs, dorsal longitudinal anastomotic vessels (DLAV) and caudal vein plexus) at 30 hpf, aqp8a.1 expression is absent in cerebral blood vessels and is restricted to the DA, CA and the ventral regions of ISVs in the trunk.After 1 dpf, aqp8a.1 expression expands to the entire ISV and DLAV.Additionally, the expression of aqp1a.1 and aqp8a.1 gradually decreases in the DA and CA so that both are absent by 4 dpf.Single cell RNA sequencing (scRNAseq) of ECs isolated at 34 hpf and 3 dpf further confirmed the endothelial expression of aqp1a.1 and aqp8a.1 mRNA (Fig. S2 A -D), revealed differential expression of aqp1a.1 and aqp8a.1 mRNA in distinct endothelial clusters and highlighted higher aqp1a.1 transcript expression in all endothelial subtypes compared to aqp8a.1 (Fig. S2 E -H).
We next examined the expression pattern of aqp1a.1 and aqp8a.1 at the beginning of sprouting angiogenesis at higher resolution using RNAscope and confocal microscopy.At 20 hpf, aqp1a.1 mRNA is highly expressed in the DA with little expression in the PCV (Fig. 1A, C and Fig. S3).Closer inspection uncovered heterogeneous expression in the DA, with higher expression in ECs that would be specified as tip cells (Fig. 1A, C and Fig. S3B).Indeed, tip cells of newly formed vascular sprouts express higher levels of aqp1a.1 compared to adjacent ECs in the DA at 20 hpf (Fig. 1A -C, Fig. S3B) and the majority of ISVs (61%) display higher aqp1a.1 expression in tip cells than in trailing stalk cells at 22 hpf (Fig. 1G).Although we previously observed aqp8a.1 mRNA in the DA at 30 hpf (Fig. S1), its expression here is largely absent at 20 hpf (Fig. 1B, D and Fig. S3C).At 22hpf, aqp8a.1 mRNA expression in the DA increases (Fig. 1E and F).
Notch signalling was inhibited using the gamma-secretase inhibitor, Dibenzazepine (DBZ), which prevents proteolytic cleavage and consequently, activation of the Notch receptor (Groth et al., 2010).Treatment of embryos with DBZ from 20 hpf for 6 and 24 hours significantly decreased the expression of hey1 and hey2, canonical targets of Notch signalling (Fig. 1J), confirming the effectiveness of the drug treatment in our experiments.When compared to DMSO-treated embryos, 6-hour treatment with DBZ did not cause a significant change in aqp1a.1 and aqp8a.1 expression levels (Fig. 1J).Longer treatment with DBZ for 24 hours, however, increased aqp8a.1 expression by 98% (p < 0.0001) while aqp1a.1 expression was relatively unchanged (Fig. 1J).These findings suggest that Notch signalling inhibits aqp8a.1, but not aqp1.a,expression.

Aqp1a.1 and Aqp8a.1 proteins are enriched at the leading front of migrating tip cells
To gain a better understanding of where within the cell Aquaporin protein functions, we generated stable Tg(fli1ep:aqp1a.1-mEmerald)rk30 and Tg(fli1ep:aqp8a.1-mEmerald)rk31 zebrafish lines in which ECs express Aqp1a.1 or Aqp8a.1 tagged with mEmerald, respectively.At 25 hpf, we observed an accumulation of Aqp1a.1 (Fig. 2A) and Aqp8a.1 (Fig. 2B) in mCherryCAAX-positive membrane clusters in the cytoplasm and filopodia of tip cells.Interestingly, many of the Aqp1a.1orAqp8a.1-positivemembrane clusters are located at the migrating front of the tip cell.Timelapse imaging capturing the process of ISV formation between 24 to 30 hpf further showed sustained enrichment of Aqp1a.1 and Aqp8a.1 in clusters at the front of the tip cell as well as in filopodia (Fig. 2C and D, Movie S1 and S2), suggesting that Aqp1a.1 and Aqp8a.1 may promote localized water flux at the migrating edge of tip cells.

