More Than 95% Reversal of Left–Right Axis Induced by Right-Sided Hypodermic Microinjection of Activin into Xenopus Neurula Embryos

Abstract

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.

Xenopus nodal related-1 is indispensable only for left-right axis determination

“…At this stage, left-right orientation of the heart and visceral organs was examined and the incidence of left-right reversal in the injected embryos was scored according to the criteria as previously described (Toyoizumi et al, 1997(Toyoizumi et al, , 2000Mogi et al, 2003). To more precisely judge the gut asymmetry, the laterality of the ventral pancreas was also checked.…”

“…Part of the embryos were photographed after penetrating with 50% glycerol solution. We attached importance to the area and intensity of the expression in the LPM as in our previous papers (Toyoizumi et al, 2000;Mogi et al, 2003). For individual specimen, both LPMs were carefully checked and scored for the laterality of gene expression.…”

XCR2, one of threeXenopusEGF-CFC genes, has a distinct role in the regulation of left-right patterning

“…On the one hand, our results indicate that XCR2-dependent activity of neither the nodal family ligands nor their antagonists is essential on the right side for correct patterning. On the other hand, ectopic expression of XCR2 is sufficient to reverse the left-right polarity, as can ectopic expression of TGF␤ family ligands or activated receptors Hanafusa et al, 2000;Hyatt et al, 1996;Sampath et al, 1997;Toyoizumi et al, 2000). Data from several laboratories, including our own, have shown that EGF-CFC proteins function as co-factors for nodal/GDF1 signaling, but are not sufficient to activate SMAD2 signaling in absence of the ligands (Fig.…”

Mosaic versus regulation development in avian blastoderms depends on the spatial distribution of Rauber's sickle material

“…Our results could suggest that early asymmetry in the node is the consequence of the early left-right mosaic development in the concavity of Rauber's sickle. In Xenopus embryos, microinjection of activin reverses the left-right axis in 95% of the cases (Toyoizumi et al, 2000). It has been shown by Ziv et al (1992) that activin can generate ectopic axial structures in chick blastoderm explants.…”