Levin M and Mercola M (1998), Evolutionary conservation of mechanisms upstrea...

XB-ART-13854

Dev Genet 1998 Jan 01;233:185-93. doi: 10.1002/(SICI)1520-6408(1998)23:3<185::AID-DVG4>3.0.CO;2-1.

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Evolutionary conservation of mechanisms upstream of asymmetric Nodal expression: reconciling chick and Xenopus.

Levin M , Mercola M .

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Recent experiments have suggested a pathway of genes that regulate left-right 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-side mesoderm 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 and lateral 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 regenerates node/notochord 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 (Shh 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 expression.

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Species referenced: Xenopus
Genes referenced: nodal nodal1 not shh tbx2

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	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.