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Establishment of left-right (L-R) asymmetry is fundamental to vertebrate development. Several genes involved in L-R asymmetry have been described. In the Xenopus embryo, Vg1/activin signals are implicated upstream of asymmetric nodal related 1 (Xnr1) and Pitx2 expression in L-R patterning. We report here that Zic3 carries the left-sided signal from the initial activin-like signal to determinative factors such as Pitx2. Overexpression of Zic3 on the right side of the embryo altered the orientation of heart and gut looping, concomitant with disturbed laterality of expression of Xnr1 and Pitx2, both of which are normally expressed in the leftlateral plate mesoderm. The results indicate that Zic3 participates in the left-sided signaling upstream of Xnr1 and Pitx2. At early gastrula, Zic3 was expressed not only in presumptive neuroectoderm but also in mesoderm. Correspondingly, overexpression of Zic3 was effective in the L-R specification at the early gastrula stage, as revealed by a hormone-inducible Zic3 construct. The Zic3 expression in the mesoderm is induced by activin (beta) or Vg1, which are also involved in the left-sided signal in L-R specification. These findings suggest that an activin-like signal is a potent upstream activator of Zic3 that establishes the L-R axis. Furthermore, overexpression of the zinc-finger domain of Zic3 on the right side is sufficient to disturb the L-R axis, while overexpression of the N-terminal domain on the left side affects the laterality. These results suggest that Zic3 has at least two functionally important domains that play different roles and provide a molecular basis for human heterotaxy, which is an L-R pattern anomaly caused by a mutation in human ZIC3.
Fig. 1. Reversal of heart and gut looping in Zic3-injected embryos.
(A,B) Wild-type Xenopus embryo (stage 47) with a rightward
looping heart and counterclockwise coiled gut. (C,D) Embryo that
was injected with 50 pg of Zic3 on the right side, with a leftward
looping heart and a clockwise coiled gut. (E) The frequency of
reversed organs. Right-sided Zic3 injection altered heart and gut
looping more frequently than left-sided injection (see also Table 1).
The inversion frequency by activin injection (0.5 pg) was determined
in a positive control.
Fig. 2. Asymmetric expression
of Xnr1 and Pitx2 is disturbed
by Zic3. In situ hybridization of
Xnr1 (A-D) and Pitx2 (E-H).
Xnr1 and Pitx2 expression was
observed in the left LPM
(A,B,E,F). Zic3 (100 pg)-
injected embryos show bilateral
(C,G) or right side (D,H)
expression. The frequency of
disturbed Xnr1 (I) or Pitx2 (J)
expression by left or right side
Zic3, Zic1 (250 pg), Zic2 (100
pg), GLI1 (1000 pg) or activin
(0.5 pg) injection.
Fig. 3. Zic3 is expressed in the mesodermal tissues at early gastrula.
Zic3 is expressed in the ring of involuting mesoderm (B,D) like the
pan-mesodermal marker, brachyury (A,C) at stage 10.5. Arrows
indicate blastopore. In C,D, embryos were cut along the broken line
shown in (I). At stage 12, Zic3 is not detected in involuting
mesoderm (F,H) in contrast to brachyury (E,G). White arrows
indicate yolk plug. (G,H) Embryos were cut along the broken line in
(J). Zic1 (K) and Zic2 (L) are also expressed in the mesodermal
tissues at early gastrula. Arrows indicate blastopore. bp, blastopore;
D, dorsal side; V, ventral side; yp, yolk plug.
Fig. 4. Zinc-finger domain or N-terminal domain of Zic3 alone can
disturb the L-R axis. (A) Deletion constructs of Zic3 used in this
experiment. Xnr1 or Pitx2 expression in Zic3- (100 pg), XZ3d4- (500
pg), XZ3d6- (1000 pg) or XZ3d7- (500 pg) injected embryos. The
frequency of the disturbed Xnr1 (B) or Pitx2 (C) expression by the
left or right side injections.
Fig. 5. Zic3
specifies the L-R laterality at early gastrula stage.
(A) Hormone-inducible construct of Zic3 used in this experiment. (B) Disturbed expression of Pitx2
in Zic3-GR (100 pg) injected embryo when dexamethasone was added at several stages. The frequency
of the disturbed Pitx2 expression by the right-sided injections. DEX, dexamethasone; hGR, human
glucocorticoid
receptor.
Fig. 6. Vg1 and activin can induce Zic3 in the mesoderm. (A) Zic3 is
expressed symmetrically in wild-type embryos. Zic3 expression was
enhanced in the mesoderm at stage 10.5 when Vg1 (1000 pg) or
activin (5 pg) was injected into the lateral side of one blastomere at
the two-cell stage. Embryos were cut along the broken line shown.
RT-PCR analysis of animal cap explants from embryos injected with
Vg1 (250 pg) (B) or activin (1 pg) (C) at stage 25. Zic3, Xnr1 and
Pitx2 are induced by both genes. Vg1 or activin induce the
mesodermal marker, m-actin, but not the neural markers, neural celladhesion
molecule (N-CAM) and neurogenin. EF1a was used to
monitor RNA recovery.
Fig. 7. A hypothetical model for the involvement of Zic3 in the L-R
axis formation in Xenopus. Zic3 mediates the left-sided signaling
pathway. Activation of the activin-like signaling pathway on the left
side induces Zic3, and Zic3 specifies the left identity by the induction
of Xnr1 and Pitx2.
