Lustig KD et al. (1996), A Xenopus nodal-related gene that acts in syner...

XB-ART-17625

Development 1996 Oct 01;12210:3275-82. doi: 10.1242/dev.122.10.3275.

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A Xenopus nodal-related gene that acts in synergy with noggin to induce complete secondary axis and notochord formation.

Lustig KD , Kroll K , Sun E , Ramos R , Elmendorf H , Kirschner MW .

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Using a paracrine assay to screen for signaling proteins that could respecify ectodermal tissue, we isolated a Xenopus gene related to the mouse gene nodal, a member of the TGFbeta superfamily. The gene is expressed in three regions in the early Xenopus embryo: first in the gastrula organizer, then in two stripes of cells flanking the posterior notochord in late neurulae, and finally in lateral plate mesoderm restricted to the left side of tailbud-stage embryos. Ectopic expression of the gene induces muscle formation in ectodermal explants and partial secondary axes in whole embryos. Together with noggin, another secreted protein also present in the organizer, it induces notochord formation in ectodermal explants and complete secondary axes in whole embryos. These results suggest that the nodal-related gene may act together with noggin to induce axial pattern during gastrulation and also may play a role in left-right asymmetry generation in the post-gastrula embryo.

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Species referenced: Xenopus laevis
Genes referenced: acta4 actc1 actl6a dnai1 gsc mtor ncam1 nodal nodal1 nog tbxt tgfb1

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	Fig. 1. Induction of tissue elongation by Xnr1 and noggin. Ectodermal explants from early gastrula stage embryos were grafted onto uninjected control oocytes or onto oocytes that had been injected with 100 pg of noggin RNA, 100 pg of Xnr1 RNA or 100 pg of both RNAs. The oocyte-explant conjugates were photographed when sibling embryos reached the early tailbud stage of development. Bar, 400 mm.
	Fig. 2. Developmental expression of Xnr1. X. laevis embryos were harvested at the indicated stage and assayed by RT-PCR for expression of Xnr1. EF1-a was also assayed to show that comparable amounts of RNA were isolated from each sample. EF1-a is expressed at low levels prior to the mid blastula transition and at higher levels thereafter. Each PCR reaction contained the cDNA equivalent of one-tenth of an embryo.
	Fig. 3. Localization of Xnr1 transcripts by whole mount in situ hybridization. Embryos were hybridized with a digoxygenin-labeled Xnr1 antisense RNA probe. (A) Dorsal view of a stage 22 embryo. (B) Side view of embryo in A. (C) Dorsal view of a stage 24 embryo. (D) Side view of embryo in C. (E) Dorsal view of stage 28 embryo. Anterior is to the left in A-E. (F) Transverse section of a stage 24 embryo, showing Xnr1 expression in lateral plate mesoderm on the left side of the embryo. Expression was restricted to the left side in ~100 early tailbud stage embryos examined.
	Fig. 4. Secondary axis formation by Xnr1 and noggin. A single ventral blastomere of a four-cell embryo was injected with 2 pg of noggin RNA, 200 pg of Xnr1 RNA or 2 pg of noggin RNA and 200 pg of Xnr1 RNA. Embryos were photographed when uninjected control embryos reached stage 31. All embryos are shown in dorsal view with anterior to the left except for the two upper embryos in the lower right panel, which are shown in anterior view.
	Fig. 5. Induction of notochord by co-expression of Xnr1 and noggin. Ectodermal explants from early gastrula stage embryos were grafted onto uninjected oocytes or onto oocytes injected with 1 ng noggin RNA, 1 ng Xnr1 RNA or 1 ng of both RNAs. Upper left panel: bright-field photograph of an explant that had been grafted onto an oocyte expressing both Xnr1 and noggin. Upper right panel: fluorescence micrograph of the same explant immunostained in whole mount with the Tor-70 notochord-specific antibody. Lower panels: differential interference contrast (DIC) and corresponding fluorescence micrographs of explant sections immunostained with Tor-70. Notochords up to 140 mm in length were detected within a single explant.
	Fig. 6. Mesoderm induction is due to non-cell-autonomous signaling by Xnr1. Ectodermal explants from early blastula or early gastrula embryos were grafted onto uninjected oocytes or onto oocytes injected with the indicated amount of Xnr1, activin, or noggin RNA. Upper panel: oocyte-explant conjugates were photographed when sibling embryos reached the early tailbud stage. