Skip to main content

Advertisement

SpringerLink
Log in

In vivo dissection of Rhoa function in vascular development using zebrafish

  • Original Paper
  • Published:
Angiogenesis Aims and scope Submit manuscript

Abstract

The small monomeric GTPase RHOA acts as a master regulator of signal transduction cascades by activating effectors of cellular signaling, including the Rho-associated protein kinases ROCK1/2. Previous in vitro cell culture studies suggest that RHOA can regulate many critical aspects of vascular endothelial cell (EC) biology, including focal adhesion, stress fiber formation, and angiogenesis. However, the specific in vivo roles of RHOA during vascular development and homeostasis are still not well understood. In this study, we examine the in vivo functions of RHOA in regulating vascular development and integrity in zebrafish. We use zebrafish RHOA-ortholog (rhoaa) mutants, transgenic embryos expressing wild type, dominant negative, or constitutively active forms of rhoaa in ECs, pharmacological inhibitors of RHOA and ROCK1/2, and Rock1 and Rock2a/b dgRNP-injected zebrafish embryos to study the in vivo consequences of RHOA gain- and loss-of-function in the vascular endothelium. Our findings document roles for RHOA in vascular integrity, developmental angiogenesis, and vascular morphogenesis in vivo, showing that either too much or too little RHOA activity leads to vascular dysfunction.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

EC:

Endothelial cell

RHOA:

Ras homolog gene family, member A

ROCK:

Rho-associated protein kinase

CCM:

Cerebral cavernous malformation

Hpf:

Hours post fertilization

ENU:

N-Ethyl-N-nitrosourea

SSLP:

Simple sequence length polymorphism

LDA:

Lateral dorsal aortae

PHBC:

Primordial hindbrain channel

CtA:

Cranial central artery

DA:

Dorsal aorta

CV:

Cardinal vein

ISV:

Intersegmental vessel

DN:

Dominant negative

CA:

Constitutively active

BBB:

Blood brain barrier

HUVEC:

Human umbilical vein endothelial cells

GFP:

Green fluorescent protein

EGFP:

Enhanced green fluorescent protein

UAS:

Upstream activating sequence

2A:

P2A viral cleavage peptide

Tg:

Transgenic

References

  1. Hu X, De Silva TM, Chen J, Faraci FM (2017) Cerebral vascular disease and neurovascular injury in ischemic stroke. Circ Res 120(3):449–471. https://doi.org/10.1161/CIRCRESAHA.116.308427

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Radeva MY, Waschke J (2018) Mind the gap: mechanisms regulating the endothelial barrier. Acta Physiol (Oxf). https://doi.org/10.1111/apha.12860

    Article  Google Scholar 

  3. Harris TJ, Tepass U (2010) Adherens junctions: from molecules to morphogenesis. Nat Rev Mol Cell Biol 11(7):502–514. https://doi.org/10.1038/nrm2927

    Article  CAS  PubMed  Google Scholar 

  4. van Buul JD, Timmerman I (2016) Small Rho GTPase-mediated actin dynamics at endothelial adherens junctions. Small GTPases 7(1):21–31. https://doi.org/10.1080/21541248.2015.1131802

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Beckers CM, van Hinsbergh VW, van Nieuw Amerongen GP (2010) Driving Rho GTPase activity in endothelial cells regulates barrier integrity. Thromb Haemost 103(1):40–55. https://doi.org/10.1160/TH09-06-0403

    Article  CAS  PubMed  Google Scholar 

  6. Wojciak-Stothard B, Ridley AJ (2002) Rho GTPases and the regulation of endothelial permeability. Vascul Pharmacol 39(4–5):187–199. https://doi.org/10.1016/s1537-1891(03)00008-9

    Article  CAS  PubMed  Google Scholar 

  7. Barlow HR, Cleaver O (2019) Building blood vessels-one Rho GTPase at a time. Cells 8:6. https://doi.org/10.3390/cells8060545

    Article  CAS  Google Scholar 

  8. Nobes C, Hall A (1994) Regulation and function of the Rho subfamily of small GTPases. Curr Opin Genet Dev 4(1):77–81

    Article  CAS  Google Scholar 

  9. Amano M, Chihara K, Kimura K, Fukata Y, Nakamura N, Matsuura Y, Kaibuchi K (1997) Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science 275(5304):1308–1311

    Article  CAS  Google Scholar 

  10. Jaffe AB, Hall A (2005) Rho GTPases: biochemistry and biology. Annu Rev Cell Dev Biol 21:247–269. https://doi.org/10.1146/annurev.cellbio.21.020604.150721

    Article  CAS  PubMed  Google Scholar 

  11. Yao L, Romero MJ, Toque HA, Yang G, Caldwell RB, Caldwell RW (2010) The role of RhoA/Rho kinase pathway in endothelial dysfunction. J Cardiovasc Dis Res 1(4):165–170. https://doi.org/10.4103/0975-3583.74258

    Article  PubMed  PubMed Central  Google Scholar 

  12. Shih YP, Yuan SY, Lo SH (2017) Down-regulation of DLC1 in endothelial cells compromises the angiogenesis process. Cancer Lett 398:46–51. https://doi.org/10.1016/j.canlet.2017.04.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. El Atat O, Fakih A, El-Sibai M (2019) RHOG activates RAC1 through CDC42 leading to tube formation in vascular endothelial cells. Cells. https://doi.org/10.3390/cells8020171

    Article  PubMed  PubMed Central  Google Scholar 

  14. Bayless KJ, Davis GE (2002) The Cdc42 and Rac1 GTPases are required for capillary lumen formation in three-dimensional extracellular matrices. J Cell Sci 115(Pt 6):1123–1136

    Article  CAS  Google Scholar 

  15. Bryan BA, Dennstedt E, Mitchell DC, Walshe TE, Noma K, Loureiro R, Saint-Geniez M, Campaigniac JP, Liao JK, D’Amore PA (2010) RhoA/ROCK signaling is essential for multiple aspects of VEGF-mediated angiogenesis. FASEB J 24(9):3186–3195. https://doi.org/10.1096/fj.09-145102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ridley AJ, Hall A (1992) The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70(3):389–399

    Article  CAS  Google Scholar 

  17. Soga N, Namba N, McAllister S, Cornelius L, Teitelbaum SL, Dowdy SF, Kawamura J, Hruska KA (2001) Rho family GTPases regulate VEGF-stimulated endothelial cell motility. Exp Cell Res 269(1):73–87. https://doi.org/10.1006/excr.2001.5295

