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It has been suggested that ILK (integrin-linked kinase) participates in integrin- and growth factor-mediated signaling pathways and also functions as a scaffold protein at cell-extracellular matrix (ECM) adhesion sites. As the recently reported ILK knockout mice were found to die at the peri-implantation stage, the stage specific to mammals, little is known about the function of ILK in early developmental processes common to every vertebrate. To address this, we isolated a Xenopus ortholog of ILK (XeILK) and characterized its role in early Xenopus embryogenesis. XeILK was expressed constitutively and ubiquitously throughout the early embryogenesis. Depletion of XeILK with morpholino oligonucleotides (XeILK MO) caused severe defects in blastopore closure and axis elongation without affecting the mesodermal specification. Furthermore, XeILK MO was found to interfere with cell-cell and cell-ECM adhesions in dorsal marginal zone explants and to result in a significant loss of cell-ECM adhesions in activin-treated dissociated animal cap cells. These results thus indicate that XeILK plays an essential role in morphogenetic movements during gastrulation.
Figure 1 Cloning and expression of Xenopus ILK. (A) Alignment of the deduced amino acid sequence of
Xenopus ILK with the human, Drosophila and C. elegans
ILK proteins. Identical residues are boxed. (B) The schematic structure of XeILK. ILK comprises an N-terminal
ankyrin repeat (ANKr) domain (blue), a C-terminal kinase domain (yellow and green) and a pleckstrin homology (PH)-like domain (red and yellow) that partially overlaps with the N-terminal region of the kinase domain. Percent identity of the three domains in ILK between Xenopus and other species are shown below. (C) XeILK mRNA is present during early embryogenesis. Total RNA isolated from indicated stages was subjected to RT-PCR.
Xenopus embryonic ornithine decarboxylase (XeODC ) is a loading control. Xbra was also examined. No signal was detected in the absence of reverse transcriptase (– RT). (D) Whole-mount in situ hybridization analysis of XeILK at the
indicated stages. XeILK sense RNA probe was used as a negative control. (E)
XeILK is expressed ubiquitously in the three germ layers
at stage 11. Ten embryos (stage 11) were dissected into dorsal mesoderm (DM), ventralmesoderm (VM), endoderm (Endo), and ectoderm (Ecto) regions as shown. Each was processed for RT-PCR. The dorsal mesoderm marker
Chordin , the endoderm marker Xsox17α, the ventralmesoderm marker Xwnt8, and the pan-mesodermal marker Xbra were also analyzed. (F) XeILK is expressed ubiquitously at stage 24. Five embryos (stage 24) were dissected into head, dorsal, and ventral regions as shown. Each was processed for RT-PCR. The forebrain marker Otx2 and the somitic muscle marker muscle actin were also analyzed.
Figure 2
XeILK is required for morphogenesis, but not for
mesoderm specification. (A)
Anti-XeILK morpholino oligonucleotide
(XeILK MO, 25 ng) specifically blocked the translation of
injected XeILK-myc mRNA (1.5 ng). XeILK MO did not block
the translation of mutated XeILK-myc mRNA (mut. XeILKmyc,
1.5 ng), in which mutations were introduced in the XeILK
MO target sequence. A standard control MO (control MO,
25 ng), a five-base mismatched MO (5-mis MO, 25 ng) and an
invert of the anti-sense MO (inv. MO, 25 ng) had no effects on the
protein level of XeILK-myc or that of mut. XeILK-myc. MAPK
is a loading control. (B–D) Injection of XeILK MO caused severe
morphogenetic defects during early embryogenesis and partially
rescued by co-injecting XeILK mRNA. MOs (25 ng) were
injected as indicated into the two dorsal blastomeres of the fourcell
stage embryos. For rescue of XeILK depletion, mut. XeILK
mRNA (0.5 ng) was co-injected with XeILK MO. (B)
During
gastrula stages, the XeILK MO-injected embryos showed delay in
blastopore lip formation and in blastopore closure. Phenotypes are
observed at stage 10.5 (top panels, vegetal view), stage 12 (middle
panels, vegetal view) and stage 14 (bottom panels, dorsal view;
control MO, 5-mis MO, inv. MO and XeILK MO +XeILK
mRNA, vegetal view; XeILK MO). Arrowheads indicate the
pigment accumulation, a site for future blastopore lip formation.
