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Vertebrate species display consistent left-right asymmetry in the arrangement of their internal organs. This asymmetry reflects the establishment of the left-right axis and the alignment of the organs along this axis during development. Members of the TGF-beta family of molecules have been implicated in both the establishment and signaling of left-right axis information. Asymmetric expression of one member, nodal (called Xnr-1 in the frog, Xenopus laevis), is highly conserved among species. The nodal-related genes are normally expressed in the leftlateral plate mesoderm prior to the development of morphologic asymmetry. Expression patterns of nodal have been correlated with heart situs in mouse, chick, and frog and our previous work has implicated the dorsal midline structures in the regulation of nodal expression and cardiac laterality. In this study, three approaches were used to address the embryologic and molecular basis of asymmetric Xnr-1 expression. First, notochord and lateral plate recombinants were performed and showed that notochord can repress Xnr-1 expression in lateral plate mesoderm explants derived from either the left or the right side. Second, lateral plate mesoderm grafts indicated that Xnr-1 expression is specified but not determined at neurula stages and can subsequently be repatterned. These experiments suggest that a repressive signal from the notochord is required for maintenance of asymmetric Xnr-1 expression and that Xnr-1 expression is regulated by signals outside of the lateral plate mesoderm. Third, candidate molecules were injected to test for their ability to alter Xnr-1 expression pattern in the lateral plate. Late injection of activin protein on the right side of the embryo induced ectopic Xnr-1 expression and randomized cardiac orientation. This suggests that activin or a related TGF-beta molecule is involved in the proximal regulation of asymmetric Xnr-1 expression.
Fig. 1. A: Schematic diagram of recombinant explants. Lateral
plate explant from a stage 15/16 embryo is wrapped around a stage 19
notochord and allowed to heal. B: Xnr-1 expression at stage 24 assayed
by in situ hybridization in isolated explants of left and right lateral
plate mesoderm [from Lohr et al., 1997] and recombinant explants.
Xnr-1 expression is repressed in 66% of recombinants from both right
and left lateral plate. LPM, lateral plate mesoderm; N, number of
explants. For left LPM recombinants, z 5 3.6, P , 0.001 vs. left LPM;
for right lateral plate recombinants, z 5 3.5, P , 0.001 vs. right LPM.
Fig. 2. Xnr-1 expression in lateral plate (LP) grafts performed at
stage 20. Embryos in A–C were fixed and assayed for Xnr-1 expression
by in situ hybridization at stage 24/25. Embryos are photographed
from a dorsal view with anterior at the top of the page. Photos are
cropped to show relevant region only. Red arrows point to grafts, black
arrows to host Xnr-1 expression, and black arrowheads to Xnr-1
expression in the grafts. A: Left LP explant is grafted onto the right
side of host embryo. There is normal Xnr-1 expression in the host left
lateral plate mesoderm (LPM) and no Xnr-1 expression in the left LPM
grafted into the right side. B: Left LP explant grafted into the left side
of the host embryo as a control for the grafting experiment in A. There
is expression in both host left LPM and the graft. C: Right LP graft
expresses Xnr-1 when grafted into the left LP. The host left LPM
expresses Xnr-1 normally. D: Percentage of LP explants that express
Xnr-1 at stage 24 by in situ hybridization when grafted into the site
shown (grafts performed with donor and host at stage 20).
Fig. 3. A–C are dorsal views of albino Xenopus embryos at stage
24/25. Anterior is at the top of the page. A: Normal left-sided Xnr-1
expression. B: Bilateral expression of Xnr-1 in the lateral plate
mesoderm and somites of an embryo injected in the right lateral plate
mesoderm with 25 ng of human recombinant activin A. C: Symmetric
expression of Xshh in the neural tube/floorplate of an embryo injected
with 25 ng of activin into the right lateral plate at stage 20. Right
lateral view of embryo (on right side of panel) indicates no ectopic Xshh
expression. D: Expression of Xenopus activinbB at stage 20. (Top
embryo - dorsal view with anterior towards the top of the page; bottom
embryo - left lateral view). No consistent asymmetries of expression