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Mogi K
,
Goto M
,
Ohno E
,
Azumi Y
,
Takeuchi S
,
Toyoizumi R
.
???displayArticle.abstract???
A variety of TGF-beta-related ligands regulate the left-right asymmetry of vertebrates but the involvement of TGF-betas in left-right specification has not been reported. We assessed whether TGF-beta signaling is involved in the left-right specification of Xenopus post-gastrula embryos by microinjecting Xenopus TGF-beta5 protein into the left or right flank of neurula-tailbud embryos. Injection on the right side of neurulae caused left-right reversal of the internal organs in 93% of the embryos, while injection on the left side caused less than 5% left-right reversal. Expression of Xenopus nodal related-1 (Xnr-1 ), Xenopus antivin and Xenopus Pitx2, which are normally expressed on the left, was unaltered by the left-side injection. In contrast, right-side injection into neurulae induced the expression of these genes predominantly on the right side. Right-side injection into tailbud embryos caused bilateral expression of these handed genes. Time course analysis of asymmetric gene expression revealed that Xnr-1 could be induced by TGF-beta5 at late neurula stage, while antivin and Pitx2 could be induced by TGF-beta5 at the latertail bud stage. Injection of the antisense morpholino oligonucleotide against Xenopus TGF-beta5 into the left dorsal blastomere inhibited the normal left-handed expression of Xnr-1 and Pitx2, and caused the organ reversal in the injected embryos. These results suggest that normal left-right balance of endogenous TGF-beta5 signaling in the neurulaembryo may be needed to determine the laterality of the asymmetric genes and to generate the correct left-right axis.
Fig. 1. Effect of TGF-β5 protein injection on the
left-right asymmetry of Xenopus internal organs.
All embryos are viewed from the ventral
side. (A) An uninjected sibling embryo at stage 42
shows normal heart looping, and the ventral pancreas
is situated in the left side. (B) An embryo
after injecting 10 pg TGF-β5 in the right flank at the
mid-neurula stage (stage 15). Both heart and
visceral organs are inverted. (C) An embryo after
injecting 10 pg TGF-β5 in the left flank at stage 15.
Left-right orientation of the internal organs is
normal.
Fig. 2. Stage-dependent transition
of the incidence of leftright
inversion caused by
TGF-β5 injection. Incidence of
left-right reversal in embryos
injected with TGF-β5 (A,B), TGF-
β1 (C,D), TGF-β2 (E,F) and TGF-
β3 (G) in the left flank (B,D,F) or
in the right flank (A,C,E,G). The
horizontal axis shows the stage
of development when the injection
took place, while the
longitudinal axis represents the
ratio of left-right inversion as percent. Asterisk (*) shows the case not tested. n, number of embryos
that reached stage 41-42 and were checked for left-right orientation.
Fig. 3. Effect of injecting TGF-β5 into one side of neurula embryos on
asymmetric gene expression. TGF-β5 (50 pg) was injected at early-mid
neurula stage (stage 13-16) and the embryos were fixed at the tailbud stage
(stage 24-28) and assessed for Xnr-1(left injection, A; right injection, B-C),
Xatv (left injection, D; right injection, E), Pitx2 (left injection, F; right
injection, G) and Smad2 (H, I) expression. After injecting in the left flank,
Xnr-1 (A), Xatv (D) and Pitx2 (F) were expressed on the left . After injecting
in the right flank, Xnr-1 was expressed predominantly on the right only (B),
or less frequently on both sides or not at all (C). After injecting in the right
side, Xatv (E) and Pitx2 (G) were also expressed predominantly on the right
only. The bilateral expression of Pitx2 around the eyes and cement gland
was not altered by either the left side or right side injection. (H) Expression
of Smad2 in the untreated embryo at stage 34. Note the absence of the
mRNA in both LPMs. (I) Expression of Smad2 after injecting TGF-β5 in the
right side. Smad2 was not induced in either LPM. The embryos in A-C are
dorsal views with the anterior of the embryo to the top of the panels. The
embryos in D-I are lateral views. Small capitals represent the left side (L)
or the right side (R). Scale bar, 1 mm.
Fig. 4. Effect of injecting TGF-β5 into the right
side of the tailbud embryos on asymmetric
gene expression patterns. TGF-β5 (50 pg) was
injected at stage 21-24 and the embryos were fixed
at stage 26-30 and assessed for the expression of
(A) Xnr-1 (dorsal view of bilateral expression), (B)
Xatv (ventral view of bilateral expression), and (C)
Pitx2 (lateral view of bilateral expression). By injecting
TGF-β5 into the right side of tailbud embryos,
most of the embryos showed the bilateral expression
of these genes. Scale bar, 1 mm.
