January 1, 1998;
Evolutionary conservation of mechanisms upstream of asymmetric Nodal expression: reconciling chick and Xenopus.
Recent experiments have suggested a pathway of genes that regulate left
asymmetry in vertebrate embryogenesis. The most downstream member of this cascade is nodal
(XNR-1 in frogs), which is expressed in the left
-side lateral mesoderm
. Previous work in the chick [Levin, 1998] suggests that an inductive interaction by Shh
(Sonic hedgehog) present at the midline was needed for the left
-sided expression of nodal
, which by default would not be expressed. Interestingly, it has been reported [Lohr et al., 1997] that in Xenopus, right
that is explanted at st. 15 and allowed to develop in culture, goes on to express nodal
, suggesting that lateral mesoderm
expresses this gene by default and that a repression of nodal
by the midline is needed to achieve asymmetry. Such a contradiction raises interesting questions about the degree of conservation of the mechanisms upstream of nodal
asymmetry and, in general, about the differences in the LR pathway among species. Thus we examined this issue directly. We show that in the chick, as in the frog, explanted mesoderm
from both sides does, indeed, go on to express nodal
, including both the medial
expression domains. Ectopic nodal
expression in the medial
domain on the right
side is not sufficient to induce an ectopic lateral
domain. We also show that explanted lateral tissue
structures exhibiting Shh
expression. Furthermore, we show that Xenopus explants done at st. 15 also regenerate notochord
by the stage at which XNR-1 would be expressed. Thus explants are not isolated from the influence of the midline. In contrast to the midline repressor model previously suggested [Lohr et al., 1997] to explain the presence of nodal
expression in explants, we propose that the expression is due to induction by signals secreted by regenerating node and notochord tissue
in the chick). Thus our results are consistent with Shh
being necessary for nodal
induction in both species, and we provide an explanation for both sets of data in terms of a single conserved mechanism upstream of nodal
[+] show captions
Fig. 1. Two competing models of
events upstream of nodal asymmetry.
Nodal (Xnr-1 in Xenopus) is expressed
in left lateral mesoderm in chicks, frogs,
and mice. Studies in the chick [Levin,
1998] support the model that Shh, present
in the left half of Hensen’s node, is
necessary to induce nodal on the left. In
the absence of such an induction, nodal
is not expressed on either side. In contrast,
recent experiments in Xenopus
[Lohr et al., 1997] have been interpreted
to suggest that both sides are
normally committed to express nodal
and that a midline repressor is needed
to prevent right-sided expression. Such
a discrepancy would be very difficult to
understand in evolutionary terms.
Fig. 2. Explant and culture conditions allow nodal expression in
presence of midline. A. Explants were made by cutting halves of
blastoderms, immediately adjacent to the primitive streak, at st. 4;
arrows indicate primitive streak. B. Embryos at this stage show
Brachyury (a mesoderm marker) stain lateral to the primitive streak.
C. Stain is also seen in the explant (arrow). Thus mesodermal
precursors are present in explants. D. When the node but not the
streak is included in left explants, the explants go on to express nodal
(E, arrow indicates expression), showing that sufficient numbers of
mesodermal cells have left the streak by st. 4 to support nodal
expression in the presence of signals from the node. F. Left lateral
explants including the streak and node made at st. 6 go on to express
nodal (G, arrow indicates expression). H. Right explants including the
streak and node at st. 6 do not express nodal I. Thus culture conditions
allow the proper sequence of events upstream of nodal expression.
Fig. 3. As in Xenopus, chick lateral tissue expressed nodal when
explanted away from the midline. A. When left lateral tissue, not
including primitive streak or Hensen’s node, is explanted at st. 4 and
grown for 12–20 hours, nodal expression can be detected in 38% of the
cases (n 5 31). B. Right side tissue likewise expresses nodal, in 40% of
the cases (n 5 44). Arrowheads indicate expression.
Fig. 4. Chick explants regenerate node without correct LR asymmetry. A. Left and right explants, when cultured for 10–20 hours, exhibit Shh
expression. The expression domains range from spots or horseshoes (node-type pattern) to straight lines (notochord-type pattern). B. Left and
(C) right explants express nodal adjacent to Shh expression domains. D. The regenerating Shh domain often exhibits no left-right asymmetry, in
contrast to endogenous w.t. expression (E), where Shh is expressed only on the left side of Hensen’s node. F. These results are consistent with
nodal expression in lateral explants being a result of induction by newly regenerating Shh expression. Red arrowheads indicate Shh expression;
black arrowheads indicate nodal expression.
Fig. 5. Nodal does not induce nodal. A. In intact embryos, there are two domains of nodal expression: a small medial domain (M arrowhead)
directly adjacent to Shh expression (S arrowhead), and a larger lateral domain some distance away (L arrowhead). B. Explants sometimes
recapitulate this pattern of expression exactly. C. To test whether the large nodal domain could result from nodal expression in the medial
domain, nodal-expressing cells were implanted on the right side of the node in st. 6 embryos. Ectopic right-sided nodal expression was never
observed outside of the nodal-expressing cell pellet (P arrowhead). D. In contrast, Shh-expressing cell pellets (positive controls) do induce ectopic
nodal domains (L arrowhead). P—Shh cell pellet.
Fig. 6. Xenopus lateral explants regenerate notochord structures.
A. Lateral tissue was explanted from st. 15/16 embryos and cultured to
st. 22 (for Xnot stain) or st. 28 (for MZ15 stain). B. MZ15, an antibody
to the notochord epitope keratan sulphate, stains the notochord
sheath in control st. 16 embryos sectioned transversely. C. Explants
fixed and stained with MZ15 immediately after explantation (i.e.,
without culture), exhibit no stain, showing that the explants contain
no notochordal cells when they are put into culture. D. In contrast,
dorsal tissue left over from the explants does show a stripe of
notochordal staining; it is also seen that the dorsal tissue not included
in the explants is approximately three times as wide as the notochord.
E. Explants cultured overnight and processed for immunohistochemistry
without the primary MZ15 antibody exhibit no staining (negative
control). In contrast, left (F) and right (G) explants clearly show MZ15
staining, showing that they regenerate notochord cells. Analogous
results were obtained with in situ hybridization to an Xnot probe.
H. Whole st. 15 embryos probed with Xnot show signal in the
notochord. I. Tissue remaining after the dorsal strip was removed (but
before the remaining embryo was divided into left and right halves)
exhibits no signal. J. Dorsal explants show signal in the midline.
K. When explants are cultured and probed with a sense probe for Xnot,
no signal is detected. In contrast, both left (L) and right (M) explants
exhibit Xnot expression. Red arrowhead indicates expression.