At (C) the tailbud and (D) the tadpole stages, the XeILK MOinjected
embryos showed anterior structure defects or dorsal open
phenotypes. (C)
(a) The control MO-injected embryos. (b)
The XeILK MO-injected embryos. Head defects (lower) or dorsal
open phenotype (upper) were apparent. (c) The 5-mis MOinjected
embryos. (d)
The inv. MO-injected embryos. (e)
The
XeILK MO plus XeILK mRNA-injected embryos. Co-injection
of XeILK mRNA partially rescued the defects caused by XeILK
MO. (D) (a) The control MO-injected embryos. (b) The XeILK
MO-injected embryos. Head defects (upper) or dorsal open phenotype
(lower) were apparent. (c) The 5-mis MO-injected embryos.
(d) The inv. MO-injected embryos. (e) The XeILK MO plus
XeILK mRNA-injected embryos. Co-injection of XeILK mRNA
partially rescued the defects caused by XeILK MO. In (C) and
(D), all the embryos are oriented with anterior to the right.
(C,a,b(lower),c–e) and (D,a,b(lower),c–e) Lateral view. (C,b(upper))
and (D,b(upper)) Dorsal view. (E) Injection of XeILK MO did not
affect the expression of mesodermal markers. MOs (25 ng) were
injected as indicated into the two dorsal blastomeres at the
four-cell stage. The total RNA isolated from stage 11 embryos
were processed for RT-PCR.
Figure 3 Involvement of XeILK in proper blastopore lip formation, blastopore closure and axis elongation. MOs (25 ng) were injected
as indicated into the two dorsal blastomeres of the four-cell stage embryos. Rhodamine-dextran was co-injected with MOs as a lineage
tracer. Embryos were observed at indicated stages. Fluorescence view of left panel is shown in right panel. The XeILK MO-injected
embryos showed defects in proper blastopore lip formation (arrowheads), blastopore closure (arrows) and anteroposterior axis elongation.
(stages 10.25–10.75 and stage 11, upper panels) Vegetal view with dorsal to the top (stage 11, lower panels and stages 11.5–14). Dorsal
view with anterior to the top. Arrowheads indicate the edges of the blastopore lip. Arrows indicate the diameter of the blastopore.
Figure 4 XeILK regulates cell–cell and cell–extracellular matrix
adhesions. (A) Cell–cell and/or cell–extracellular matrix adhesions
were disrupted in the XeILK deficient cells and partially rescued
by co-injecting XeILK mRNA. MOs (25 ng) were injected as
indicated into the animal pole region at the four-cell stage. For
rescue of XeILK depletion, mut. XeILK mRNA (0.5 ng) was coinjected
with XeILK MO. Rhodamine-dextran was co-injected
as a lineage tracer. Animal cap explants were excised at stage 8.5
and cultured with or without 10 ng/mL recombinant activin until
stage 19. Arrows indicate weak defects in the 5-mis MO-treated animal
caps. Arrowheads indicate the rescued animal caps. (B) Activininduced
mesodermal differentiation was not affected by the
injection of XeILK MO. Embryos were injected as indicated
(25 ng) into the animal pole region at the four-cell stage. Animal
cap explants were excised at stage 8.5, cultured with or without
10 ng/mL recombinant activin until stage 10.5 for RT-PCR
analysis of Xbra and Chordin expression. (C) Cell–cell and cell–
extracellular matrix adhesions were disrupted in the XeILKdeficient
dorsal marginal zone (DMZ) explants and partially
rescued by co-injecting XeILK mRNA. MOs (25 ng) were
injected as indicated into the two dorsal blastomeres at the fourcell
stage. For rescue of XeILK depletion, mut. XeILK mRNA
(0.5 ng) was co-injected with XeILK MO. Rhodamine-dextran
was co-injected as a lineage tracer. At stage 10, DMZ explants
were dissected, mounted on to fibronectin-coated coverslips and
cultured until stage 19. Magnified images of the middle panels are
shown in the bottom panels.
ilk (integrin-linked kinase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anteriorleft, dorsal up.
