Sampath K et al. (1997), Functional differences among Xenopus nodal-rela...

XB-ART-15998

Development 1997 Sep 01;12417:3293-302. doi: 10.1242/dev.124.17.3293.

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Functional differences among Xenopus nodal-related genes in left-right axis determination.

Sampath K , Cheng AM , Frisch A , Wright CV .

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An association has been noted previously in chick, mouse and frog embryos between asymmetric nodal-related gene expression and embryonic situs, implying an evolutionarily conserved role in left-right specification. Of the four Xenopus nodal-related genes expressed during gastrulation, only Xnr-1 is re-expressed unilaterally in the left lateral plate mesoderm at neurula/tailbud stages. Here, we show that the asymmetric expression of Xnr-1 can be made bilaterally symmetric by right-sided microinjection of RNA encoding active Xenopus hedgehog proteins. Moreover, we provide the first evidence that Xnr-1 expression per se is a causal factor in left-right axis determination. When plasmids expressing Xnr-1 were delivered unilaterally to the right side of Xenopus embryos, a reversed laterality of both the heart and gut (homotaxic reversal) was induced in 40% of surviving embryos, while an additional 10-20% showed reversal of the heart or gut alone (heterotaxia). This effect on laterality was specific to Xnr-1, since neither Xnr-2 nor Xnr-3 plasmids had this activity. In addition, we find that Xnr-1 and Xnr-2, which have both been defined as mesoderm inducers from overexpression studies, show quantitative differences in their ability to induce dorsal mesoderm. Together, these findings suggest that the various Xnrs perform substantially different functions during Xenopus embryogenesis. Moreover, they strongly support the hypothesis that left lateral plate expression of nodal-related genes is a causative factor in the determination of asymmetry in vertebrate embryos.

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Species referenced: Xenopus
Genes referenced: gsc ihh mt-tr nodal nodal1 nodal2 shh tbxt trna

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	Fig. 1. In situ hybridization for Xnr-1 in Xenopus hedgehog RNAinjected embryos. The embryos shown are dorsal views at stage 21, left and right are indicated in (A). (A) Xnr-1 expression in the left lateral plate mesoderm, as seen in normal embryos. (B) Bilateral Xnr-1 expression in the left and right lateral plate mesoderm caused by right side injection of Xbhh-N RNA. (C) Right side expression of Xnr-1 caused by Xshh-N RNA. (D) Xnr-1 expression absent from both sides of an Xbhh-N RNA-injected embryo.
	Fig. 2. Endogenous versus plasmid-encoded Xnr-1 expression at neurula/tailbud stages. RNase protection assays were used to analyze Xnr-1 transcript levels at neurula (stage 15) and tailbud (stage 19), either in uninjected embryos (U), or in embryos injected with 20 pg or 100 pg of pXEX/Xnr-1 (lanes labeled 20 and 100). The 420 nucleotide product represents endogenous transcripts and the 370 product (arrowhead) those derived from pXEX/Xnr-1. EF-1a was used as a loading control. P, 480 bp Xnr-1 probe; T, tRNA control.
	Fig. 3. Reversal of heart and gut looping in pCSKA/Xnr-1 injected embryos. The upper panels are ventral views of stage 45 embryos injected with pCSKA/Xnr-1 with normal looping of the heart (A), or reversed looping (B), visualized by indirect immunofluorescence with the muscle-specific MF20 antibody. The arrowhead indicates the outflow tract; v indicates the ventricle. The shape of the heart is diagrammatically represented by the outline drawings. The lower two panels are ventral views of the same embryos showing intestinal looping in pCSKA/Xnr-1-injected embryos: (C) normal looping, (D) reversed looping. Arrows indicate the direction of looping on the outline drawings.
	Fig. 4. Differences in mesoderm induction by Xnr-1 and Xnr-2. RTPCR analysis of animal cap explants from embryos injected with 10, 50 or 150 pg of Xnr-1, compared to 10 or 50 pg of Xnr-2 RNA, showing expression of the pan-mesodermal marker, Xbra, the dorsal mesodermal marker, Gsc, and the constitutive marker, FGFR. NAM lane represents uninjected explants cultured in normal amphibian medium.