    Article  CAS  PubMed  Google Scholar 

  18. Pronk MCA, van Bezu JSM, van Nieuw Amerongen GP, van Hinsbergh VWM, Hordijk PL (2017) RhoA, RhoB and RhoC differentially regulate endothelial barrier function. Small GTPases. https://doi.org/10.1080/21541248.2017.1339767

    Article  PubMed  PubMed Central  Google Scholar 

  19. van Nieuw Amerongen GP, Koolwijk P, Versteilen A, van Hinsbergh VW (2003) Involvement of RhoA/Rho kinase signaling in VEGF-induced endothelial cell migration and angiogenesis in vitro. Arterioscler Thromb Vasc Biol 23(2):211–217

    Article  Google Scholar 

  20. van Nieuw Amerongen GP, van Delft S, Vermeer MA, Collard JG, van Hinsbergh VW (2000) Activation of RhoA by thrombin in endothelial hyperpermeability: role of Rho kinase and protein tyrosine kinases. Circ Res 87(4):335–340

    Article  Google Scholar 

  21. Oldenburg J, de Rooij J (2014) Mechanical control of the endothelial barrier. Cell Tissue Res 355(3):545–555. https://doi.org/10.1007/s00441-013-1792-6

    Article  CAS  PubMed  Google Scholar 

  22. Gavard J, Patel V, Gutkind JS (2008) Angiopoietin-1 prevents VEGF-induced endothelial permeability by sequestering Src through mDia. Dev Cell 14(1):25–36. https://doi.org/10.1016/j.devcel.2007.10.019

    Article  CAS  PubMed  Google Scholar 

  23. Xu M, Waters CL, Hu C, Wysolmerski RB, Vincent PA, Minnear FL (2007) Sphingosine 1-phosphate rapidly increases endothelial barrier function independently of VE-cadherin but requires cell spreading and Rho kinase. Am J Physiol Cell Physiol 293(4):C1309-1318. https://doi.org/10.1152/ajpcell.00014.2007

    Article  CAS  PubMed  Google Scholar 

  24. Zhang XE, Adderley SP, Breslin JW (2016) Activation of RhoA, but not Rac1, mediates early stages of S1P-induced endothelial barrier enhancement. PLoS ONE 11(5):e0155490. https://doi.org/10.1371/journal.pone.0155490

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Pedersen E, Brakebusch C (2012) Rho GTPase function in development: how in vivo models change our view. Exp Cell Res 318(14):1779–1787. https://doi.org/10.1016/j.yexcr.2012.05.004

    Article  CAS  PubMed  Google Scholar 

  26. Kaunas R, Nguyen P, Usami S, Chien S (2005) Cooperative effects of Rho and mechanical stretch on stress fiber organization. Proc Natl Acad Sci USA 102(44):15895–15900. https://doi.org/10.1073/pnas.0506041102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Shikata Y, Rios A, Kawkitinarong K, DePaola N, Garcia JG, Birukov KG (2005) Differential effects of shear stress and cyclic stretch on focal adhesion remodeling, site-specific FAK phosphorylation, and small GTPases in human lung endothelial cells. Exp Cell Res 304(1):40–49. https://doi.org/10.1016/j.yexcr.2004.11.001

    Article  CAS  PubMed  Google Scholar 

  28. Yamazaki Y, Kanekiyo T (2017) Blood-brain barrier dysfunction and the pathogenesis of Alzheimer’s disease. Int J Mol Sci 18:9. https://doi.org/10.3390/ijms18091965

    Article  CAS  Google Scholar 

  29. Zafar A, Quadri SA, Farooqui M, Ikram A, Robinson M, Hart BL, Mabray MC, Vigil C, Tang AT, Kahn ML, Yonas H, Lawton MT, Kim H, Morrison L (2019) Familial cerebral cavernous malformations. Stroke 50(5):1294–1301. https://doi.org/10.1161/STROKEAHA.118.022314

    Article  PubMed  PubMed Central  Google Scholar 

  30. Stockton RA, Shenkar R, Awad IA, Ginsberg MH (2010) Cerebral cavernous malformations proteins inhibit Rho kinase to stabilize vascular integrity. J Exp Med 207(4):881–896. https://doi.org/10.1084/jem.20091258

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Borikova AL, Dibble CF, Sciaky N, Welch CM, Abell AN, Bencharit S, Johnson GL (2010) Rho kinase inhibition rescues the endothelial cell cerebral cavernous malformation phenotype. J Biol Chem 285(16):11760–11764. https://doi.org/10.1074/jbc.C109.097220

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Whitehead KJ, Chan AC, Navankasattusas S, Koh W, London NR, Ling J, Mayo AH, Drakos SG, Jones CA, Zhu W, Marchuk DA, Davis GE, Li DY (2009) The cerebral cavernous malformation signaling pathway promotes vascular integrity via Rho GTPases. Nat Med 15(2):177–184. https://doi.org/10.1038/nm.1911

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. McDonald DA, Shi C, Shenkar R, Stockton RA, Liu F, Ginsberg MH, Marchuk DA, Awad IA (2012) Fasudil decreases lesion burden in a murine model of cerebral cavernous malformation disease. Stroke 43(2):571–574. https://doi.org/10.1161/STROKEAHA.111.625467

    Article  CAS  PubMed  Google Scholar 

  34. Shenkar R, Shi C, Austin C, Moore T, Lightle R, Cao Y, Zhang L, Wu M, Zeineddine HA, Girard R, McDonald DA, Rorrer A, Gallione C, Pytel P, Liao JK, Marchuk DA, Awad IA (2017) RhoA kinase inhibition with fasudil versus simvastatin in murine models of cerebral cavernous malformations. Stroke 48(1):187–194. https://doi.org/10.1161/STROKEAHA.116.015013

    Article  CAS  PubMed  Google Scholar 

  35. Shenkar R, Peiper A, Pardo H, Moore T, Lightle R, Girard R, Hobson N, Polster SP, Koskimaki J, Zhang D, Lyne SB, Cao Y, Chaudagar K, Saadat L, Gallione C, Pytel P, Liao JK, Marchuk D, Awad IA (2019) Rho kinase inhibition blunts lesion development and hemorrhage in murine models of aggressive Pdcd10/Ccm3 disease. Stroke 50(3):738–744. https://doi.org/10.1161/STROKEAHA.118.024058

