1.
P. Madaule, R. Axel, A novel ras-related gene family, Cell 41 (1985) 31–40.
2.
A. Boureux, E. Vignal, S. Faure, P. Fort, Evolution of the Rho family of ras-like GTPases in
eukaryotes, Mol. Biol. Evol. 24 (2007) 203–216.
3.
R.G. Hodge, A.J. Ridley, Regulating Rho GTPases and their regulators, Nat. Rev. Mol. Cell
Biol. 17 (2016) 496–510.
4.
O. Nakagawa, K. Fujisawa, T. Ishizaki, Y. Saito, K. Nakao, S. Narumiya, ROCK-I and
ROCK-II, two isoforms of Rho-associated coiled-coil forming protein serine/threonine
kinase in mice, FEBS Lett. 392 (1996) 189–193.
5.
K. Riento, A.J. Ridley, Rocks: multifunctional kinases in cell behavior, Nat. Rev. Mol. Cell
Biol. 4 (2003) 446–456.
6.
L. Wei, W. Roberts, L. Wang, M. Yamada, S. Zhang, Z. Zhao, S.A. Rivkees, R.J. Schwartz ,
K. Imanaka-Yoshida, Rho kinases play an obligatory role in vertebrate embryonic
organogenesis, Development 128 (2001) 2953–2962.
7.
Z. Zhao, S.A. Rivkees, Rho-associated kinases play an essential role in cardiac
morphogenesis and cardiomyocyte proliferation, Dev. Dynam. 226 (2003) 24–32.
8.
Y. Shimizu, D. Thumkeo, J. Keel, T. Ishizaki, H. Oshima, M. Oshima, Y. Noda, F.
Matsumura, M.M. Taketo, S. Narumiya, ROCK-I regulates closure of the eyelids and ventral
body wall by inducing assembly of actomyosin bundles, J. Cell. Biol. 168 (2005) 941–953.
9.
D. Thumkeo, J. Keel, T. Ishizaki, M. Hirose, K. Nonomura, H. Oshima, M. Oshima, M.M.
Taketo, S. Narumiya, Targeted disruption of the mouse rho-associated kinase 2 gene results
in intrauterine growth retardation and fetal death, Mol. Cell. Biol. 23 (2003) 5043–5055.
10. C.B. Kimmel, W.W. Ballard, S.R. Kimmel, B. Ullmann, T.F. Schilling, Stages of embryonic
development of the zebrafish, Dev. Dynam. 203 (1995) 253–310.
11. G. Wang, A.B. Cadwallader, D.S. Jang, M. Tsang, H.J. Yost, J.D. Amack, The Rho kinase
Rock2b establishes anteroposterior asymmetry of the ciliated Kupffer's vesicle in zebrafish,
Development 138 (1995) 45–54.
12. J. Lisowska, C.J. Rödel, S. Manet, Y.A. Miroshnikova, C. Boyault, E. Planus, R. De Mets,
H.H. Lee, O. Destaing, H. Mertani, G. Boulday, E. Tournier-Lasserve, M. Balland, S.
Abdelilah-Seyfried, C. Albiges-Rizo, E. Faurobert, Cerebral cavernous malformation 1/2
complex controls ROCK1 and ROCK2 complementary functions for endothelial integrity, J.
Cell Sci. 131 (2018) 15.
13. F. Marlow, J. Topczewski, D. Sepich, L. Solnica-Krezel, Zebrafish Rho kinase 2 acts
downstream of Wnt11 to mediate cell polarity and effective convergence and extension
movements, Curr. Biol. 12 (2002) 876–884.
14. K. Asakawa, M.L. Suster, K. Mizusawa, S. Nagayoshi, T. Kotani, A Urasaki, Y. Kishimoto,
M. Hibi, K. Kawakami, Genetic dissection of neural circuits by Tol2 transposon-mediated
Gal4 gene and enhancer trapping in zebrafish, Proc. Natl. Acad. Sci (USA) 105 (2008)
1255–1260.
15. N.D. Lawson, B.M. Weinstein, In vivo imaging of embryonic vascular development using
transgenic zebrafish, Dev Biol 248 (2002) 307–318.
16. T. Zygmunt, C.M. Gay, J. Blondelle, M.K. Singh, K.M. Flaherty, P.C. Means, L. Herwig, A.
Krudewig, H.G. Belting, M. Affolter, J.A. Epstein, J. Torres-Vázquez, Semaphorin-
PlexinD1 signaling limits angiogenic potential via the VEGF decoy receptor sFlt1, Dev. Cell
21 (2011) 301–314.
17. R.M. White, A. Sessa, C. Burke, T. Bowman, J. LeBlanc, C. Ceol, C. Bourque, M. Dovey,
W. Goessling, C.E. Burns, L.I. Zon, Transparent adult zebrafish as a tool for in vivo
transplantation analysis, Cell Stem Cell, 7 (2008) 183–189.
18. G. D'Agati, R. Beltre, A. Sessa, A. Burger, Y. Zhou, C. Mosimann, R.M. White, A defect in
the mitochondrial protein Mpv17 underlies the transparent casper zebrafish, Dev. Biol. 430
(2017) 11–17.
19. M. Amano, K. Chihara, N. Nakamura, Y. Fukata, T. Yano, M. Shibata, M. Ikebe, K.
Kaibuchi, Myosin II activation promotes neurite retraction during the action of Rho and
Rho-kinase, Genes Cells 3 (1998) 177–188.