Loss of Aqp1a.1 and Aqp8a.1 function leads to defective trunk blood vessel formation
We subsequently proceeded to generate zebrafish aqp1a.1 and aqp8a.1 mutants to investigate the function of Aquaporin-mediated water flow in blood vessel development.Using the CRISPR/Cas9 genome editing technique, we targeted the first exons of aqp1a.1 and aqp8a.1 genes.The CRISPRgenerating allele aqp1a.1 rk28 carries a 14-bp deletion and an 8-bp insertion in the 5' end of the gene leading to a premature termination codon at amino acid 76 after 1 missense amino acid (Fig. S4A-B).The aqp8a.1 rk29 allele carries a 4-bp deletion in the 5' end of the gene that leads to a frameshift after T15 and premature termination codon at amino acid 73 after 58 missense amino acids (Fig. S5A-B).Premature termination codons are often more deleterious than missense mutations because they result in the loss of protein expression.Nonsense-mediated mRNA decay is a qualitycontrol mechanism that selectively degrades mRNAs harboring premature termination codons (Chang et al., 2007).Using whole-mount in situ hybridization and qPCR analysis, we demonstrated that aqp1a.1 and aqp8a.1 mRNA expression is significantly decreased in aqp1a.1 rk28/rk28 (Fig. S4C-D) and aqp8a.1 rk29/rk29 (Fig. S5C-D) zebrafish embryos, respectively, suggesting a rapid degradation of mutant mRNAs and as a result, the loss of Aquaporin protein expression.
Comparison of ISV length in aqp1a.1 rk28/rk28 , aqp8a.1 rk29/rk29 and aqp1a.1 rk28/rk28 ;aqp8a.1 rk29/rk29 embryos shows that the loss of aqp1a.1 leads to a greater decrease in ISV length than the loss of aqp8a.1 (Fig. 3E, Fig. S6I and J), and that the loss of both aqp1a.1 and aqp8a.1 resulted in the greatest reduction.These observations indicate an additive effect of Aqp1a.1 and Aqp8a.1 function in blood vessel development and implicate Aqp1a.1 as the more dominant Aquaporin protein in EC function, in line with the higher expression of aqp1a.1 mRNA in tip cells.
We next examined the embryos at 3 dpf, a stage when arterial ISVs (aISVs) and venous (vISVs) are fully established and lumenized, connecting the DLAV to either the DA or PCV (Fig. 3F and G), respectively.Such fully established ISVs are significantly reduced in embryos when aqp1a.1, aqp8a.1 or both aqp1a.1 and aqp8a.1 are deleted.Instead, these embryos display an increased number of aISVs and vISVs that fail to establish a connection to the DLAV, DA or PCV (Fig. 3H -J).Quantification shows that while 19.1% of wildtype embryos display at least 1 incomplete ISV per embryo, this increased to 65.8% in aqp8a.1 rk29/rk29 embryos, 83.3% in aqp1a.1 rk28/rk28 embryos and 92.2% in aqp1a.1 rk28/rk28 ;aqp8a.1 rk29/rk29 embryos (Fig. 3K).The number of incomplete ISVs per embryo in aquaporin mutants was variable, between 1 to 6, 1 to 16 and 1 to 22 incomplete ISVs in aqp8a.1 rk29/rk29 , aqp1a.1 rk28/rk28 and aqp1a.1 rk28/rk28 ;aqp8a.1 rk29/rk29 embryos, respectively (Fig. S6A-D).We also found that incomplete ISVs appear with higher frequency in the zebrafish trunk (ISV number 6 to 15) rather than in the tail (ISV number 16 to 27, Fig. S6E).Detailed analysis revealed that most of the incomplete ISVs are vISVs that are connected to the PCV but not the DLAV (Class I, 75.2%), followed by the absence of aISV or vISV (Class II, 15.8%, Fig. 3L and Fig. S7A and B).A small fraction of the incompletely formed vessels are aISVs that are not connected to the DA (Class IV, 2.2%, Fig. S7D) or DLAV (Class V, 2.9%, Fig. S7E), or vISVs that are not connected to the DLAV (Class VII, 2.68%, Fig. S7F).At some regions, there is an aISV that is not connected to the DA and a vISV that is not connected to the DLAV (Class III, 1.2%, Fig. S7C).
In summary, our analyses suggest that in the absence of Aqp1a.1 and/or Aqp8a.1 function, there is an initial delay in ISV formation during primary sprouting angiogenesis.By 3 dpf, some ISVs are completely formed (Fig. S8K and M), connecting the DA or PCV to the DLAV.However, the number of completely formed ISVs is significantly decreased, with the loss of Aqp1a.1 having a more severe impact compared to the loss of Aqp8a.1, and the combined loss of Aqp1a.1 or Aqp8a.1 having a greater deleterious effect.
