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Dev Growth Differ
2004 Apr 01;462:139-52. doi: 10.1111/j.1440-169X.2004.00733.x.
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Calcineurin inhibitors block dorsal-side signaling that affect late-stage development of the heart, kidney, liver, gut and somitic tissue during Xenopus embryogenesis.
Yoshida Y
,
Kim S
,
Chiba K
,
Kawai S
,
Tachikawa H
,
Takahashi N
.
???displayArticle.abstract??? Calcineurin, a calcium/calmodulin-dependent serine/threonine protein phosphatase, is a key constituent of signaling pathways involved in antigen-dependent T-cell activation and development of the mammalian heart. In addition, calcineurin constitutes a part of the Wnt/calcium-signaling pathway that regulates early stages of dorsoventral axis formation in Xenopus embryos. Although some of the Wnt family members are involved in organ formation at relatively late stages of Xenopus development, the involvement of calcineurin in the development of those organs remains unclear. In the present study, we demonstrate that calcineurin inhibitors (cyclosporine A, FK506, and FK520), but not non-calcineurin inhibitors (rapamycin and GPI1046) that bind the same intracellular receptor as that for FK506, induce edema and gut coiling disruption and exhibit teratogenesis in the kidney, heart, gut, liver, and somitic tissue during Xenopus development. The same effects were observed by injecting the calcineurin inhibitors into the dorsal side, but not ventral side, of blastomeres at the 4-cell stage, although the inhibitors did not affect dorsoventral axis formation. These results suggest that calcineurin is involved in dorsal-side signaling that leads to the formation of the heart, kidney, liver, gut and somitic tissue during Xenopus embryogenesis.
Fig. 1 Effects of calcineurin inhibitors and non-calcineurin inhibitors on the development of Xenopus embryos (morphological appearance).
(A) Effects of the calcineurin inhibitors. Stage 29/30 embryos were treated with either the control solution (0.083% ethanol and
0.017% Tween 20), or this solution containing 500 nM cyclosporine A (CsA), 50 nM FK506, 500 nM FK520, 10 μM GPI1046, or 10 μM
rapamycin, and observed at the stage 46. Left side is the lateral view. Right side is the ventral view of the gut after removal of the
abdominal skin. Bars, 1 mm. (B) Effect of the non-calcineurin inhibitors. Stage 29/30 embryos were treated with 10 μM GPI1046 or
10 μM rapamycin, and then observed 5 days later. In each photograph, a control embryo (top) is compared with embryos treated with
the respective non-calcineurin inhibitor (lower two embryos). Left, lateral view; right, ventral view. Bars, 1 mm.
Fig. 2 Histological analyses of
calcineurin inhibitor-treated embryos.
Effects of the calcineurin
inhibitors on heart formation
(A–D) Stage 29/30 embryos were
treated with either 0.083% ethanol
and 0.017% Tween 20 (control),
or this solution containing 500 nM
CsA, 50 nM FK506, 500 nM
FK520, or 10 μM GPI1046, and
observed at stage 46. All sections
were stained with hematoxylin
and eosin, and observed under
light microscopy. (A) 10 μm vertical
heart sections. Arrowheads
indicates a valve. at, atrium; ve,
ventricle. Bar, 200 μm. (B) Transverse
10 μm heart sections.
Arrows indicate endocardium,
and arrowheads trabeculae carneae.
Bar, 300 μm. (C) Effects of
the calcineurin inhibitors on liver
formation. Transverse liver sections
of 10 μm. LV, liver; int,
intestine. In embryos treated with
CsA, FK506 or FK520, but not
with GPI1046, livers were swollen
and displayed many holes. Bar,
300 μm. (D) Effects of the
calcineurin inhibitors on the
pronephros and somitic tissue
formations. Transverse sections
of 10 μm around pronephros and
somitic tissue. Embryos treated
with CsA, FK506 or FK520
showed a clear decrease in pronephric
tubules, and the margin
of the somitic tissue is not clearly
defined. pt, pronephric tubules;
nc, notochord; sc, spinal cord; s,
somitic tissue. Bar, 300 μm.
Fig. 3 Dependence of the side of calcineurin inhibitor injection
on induction of teratogenesis of internal organs, and effects of
FK506 on the development of pronephric tissues from animal
explants. (A) Embryos were injected with 100 μM FK506 in corn
oil on each of two dorsal or ventral blastomeres at the 4-cell
stage (4 nL per cell), and were observed at stage 46. The transverse
sections (10 μm) were stained with hematoxylin and eosin,
and observed under light microscopy. Control (dorsal), injected
with corn oil on dorsal blastomeres; FK506 (dorsal), injected with
FK506 on dorsal blastomeres; FK506 (ventral), injected with
FK506 on ventral blastomeres. Arrow, thickening of endocardium;
arrowhead, trabeculated; LV, liver; pt, pronephric tubules;
s, somite. Bar, 300 μm (B) Animal caps were isolated from late blastula
embryos, and were immediately treated with activin and
retinoic acid. Pronephrogen-treated explants were cultured in
the presence (A) or the absence (B) of FK506 for 4 days, and
were then fixed for histological analysis. The explants were cut
into 6 μm sections and stained with hematoxylin and eosin.
Arrowheads indicate pronephric tubules.