20. M. Iizuka, K. Kimura, S. Wang, K. Kato, M. Amano, K. Kaibuchi, A. Mizoguchi, Distinct
distribution and localization of Rho-kinase in mouse epithelial, muscle and neural tissues,
Cell Struct. Func. 37 (2012) 155–175.
21. A. Iida, Z. Wang, H. Hirata, A. Sehara-Fujisawa, Integrin β1 activity is required for
cardiovascular formation in zebrafish, Genes Cells 23 (2018) 938–951.
22. S.F. Retta, F. Balzac, P. Ferraris, A.M. Belkin, R. Fässler, M.J. Humphries, G. Tarone. β1integrin cytoplasmic subdomains involved in dominant negative function. Mol. Biol. Cell, 9
(1998) 715–731.
Figure and Legends
Figure 1. Schematic of the dominant negative ROCK-2 used for the generation of
transgenic zebrafish and its proposed mechanism
(A) The colored boxes represent the different sequences of the domains in endogenous zebrafish
Rock2a. The red line indicates the active site. In the dominant negative (dnROCK-2; DN)
construct the kinase domain was replaced with mCherry, whereas the Rho-binding domain was
conserved. The intact mCherry construct, used as a negative control, was obtained by deleting
the functional domain from dnROCK-2. (B) Cartoon showing the predicted molecular
mechanism of the dnROCK-2-mediated ROCK-2 inhibition. The native ROCK-2 protein is
activated via binding to the GTP form of a Rho GTPase. The dnROCK-2 competes with the
native ROCK-2 protein for the Rho-GTP but cannot be activated since it lacks the kinase
domain. The removal of Rho-GTP from native ROCK-2 leads to its inactivation.
Figure 2. Ubiquitous expression of dnROCK-2 in zebrafish embryos
Typical images of transgenic zebrafish embryos ubiquitously expressing mCherry-dnROCK-2
(mild or severe phenotypes) or mCherry (control line) at 28 hpf (A) and 48 hpf (B). Both the
Tg(UAS: mCherry-dnROCK-2)ko114Tg and Tg(UAS: mCherry)ko115Tg strains were crossed with the
SAGFF(LF)73A transgenic line. GFP is used as a Gal4-UAS expression marker. mCherry
expression is indicated by RFP and shows the expression of mCherry-tagged dnROCK-2. The
black arrows indicate eyes. The black arrowheads indicate edema in the heart and yolk. Scale
bar, 500 μm. n.e., not examined. (C) Confocal image of muscle fibers in the trunk region of the
transgenic lines. Phalloidin staining reveals the shapes of the muscle fibers through labelling of
filamentous actin. The yellow squares indicate fast muscle fibers. The magenta squares indicate
muscle pioneer cells. The white arrowheads indicate Hensen’s zone observed on the myofibers.
DAPI was used to stain cell nuclei. RFP indicates expression of the mCherry tagged dnROCK-2.
Scale bar, 20 μm. (D) Cartoon of a hypothetical model to explain the confocal observation in C.
The upper picture shows the molecular structure of the myofiber. The lower pictures provide a
cartoon to explain the phalloidin fluorescent signals seen in the control or DN fish. Hensen’s
zone is an interspace between actin filaments, thus; it is visualized as a phalloidin-negative
region on the myofibers. In the DN fish, Hensen’s zone was unclear and the phalloidin-labeled
fibers ended to be narrower.
Figure 3. Endothelial cell-specific expression of dnROCK-2 in zebrafish embryos
(A) Typical phenotypes of hemorrhage, congestion, and edema seen in transgenic zebrafish
embryos expressing an endothelial cell-specific mCherry-dnROCK-2 compared to mCherry
(control) at 30 hpf. The Tg(UAS: mCherry-dnROCK-2)ko114Tg and Tg(UAS: mCherry)ko115Tg
strains were crossed with the Tg(fli1a: Gal4FF)ubs4 transgenic line. The black arrowheads indicate
abnormalities in the DN embryo. E, eye. Y, yolk. H, heart. (B) Quantitation of the incidence rate
of cardiovascular defects observed in transgenic zebrafish at 54-hpf. Student’s t test was used for
statistical analyses. The p values for Con vs. DN are 2.99 x 10-3 (hemorrhage), 5.72 x 10-3
(congestion) and 4.8 x 10-5 (edema). **p < 0.05. Con, control. DN, dominant-negative. (C) GFP
indicates the endothelial cells labelled by fli1a:EGFP. The enlarged images indicate the trunk
region. The white arrowhead indicates an intersegmental vessel. The illustrations are cartoons to
explain the observed phenotype. DA, dorsal aorta. PCV, posterior cardinal vein. ISV,
intersegmental vessel. Scale bars, 500 μm (whole embryo) and 100 μm (enlarged trunk). (D)
Images of the transgenic zebrafish embryos shown in A at 54 hpf. The white arrowhead indicates
a dorsal longitudinal anastomotic vessel (DLAV). The yellow arrowhead indicates a parachordal
lymphangioblast (PL). The illustrations are a cartoon to explain the observed phenotype. Scale
bars, 500 μm (whole embryo) and 100 μm (enlarged trunk).
...
論文の公開元へ
