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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.
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, anteriorleft, dorsal up.
