Callanan M et al. (2000), Developmentally regulated activity of CRM1/XPO1...

XB-ART-11635

J Cell Sci 2000 Feb 01;113 ( Pt 3):451-9. doi: 10.1242/jcs.113.3.451.

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Developmentally regulated activity of CRM1/XPO1 during early Xenopus embryogenesis.

Callanan M , Kudo N , Gout S , Brocard M , Yoshida M , Dimitrov S , Khochbin S .

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In this work, we have investigated the role of CRM1/XPO1, a protein involved in specific export of proteins and RNA from the nucleus, in early Xenopus embryogenesis. The cloning of the Xenopus laevis CRM1, XCRM1, revealed remarkable conservation of the protein during evolution (96.7% amino acid identity between Xenopus and human). The protein and mRNA are maternally expressed and are present during early embryogenesis. However, our data show that the activity of the protein is developmentally regulated. Embryonic development is insensitive to leptomycin B, a specific inhibitor of CRM1, until the neurula stage. Moreover, the nuclear localization of CRM1 changes concomitantly with the appearance of the leptomycin B sensitivity. These data suggest that CRM1, present initially in an inactive form, becomes functional before the initiation of the neurula stage during gastrula-neurula transition, a period known to correspond to a critical transition in the pattern of gene expression. Finally, we confirmed the gastrula-neurula transition-dependent activation of CRM1 by pull-down experiments as well as by the study of the intracellular localization of a green fluorescent protein tagged with a nuclear export signal motif during early development. This work showed that the regulated activity of CRM1 controls specific transitions during normal development and thus might be a key regulator of early embryogenesis.

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Species referenced: Xenopus laevis
Genes referenced: actl6a xpo1

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	Fig. 1. CRM1 is a highly conserved protein in vertebrates. Xenopus leavis CRM1 encoding cDNA was cloned and the deduced amino acid sequence was compared with that of the human CRM1. Stars indicate amino acid identity.
	Fig. 2. CRM1 is constitutively expressed during Xenopus embryogenesis. (A) 20 mg of total RNA isolated from embryos taken at the indicated stages were used to obtain a northern blot. The blot was then probed with 32P-labeled XCRM1 (upper panel) and Cardiac actin probe (lower panel). The ethidium bromide stained gel before the transfer of RNAs onto the membrane is also shown. (B) Equivalent amount of proteins extracted from embryos at the indicated stages of development was analyzed on a 8% polyacrylamide gel (stained gel, lower panel) and then transferred to a membrane that was used to detect CRM1 using a polyclonal anti- CRM1 antibody (upper panel).
	Fig. 3. Intranuclear localization of CRM1 changes during Xenopus embryonic development. (A) Cryosections obtained from embryos taken at the indicated stages were used for the immunodetection of CRM1. DNA column represents Hoechst labeled nuclei shown in the CRM1 column. Arrows indicate the specific intranuclear localization of CRM1 discussed in the text. (B) The ectopically expressed CRM1 becomes rapidly associated with nuclear membrane region. A plasmid containing the human CRM1 cDNA fused to GFP under CMV promoter was microinjected into stage 1 embryos. Embryos were then taken at the stage 10, fixed, cryosectioned and analyzed for GFP fluorescence.
	Fig. 4. Ectopic expression of CRM1 interferes with normal embryonic development. (A) 1 ng of RNA encoding either human or yeast (S. pombe) CRM1 (HuCRM1 and YCRM1, respectively), was microinjected into stage 1 embryos. Embryos were photographed when control embryos reached stage 18/19. (B) Stage 1 embryos were microinjected with 0.1 ng human or yeast CRM1-encoding RNA and embryos were photographed when control embryos reached stage 29/31 or stages 35/36 of development.
	Fig. 5. Leptomycin B treatment blocks embryonic development at the neurula stage of development. (A) Stage 2 embryos were placed in the presence of 1 mg/ml of leptomycin B and were photographed when control embryos (untreated) reached stage 24/26 of development. (B) Leptomycin B treatment disturbs the specific nuclear localization of CRM1. Untreated (control) and LMB-treated embryos taken at the indicated stages were collected and fixed. Cryosections from these embryos were used for immunolocalization of CRM1 as in Fig. 3. The DNA column represents Hoechst-labeled nuclei shown also in the CRM1 column (CRM1-related immunofluorescence). Arrows indicate the specific nuclear localization of CRM1 discussed in the text.
	Fig. 6. Developmentally regulated activity of CRM1. (A) 100 Xenopus embryos were collected at indicated stages to prepare an extract. The extracts were then incubated with glutathione beads coupled to a GST-GFP-NES fusion protein. After incubation, beads were washed and the presence of CRM1 and the fusion GST proteins were determined by western blotting (pull-down panel). 10 ml fraction of the extract from each stage was analyzed for the presence of CRM1 before the pull-down procedure (input panel). In parallel, a pull-down was also performed from stage 13 embryos in presence of 100 nM leptomycin (+LMB line). (B) A plasmid encoding a GSTSV40 T NLS-GFP-Rev NES fusion protein was microinjected into stage 1 Xenopus embryos. Cryosections were prepared from embryos taken at stages 10 and 13, fixed and counterstained with Hoechst to visualize the nuclei (DNA panel). The corresponding GFP-related fluorescence is shown in the GFP panels.