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Mikelis CM, Simaan M, Ando K, Fukuhara S, Sakurai A, Amornphimoltham P, Masedunskas A, Weigert R, Chavakis T, Adams RH, Offermanns S, Mochizuki N, Zheng Y, Gutkind JS (2015) RhoA and ROCK mediate histamine-induced vascular leakage and anaphylactic shock. Nat Commun 6:6725. https://doi.org/10.1038/ncomms7725

    Article  CAS  PubMed  Google Scholar 

  37. Hoang MV, Whelan MC, Senger DR (2004) Rho activity critically and selectively regulates endothelial cell organization during angiogenesis. Proc Natl Acad Sci U S A 101(7):1874–1879. https://doi.org/10.1073/pnas.0308525100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Park HJ, Kong D, Iruela-Arispe L, Begley U, Tang D, Galper JB (2002) 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors interfere with angiogenesis by inhibiting the geranylgeranylation of RhoA. Circ Res 91(2):143–150. https://doi.org/10.1161/01.res.0000028149.15986.4c

    Article  CAS  PubMed  Google Scholar 

  39. Barry DM, Koo Y, Norden PR, Wylie LA, Xu K, Wichaidit C, Azizoglu DB, Zheng Y, Cobb MH, Davis GE, Cleaver O (2016) Rasip1-mediated Rho GTPase signaling regulates blood vessel tubulogenesis via nonmuscle myosin II. Circ Res 119(7):810–826. https://doi.org/10.1161/CIRCRESAHA.116.309094

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Zahra FT, Sajib MS, Ichiyama Y, Akwii RG, Tullar PE, Cobos C, Minchew SA, Doci CL, Zheng Y, Kubota Y, Gutkind JS, Mikelis CM (2019) Endothelial RhoA GTPase is essential for in vitro endothelial functions but dispensable for physiological in vivo angiogenesis. Sci Rep 9(1):11666. https://doi.org/10.1038/s41598-019-48053-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Gore AV, Monzo K, Cha YR, Pan W, Weinstein BM (2012) Vascular development in the zebrafish. Cold Spring Harb Perspect Med 2(5):a006684. https://doi.org/10.1101/cshperspect.a006684

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Butler MG, Gore AV, Weinstein BM (2011) Zebrafish as a model for hemorrhagic stroke. Methods Cell Biol 105:137–161. https://doi.org/10.1016/B978-0-12-381320-6.00006-0

    Article  CAS  PubMed  Google Scholar 

  43. Stratman AN, Weinstein BM (2021) Assessment of vascular patterning in the zebrafish. Methods Mol Biol 2206:205–222. https://doi.org/10.1007/978-1-0716-0916-3_15

    Article  CAS  PubMed  Google Scholar 

  44. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203(3):253–310. https://doi.org/10.1002/aja.1002030302

    Article  CAS  PubMed  Google Scholar 

  45. Yarrow JC, Totsukawa G, Charras GT, Mitchison TJ (2005) Screening for cell migration inhibitors via automated microscopy reveals a Rho-kinase inhibitor. Chem Biol 12(3):385–395. https://doi.org/10.1016/j.chembiol.2005.01.015

    Article  CAS  PubMed  Google Scholar 

  46. Sehnert AJ, Huq A, Weinstein BM, Walker C, Fishman M, Stainier DY (2002) Cardiac troponin T is essential in sarcomere assembly and cardiac contractility. Nat Genet 31(1):106–110. https://doi.org/10.1038/ng875

    Article  CAS  PubMed  Google Scholar 

  47. Prince VE, Moens CB, Kimmel CB, Ho RK (1998) Zebrafish hox genes: expression in the hindbrain region of wild-type and mutants of the segmentation gene, valentino. Development 125(3):393–406

    Article  CAS  Google Scholar 

  48. Thisse C, Thisse B, Schilling TF, Postlethwait JH (1993) Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos. Development 119(4):1203–1215

    Article  CAS  Google Scholar 

  49. Traver D, Paw BH, Poss KD, Penberthy WT, Lin S, Zon LI (2003) Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants. Nat Immunol 4(12):1238–1246. https://doi.org/10.1038/ni1007

    Article  CAS  PubMed  Google Scholar 

  50. Jin SW, Beis D, Mitchell T, Chen JN, Stainier DY (2005) Cellular and molecular analyses of vascular tube and lumen formation in zebrafish. Development 132(23):5199–5209. https://doi.org/10.1242/dev.02087

    Article  CAS  PubMed  Google Scholar 

  51. Lawson ND, Weinstein BM (2002) In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol 248(2):307–318. https://doi.org/10.1006/dbio.2002.0711

    Article  CAS  PubMed  Google Scholar 

  52. Roman BL, Pham VN, Lawson ND, Kulik M, Childs S, Lekven AC, Garrity DM, Moon RT, Fishman MC, Lechleider RJ, Weinstein BM (2002) Disruption of acvrl1 increases endothelial cell number in zebrafish cranial vessels. Development 129(12):3009–3019

    Article  CAS  Google Scholar 

  53. Venero Galanternik M, Castranova D, Gore AV, Blewett NH, Jung HM, Stratman AN, Kirby MR, Iben J, Miller MF, Kawakami K, Maraia RJ, Weinstein BM (2017) A novel perivascular cell population in the zebrafish brain. Elife. https://doi.org/10.7554/eLife.24369

    Article  PubMed  Google Scholar 

  54. Kawakami K, Abe G, Asada T, Asakawa K, Fukuda R, Ito A, Lal P, Mouri N, Muto A, Suster ML, Takakubo H, Urasaki A, Wada H, Yoshida M (2010) zTrap: zebrafish gene trap and enhancer trap database. BMC Dev Biol 10:105. https://doi.org/10.1186/1471-213X-10-105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Asakawa K, Suster ML, Mizusawa K, Nagayoshi S, Kotani T, Urasaki A, Kishimoto Y, Hibi M, Kawakami K (2008) Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish. Proc Natl Acad Sci U S A 105(4):1255–1260. https://doi.org/10.1073/pnas.0704963105

    Article  PubMed  PubMed Central  Google Scholar 

  56. Zhang Y, Werling U, Edelmann W (2012) SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Res 40(8):e55. https://doi.org/10.1093/nar/gkr1288

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Kim JH, Lee SR, Li LH, Park HJ, Park JH, Lee KY, Kim MK, Shin BA, Choi SY (2011) High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS ONE 6(4):e18556. https://doi.org/10.1371/journal.pone.0018556