Furthermore, in some aqp1a.1 rk28/rk28 ;aqp8a.1 rk29/rk29 embryos, tip cells retract whereby membrane protrusions do not extend dorsally in the direction of migration but withdraw into the DA so that a primary ISV does not form at this location (Fig. 4C, Movie S4).These observations indicate that tip cell protrusion from the DA is significantly delayed in the absence of Aqp1a.1 and Aqp8a.1 function.
In conclusion, Aqp1a.1 and Aqp8a.1 are required for sprouting angiogenesis by promoting endothelial tip cell emergence from the DA, the formation of membrane protrusions and endothelial cell migration.

Tip cell volume regulation depends on Aquaporin-mediated water influx
Aquaporin channels permit bidirectional flow of water across the plasma membrane, with the direction of flow determined by the osmotic gradient between the extracellular environment and cell cytoplasm (Agre et al., 2002).We next sought to determine whether water flows in or out of tip cells during sprouting angiogenesis.We hypothesize that water flux across the cell membrane can lead to changes in tip cell volume, with water influx increasing the volume while outflow decreases.To quantify tip cell volume, we injected a plasmid encoding kdrl:mEmerald transgene into 1-cell stage wildtype or aqp1a.1 rk28/rk28 ;aqp8a.1 rk29/rk29 embryos to label ECs in a mosaic manner.mEmerald-positive tip cells were imaged and their volume measured at 24 to 25 hpf.
Wildtype ECs are on average 946.6 ± 299.8 µm 3 in size (Fig. 5A and C).In the absence of both Aqp1a.1 and Aqp8a.1 function (Fig. 5B), tip cell volume significantly decreased by about 24% to 718.4 ± 308.5 µm 3 (p = 0.0176, Fig. 5C) when compared to wildtype tip cells.This finding supports water influx as the direction of water flow in tip cells during migration, and that this is mediated through Aqp1a.1 and Aqp8a.1.
It has been proposed that water influx through Aquaporin increases the diffusion of actin monomers and increases actin polymerisation (Loitto et al., 2007;Papadopoulos et al., 2008) .
Given that filopodia formation is dependent on actin polymerization (Mallavarapu and Mitchison, 1999;Ridley, 2011), we examined whether actin polymerization is perturbed in the absence of Aqp1a.1 and Aqp8a.1 by quantifying the growth rate of actin bundles in tip cell filopodia by tracking Lifeact-mCherry signal in the filopodia.In wildtype embryos, the average growth rate of actin is 0.054 ± 0.015 µm per second whereas in aqp1a.1 rk28/rk28 ;aqp8a.1 rk29/rk29 embryos, the growth rate is significant decreased by 47% to 0.028 ± 0.010 µm per second (p < 0.0001, Fig. 5D).
In summary, our results demonstrate a role of endothelial Aquaporins in increasing water influx and as a consequence, endothelial tip cell volume and hydrostatic pressure, as well as in promoting actin polymerization.

Additive effects of actin polymerization and water influx in driving EC migration and sprouting angiogenesis
We have previously observed that ECs are still able to migrate to generate ISVs after the inhibition of actin polymerization (Phng et al., 2013).In these experiments, embryos were treated with a low concentration of Latrunculin B (Lat.B), an inhibitor of actin polymerization, that resulted in the suppression of filopodia formation.Under such conditions, ECs can generate small membrane protrusions and migrate more slowly in a directed manner to generate ISVs.How ECs are still able to migrate after the inhibition of actin polymerization was unclear.As our current study demonstrates a role of water inflow and hydrostatic pressure as another mechanism of cell migration, we hypothesize that ECs employ water influx to migrate when actin polymerization is compromised and that the depletion of both water influx and actin polymerization will result in a greater inhibition of EC migration.
To test this hypothesis, we examined ISV development in embryos with decreased actin polymerization and water inflow by treating aqp1a.1 rk28/rk28 ;aqp8a.1 rk29/rk29 embryos with Lat.B.
These results therefore demonstrate additive functions of actin polymerization and water influx in driving EC migration during sprouting angiogenesis (Fig. 6F).