Fig. 5. Incidence of altered asymmetric
gene expression and the predicted incidence
of left-right organ reversal. The blue,
green, and red bars represent the expression
of Xnr-1, Xatv and Pitx2, respectively. L, expression
on the left only; L>R, bilateral expression
that predominates on the left; L=R,
symmetrical bilateral expression; L<R, bilateral
expression that predominates on the
right; R, expression on the right only; Absence,
absence of expression. (A) Scoring of
the embryos injected on the left at the neurula
stage. (B) Scoring of the embryos injected
on the right at the neurula stage. Rightside
but not left-side injection induced gene
expression in the right LPM and simultaneously
repressed the normal left expression
of these genes. (C) Scoring of the embryos
injected on the right at the tailbud
stage. Gene expression was induced in the
right LPM but the normal left expression was
not suppressed. (D) Calculation of the predicted
incidence of left-right reversal on the
basis of the altered expression patterns of
the asymmetric genes caused by the injection
of TGF-β5 on the right.
Fig. 6. Temporal expression of
the asymmetric genes after the
injection of TGF-β5 into the left
or right side of early neurulae.
Embryos were injected on either
side at the early neurula stage,
fixed at various subsequent stages
and assessed for the expression
of Xnr-1( (A,B) left injection; (C,D)
right injection), Xatv ((E) right injection),
or Pitx2 ((F), right injection). TGF-β5 injection could not induce the
precocious expression of any of the genes in the mid-neurula embryos (stage
16; see A,C,E,F) and expression commenced at the normal time points (e.g.
Xnr-1 was expressed at stage 19, see B,D). (G) Injection did not alter Shh
expression along the midline or induce the ectopic expression of Shh.
Fig. 7. Timing of expression of the asymmetric genes after TGF-β5 injection. Early
neurulae were injected with TGF-β5 and then fixed at various time points and their gene
expression was scored. The scoring was focused on the expression in the LPM ipsilateral
to the injection side. Significant (more than 10%) expression is colored pink; gray zone,
not tested. Colored bars represent the time course of the expression of the handed
genes in normal embryos (Lowe et al., 1996; Ryan et al., 1998; Cheng et al., 2000).
Fig. 8. Effect of injecting antisense morpholino oligonucleotide against
TGF-β5. (A,B) Right-side injection of TGF-β5 MO had no effect on left-right
orientation of the internal organs (A, normal situs), whereas left-side
injection reversed the orientation (B, the case of heart-alone reversal).
(C,D,E) Injection of TGF-β5 MO into the dorsal blastomere inhibited the
expression of TGF-β5 in somites in the early tailbud embryo. Bilateral
expression of TGF-β5 (C, a control embryo) was down-regulated only on
the injection side by injecting the MO. D, right side injection of the MO; the
expression in somites was reduced or ceased in 60% of the embryos
(n=18/30). E, left side injection; the paraxial expression was reduced or
ceased in 57% of the embryos (n=20/35). (F) Shown are the ratios (%) of
left-right reversal after injecting Xenopus TGF-β5 MO, human globin MO,
and RNase-free water. The bottom numbers show the ratio of embryos
with organ reversal/survived embryos. Only the case of left-side injection
of TGF-β5 MO elicited significant left-right reversal. (G,H,I) TGF-β5 expression
in the eye primordia and Morpholino knock-down of the pigmented
retina differentiation. G, lateral view of a stage 23 embryo, which does not
express TGF-β5 in the eye but expresses in somites. In later tailbud
embryos, TGF-β5 is expressed in the inner margin of the retina primordia
and the forming lens, nasal plate and branchial region (H). I, the larval âfree
lensâ without retinal differentiation was frequently observed after the
injection of the TGF-β5 MO.
Fig. 9. Inhibition of handed gene expression by the injection of TGF-β5
MO. (A,B) Xnr-1 expression in embryos injected with TGF-β5 MO. The upper
one was injected in the dorsal left blastomere, and ceases left-handed Xnr-
1 expression (A, viewed from left side). In the lower one which was injected
in the dorsal right blastomere, the expression of Xnr-1 in the left LPM is not
affected. (C,D) Pitx2 expression in embryos injected with TGF-β5 MO. The
upper one was injected in the dorsal left blastomere and, similarly to the case
of Xnr-1, the embryo ceases left-handed Pitx2 expression (C, viewed from
left side). The lower one was injected in the dorsal right blastomere, and left
Pitx2 expression is not affected. (E,F) Embryos were scored for the expression
of Xnr-1 (blue bar) and Pitx2 (red bar) after injecting TGF-β5 MO into the
right (E) or left (F) blastomere. When injected on the left side, TGF-β5 MO
downregulates the left-handed expression of Xnr-1 and Pitx2.