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Provost E, Rhee J, Leach SD (2007) Viral 2A peptides allow expression of multiple proteins from a single ORF in transgenic zebrafish embryos. Genesis 45(10):625–629. https://doi.org/10.1002/dvg.20338

    Article  CAS  PubMed  Google Scholar 

  59. Yokogawa T, Hannan MC, Burgess HA (2012) The dorsal raphe modulates sensory responsiveness during arousal in zebrafish. J Neurosci 32(43):15205–15215. https://doi.org/10.1523/JNEUROSCI.1019-12.2012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Horstick EJ, Jordan DC, Bergeron SA, Tabor KM, Serpe M, Feldman B, Burgess HA (2015) Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish. Nucleic Acids Res 43(7):e48. https://doi.org/10.1093/nar/gkv035

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Lawson ND, Mugford JW, Diamond BA, Weinstein BM (2003) phospholipase C gamma-1 is required downstream of vascular endothelial growth factor during arterial development. Genes Dev 17(11):1346–1351. https://doi.org/10.1101/gad.1072203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Neff MM, Turk E, Kalishman M (2002) Web-based primer design for single nucleotide polymorphism analysis. Trends Genet 18(12):613–615. https://doi.org/10.1016/s0168-9525(02)02820-2

    Article  CAS  PubMed  Google Scholar 

  63. Neff MM, Neff JD, Chory J, Pepper AE (1998) dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. Plant J 14(3):387–392. https://doi.org/10.1046/j.1365-313x.1998.00124.x

    Article  CAS  PubMed  Google Scholar 

  64. Michaels SD, Amasino RM (1998) A robust method for detecting single-nucleotide changes as polymorphic markers by PCR. Plant J 14(3):381–385. https://doi.org/10.1046/j.1365-313x.1998.00123.x

    Article  CAS  PubMed  Google Scholar 

  65. Meeker ND, Hutchinson SA, Ho L, Trede NS (2007) Method for isolation of PCR-ready genomic DNA from zebrafish tissues. Biotechniques 43(5):610–614. https://doi.org/10.2144/000112619

    Article  CAS  PubMed  Google Scholar 

  66. Gagnon JA, Valen E, Thyme SB, Huang P, Akhmetova L, Pauli A, Montague TG, Zimmerman S, Richter C, Schier AF (2014) Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS ONE 9(5):e98186. https://doi.org/10.1371/journal.pone.0098186

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Labun K, Montague TG, Krause M, Torres Cleuren YN, Tjeldnes H, Valen E (2019) CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res 47(W1):W171–W174. https://doi.org/10.1093/nar/gkz365

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Davis AE, Castranova D, Weinstein BM (2021) Rapid generation of pigment free, immobile zebrafish embryos and larvae in any genetic background using CRISPR-Cas9 dgRNPs. Zebrafish 18(4):235–242. https://doi.org/10.1089/zeb.2021.0011

    Article  CAS  PubMed  Google Scholar 

  69. Hoshijima K, Jurynec MJ, Klatt Shaw D, Jacobi AM, Behlke MA, Grunwald DJ (2019) Highly efficient CRISPR-Cas9-based methods for generating deletion mutations and F0 embryos that lack gene function in Zebrafish. Dev Cell 51(5):645-657e644. https://doi.org/10.1016/j.devcel.2019.10.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Sood R, Carrington B, Bishop K, Jones M, Rissone A, Candotti F, Chandrasekharappa SC, Liu P (2013) Efficient methods for targeted mutagenesis in zebrafish using zinc-finger nucleases: data from targeting of nine genes using CompoZr or CoDA ZFNs. PLoS ONE 8(2):e57239. https://doi.org/10.1371/journal.pone.0057239

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Norrman K, Fischer Y, Bonnamy B, Wolfhagen Sand F, Ravassard P, Semb H (2010) Quantitative comparison of constitutive promoters in human ES cells. PLoS ONE 5(8):e12413. https://doi.org/10.1371/journal.pone.0012413

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Liu JW, Pernod G, Dunoyer-Geindre S, Fish RJ, Yang H, Bounameaux H, Kruithof EK (2006) Promoter dependence of transgene expression by lentivirus-transduced human blood-derived endothelial progenitor cells. Stem Cells 24(1):199–208. https://doi.org/10.1634/stemcells.2004-0364

    Article  PubMed  Google Scholar 

  73. Turner DL, Weintraub H (1994) Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev 8(12):1434–1447. https://doi.org/10.1101/gad.8.12.1434

    Article  CAS  PubMed  Google Scholar 

  74. Michaelson D, Silletti J, Murphy G, D’Eustachio P, Rush M, Philips MR (2001) Differential localization of Rho GTPases in live cells: regulation by hypervariable regions and RhoGDI binding. J Cell Biol 152(1):111–126. https://doi.org/10.1083/jcb.152.1.111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Michaelson D, Philips M (2006) The use of GFP to localize Rho GTPases in living cells. Methods Enzymol 406:296–315. https://doi.org/10.1016/S0076-6879(06)06022-8

    Article  CAS  PubMed  Google Scholar 

  76. Stratman AN, Burns MC, Farrelly OM, Davis AE, Li W, Pham VN, Castranova D, Yano JJ, Goddard LM, Nguyen O, Galanternik MV, Bolan TJ, Kahn ML, Mukouyama YS, Weinstein BM (2020) Chemokine mediated signalling within arteries promotes vascular smooth muscle cell recruitment. Commun Biol 3(1):734. https://doi.org/10.1038/s42003-020-01462-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Breslin JW, Zhang XE, Worthylake RA, Souza-Smith FM (2015) Involvement of local lamellipodia in endothelial barrier function. PLoS ONE 10(2):e0117970. https://doi.org/10.1371/journal.pone.0117970

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Sharma SK, Carew TJ (2002) Inclusion of phosphatase inhibitors during Western blotting enhances signal detection with phospho-specific antibodies. Anal Biochem 307(1):187–189. https://doi.org/10.1016/s0003-2697(02)00008-8

    Article  CAS  PubMed  Google Scholar 

  79. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. https://doi.org/10.1038/nmeth.2019

    Article  CAS  PubMed  Google Scholar 

  80. Ferreira T, Miura K, Chef B, Eglinger J (2015) Scripts: bar 1.1.6. Zenodo. https://doi.org/10.5281/ZENODO.28838

  81. Ihara K, Muraguchi S, Kato M, Shimizu T, Shirakawa M, Kuroda S, Kaibuchi K, Hakoshima T (1998) Crystal structure of human RhoA in a dominantly active form complexed with a GTP analogue. J Biol Chem 273(16):9656–9666

