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Figure 1. cmXnr5 induced a hyperplastic cement gland or an ectopic head structure formation with a single eye and a cement gland. A: Scheme of the injection points of cmXnr5 RNA. The injection point B is the marginal region of two ventral-vegetal blastomeres near the ventral cross point of the first and the third cleavage lines. The injection point C/D/E is the approximately half region between the vegetal cross point of the first and the second cleavage lines and the ventral cross point of the first and the third cleavage lines in both ventral-vegetal blastomeres at 8-cell stage. Each injection point is correspondent to B or C/D/E, respectively. B: Embryos were injected with 500 pg of cmXnr5 RNA into the marginal region (injection point B). Embryos had shortened body axis and a hyperplastic cement gland (arrowhead). C: Embryos were injected with 1 ng of cmXnr5 RNA into the prospective ventral endoderm region (injection point C/D/E). Embryos had an ectopic eye (red arrow) and a cement gland (red arrowhead) at stage 40. The original eye and cement gland are indicated by the black arrow and arrowhead, respectively. D: Histological section of the embryo injected with 1 ng of cmXnr5 RNA into the prospective ventral endoderm region. An ectopic eye (red arrow) and a cement gland (red arrowhead) were observed in the ventral region. E: The embryo in D at high magnification. The ectopic eye showed normal structure with retinal pigment epithelium (yellow arrowhead), neural retina (nr), and lens. F-H: The expression of cement gland marker XCG1. F: An uninjected albino embryo stage 35. G: Embryos injected with 1 ng of cmXnr5 RNA into the marginal region (injection point B). H: Embryos injected with 1 ng of cmXnr5 RNA into the prospective ventral endoderm region (injection point C/D/E). Whole-mount in situ hybridization was performed according to Harland ([1991]).
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Fig. 2. cmXnrs caused neural formation and inhibited BMP signaling in animal cap explants.
A: Two-cell-stage embryos were injected into both blastomeres with 2 ng RNA of Xnr3 (3), cmXnr2
(c2), cmXnr5 (c5), or cmActivin (cA). Animal cap explants were processed for RT-PCR at stage 30.
Xnr3, cmXnr2, or cmXnr5 induced the expression of pan-neural marker, NCAM, but never induced
the expression of dorsal mesodermal marker, ms-actin. cmActivin did not induce the expression of
either marker gene. B: Two-cell-stage embryos were injected into both blastomeres with 1 ng of
Bmp4 RNA plus 1 ng of Xnr3 (3), cmXnr2 (c2), or cmXnr5 (c5) RNA. Animal cap explants were processed
for RT-PCR at stage 10.5. All three RNAs suppressed the expression of Xbra, Xwnt8, and msx-1
induced by Bmp4. Injection of 1 ng of cmXnrs RNA did not induce Xbra, Xwnt8, or msx-1 expression.
un, uninjected explants control; WE, whole embryo positive control; (-), RT-negative control.
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Fig. 3. cmXnr2 and cmXnr5 inhibited the activity
of Xwnt8. A: RT-PCR of animal caps co-injected
with Xwnt8 (2 pg), mouse dishevelled
(500 pg), or -catenin (500 pg) RNA and 1 ng of
cmXnr5 RNA. cmXnr5 suppressed the expression
of siamois and Xnr3 induced by Xwnt8, but
did not inhibit it induced by dishevelled and
-catenin. B: RT-PCR of animal caps co-injected
with 2 pg of Xwnt8 RNA and 1 ng of
cmXnr2 or cmXnr5 RNA. Both cmXnr2 and
cmXnr5 blocked the expression of siamois and
Xnr3 induced by Xwnt8. Both cmXnrs did not
induce the expression of Wnt ligand antagonists,
cerberus, Frzb1, or dkk1 in the animal cap
explants. Xwnt8 induced the expression of dkk1
in animal caps, and both cmXnrs also inhibited
the expression. C: RT-PCR of animal caps coinjected
5 pg of Xwnt8 and 4 ng of cmXnr1,
cmXnr2, cmXnr5, cmXnr6, cmNodal, cmderrie`
re, cmActivin and EGFP. cmXnr1, cmXnr6,
cmNodal, cmderrie` re, cmActivin, or EGFP, did
not inhibit the expression of siamois and Xnr3
induced by Xwnt8, while cmXnr2 and cmXnr5
did inhibit Xwnt8 activity. D: Intact Xnr2 and
Xnr5 did not inhibit Xwnt8 activity. One nanogram
of lacZ, Xnr2, or Xnr5 RNA was coinjected
with 2 pg of Xwnt8 RNA. w8, Xwnt8; dvl, dishevelled;
-cat, -catenin; c1, cmXnr1; c2,
cmXnr2; c5, cmXnr5; c6, cmXnr6; cN, cmNodal;
cd, cmderrie` re; cA, cmActivin; G, EGFP; un,
uninjected explants control; WE, whole embryo
positive control; (-), RT-negative control.
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Fig. 4. The pro domain is necessary, but not sufficient for inhibitory activity on Wnt signaling.