DISCUSSION
In this study, we demonstrate that endothelial tip cells utilize two mechanisms of migration concurrently during sprouting angiogenesis in the zebrafish -actin polymerization and hydrostatic pressure.The increase in hydrostatic pressure in endothelial tip cells is generated by Aqp1a.1-andAqp8a.1-mediatedwater inflow.By performing detailed expression analyses, we showed that aqp1a.1 and aqp8a.1 are differentially expressed in newly formed vascular sprouts.Aqp1a.1 is expressed earlier in the DA than aqp8a.1 during development and in ECs destined to be tip cells.
In newly formed ISVs, endothelial tip cells express higher aqp1a.1 mRNA levels while stalk cells express higher aqp8a.1 mRNA levels.In zebrafish harbouring mutations in both aqp1a.1 and aqp8a.1,there is delayed tip cell protrusion from the DA, decreased tip cell migration and increased occurrence of truncated ISVs at 3 dpf.We further discovered that aqp1a.1 and aqp8a.1 expression is upregulated by VEGFR2 activity, proposing that VEGFA-VEGFR2 signalling employs Aquaporin function to drive sprouting angiogenesis.
Several lines of evidence implicate Aquaporins in facilitating water flow into endothelial tip cells during sprouting angiogenesis.First, timelapse imaging showed enriched localization of Aqp1a.1 and Aqp8a.1 proteins at the migrating front of tip cells, suggesting that water flow occurs locally at the leading edge of the cell.Secondly, single cell analyses revealed Aqp1a.1-andAqp8a.1-deficienttip cells have decreased cell volume, impaired expansion of cell membranes at the leading edge and decreased generation of stable cell protrusions.These observations implicate water flow into tip cells, leading to increased cell volume.Due to the incompressible property of water within an enclosed compartment such as the cell, water influx will lead to a rise in hydrostatic pressure.At the leading edge of tip cells, increased hydrostatic pressure would expand and deform membranes to generate protrusions.Additionally, water influx can reduce cytoplasmic viscosity and increase spacing between the plasma membrane and the underlying actin cytoskeleton, promoting actin monomer diffusion, actin polymerization and the generation of membrane protrusions to facilitate cell migration (Boer et al., 2023;Loitto et al., 2009).
A key finding in this study is the demonstration that endothelial tip cells employ hydrostatic pressure as a second mechanism of cell migration in vivo.In a previous study, we discovered that ECs continue to migrate when actin polymerization is inhibited and in the absence of filopodia (Phng et al., 2013).Here, we show that hydrostatic pressure is the driving force for EC migration in the absence of actin-based force generation.When both mechanisms are perturbed, there is a greater impairment in EC migration (Fig. 6D and E).This finding highlights the importance of hydrostatic pressure as an additional mechanism that reinforces actin-based cell shape changes and motility to ensure robust migration and formation of new blood vessels in complex threedimensional environments.Such dual mode migration is advantageous when blood vessels develop in physically confined spaces such as in the zebrafish trunk, where ECs must emerge from the DA and migrate within somite boundaries and over tissues such as the notochord and neural tube to form ISVs.Such dependence on water flow in cell migration under confined microenvironment has been reported in human MDA-MB-231 breast cancer cells and mouse S180 sarcoma cells, which show cell migration persistence in narrow channels after inhibition of actin polymerization or myosin II-mediated contractility (Stroka et al., 2014).In this context, AQP5 mediates water flow and the decrease or increase in AQP5 expression suppresses or enhances, respectively, cancer cell migration (Chae et al., 2008;Jung et al., 2011;Stroka et al., 2014).
Our findings on the endothelial function of zebrafish Aqp1a.1, which is homologous to mammalian AQP1, corroborate previous studies demonstrating a function of AQP1 in promoting cell migration.In tumour bearing AQP1-null mice, there is reduced tumour angiogenesis and AQP1-deficient ECs display reduced migration in an in vitro wound healing assay (Maltaneri et al., 2020;Saadoun et al., 2005).In chick, AQP1 expression levels affect neural crest cell migration speed and direction by regulating filopodia length and stability (McLennan et al., 2019).The regulation of membrane protrusions has also been demonstrated for other Aquaporin proteins.For example, AQP9 weakens membrane-cytoskeleton anchorage and promote formation of membrane protrusions such as filopodia and blebs (Karlsson et al., 2013;Loitto et al., 2007).We also demonstrate for the first time a role of Aqp8a.1 in promoting EC migration and sprouting angiogenesis in the zebrafish.However, its mammalian ortholog, AQP8, was shown to inhibit migration of colorectal cancer cell lines in vitro (Wu et al., 2017), suggesting cell-and contextdependent function of AQP8 in the regulation of cell migration.