    Article  CAS  Google Scholar 

  82. Wei Y, Zhang Y, Derewenda U, Liu X, Minor W, Nakamoto RK, Somlyo AV, Somlyo AP, Derewenda ZS (1997) Crystal structure of RhoA-GDP and its functional implications. Nat Struct Biol 4(9):699–703

    Article  CAS  Google Scholar 

  83. Isogai S, Horiguchi M, Weinstein BM (2001) The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. Dev Biol 230(2):278–301. https://doi.org/10.1006/dbio.2000.9995

    Article  CAS  PubMed  Google Scholar 

  84. Coso OA, Chiariello M, Yu JC, Teramoto H, Crespo P, Xu N, Miki T, Gutkind JS (1995) The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell 81(7):1137–1146

    Article  CAS  Google Scholar 

  85. Haidari M, Zhang W, Chen Z, Ganjehei L, Warier N, Vanderslice P, Dixon R (2011) Myosin light chain phosphorylation facilitates monocyte transendothelial migration by dissociating endothelial adherens junctions. Cardiovasc Res 92(3):456–465. https://doi.org/10.1093/cvr/cvr240

    Article  CAS  PubMed  Google Scholar 

  86. Wojciak-Stothard B, Potempa S, Eichholtz T, Ridley AJ (2001) Rho and Rac but not Cdc42 regulate endothelial cell permeability. J Cell Sci 114(Pt 7):1343–1355

    Article  CAS  Google Scholar 

  87. Chrzanowska-Wodnicka M, Burridge K (1996) Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J Cell Biol 133(6):1403–1415

    Article  CAS  Google Scholar 

  88. Huveneers S, Oldenburg J, Spanjaard E, van der Krogt G, Grigoriev I, Akhmanova A, Rehmann H, de Rooij J (2012) Vinculin associates with endothelial VE-cadherin junctions to control force-dependent remodeling. J Cell Biol 196(5):641–652. https://doi.org/10.1083/jcb.201108120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. van Geemen D, Smeets MW, van Stalborch AM, Woerdeman LA, Daemen MJ, Hordijk PL, Huveneers S (2014) F-actin-anchored focal adhesions distinguish endothelial phenotypes of human arteries and veins. Arterioscler Thromb Vasc Biol 34(9):2059–2067. https://doi.org/10.1161/ATVBAHA.114.304180

    Article  CAS  PubMed  Google Scholar 

  90. Calderwood DA, Shattil SJ, Ginsberg MH (2000) Integrins and actin filaments: reciprocal regulation of cell adhesion and signaling. J Biol Chem 275(30):22607–22610. https://doi.org/10.1074/jbc.R900037199

    Article  CAS  PubMed  Google Scholar 

  91. Millan J, Cain RJ, Reglero-Real N, Bigarella C, Marcos-Ramiro B, Fernandez-Martin L, Correas I, Ridley AJ (2010) Adherens junctions connect stress fibres between adjacent endothelial cells. BMC Biol 8:11. https://doi.org/10.1186/1741-7007-8-11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Burridge K, Fath K, Kelly T, Nuckolls G, Turner C (1988) Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol 4:487–525. https://doi.org/10.1146/annurev.cb.04.110188.002415

    Article  CAS  PubMed  Google Scholar 

  93. Huveneers S, de Rooij J (2013) Mechanosensitive systems at the cadherin-F-actin interface. J Cell Sci 126(Pt 2):403–413. https://doi.org/10.1242/jcs.109447

    Article  CAS  PubMed  Google Scholar 

  94. Wu MH (2005) Endothelial focal adhesions and barrier function. J Physiol 569(Pt 2):359–366. https://doi.org/10.1113/jphysiol.2005.096537

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Bachir AI, Horwitz AR, Nelson WJ, Bianchini JM (2017) Actin-based adhesion modules mediate cell interactions with the extracellular matrix and neighboring cells. Cold Spring Harb Perspect Biol 9:7. https://doi.org/10.1101/cshperspect.a023234

    Article  CAS  Google Scholar 

  96. Le Boeuf F, Houle F, Huot J (2004) Regulation of vascular endothelial growth factor receptor 2-mediated phosphorylation of focal adhesion kinase by heat shock protein 90 and Src kinase activities. J Biol Chem 279(37):39175–39185. https://doi.org/10.1074/jbc.M405493200

    Article  CAS  PubMed  Google Scholar 

  97. Bowers SLK, Kemp SS, Aguera KN, Koller GM, Forgy JC, Davis GE (2020) Defining an upstream VEGF (vascular endothelial growth factor) priming signature for downstream factor-induced endothelial cell-pericyte tube network coassembly. Arterioscler Thromb Vasc Biol 40(12):2891–2909. https://doi.org/10.1161/ATVBAHA.120.314517

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. van Wetering S, van Buul JD, Quik S, Mul FP, Anthony EC, ten Klooster JP, Collard JG, Hordijk PL (2002) Reactive oxygen species mediate Rac-induced loss of cell-cell adhesion in primary human endothelial cells. J Cell Sci 115(Pt 9):1837–1846

    Article  Google Scholar 

  99. Pirone DM, Liu WF, Ruiz SA, Gao L, Raghavan S, Lemmon CA, Romer LH, Chen CS (2006) An inhibitory role for FAK in regulating proliferation: a link between limited adhesion and RhoA-ROCK signaling. J Cell Biol 174(2):277–288. https://doi.org/10.1083/jcb.200510062

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Strassheim D, Porter RA, Phelps SH, Williams CL (2000) Unique in vivo associations with SmgGDS and RhoGDI and different guanine nucleotide exchange activities exhibited by RhoA, dominant negative RhoA(Asn-19), and activated RhoA(Val-14). J Biol Chem 275(10):6699–6702. https://doi.org/10.1074/jbc.275.10.6699

    Article  CAS  PubMed  Google Scholar 

  101. Feig LA (1999) Tools of the trade: use of dominant-inhibitory mutants of Ras-family GTPases. Nat Cell Biol 1(2):E25-27. https://doi.org/10.1038/10018

    Article  CAS  PubMed  Google Scholar 

  102. Feig LA, Cooper GM (1988) Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol 8(8):3235–3243. https://doi.org/10.1128/mcb.8.8.3235

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Prendergast GC, Khosravi-Far R, Solski PA, Kurzawa H, Lebowitz PF, Der CJ (1995) Critical role of Rho in cell transformation by oncogenic Ras. Oncogene 10(12):2289–2296