A: Schema of chimera constructs: pXnr5 encodes the pro domain of Xnr5, and the C-terminus
sequence is “RRHRR” (putative cleavage site of Xnr5). pXnr2 encodes the pro domain of Xnr2, and
the C-terminus sequence is “RRPRR” (putative cleavage site of Xnr2). pcmXnr5 also encodes the
pro domain of Xnr5, and the C-terminus sequence “RRHRR” was changed to “GVDGG,” corresponding
to cmXnr5. cmActivin-Xnr2, and cmActivin-Xnr5, which encode the pro domain of
Activin B, the mutated cleavage site, “GVDGG,” and the mature domain of Xnr2 or Xnr5, respectively.
cmXnr6-Xnr5 encodes the pro domain of Xnr6, the mutated cleavage site, “GVDGG,” and the
mature domain of Xnr5. cmXnr5-Xnr6 encodes the pro domain of Xnr5, the mutated cleavage site,
“GVDGG,” and the mature domain of Xnr6. B: RT-PCR of animal caps co-injected with 2 pg of
Xwnt8 RNA plus 1 ng of pXnr5 (p5) or pcmXnr5 (pc5) RNA. Neither pXnr5 nor pcmXnr5 suppressed
the expression of siamois and Xnr3 induced by Xwnt8. C: RT-PCR of animal caps co-injected with
2 pg of Xwnt8 RNA plus 1 ng of cmXnr2 (c2), cmActivin-Xnr2 (cA2), or cmActivin (cA) RNA. cmXnr2
inhibited the expression of siamois and Xnr3 induced by Xwnt8, but cmActivin-Xnr2 and cmActivin
did not inhibit the expression of these genes. D: RT-PCR of animal caps co-injected with 2 pg of
Xwnt8 RNA plus 1 ng of lacZ, cmXnr6-Xnr5, cmActivin-Xnr5, pXnr2, chordin, cmBMP7, dickkopf
(dkk), or cmXnr5 RNA. cmXnr6-Xnr5, cmActivin-Xnr5, and pXnr2 did not suppress Xwnt8 activity.
BMP inhibitor chordin and cmBMP7 did not suppress Xwnt8 activity. cmXnr5 and Wnt inhibitor dkk
suppressed Xwnt8 activity. E: RT-PCR of animal caps co-injected with 2 pg of Xwnt8 RNA plus 1
ng of lacZ or cmXnr5-Xnr6. cmXnr5-Xnr6 did not suppress Xwnt8 activity. un, uninjected explants
control; WE, whole embryo positive control; (-), RT-negative control.
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Fig. 5. Secretion and stability of unprocessed Xnr5 protein, tagged with myc epitope. A: Schema of four myc-tagged Xnr5 constructs. Xnr5-myc and
cmXnr5-myc encode each single c-myc-epitope in the pro domain. Xnr5-6myc encodes six c-myc-epitopes on the C-terminus of mature domain.
pXnr5-6myc encodes six c-myc-epitopes on the C-terminus of pro domain. B: Western blot analysis of culture medium of each of 10 oocytes injected
50 ng of Xnr5-myc, cmXnr5-myc, and pXnr5-6myc RNA. Each oocyte was cultured in 10 l of OR2 medium containing 0.05% BSA. Molecular masses
of standard proteins are indicated in kilodaltons at the side. C: Each oocyte was injected with 40 ng mRNA and cultured in 15 l of OR2 medium.
Unprocessed (68 kDa) and processed (30 kDa) forms of Xnr5-6myc were detected in both culture medium and oocyte lysate. Additionally 50 kDa of
band was detected in the lysate sample. Actin was used as a loading control. D: Each embryo was injected with 1 ng RNA and blastocoel fluid was
collected at stage 10.5. Unprocessed and processed forms of Xnr5-6myc were detected in both whole embryo lysate and the blastocoel fluid. -Actin
was used as a loading control. E: Western blot analysis of extract of embryos at stage 10. Injected amounts of Xnr5-6myc RNA are indicated at the
top. Both the processed mature protein (30 kDa) and the unprocessed full-length protein (68 kDa) were detected from 100 pg RNA injection. F:
Embryos were co-injected with 250 pg of cmXnr5-myc RNA and 250 pg of lineage-tracer NLS-lacZ RNA into one marginal blastomere at the 32- to
64-cell stage, and were stained with the RedGal substrate then immunostained at stage 10. Immunostain signal (purple) was detected at the
extracellular space (black arrows) outside of the region of RedGal labeling (red). F’: The high-magnification image of sample in F. The RedGal labeled
region is surrounded with a black broken line. G: Uninjected control embryo immunostained with anti-c–myc antibody showed no significant signal.
H: Embryos were co-injected with 250 pg of pXnr5-6myc RNA and 250 pg of lineage-tracer NLS-lacZ RNA into one marginal blastomere at the 32-
to 64-cell stage, and were stained with the RedGal substrate then immunostained at stage 10. Immunostain signal (purple) was detected at the
extracellular space outside of the region of RedGal labeling (red). H’: The high-magnification image of sample in H. The RedGal labeled region is
surrounded with a red broken line.
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