A central determinant of water movement is the osmotic gradient across the membrane, with water flowing in the direction of higher solute concentration.Given a crucial role of water flow in regulating cell volume, shape and migration, it is also important to understand the function and distribution of ion channels, exchangers and transporters and water channels that generate an osmotic gradient across the cell.This is reflected in a growing number of studies demonstrating a role of ion transporters in cell migration.In neutrophils, the increase in cell volume and potentiation of cell migration depend on the sodium-proton exchanger 1 (NHE1) and the chloridebicarbonate exchanger 2 (AE2) (Nagy et al., 2023) .In T cells, chemokine-induced migration depends on ion influx via SLC12A1, and water influx via AQP3 (Boer et al., 2023).In tumour cells migrating in narrow channels, the establishment of a polarized distribution of Na + /H + pumps and Aquaporin proteins in cell membranes is required for net inflow of ions and water at the leading edge and net outflow at the trailing edge (Stroka et al., 2014).We have also observed enrichment of Aqp1a.1 and Aqp8a.1 proteins in ECs, at the leading edge of migrating endothelial tip cells as well as apical membranes during lumen formation (not shown).This leads to the question of how Aquaporin localisation is regulated as this will determine the site of increased water flow and hydrostatic pressure.The rapid changes in subcellular localization of mammalian Aquaporins upon stimulation occurs mainly via trafficking to and from the plasma membrane (Markou et al., 2022), and can lead to changes in the amount of Aquaporin protein in the plasma membrane.The regulation of AQP subcellular relocalization via calmodulin-and/or phosphorylation-dependent mechanisms has been implicated for AQP0-5 and AQP7-9 (Markou et al., 2022).For example, rapid translocation of AQP1 to plasma membrane upon a hypotonic stimulus is dependent on calmodulin activation and phosphorylation by protein kinases C (PKC) (Conner et al., 2012).Phosphorylation of AQP2 by protein kinase A (PKA) is required for vasopressin-stimulated relocalization of AQP2 from storage vesicles to the plasma membrane (Noda and Sasaki, 2005).Subcellular localization of AQP8 in hepatocytes is also regulated by PKA and PI3K signalling (Gradilone et al., 2005(Gradilone et al., , 2003)).In mouse neutrophils, exposure to a chemotactic gradient caused translocation of AQP9 to the leading edge of the cell that is mediated by PKC-dependent phosphorylation (Karlsson et al., 2011).Further work is needed to elucidate the mechanism of Aqp1a.1 and Aqp8a.1 protein distribution in ECs and which ion transporters are expressed in ECs to regulate the ion movement and cell migration.
AQP8 is permeable to ammonia (Jahn et al., 2004;Liu et al., 2006) and is present in the inner mitochondrial membrane of different cells (Calamita et al., 2005), where it is suggested to mediate ammonia transport rather than water fluxes (Soria et al., 2010).Among the three Aqp8 paralogs in teleost, Aqp8a.1 and Aqp8a.2 are also permeable to urea (Tingaud-Sequeira et al., 2010) and Aqp8b was shown to act mainly as a mitochondrial H2O2 channel (peroxiporin) in activated marine and freshwater teleost spermatozoa (Chauvigné et al., 2021(Chauvigné et al., , 2015)).Nevertheless, because water movement flows up an osmotic gradient, increased entry of ions into the cell through Aquaporins will also trigger water inflow.
In summary, our study highlights the role of water influx and hydrostatic pressure as another force-generating mechanism utilized by cells to build tissues during animal development.
We demonstrate that endothelial tip cells employ both actin polymerization and hydrostatic pressure for robust sprouting angiogenesis.As morphogenetic events are governed by a coordination of cell shape changes, cell migration and rearrangements, we envision that the shaping and formation of other tissues will also depend on Aquaporin function and changes in hydrostatic pressure.