    CAS  PubMed  Google Scholar 

  104. Self AJ, Hall A (1995) Measurement of intrinsic nucleotide exchange and GTP hydrolysis rates. Methods Enzymol 256:67–76. https://doi.org/10.1016/0076-6879(95)56010-6

    Article  CAS  PubMed  Google Scholar 

  105. Burridge K, Doughman R (2006) Front and back by Rho and Rac. Nat Cell Biol 8(8):781–782. https://doi.org/10.1038/ncb0806-781

    Article  CAS  PubMed  Google Scholar 

  106. Reinhard NR, Mastop M, Yin T, Wu Y, Bosma EK, Gadella TWJ Jr, Goedhart J, Hordijk PL (2017) The balance between Galphai-Cdc42/Rac and Galpha12/13-RhoA pathways determines endothelial barrier regulation by sphingosine-1-phosphate. Mol Biol Cell 28(23):3371–3382. https://doi.org/10.1091/mbc.E17-03-0136

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Nakamura F (2013) FilGAP and its close relatives: a mediator of Rho-Rac antagonism that regulates cell morphology and migration. Biochem J 453(1):17–25. https://doi.org/10.1042/BJ20130290

    Article  CAS  PubMed  Google Scholar 

  108. Somlyo AP, Somlyo AV (2000) Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol 522(Pt 2):177–185. https://doi.org/10.1111/j.1469-7793.2000.t01-2-00177.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K (1996) Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273(5272):245–248. https://doi.org/10.1126/science.273.5272.245

    Article  CAS  PubMed  Google Scholar 

  110. Abraham S, Yeo M, Montero-Balaguer M, Paterson H, Dejana E, Marshall CJ, Mavria G (2009) VE-Cadherin-mediated cell-cell interaction suppresses sprouting via signaling to MLC2 phosphorylation. Curr Biol 19(8):668–674. https://doi.org/10.1016/j.cub.2009.02.057

    Article  CAS  PubMed  Google Scholar 

  111. Mavria G, Vercoulen Y, Yeo M, Paterson H, Karasarides M, Marais R, Bird D, Marshall CJ (2006) ERK-MAPK signaling opposes Rho-kinase to promote endothelial cell survival and sprouting during angiogenesis. Cancer Cell 9(1):33–44. https://doi.org/10.1016/j.ccr.2005.12.021

    Article  CAS  PubMed  Google Scholar 

  112. Hirano M, Hirano K (2016) Myosin di-phosphorylation and peripheral actin bundle formation as initial events during endothelial barrier disruption. Sci Rep 6:20989. https://doi.org/10.1038/srep20989

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Iyer S, Ferreri DM, DeCocco NC, Minnear FL, Vincent PA (2004) VE-cadherin-p120 interaction is required for maintenance of endothelial barrier function. Am J Physiol Lung Cell Mol Physiol 286(6):L1143-1153. https://doi.org/10.1152/ajplung.00305.2003

    Article  CAS  PubMed  Google Scholar 

  114. Sun H, Breslin JW, Zhu J, Yuan SY, Wu MH (2006) Rho and ROCK signaling in VEGF-induced microvascular endothelial hyperpermeability. Microcirculation 13(3):237–247. https://doi.org/10.1080/10739680600556944

    Article  CAS  PubMed  Google Scholar 

  115. Bhadriraju K, Yang M, Alom Ruiz S, Pirone D, Tan J, Chen CS (2007) Activation of ROCK by RhoA is regulated by cell adhesion, shape, and cytoskeletal tension. Exp Cell Res 313(16):3616–3623. https://doi.org/10.1016/j.yexcr.2007.07.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. van Buul JD, Geerts D, Huveneers S (2014) Rho GAPs and GEFs: controling switches in endothelial cell adhesion. Cell Adh Migr 8(2):108–124. https://doi.org/10.4161/cam.27599

    Article  PubMed  PubMed Central  Google Scholar 

  117. Salas-Vidal E, Meijer AH, Cheng X, Spaink HP (2005) Genomic annotation and expression analysis of the zebrafish Rho small GTPase family during development and bacterial infection. Genomics 86(1):25–37. https://doi.org/10.1016/j.ygeno.2005.03.010

    Article  CAS  PubMed  Google Scholar 

  118. Yanakieva I, Erzberger A, Matejcic M, Modes CD, Norden C (2019) Cell and tissue morphology determine actin-dependent nuclear migration mechanisms in neuroepithelia. J Cell Biol 218(10):3272–3289. https://doi.org/10.1083/jcb.201901077

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. Lai SL, Chang CN, Wang PJ, Lee SJ (2005) Rho mediates cytokinesis and epiboly via ROCK in zebrafish. Mol Reprod Dev 71(2):186–196. https://doi.org/10.1002/mrd.20290

    Article  CAS  PubMed  Google Scholar 

  120. Weiser DC, Pyati UJ, Kimelman D (2007) Gravin regulates mesodermal cell behavior changes required for axis elongation during zebrafish gastrulation. Genes Dev 21(12):1559–1571. https://doi.org/10.1101/gad.1535007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. Weiser DC, Kimelman D (2012) Analysis of cell shape and polarity during zebrafish gastrulation. Methods Mol Biol 839:53–68. https://doi.org/10.1007/978-1-61779-510-7_5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Julian L, Olson MF (2014) Rho-associated coiled-coil containing kinases (ROCK): structure, regulation, and functions. Small GTPases 5:e29846. https://doi.org/10.4161/sgtp.29846

    Article  PubMed  PubMed Central  Google Scholar 

  123. Thumkeo D, Keel J, Ishizaki T, Hirose M, Nonomura K, Oshima H, Oshima M, Taketo MM, Narumiya S (2003) Targeted disruption of the mouse rho-associated kinase 2 gene results in intrauterine growth retardation and fetal death. Mol Cell Biol 23(14):5043–5055

    Article  CAS  Google Scholar 

  124. Shimizu Y, Thumkeo D, Keel J, Ishizaki T, Oshima H, Oshima M, Noda Y, Matsumura F, Taketo MM, Narumiya S (2005) ROCK-I regulates closure of the eyelids and ventral body wall by inducing assembly of actomyosin bundles. J Cell Biol 168(6):941–953. https://doi.org/10.1083/jcb.200411179

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. Liao JK, Seto M, Noma K (2007) Rho kinase (ROCK) inhibitors. J Cardiovasc Pharmacol 50(1):17–24. https://doi.org/10.1097/FJC.0b013e318070d1bd

