Toyoizumi R et al. (2000), More than 95% reversal of left-right axis induc...

XB-ART-11093

Dev Biol 2000 May 15;2212:321-36. doi: 10.1006/dbio.2000.9666.

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More than 95% reversal of left-right axis induced by right-sided hypodermic microinjection of activin into Xenopus neurula embryos.

Toyoizumi R , Mogi K , Takeuchi S .

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In recent years, genes that show left-right (L-R) asymmetric expression patterns have been identified one after another in vertebrate gastrula-neurula embryos. However, we still have little information about when the irreversible L-R specification is established in vertebrate embryos. In this report, we show that almost 100% of the embryos develop to be L-R-inverted larvae after microinjection of activin molecules into the right lateral hypodermic space of Xenopus neurula embryos. After right-side injection of 10-250 pg activin protein, both early neurulae just after gastrulation movement (stage 13-14) and late neurulae just before neural tube closure (stage 17-18) showed almost 100% reversal of the heart and gut L-R axes. At higher doses of activin, more than 90% of the L-R-inverted embryos showed L-R reversal of both heart and gut. The survival ratio of the right-injected 4-day embryos was 90% on average. In the left-injected embryos, the occurrence of L-R inversion was less than 2% as observed in normal untreated siblings (1.7%). When the same amount of activin (1-50 pg) was microinjected into both sides of neurula embryos, the incidence of L-R inversion was reduced to 58%. The injection of activin along the dorsal midline in the trunk region also randomized the visceral L-R axis. Injection of activin into the right side changed normal left-handed expression of Xnr-1 to right-handed or bilateral expression. In contrast, left-handed expression of Pitx2 was switched to the right side by right activin injection. This is the first report of a method that achieves complete inversion of the visceral L-R axis by treatment of embryos at the neurula stage. Activin not only acts on the neurulae to cancel the original L-R specification up to the late neurula stage, but also rebuilds a new L-R axis whose left side coincides with the injection side. It is suggested that the left and right halves of neurulae have equal potential for L-R differentiation.

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Species referenced: Xenopus laevis
Genes referenced: inhba nodal1 pitx2

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	FIG. 1. Procedure for the hypodermic injection of activin into Xenopus neurula embryos. (A) Schematic illustration of the experimental procedures. In order not to wound the lateral plate mesoderm and to administer activin effectively, the hypodermic injection was carried out by inserting the capillary at a low angle toward the center of the lateral side. The tip of the capillary was thrust into the space beneath the epidermis covering the lateral plate mesoderm, and then the solution was injected. (B) Photograph of a hypodermic microinjection of activin into the right side of a stage 17 embryo. Activin A mixed with 1% Nile blue (vital dye) was microinjected into the lateral hypodermic space of Xenopus neurula embryos at a dose of 0–250 pg. Scale bar, 1 mm.
	FIG. 2. Mirror-image reversal of the visceral organs induced by the hypodermic injection of activin into the right side of a neurula embryo. Left, a stage 42 embryo with normal situs after left-side injection of activin (5 nl of 2 mg/ml, i.e., 10 pg) at stage 15. Right, a stage 42 embryo with inverted heart and gut situs after right-side injection of activin (5 nl of 2 mg/ml, i.e., 10 pg) at stage 15. Note that the blue spots and situs of the whole organs make mirror images of each other. Scale bar, 1 mm.
	FIG. 3. Stage-dependent change of the incidence of both heart and gut L-R reversal, heart-alone L-R reversal, and gut-alone L-R reversal, by the injection of 10–250 pg of activin. The height of each bar represents the total incidence of the three types of L-R reversal, meaning that at least one of the two organs was inverted. In stage 13–18 embryos, from 80 to 90% of the L-R-inverted embryos were classified as “L-R reversal of both heart and gut.” In the embryos after stage 18, it became difficult to invert the L-R axis completely, and the relative incidence of heterotaxia increased, compared with that of both heart and gut reversal.
	FIG. 4. Long-term cultivation of activin-injected embryos. (A) A larva with L-R-inverted heart looping and gut coiling that was reared for 3 weeks after right injection of activin (10 pg) at the midneurula stage (stage 15–16). (B) Magnified view of the thoracic region of the embryo in (A). The shape of the asymmetric heart and the distribution pattern of the neighboring blood vessels are normal except for laterality. (C) Metamorphosis of L-R-inverted larvae produced by right-side injection of 10 pg activin at stage 15–16. Some of the activin-induced L-R-inverted embryos were reared up to the stage of metamorphosis. Like this larva, activin-injected embryos underwent the normal metamorphic process. Limb formation was quite symmetrical with normal morphology. (D) A cross section of a L-R-inverted 4-day embryo after right injection of activin (10 pg, at stage 15–16). Shapes of the organs are quite symmetrical except for the visceral organs. Abbreviations: e, esophagus; g, gill chamber; i, intestine; l, liver; n, notochord; p, pancreas; t, trachea. Scale bars, 1 mm (A–C) or 100 mm (D).
	FIG. 5. Left-handed expression of Xnr-1 and Xenopus Pitx2 are altered by the hypodermic injection of activin into the right flank. (A) An untreated embryo. Normal expression of Xnr-1 in the left LPM is observed (lateral view). (B) A case of bilateral expression of Xnr-1 in both LPMs induced by right injection of 10 pg activin (dorsal view). (C) Left expression of Pitx2 after left injection of 10 pg activin at stage 15â€“16. (D) Bilateral expression of Pitx2 after right injection of 10 pg of activin at stage 15â€“16. (E) A cross section of a tailbud-stage embryo showing bilateral expression of Xnr-1 after the right injection. (F) A section of a tailbud-stage embryo showing bilateral expression of Pitx2 after the right injection. Scale bars, 1 mm (Aâ€“D), 100 mm (E, F).
	FIG. 6. Xnr-1 expression pattern in Xenopus embryos after activin injection into the hypodermic space of the right flanks. 10 pg of activin protein was injected at stage 15–16, and the embryos were fixed and stained at stage 23–26. (A) Left-sided expression of Xnr-1 after the right injection. (B) Right-sided expression of Xnr-1 after the right injection. (C) Bilateral expression of Xnr-1 after the right injection. In the right-injected embryos, right-sided expression (B) or bilateral expression (C) in the LPMs was most frequently observed. Laterality of Xnr-1 expression seemed to be randomized by right injection of activin. Scale bar, 1 mm.
	FIG. 7. Switch of the expression side of Pitx2 after right hypodermic injection of activin. 10 pg of activin protein was injected at stage 15–16, and the embryos were fixed and stained at stage 26–30. (A) Left-sided expression of Pitx2 after the left injection. (B) Left-sided expression of Pitx2 after the right injection. (C) Right-sided expression of Pitx2 after the right injection. (D) Bilateral expression of Pitx2 after the right injection. In left-injected embryos, Pitx2 expression was restricted to the left LPM (A), whereas in right-injected embryos 69% of the embryos showed right-sided expression of Pitx2 (C). All the presented embryos are viewed from the ventral side. Scale bar, 1 mm.
	FIG. 8. Ectopic expression of Xnr-1 in the midneurula LPM (stage 15) after hypodermic injection of activin into the right side of early neurula embryos (stage 13). Note that Xnr-1 was ectopically induced around the scratch of the injection point (arrow), showing that activin can induce Xnr-1 long before the onset of its normal expression on the left side at stage 19. In case of right activin injection at the early neurula, 22 of 35 midneurula embryos (63%) were observed to show ectopic Xnr-1 expression. A, anterior side; P, posterior side. Scale bar, 1 mm.