Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Dev Biol
2007 Aug 15;3082:485-93. doi: 10.1016/j.ydbio.2007.06.001.
Show Gene links
Show Anatomy links
IQGAP2 is required for the cadherin-mediated cell-to-cell adhesion in Xenopus laevis embryos.
Yamashiro S
,
Abe H
,
Mabuchi I
.
???displayArticle.abstract???
We have previously identified two Xenopus homologues of mammalian IQGAP, XIQGAP1 and XIQGAP2, which show high homology with human IQGAP1 and IQGAP2, respectively. In order to clarify function of the IQGAPs during development, we performed knock-down experiments on the XIQGAPs in Xenopus laevis embryos by microinjecting morpholino antisense oligonucleotides into blastomeres at the two-cell stage. Suppression of XIQGAP2 expression caused ectodermal lesions in the neurula stage embryos. While suppression of XIQGAP1 expression alone did not show any obvious defect in subsequent developmental processes, simultaneous knock-down of both XIQGAPs caused the ectodermal lesions during the gastrula stage. Histological studies suggested that a loss of cell adhesion in the ectodermal and mesodermal layers of the embryos caused the defect. The suppression of XIQGAP2 expression resulted in loss of actin filaments, beta-catenin, and XIQGAP1 from cell borders in the ectoderm, although it did not affect the expression levels of these proteins. Furthermore, it inhibited Ca(2+)-induced reaggregation of embryonic cells which had been dissociated in a Ca(2+)/Mg(2+)-free medium. These results strongly suggest that XIQGAP2 is crucial for cell adhesion during early development in Xenopus.
Fig. 1. Effects of injection of morpholino antisense oligonucleotides into two-cell stage embryos on the expression of XIQGAPs. (A) Reduction of XIQGAP1 at the late gastrula stage (stage 11) caused by injection of XIQGAP1-AS into the two-cell stage embryo as shown by immunoblotting with anti-XIQGAP1 antibodies. (B) Reduction of XIQGAP2 at the mid-gastrula stage (stage 10) and the early neurula stage (stage 13) caused by injection of XIQGAP2-AS into the two-cell stage embryo as shown by immunoblotting with anti-XIQGAP2 antibodies. α-Tubulin was used as a loading control.
Fig. 2. Injection of XIQGAP2-AS into two-cell stage embryos lead to lesions. XIQGAP2-AS (20 ng) or control oligonucleotide (20 ng) was injected into one blastomere of the two-cell stage embryos. The injected embryos were cultured in buffer A (see Materials and methods) after the neurula stage to maintain Ca2+-dependent cell-to-cell adhesion. (A) An XIQGAP2-AS-injected embryo developed normally until early neurula (a, stage 13), but lesions were observed during the neurula stage. Finally, the ectodermal layer and the mesodermal layer of the half of the embryo were disintegrated at the early tailbud stage (b, stage 24, arrow). (c, d) Control oligonucleotide-injected embryos developed normally. Dorsal views are shown. The embryos are placed such that their anterior faces top (a, c) or left (b, d). (B) Transverse sections of embryos at the neurula stage (stage 20) and the early tailbud stage (stage 24). (a) An embryo at the neurula stage previously injected with XIQGAP2-AS. The arrow indicates cells detached from the ectoderm. (b) An embryo at the early tailbud stage previously injected with XIQGAP2-AS. The ectodermal layer and the mesodermal layer of the right side of the embryo (arrow) were disintegrated. (c) An embryo at the neurula stage previously injected with control oligonucleotide. (d) An embryo at the early tailbud stage injected with control oligonucleotide. ec, ectoderm; me, mesoderm; en, endoderm; no, notochord; nt, neural tube.
Fig. 3. Immunoblot analysis of XIQGAPs and adherens junction proteins in extracts from stage 20 embryos. The two blastomeres of the two-cell stage embryos were injected with XIQGAP2-AS (20 ng each) or control oligonucleotide (20 ng each). The blots were probed with antibodies against the proteins indicated in the figure. The injection of XIQGAP2-AS efficiently caused reduction of XIQGAP2 (top panel), while expression levels of the other proteins were not altered (lower panels). α-Tubulin was used as a loading control.
Fig. 4. Loss of actin filaments, β-catenin and XIQGAP1 at the ectodermal cell borders in XIQGAP2-AS-injected embryos. XIQGAP2-AS (13 ng) was injected into one blastomere of two-cell stage embryo together with a fixable dextran conjugated with Oregon Green, which was used as a lineage tracer. (A) Embryos at the neurula stage (stage 18) were fixed, sectioned transversely, and then stained with rhodamine–phalloidin (a), anti-β-catenin antibodies (c) or anti-XIQGAP1 antibodies (e). a′ and a′′, enlarged images of boxed regions in a. c′ and c′′, enlarged images of boxed regions in c. e′ and e′′, enlarged images of boxed regions in e. b, d and f, merged images of Oregon Green–dextran (green) and Rhodamine–phalloidin (b), anti-β-catenin (d) or anti-XIQGAP1 (f). (B) Control oligonucleotide (13 ng) was injected as in panel A. Embryos at the neurula stage (stage 20) were fixed, sectioned and stained with rhodamine–phalloidin (a) or anti-XIQGAP1 antibodies (c). b and d, merged images of Oregon Green–dextran and rhodamine–phalloidin (b) or anti-XIQGAP1 antibodies (d). ec, ectoderm; no, notochord; so, somites; nu, neural plate and neural tube. Bars = 100 μm.
Fig. 5. Injection of XIQGAP2-AS inhibits Ca2+-dependent cell reaggregation. Control oligonucleotide (13 ng) or XIQGAP2-AS (13 ng) was injected into each blastomere of two-cell stage embryos together with fluorescein–dextran (Fdx) or rhodamine–dextran (Rdx) which was used as a lineage tracer. Animal cap cells dissociated from control oligonucleotide-injected embryos reaggregated in the Steinberg's solution which contained Ca2+ (a), while those of XIQGAP2-AS-injected embryos did not reaggregate (b). (c–e) Mixture of two groups of animal cap cells dissociated from control oligonucleotide-injected embryos one contained Fdx and the other contained Rdx formed large clumps in the Steinberg's solution. (f–h) When animal cap cells dissociated from XIQGAP2-AS and Fdx-co-injected embryos and those from control oligonucleotide and Rdx-co-injected embryos were mixed, the cells containing control oligonucleotide and Rdx-formed clumps, while those containing XIQGAP2-AS and Fdx did not reaggregate. Bar = 100 μm.
Supplemental Fig. 1. Time-lapse images of a control (buffer-injected, left) and an XIQGAP2 mRNA-injected (right) embryos. The injections were done into both blastomeres at the two-cell stage. Top photographs (time, 0 min) are the embryos at stage 10.5. Arrowheads show the closing dorsal lip formed in the control embryo. The XIQGAP2 mRNA-injected embryo showed exogastrulation (arrow).