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  126. Kaneko T, Amano M, Maeda A, Goto H, Takahashi K, Ito M, Kaibuchi K (2000) Identification of calponin as a novel substrate of Rho-kinase. Biochem Biophys Res Commun 273(1):110–116. https://doi.org/10.1006/bbrc.2000.2901

    Article  CAS  PubMed  Google Scholar 

  127. Hartmann S, Ridley AJ, Lutz S (2015) The function of Rho-associated kinases ROCK1 and ROCK2 in the pathogenesis of cardiovascular disease. Front Pharmacol 6:276. https://doi.org/10.3389/fphar.2015.00276

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  128. Lisowska J, Rodel CJ, Manet S, Miroshnikova YA, Boyault C, Planus E, De Mets R, Lee HH, Destaing O, Mertani H, Boulday G, Tournier-Lasserve E, Balland M, Abdelilah-Seyfried S, Albiges-Rizo C, Faurobert E (2018) The CCM1-CCM2 complex controls complementary functions of ROCK1 and ROCK2 that are required for endothelial integrity. J Cell Sci. https://doi.org/10.1242/jcs.216093

    Article  PubMed  Google Scholar 

  129. El-Brolosy MA, Kontarakis Z, Rossi A, Kuenne C, Gunther S, Fukuda N, Kikhi K, Boezio GLM, Takacs CM, Lai SL, Fukuda R, Gerri C, Giraldez AJ, Stainier DYR (2019) Genetic compensation triggered by mutant mRNA degradation. Nature 568(7751):193–197. https://doi.org/10.1038/s41586-019-1064-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  130. Ma Z, Zhu P, Shi H, Guo L, Zhang Q, Chen Y, Chen S, Zhang Z, Peng J, Chen J (2019) PTC-bearing mRNA elicits a genetic compensation response via Upf3a and COMPASS components. Nature 568(7751):259–263. https://doi.org/10.1038/s41586-019-1057-y

    Article  CAS  PubMed  Google Scholar 

  131. Rossi A, Kontarakis Z, Gerri C, Nolte H, Holper S, Kruger M, Stainier DY (2015) Genetic compensation induced by deleterious mutations but not gene knockdowns. Nature 524(7564):230–233. https://doi.org/10.1038/nature14580

    Article  CAS  PubMed  Google Scholar 

  132. Song JW, Daubriac J, Tse JM, Bazou D, Munn LL (2012) RhoA mediates flow-induced endothelial sprouting in a 3-D tissue analogue of angiogenesis. Lab Chip 12(23):5000–5006. https://doi.org/10.1039/c2lc40389g

    Article  CAS  PubMed  Google Scholar 

  133. Shasby DM, Stevens T, Ries D, Moy AB, Kamath JM, Kamath AM, Shasby SS (1997) Thrombin inhibits myosin light chain dephosphorylation in endothelial cells. Am J Physiol 272(2 Pt 1):L311-319. https://doi.org/10.1152/ajplung.1997.272.2.L311

    Article  CAS  PubMed  Google Scholar 

  134. Essler M, Amano M, Kruse HJ, Kaibuchi K, Weber PC, Aepfelbacher M (1998) Thrombin inactivates myosin light chain phosphatase via Rho and its target Rho kinase in human endothelial cells. J Biol Chem 273(34):21867–21874. https://doi.org/10.1074/jbc.273.34.21867

    Article  CAS  PubMed  Google Scholar 

  135. Zeng H, Zhao D, Mukhopadhyay D (2002) KDR stimulates endothelial cell migration through heterotrimeric G protein Gq/11-mediated activation of a small GTPase RhoA. J Biol Chem 277(48):46791–46798. https://doi.org/10.1074/jbc.M206133200

    Article  CAS  PubMed  Google Scholar 

  136. McKenzie JA, Ridley AJ (2007) Roles of Rho/ROCK and MLCK in TNF-alpha-induced changes in endothelial morphology and permeability. J Cell Physiol 213(1):221–228. https://doi.org/10.1002/jcp.21114

    Article  CAS  PubMed  Google Scholar 

  137. van Nieuw Amerongen GP, Beckers CM, Achekar ID, Zeeman S, Musters RJ, van Hinsbergh VW (2007) Involvement of Rho kinase in endothelial barrier maintenance. Arterioscler Thromb Vasc Biol 27(11):2332–2339. https://doi.org/10.1161/ATVBAHA.107.152322

    Article  CAS  PubMed  Google Scholar 

  138. Szulcek R, Beckers CM, Hodzic J, de Wit J, Chen Z, Grob T, Musters RJ, Minshall RD, van Hinsbergh VW, van Nieuw Amerongen GP (2013) Localized RhoA GTPase activity regulates dynamics of endothelial monolayer integrity. Cardiovasc Res 99(3):471–482. https://doi.org/10.1093/cvr/cvt075

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  139. Yalcin HC, Amindari A, Butcher JT, Althani A, Yacoub M (2017) Heart function and hemodynamic analysis for zebrafish embryos. Dev Dyn 246(11):868–880. https://doi.org/10.1002/dvdy.24497

    Article  PubMed  Google Scholar 

  140. Quinonez-Silvero C, Hubner K, Herzog W (2020) Development of the brain vasculature and the blood-brain barrier in zebrafish. Dev Biol 457(2):181–190. https://doi.org/10.1016/j.ydbio.2019.03.005

    Article  CAS  PubMed  Google Scholar 

  141. Eisa-Beygi S, Benslimane FM, El-Rass S, Prabhudesai S, Abdelrasoul MKA, Simpson PM, Yalcin HC, Burrows PE, Ramchandran R (2018) Characterization of endothelial cilia distribution during cerebral-vascular development in Zebrafish (Danio rerio). Arterioscler Thromb Vasc Biol 38(12):2806–2818. https://doi.org/10.1161/ATVBAHA.118.311231

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  142. Wang Y, Pan L, Moens CB, Appel B (2014) Notch3 establishes brain vascular integrity by regulating pericyte number. Development 141(2):307–317. https://doi.org/10.1242/dev.096107

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  143. Ando K, Fukuhara S, Izumi N, Nakajima H, Fukui H, Kelsh RN, Mochizuki N (2016) Clarification of mural cell coverage of vascular endothelial cells by live imaging of zebrafish. Development 143(8):1328–1339. https://doi.org/10.1242/dev.132654

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  144. Hellstrom M, Kalen M, Lindahl P, Abramsson A, Betsholtz C (1999) Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development 126(14):3047–3055