To inhibit pigmentation in embryos older than 24 hpf, 0.003% N-Phenylthiourea (Sigma-Aldrich) in E3 medium was used.All animal experiments were approved by the Institutional Animal Care and Use Committee at RIKEN Kobe Branch (IACUC).

Plasmids and oligonucleotides.
All DNA constructs used in this study were generated by using In-Fusion HD Cloning kit (Takara Bio Inc.).Plasmid mEmerald-N1 was a gift from Michael Davidson (Addgene plasmid #53976).
Plasmids pDsChLexOG (Emelyanov and Parinov, 2008) containing terminal cis-required sequences of the Ds transposon from maize was a generous gift from Sergei Parinov.A detailed information regarding plasmids and primers used in this study can be found in Supplementary Tables 1 and 2, respectively.
Cloning of aqp1a.1 and aqp8a.1 genes.Total RNA was isolated from 1-day old zebrafish embryos with TRI Reagent using Direct-zol TM RNA MicroPrep kit (Zymo Research) according to the manufacturer's protocol.The first-strand cDNA was synthesized from 1 µg of a total RNA by oligo(dT) priming using the SuperScript III First-Strand synthesis system (Invitrogen) according to the manufacturer's protocol.Amplification of cDNAs was performed using a high-fidelity KOD-Plus-Neo DNA polymerase (Toyobo, Japan) and resulting PCR products were cloned using NEB ® PCR Cloning kit (New England BioLabs).Positive clones and plasmids were verified by DNA sequencing.

In vivo cell volume analysis.
Single-cell labelling of ECs in ISVs was achieved by mosaic expression of pDs-kdrl:mEmerald plasmid in wild type or aqp1a.1 rk28/rk28 ;aqp8a.1 rk29/rk29 mutant Tg(fli1:Lifeact-mCherry) ncv7 transgenic embryos.Embryos were imaged at 25-26 hpf with an Olympus UPLSAPO 60x/NA 1.2 water immersion objective with optical Z planes interval of 0.26 μm.Cell volume was measured with Huygens Essential 22.10 software (Scientific Volume Imaging B.V.) using object analyzer tool with manual adjustment of threshold.
The gRNAs were in vitro transcribed using MEGAScript SP6 kit (Invitrogen), DNase treated and purified with RNA Clean and Concentrator kit (Zymo Research).The gRNAs were qualitychecked by running 1 µg of the product on a 2% TBE-agarose gel.Cas9/gRNA RNP complex was assembled just prior to injection and after 5 min incubation at room temperature 200 pg of Cas9 protein (Invitrogen) and 200 pg of gRNAs (100 pg of each, aqp1a.1 gRNA and aqp8a.1 gRNA) were co-injected into one-cell stage Tg(fli1:myr-mCherry) ncv1 embryos.Such dose produced over 70% embryos survival post-injection showed CRISPR/Cas9-induced somatic aqp1a.1 and aqp8a.1 gene mutations.F0 founders were identified by outcrossing CRISPR/Cas9-injected fish with wild type fish and screening the offspring for mutations at 2 dpf using Sanger sequencing.

Quantitative real-time PCR.
Total RNA from whole embryos was isolated with TRI reagent using Direct-zol RNA MicroPrep kit (Zymo Research) according to the manufacturer's protocol, including on-column DNA digestion.RNA was quantified using a NanoDrop 1000 spectrophotometer (ThermoFisher Scientific, USA).cDNA was synthesized from 300 ng of purified RNA using LunaScript RT SuperMix kit (New England BioLabs) according to the manufacturer's protocol.Amplification of target cDNA was performed in technical triplicate of three biological replicates using the SYBR green methods.Each qPCR reaction mixture contained 5 µL 2x Luna Universal qPCR master mix (New England BioLabs), 1 µL cDNA (2-fold dilution), 0.2 µL antarctic thermolabile UDG (New England BioLabs) and 250 nM each primer to a final volume of 10 µL.Reactions were run in 384well plates (Applied Biosystems) using the QuantStudio 5 Real-Time PCR system (Applied Biosystems) with the following thermal cycling conditions: initial UDG treatment at 25 0 C for 2 minutes, UDG inactivation at 50 0 C for 5 min and denaturation at 95 0 C for 1 minutes, followed by 45 cycles of 15 sec at 95 0 C, 30 sec at 60 0 C with a plate read at the end of the extension step.
