Peng Y et al. (2006), Cold-inducible RNA binding protein is required ...

XB-ART-486

Biochem Biophys Res Commun 2006 May 26;3441:416-24. doi: 10.1016/j.bbrc.2006.03.086.

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Cold-inducible RNA binding protein is required for the expression of adhesion molecules and embryonic cell movement in Xenopus laevis.

Peng Y , Yang PH , Tanner JA , Huang JD , Li M , Lee HF , Xu RH , Kung HF , Lin MC .

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We have previously shown that the Xenopus homologue of cold-inducible RNA binding protein, XCIRP-1, is required for the morphogenetic migration of the pronephros during embryonic development. However, the underlying molecular mechanisms remain elusive. Here, we report that XCIRP is essential for embryonic cell movement, as suppression of XCIRP by microinjection of anti-sense mRNA and morpholino antisense oligonucleotides (MOs) significantly reduced protein expression, inhibited the cell migration rate, and inhibited eFGF and activin-induced animal cap elongation. By immunoprecipitation and RT-PCR, we further showed that the mRNA of a panel of adhesion molecules, including alphaE- and beta-catenin, C- and E-cadherin, and paraxial proto-cadherin, are the targets of XCIRP. Consistently, in animal cap explant studies, suppression of XCIRP by MOs inhibited the expression of these adhesion molecules, while over-expression of sense XCIRP-1 mRNA fully rescued this inhibition. Taken together, these results suggest for the first time that XCIRP is required to maintain the expression of adhesion molecules and cell movement during embryonic development.

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Species referenced: Xenopus laevis
Genes referenced: arf6.2 bmp4 cirbp eef1a2 fgf2 fgf4 ncam1 sox2
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	Fig. 1. Suppression of XCIRP by MOs induces gastrulation defect. The two blastomeres of the 2-cell stage embryos were injected with 2 ng of sense XCIRP, 5 nM MOa XCIRP, 5 nMMOb XCIRP, and (s + MOa) XCIRP transcripts or equal amount of b-gal mRNA (control, c). (A) The embryos were allowed to develop to stage 11, and the embryos were then homogenized and subjected to Western blotting analysis. Lane 1, MOa; lane 2, MOb; lane 3, b-gal; lane 4, sXCIRP +MOa; and lane 5, sXCIRP. (B,C) Embryos were allowed to develope to stages 11, 18, 32, and 45, as indicated. (D–F) Histological analyses of the control and defected embryos stained with H and E. (D) At stage 11, sagittal sections showed defects in the movement of cells. (E) At stage 18, parasagittal section showed malformations of neurectoderm (N), mesoderm (M), brain, and eye anlages (BE). (F) At stage 18, anterior transversal section showed defect in neural crest (NC), neural tube (NT), and notochord (Noto).
	Fig. 2. Suppression of XCIRP inhibited cell movement and eFGF and activin-induced animal cap elongation. (A) Four-cell stage embryos were microinjected at the Dorsal Marginal Zone with a single injection of 2 ng of b-gal (control), 2 ng of sense XCIRP-1 (XCIRP(s)), 5 nM MOa, and combination of XCIRP(s) +MOa as indicated. The embryos were allowed to develop until the control group reached stage 11 and then fixed for X-gal whole-mount staining of the b-gal. (B) The two blastomeres of the 2-cell stage embryos were injected with 2 ng of b-gal mRNA (control), 2 ng of sense XCIRP(s), 5 nMMOa, or XCIRP(s) + MOa. Embryos were developed to stage 8.5, animal cap explants prepared, and an elongation assay was conducted as described in Materials and methods.
	Fig. 3. Suppression of XCIRP disrupted the morphogenetic lineage migration of A1 blastomeres. The morphogenetic lineage migration of A1 blastomeres was examined by injecting to one of the A1 blastomeres (at 32-cell stage) with the following mRNAs. (A,C) 0.05 ng of b-galactosidase (b-gal); (B,D) b-gal with 5 nM of MOa. The morphogenetic migration pattern of the A1 blastomeres was visualized by X-gal whole-mount staining (A,B) and histological analysis of the dorsal section (C,D) of the stages 28–30 tailbuds.
	Fig. 4. Identification of a panel of adhesion molecules as XCIRP target mRNAs in the embryos. (A) Xenopus embryo lysates were immunoprecipitated with preimmune serum (lane 1) or the anti-XCIRP serum (lane 2), followed by RT-PCR with specific primer sets as indicated. (B,C) Embryos were injected with various mRNA and MOs, and animal caps were prepared as indicated. Total RNA was extracted from the animal caps when the control embryos were developed to stage 22. The expression of XCIRP, cyclin B1, Nek2B, ARF6, EF1a, NCAM, SOX2, BMP4, aE- and b-catenin, C- and E-cadherin, as well as paraxial proto-cadherin (PAPC) were determined by RT-PCR as described in Materials and methods.