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Development of asymmetry along the left-right axis is a critical step in the formation of the vertebrate body plan. Disruptions of normal left-right patterning are associated with abnormalities of multiple organ systems, including significant congenital heart disease. The mouse nodal gene, and its homologues in chick and Xenopus, are among the first genes known to be asymmetrically expressed along the left-right axis before the development of organ asymmetry. Alterations in the expression pattern of mouse nodal and the chick homologue (cNR-1) have been associated with defects in the development of left-right asymmetry and cardiac looping (Levin, M., Johnson, R. L., Stern, C. D., Kuehn, M. and Tabin, C. (1995) Cell 82, 803-814; Collignon, J., Varlet, I. and Robertson, E. J. (1996) Nature 381, 155-158; Lowe, L. A., Supp, D. M., Sampath, K., Yokoyama, T., Wright, C. V. E., Potter, S. S., Overbeek, P. and Kuehn, M. R. (1996) Nature 381, 158-161). Here, we show that the normal expression patterns of the Xenopus nodal-related gene (Xnr-1) are variable in a large population of embryos and that Xnr-1 expression is altered by treatments that perturb normal left-right development. The incidence of abnormal Xnr-1 expression patterns correlates well with cardiac reversal rates in both control and experimentally treated Xenopus embryos. Furthermore, dorsal midline structures, including notochord and/or hypochord and neural floorplate, regulate Xnr-1 expression prior to the specification of cardiac left-right orientation by repression of Xnr-1 expression in the rightlateral plate mesoderm during closure of the neural tube. The correlation of Xnr-1 expression and orientation of cardiac looping suggests that Xnr-1 is a component of the left-right signaling pathway required for the specification of cardiac orientation in Xenopus, and that dorsal midline structures normally act to repress the signaling pathway on the right side of the embryo.
Fig. 1. Xnr-1 expression
in control embryos,
assayed by whole-mount
RNA in situ hybridization.
Purple color indicates
expression of Xnr-1 RNA.
(A) Expression in the left
lateral middle and
posterior region of stage
21 embryo (dorsal view).
(B) Left lateral view of
Xnr-1 expression pattern
in the same stage 21
embryo. (C) Dorsal view
of a stage 24 embryo, and
(D) left lateral view of the
same embryo. Xnr-1 is
expressed in the left
anterior lateral region,
sweeping from the
somites to the cardiac
primordia along the
dorsoventral axis.
Posteriorly, Xnr-1 is
expressed just to the left
of somites. (E) Xnr-1
expression is apparent in
the left lateral plate
mesoderm, in a transverse
section through stage 24
embryo. Scale bar, 0.2
mm. (F) Xnr-1 expression
becomes limited to the left
lateral plate mesoderm, fading posteriorly, in a stage 26 embryo.
Arrows indicate Xnr-1 expression. Anterior at top of page except in
E, where d, dorsal; v, ventral; l, left; r, right.
Fig. 2. Xnr-1 expression patterns in UV-treated embryos is dependent
on dorsoanterior development as scored by the Dorsoanterior Index
(Kao and Elinson, 1988; Danos and Yost, 1995). As dorsoanterior
development was diminished, the frequency of normal (left-sided,
black bars) Xnr-1 expression patterns decreased. The predominant
classes of aberrant Xnr-1 expression were bilateral expression
(hatched bars) and no expression (vertical lined bars). For each DAI,
DAI 5 (n=21), DAI 4 (n=16), DAI 3 (n=35), DAI 2 (n=25), DAI 0-1
(n=12), embryos were fixed and analyzed for Xnr-1 RNA expression
patterns by whole-mount in situ hybridization.
Fig. 3. Representative Xnr-1 expression patterns in Xenopus embryos
ventralized by UV irradiation. (A) Normal left expression of Xnr-1 in
a DAI 5 embryo. (B) Bilateral expression in the top embryo and left
expression in the lower embryo, both at DAI 4. (C) Bilateral
expression in two embryos at DAI 3. (D) Bilateral expression in
embryos at DAI 2. Dorsal views, anterior at the top in all panels.
Fig. 4. Xnr-1 expression pattern in Xenopus embryos after
extirpation of midline dorsoanterior structures depends on the stage
of extirpation. (A) Bilateral Xnr-1 expression in a stage 24 embryo
from which dorsal cells had been extirpated at stage 15. (B) Normal
left-sided Xnr-1 expression in a stage 24 embryo from which dorsal
cells had been extirpated at stage 20. Dorsal view, anterior at top in
both panels. (C) Xnr-1 expression patterns are predominately
bilateral (red bars) in embryos from which dorsal midline cells
were extirpated at stage 15, either in broad extirpations (75%
bilateral, s.d.=8%; n=24) or in narrow extirpations (72% bilateral,
s.d.=8%; n=27) focused on notochord and prospective floorplate
(see Methods), but normal (black bars) in embryos from which
dorsal midline cells were extirpated at stage 20 (95% left only,
s.d.=8%; n=26). Untreated control embryos from the same
experiments demonstrated Xnr-1 expression on the left only in 86%
of cases (s.d.=11%; n=52). Results from broad extirpations were
statistically identical to those from narrow extirpations at stage 15
(z=0.375, P>0.16) and were significantly different from those at
stage 20 (z=5.75, P<0.001).
Fig. 5. Xnr-1 asymmetric expression is specified in stage 20 lateral
plate mesoderm. (A) Left lateral mesoderm explanted at stage 15 (B)
Right lateral mesoderm explanted at stage 15. (C) Left lateral
mesoderm explanted at stage 20. (D) Right lateral mesoderm
explanted at stage 20. Note no Xnr-1 expression in (D) and strong
Xnr-1 expression in (A-C). All explants were cultured until sibling
embryos were at stage 24, and then fixed and assayed in parallel by
in situ hybridization for Xnr-1 expression. Scale bar, 1 mm.
Fig. 6. Comparison of predicted cardiac reversal rates (based on the
equation as defined in the text) versus observed cardiac reversals in
control embryos (diamonds), UV-treated embryos (DAI 5-DAI 3)
(circles), and midline extirpations (noto) (triangles). The predicted
cardiac reversal rates in the data set correlate positively with the
observed cardiac reversal rates as defined by the equation y=0.866x -
0.012, r2=0.839.
