Kamata T et al. (1998), Xenopus CRMP-2 is an early response gene to neu...

XB-ART-14561

Brain Res Mol Brain Res 1998 Jun 15;572:201-10. doi: 10.1016/s0169-328x(98)00082-5.

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Xenopus CRMP-2 is an early response gene to neural induction.

Kamata T , Daar IO , Subleski M , Copeland T , Kung HF , Xu RH .

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A neural specific protein, CRMP-2 (for Collapsin Response Mediator Protein-2), is considered to mediate collapsin-induced growth cone collapse during neural development. We have isolated the Xenopus homologue of the CRMP-2 (XCRMP-2) cDNA and studied the expression of XCRMP-2 mRNA and protein during neural induction. Induction of XCRMP-2 mRNA and protein expression, like N-CAM, occurred at the midgastrula stage and increased through early neural developmental stages. Whole mount in situ hybridization demonstrated that expression of XCRMP-2 mRNA was localized in neural tissues such as the neural plate and tube at early stages, while its expression in the brain, spinal cord, and eyes was observed at later stages. Immunostaining of Xenopus embryos with the antibody against CRMP-2 also showed that the protein was specifically expressed in the neural tissues at early stages. XCRMP-2 expression was induced by neural inducers such as noggin and chordin which antagonize a neural inhibitor, BMP4. A dominant negative BMP receptor also induced XCRMP-2 expression, suggesting that transcription of XCRMP-2 gene was negatively regulated by the BMP4 signaling. These results indicate that expression of XCRMP-2 is an early response marking neural commitment, and that transcriptional control of XCRMP-2 gene, is one of the targets of BMP4 signaling.

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Species referenced: Xenopus
Genes referenced: bmp4 chrd dpysl2 dpysl3 emb nog

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	Fig. 1. Amino acid alignments of XCRMP-2 and CRMP-related proteins. A partial length XCRMP-2 cDNA of 2.5 kb contained an open reading frame encompassing 563 amino acids, but the NH2-terminus has not been assigned yet. The amino acid sequence of XCRMP-2 was aligned with the corresponding amino acid sequences of bovine CRMP-2, chicken CRMP-62, rat TOAD-64, rCRMP-1, rCRMP-2, rCRMP-3, rCRMP-4, Ulip and C. elegans unc-33. Dots indicate that the amino acids are identical. The solid lines indicate gaps in the alignment. The numbers are counted from each NH2-terminus of bCRMP-2, CRMP62, ULIP, rCRMP-1, rCRMP-2, rCRMP-3, and rCRMP-4. Homology comparison were performed using the BLAST system.
	Fig. 2. Expression of XCRMP-2 mRNA and proteins. A. Total RNA was prepared from eggs, midgastrula stage 11., neural plate stage stage 14., late tailbud stage stages 27. and tadpole stage stage 37. embryos. The mRNA levels of XCRMP-2 were analyzed by RT-PCR See Section 2.. A 1.6-kb cDNA fragment was generated. The amount of RNA was quantitated by ethidium bromide staining of 28 s and 18 s rRNA. B. Indicated amounts of cytoplasmic extracts prepared from eggs, midgas- trula stage 11., late tailbud stage stage 27., and adult frog brain Ad. were loaded onto SDS-10% PAGE and immunoblotted with the anti- CRMP-2 antibody N17.
	Fig. 3. Localization of XCRMP-2 mRNA in Xenopus embryos. Whole mount in situ hybridization of Xenopus embryos with digoxigenin-labeled XRMP-2 antisense RNA reveals expression of XCRMP-2 transcripts during neurulation. Stages according to Nieuwkoop and Faber w21x refer to A. early gastrula stage 10., B. early neural plate stage 13., C. midneurula stage 16., D. late neurula stage 20., E. lateral view of early tailbud stage 23., F. dorsal view of early tailbud stage 23., G. late tailbud stage 27., and H. tadpole stage 36..
	Fig. 4. RT-PCR analysis of XCRMP-2 mRNA expression in neural tissues. The middle 1r3 of the dorsal tissue was excised from stage 17 embryos. The explants contained neural fold NF. and dorsal mesoderm DM. tissues such as notochord and somite. NF and DM were separated from some of the explants. Molecular markers were analyzed in NF, DM or NFqDM, using RT-PCR. Whole embryo Emb. as a positive control was included. EFI-a expression indicates that comparable amounts of RNA were used in each set.
	Fig. 5. Distribution of XCRMP-2 protein in Xenopus embryos. XCRMP-2 immunoreactivity in the central nervous system spinal cord and brain. in embryo at tadpole stage stage 37. was visualized by whole-mount staining with the anti-CRMP-2 antibody N17.
	Fig. 6. Induction of XCRMP-2 expression by neuralizing molecules. The animal pole of 2-cell stage embryos was injected with RNAs encoding b-galactosidase 2 ng., noggin 0.2 ng., chordin 1.5 ng., BMP-4 2 ng. and dominant negative BMP receptor DN-BR. 1 ng.. Animal caps were explanted at blastula stage and cultured to stage 24 before harvest. RNAs were extracted and gene expression was analyzed by RT-PCR as described in Fig. 4.
	dpysl2 (dihydropyrimidinase like 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, horizontal view, animal up.
	dpysl2 (dihydropyrimidinase like 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, lateral view, anterior right, dorsal up.
	dpysl2 (dihydropyrimidinase like 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior right, dorsal up.
	dpysl2 (dihydropyrimidinase like 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 36, lateral view, anterior right, dorsal up.