Control reactions included a no-template control (NTC) and a no-reverse transcriptase control (NRT).Dissociation analysis of the PCR products was performed by running a gradient from 60 to 95 0 C to confirm the presence of a single PCR product.Amplification data was analyzed using QuantStudio Design and Analysis software v1.5.3 (Applied Biosystems) and relative fold change was calculated using Pfaffl method (Pfaffl, 2001) and normalized to gapdh expression (as an internal control).Primers are listed in Supplementary Table 2. Two embryos were used in each biological replicate to generate an average value that was used to calculate the final mean ± SD from two or three independent experiments.
RNA in situ hybridization.
RNAscope in situ hybridization.RNAscope ® in situ hybridization was conducted by using RNAscope Multiplex Fluorescent Reagent kit v2 (Advanced Cell Diagnostics).We adapted manufacture's protocol designed for samples mounted on slides and protocol developed for wholemount embryo samples (Gross-Thebing et al., 2014).For each experimental point, 5 embryos were processed in one 1.5 ml Eppendorf tube.Briefly, 20 and 22 hpf old embryos were manually dechorionated and fixed in freshly prepared 4% methanol-free PFA in PBS for 1 hour at room temperature.After fixation embryos were washed three times in PBS containing 0.01% Tween-20 (PBST), dehydrated stepwise in 5-min washes in a series of increasing methanol concentrations (25%, 50%, 75%, 100%) in PBS and then stored in 100% methanol at -20 0 C before use them for hybridization.The methanol-stored embryos were incubated with 5% H2O2 in methanol for 20 min at room temperature and then rehydrated stepwise in 5-min washes in series of decreasing methanol concentrations (75%, 50%, 25%) in PBS, followed by three times 5-min washes in PBST.Chromogenic in situ hybridization.Chromogenic in situ hybridization was conducted according to standard protocol (Thisse and Thisse, 2007) with minor modifications.For each experimental point, 10 embryos were processed in one 1.5 ml Eppendorf tube.Briefly, 30, 48, 72 and 96 hpf old embryos were manually dechorionated and fixed in freshly prepared 4% methanol-free PFA in PBS (pH 7.4) at 4 0 C overnight.After fixation embryos were washed three times in PBS containing 0.1% Tween-20 (PBST), dehydrated stepwise in 5-min washes in a series of increasing methanol concentrations (25%, 50%, 75%, 100%) in PBS and then stored in 100% methanol at -20 0 C before use them for hybridization.The methanol-stored embryos were rehydrated stepwise in 5-min washes in series of decreasing methanol concentrations (75%, 50%, 25%) in PBS followed by three times 5-min washes in PBST, and permeabilized 20 min with 10 µg/mL proteinase K (ThermoFisher Scientific).Hybridization was carried out in buffer [50% formamide, 5x SSC, 50 background) and (ii) ratio, R=CTCF of tip cell/CTCF of stalk cell.We consider that if R <0.9, expression is lower in tip cell, if R=0.9-1.1, expression is equal in tip and stalk cells, if R>1.1, expression is higher in tip cell.

Chemical treatment.
Latrunculin B (Merck Millipore) was dissolved in DMSO to 1 mg/ml and stored at -20 0 C.
Dibenzazepine (YO-01027) (Selleck Chemicals) was prepared as 10 mM solution in DMSO and stored at -80 0 C. Ki8751 (Selleck Chemicals) was prepared as 5 mM solution in DMSO and stored at -80 0 C.All compounds were diluted to the desired concentration in E3 medium.

Statistical analysis.
Statistical analysis was performed using Prism software version 10.2.0 (GraphPad).The variance between the mean values of two groups was evaluated using the unpaired Student's t-test.For assessment of more than three groups, we used one-way analysis of variance (ANOVA) test.A P value of <0.05 was considered statistically significant.Statistic details can be found in each figure legend.
Single-cell RNA sequencing.
ECs were isolated from Tg(kdrl:EGFP) s843 transgenic embryos.Cell sorting was carried out with FACSAriaII Cell Sorter (BD Bioscience).Single-cell suspension was loaded into the 10X Chromium system and cDNA libraries were constructed using Chromium Next GEM Single Cell 3′ GEM, Library and Gel Bead Kit v2 (10X Genomics) according to manufacturer's protocol.
