XB-ART-2582Dev Growth Differ October 1, 2004; 46 (5): 405-12.
Identification and characterization of Xenopus OMP25.
This study describes the isolation of mitochondrial outer membrane protein 25 (OMP25) from Xenopus laevis and an analysis of its role in early development. X. laevis OMP25 (xOMP25) is a transmembrane protein of the mitochondrial outer membrane with a PDZ domain in the cytoplasmic tail, and an approximate molecular size of 25 kDa. We isolated xOMP25 from a cDNA library of X. laevis tailbud embryos. Amino acid sequence analysis of xOMP25 showed 57% identity to mouse OMP25, with 73% identity in the PDZ domains. XOMP25 mRNA is expressed maternally, and at a constant level throughout early development. The transcript is localized to eye, otic vesicle, branchial arch and neural tube. Mitochondrial targeting of an EGFP-fusion protein of xOMP25 was visualized using a mitochondria-specific fluorescent dye. Overexpression of xOMP25 in embryos caused curved axes, small eyes and disorganized head structures. Knockdown of xOMP25 protein using antisense morpholino oligonucleotides resulted in slightly shortened axes and decreased neural tissue. Although the mechanism remains unclear, our results implicate xOMP25 protein in the formation of the intact neural tube.
PubMed ID: 15606486
Article link: Dev Growth Differ
Species referenced: Xenopus laevis
Genes referenced: synj2bp
Morpholinos: synj2bp MO1
Article Images: [+] show captions
|Fig. 2. Expression pattern of xOMP25 mRNA. (A) The temporal expression pattern of xOMP25 was determined by reverse transcription-polymerase chain reaction (RT-PCR) analysis. XOMP25 mRNA exists maternally and expression is sustained until the tadpole stage. (B�F) The spatial expression pattern of xOMP25 mRNA is shown by whole-mount in situ hybridization. Signals are observed in the animal pole at the 8 cell stage (B), in the anterior neural fold at the stage 18 (C,D), and in eye, otic vesicle, branchial arch and neural tube in stages 28 and 35 (E,F). xOMP25 expression in the otic vesicle and neural tube was also seen in transverse section (G). Anterior upward view (C).|
|Fig. 3. Cellular localization of xOMP25 protein in MC3T3 cells. (A,F) Phase contrast images of transfected cells. (B,G) Visualization of transfected protein. Signals of EGFP-xOMP25-mt are seen as puncta within the cytosol whereas the EGFP signal was distributed throughout the cytosol. (C,H) Mitotracker Red staining. All cells showed puncta of red fluorescence, regardless of transfection. (D,E,I,J) Merged images. Clear co-localization was seen between Mitotracker Red and EGFP-xOMP25, but not between mitotracker and pEGFP vector. Bar, 25 μm.|
|Fig. 4. The effect of xOMP25 overexpression in embryos. (A,B) Stage 37 embryos injected with xOMP25 ORF mRNA showed curved axes, small eyes and disorganized heads. (C,D) Transverse sections of the embryos have all the components of anterior structure including eyes, notochord, pharynx and forebrain, but the order and maturation were disrupted compared with the control embryos. Bar, 100 μm. (E) Relationship between mRNA dose and the frequency of phenotypes. Phenotypes become more severe and more frequent in a dosedependent manner. (F) The effect of xOMP25 on convergent extension. xOMP25-overexpressing animal caps changed shape in response to activin treatment, but did not elongate as was the case in the control animal caps.|
|Fig. 5. Loss-of-function analysis of xOMP25 using morpholino antisense oligos. (A) Schematic diagram of xOMP25AS and xOMP25 mRNAs. XOMP25 5 UTR + ORF mRNA has the target sequence of xOMP25AS, but xOMP25 ORF mRNA does not. (B) XOMP25AS can block the translation from xOMP25 5 UTR + ORF mRNA, but not the translation from xOMP25 ORF mRNA. (C�E) The phenotypes of stage 37 embryos injected with xOMP25AS are shown in external views. Injection of xOMP25AS resulted in small or no eye and slightly shortened axes (C) compared with control embryos (D). These phenotypes were rescued by co-injection of 200 pg of xOMP25 ORF mRNA and 100 pg of pCS2-xOMP25 ORF plasmid (E). (F) Relationship between the dose of xOMP25AS and phenotype frequency. The frequency and severity of the phenotypes increased in a dosedependent manner. (G�I,K�L) Transverse sections of the embryos shown in (C) and (D). XOMP25AS-injected embryo shows reduced neural tissue from the forebrain (K), hindbrain (L) and spinal cord (M) compared with control embryos (G�I). (J,N) Terminal deoxyribonucleotidyl transferase-mediated dUTP�digoxigenin nick end-labeling (TUNEL) analysis on section of embryos shown in (C) and (D). Slightly more TUNEL-positive cells (arrow) are seen in the xOMP25AS-injected embryo (N) than in the control embryo (J), but not in neural tissue. N, neural tube; No, notochord; ov, otic vesicle. Bar, 100 μm.|
|synj2bp (synaptojanin 2 binding protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.|