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Vertebrates appear bilaterally symmetrical but have considerable left-right (LR) asymmetry in the anatomy and placement of internal organs such as the heart. Although a number of asymmetrically expressed genes are known to affect LR patterning, both the initial source of asymmetry and the mechanism that correctly orients the LR axis remain controversial. In this study, we show that the induction of dorsal organizing centers in the embryo can orient LR asymmetry. Ectopic organizing centers were induced by microinjection of mRNA encoding a variety of body axis duplicating proteins, including members of the Wnt signal transduction pathway. The ectopic and primary body axes form side-by-side conjoined twins, with the secondary axis developing as either the left or right sibling. In all cases, correct LR asymmetry was observed in the lefttwin, regardless of whether it was derived from the primary axis or induced de novo by injection of Xwnt-8, beta-catenin, or Siamois mRNA. In contrast, the righttwin was generally unbiased, regardless of the origin of the left body axis, as seen in many instances of experimentally induced and spontaneous conjoined twins. An unanticipated exception was that right twins induced by beta-catenin and Siamois, two downstream effectors of Wnt signaling, exhibited predominately normal heart looping, even when they formed the righttwin. Taken together, these results indicate that LR asymmetry is locally oriented as a consequence of Wnt signaling through beta-catenin and Siamois. We discuss the possibility that signals upstream of beta-catenin and Siamois might be required in order for a right sibling to be randomized.
FIG. 1. Creation of conjoined twins derived from the dorsal (D)
and ventral (V) hemispheres of the embryo. Axis-duplicating
mRNA is co-injected with a lineage tracer (RLDx, red) at the 4- to
8- or 16- to 32-cell stage into the left (L) or right (R) ventral vegetal
blastomere. This manipulation induces a second organizer approximately
1807 opposite the original dorsal blastopore lip. Later morphogenetic
movements cause the developing secondary axis (27) to
become aligned to either the left or the right of the primary body
axis (17). The resultant tadpole stage twins are oriented side-byside,
possessing either a left or right secondary axis as indicated by
the position of the RLDx label.
FIG. 2. In situ hybridization showing Xnr-1 expression in conjoined twins with secondary axes induced by ectopic Xwnt-8. (A) A partial left secondary axis suppresses Xnr-1 expression in the adjoining left flank of the stage 23–24 primary axis. (B) Xnr-1 expression in an uninjected control embryo, stage 23–24. (C) The presence of a complete right secondary axis does not affect Xnr-1 expression (arrow) in the left twin’s leftlateralmesoderm (stage 23–24). (D) Fluorescent micrograph of embryo in C, showing that the righttwin is the induced axis. Faint fluorescence in the flank is due to autofluorescence. Positions of left (L) and right (R) twins are indicated.
FIG. 3. Three predicted outcomes for the asymmetric development of ventrally induced secondary axes. If LR orientation is patterned
globally (depicted as solid shading on left side) and only with respect to the primary axis, then the asymmetry of the secondary axis will
be inverted (A). Alternatively, if the embryo’s LR information is restricted to the vicinity of the primary axis, and the induced organizer
is unable to establish its own LR pattern, then the secondary axis will develop an unbiased, random asymmetry (B). In contrast, if each
organizer independently determines LR pattern, then the secondary axis will develop with normal handedness (C). D, V, L, R, 17, and 27
are as in Fig. 1.
FIG. 4. Cardiac situs in conjoined twins with a complete left secondary
axis. (A) Ventral view of heart region of Xwnt-8-induced
twinned embryo at tadpole stage; the hearts of the right and the
left twins are indicated. The arrows trace the direction of looping
in each heart. In this example, the righttwin (primary axis) exhibits
inverted looping, while the lefttwin (induced axis) has a normally
looped
heart (i.e., toward the embryo’s right). (B) Fluorescent micro-
graph of embryo shown in A. The RLDx fluorescence identifies the lefttwin as the induced axis.
FIG. 5. Xnr-1 expression in conjoined twins with complete left
secondary axes. Secondary axes were induced ventrally with synthetic mRNA encoding Xwnt-8 (A, B), b-catenin (C, D), or Siamois
(E, F) and fixed at stage 23–25. In situ hybridization was performed
with an antisense probe for Xnr-1. (A, C, E) Xnr-1 expression (purple)
in the left twin’s lateralmesoderm is indicated by the arrow
(anterior is toward top of figure, dorsal is to right). The more anterior
Xnr-1 expression in embryos shown in C and E is normal for
their slightly later stage (Lowe et al., 1996). (B, D, F) Fluorescent
micrographs of dorsal views of the same embryos as in A, C, and
E showing that the lefttwin is the induced axis. Position of left
(L) and right (R) twins is indicated.