Library was sequenced on the Illumina HiSeq 1500 Sequencer (Illumina, USA).Cell Ranger v2.1 was used to de-multiplex raw base call (BCL) files generated by Illumina sequencers into FASTQ files, perform the alignment, barcode counting, and UMI counting.Sequencing reads were mapped to the zebrafish genome assembly (GRCz11, Ensembl release 92).Further analyses were performed using Seurat package49 (version 4.3.0) in R software (https://www.r-project.org).The expression matrices were first filtered by keeping genes that are expressed in a minimum of 3 cells and cells that expressed a minimum of 200 genes for downstream analysis.The data were then normalized using NormalizeData function which normalizes the gene expression for each cell by the total expression counts.To correct for batch effect between the data from the two-time points, the rpca method in the Seurat package was applied to integrate the data.The top 2000 variable genes identified using the vst method in FindVariableFeatures function were used for principal component analysis in RunPCA function, and the first 30 principal components were used for visualization analysis with Uniform Manifold Approximation and Projection (UMAP) method, and in FindNeighbors function analysis.The cell clustering resolution was set at 0.5 in FindClusters function.6 hours treatment 24 hours treatment a q p 1 a . 1 h e y 1 h e y 2 a q p 8 a . 1

Relative expression
a q p 1 a . 1 h e y 1 h e y 2 a q p 8 a . 1 a q p 1 a . 1 r k 2 8 /r k 2 8 a q p 8 a . 1 r k 2 9 /r k 2 9 a q p 1 a . 1 r k 2 8 /r k 2 ; a q p 8 a .; a q p 8 a . 1 + /r k 2 9 a q p 1 a . 1 rk 2 8 /r k 2 8 ; a q p 8 a . 1 rk 2 9 /r k 2 9 a q p 1 a . 1 + /+ ; a q p 8 a . 1 + /+ Av. no. of filopodia / 100µm Length of filopodia (µm) A B C K J I H G F a q p 1 a . 1 + /r k 2 8 ; a q p 8 a . 1 + /r k 2 9 a q p 1 a . 1 rk 2 8 /r k 2 8 ; a q p 8 a . 1 rk 2 9 /r k 2 9 a q p 1 a . 1 + /+ ; a q p 8 a . 1 + /+ aqp1a.1 rk28/rk28 aqp1a.1 +/rk28 ;aqp8a.1 +/rk29 aqp1a.1 rk28/rk28 ; aqp8a.1 rk29/rk29 a q p 1 a . 1 + /r k 2 8 ; a q p 8 a . 1 + /r k 2 9 a q p 1 a . 1 rk 2 8 /r k 2 8 were permeabilized in 2 drops of RNAscope ® Protease III for 15 min, rinsed three times with PBST and hybridized with RNAscope target probes overnight at 40 0 C in water bath.After hybridization probes were recovered and embryos were washed three times with 0.2x SSCT [0.01%Tween-20 in SSC] at room temperature, re-fixed with 4% PFA for 10 min, and washed three times with 0.2x SSCT.For RNA detection, the embryos were first hybridized with three different amplifier solution for 30 min (Amp1, and Amp2) and 15 min (Amp3) in a water bath at 40 0 C.After each hybridization step, the embryos were washed three times with 0.2x SSCT for 15 min at room temperature.To develop signal of each probe, the embryos were sequentially incubated in a water bath at 40 0 C with: (i) the horseradish peroxidase (HRP) for 15 min, (ii) TSA fluorophore for 30 min and (iii) the HRP blocker for 15 min.After each incubation step, the embryos were washed three times with 0.2x SSCT for 15 min at room temperature.HRP step is linked to a probe channel, we first developed C1 probe, Dr-aqp1a.1 (Advanced Cell Diagnostics) using HRP-C1 and TSA Vivid Fluorophore 520 (Tocris, dilution 1:1500), and then C3 probe, Dr-aqp8a.1 (Advanced Cell Diagnostics) using HRP-C3 and TSA Vivid Fluorophore 650 (Tocris, dilution 1:1500).Nuclei were counterstained with DAPI ready-to-use solution overnight at 4 0 C.Prior to imaging embryos were rinsed in PBST and kept in 70% glycerol in PBS.Images were acquired using Olympus FV3000 confocal microscope and an Olympus UPlanXApo 60x/NA 1.42 oil immersion objective.