    Article  CAS  Google Scholar 

  145. Torres-Vazquez J, Gitler AD, Fraser SD, Berk JD, Van NP, Fishman MC, Childs S, Epstein JA, Weinstein BM (2004) Semaphorin-plexin signaling guides patterning of the developing vasculature. Dev Cell 7(1):117–123. https://doi.org/10.1016/j.devcel.2004.06.008

    Article  CAS  PubMed  Google Scholar 

  146. Barry DM, Xu K, Meadows SM, Zheng Y, Norden PR, Davis GE, Cleaver O (2015) Cdc42 is required for cytoskeletal support of endothelial cell adhesion during blood vessel formation in mice. Development 142(17):3058–3070. https://doi.org/10.1242/dev.125260

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  147. Tan W, Palmby TR, Gavard J, Amornphimoltham P, Zheng Y, Gutkind JS (2008) An essential role for Rac1 in endothelial cell function and vascular development. FASEB J 22(6):1829–1838. https://doi.org/10.1096/fj.07-096438

    Article  CAS  PubMed  Google Scholar 

  148. Yoneda A, Multhaupt HA, Couchman JR (2005) The Rho kinases I and II regulate different aspects of myosin II activity. J Cell Biol 170(3):443–453. https://doi.org/10.1083/jcb.200412043

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  149. Iida A, Wang Z, Hondo E, Sehara-Fujisawa A (2020) Generation and evaluation of a transgenic zebrafish for tissue-specific expression of a dominant-negative Rho-associated protein kinase-2. Biochem Biophys Res Commun. https://doi.org/10.1016/j.bbrc.2020.02.055

    Article  PubMed  PubMed Central  Google Scholar 

  150. Ikeda S, Satoh K, Kikuchi N, Miyata S, Suzuki K, Omura J, Shimizu T, Kobayashi K, Kobayashi K, Fukumoto Y, Sakata Y, Shimokawa H (2014) Crucial role of rho-kinase in pressure overload-induced right ventricular hypertrophy and dysfunction in mice. Arterioscler Thromb Vasc Biol 34(6):1260–1271. https://doi.org/10.1161/ATVBAHA.114.303320

    Article  CAS  PubMed  Google Scholar 

  151. Wang G, Cadwallader AB, Jang DS, Tsang M, Yost HJ, Amack JD (2011) The Rho kinase Rock2b establishes anteroposterior asymmetry of the ciliated Kupffer’s vesicle in zebrafish. Development 138(1):45–54. https://doi.org/10.1242/dev.052985

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. Strassheim D, Gerasimovskaya E, Irwin D, Dempsey EC, Stenmark K, Karoor V (2019) RhoGTPase in vascular disease. Cells. https://doi.org/10.3390/cells8060551

    Article  PubMed  PubMed Central  Google Scholar 

  153. Shaw RJ, Henry M, Solomon F, Jacks T (1998) RhoA-dependent phosphorylation and relocalization of ERM proteins into apical membrane/actin protrusions in fibroblasts. Mol Biol Cell 9(2):403–419. https://doi.org/10.1091/mbc.9.2.403

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  154. Simo-Servat O, Hernandez C, Simo R (2020) The ERM complex: a new player involved in diabetes-induced vascular leakage. Curr Med Chem 27(18):3012–3022. https://doi.org/10.2174/0929867325666181016162327

    Article  CAS  PubMed  Google Scholar 

  155. Adyshev DM, Dudek SM, Moldobaeva N, Kim KM, Ma SF, Kasa A, Garcia JG, Verin AD (2013) Ezrin/radixin/moesin proteins differentially regulate endothelial hyperpermeability after thrombin. Am J Physiol Lung Cell Mol Physiol 305(3):L240-255. https://doi.org/10.1152/ajplung.00355.2012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  156. Zaman R, Lombardo A, Sauvanet C, Viswanatha R, Awad V, Bonomo LE, McDermitt D, Bretscher A (2021) Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain. J Cell Biol. https://doi.org/10.1083/jcb.202007146

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors would like to thank members of the Weinstein laboratory for their critical comments on this manuscript. Schematics of larval zebrafish were created with BioRender.com.

Funding

This work was supported by the intramural program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (ZIA-HD001011 and ZIA-HD008915, to BMW).

Author information

Author notes

  1. Joseph J. Yano

    Present address: Department of Cell and Molecular Biology, University of Pennsylvania, 440 Curie Blvd, Philadelphia, PA, 19104, USA

  2. Amber N. Stratman

    Present address: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, 63110, USA

Authors and Affiliations

  1. Division of Developmental Biology, National Institute of Child Health and Human Development, National Institutes of Health, 6 Center Dr. Bethesda, Bethesda, MD, 20892, USA

    Laura M. Pillay, Joseph J. Yano, Andrew E. Davis, Matthew G. Butler, Megan O. Ezeude, Jong S. Park, Keith A. Barnes, Vanessa L. Reyes, Daniel Castranova, Aniket V. Gore, Matthew R. Swift, James R. Iben, Madeleine I. Kenton, Amber N. Stratman & Brant M. Weinstein

Authors
  1. Laura M. Pillay
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joseph J. Yano
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andrew E. Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Matthew G. Butler
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Megan O. Ezeude
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jong S. Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Keith A. Barnes
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Vanessa L. Reyes
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Daniel Castranova
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Aniket V. Gore
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Matthew R. Swift
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. James R. Iben
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Madeleine I. Kenton
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Amber N. Stratman
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Brant M. Weinstein
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Brant M. Weinstein.

Ethics declarations

Conflict of interest

The authors declare no competing interests or disclosures.

Ethical approval

Zebrafish husbandry and research protocols were reviewed and approved by the NICHD Animal Care and Use Committee, in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Availability of data and material

All data and materials reported in this manuscript are available from the Zebrafish International Resource Center (https://zebrafish.org) or upon request by contacting the corresponding author.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 46512 kb)

Supplementary file2 (MP4 24212 kb)

Supplementary file3 (MP4 17891 kb)

Supplementary file4 (MP4 33890 kb)

Supplementary file5 (MP4 8242 kb)

Supplementary file6 (MP4 6310 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pillay, L.M., Yano, J.J., Davis, A.E. et al. In vivo dissection of Rhoa function in vascular development using zebrafish. Angiogenesis 25, 411–434 (2022). https://doi.org/10.1007/s10456-022-09834-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10456-022-09834-9

Keywords

Search

Navigation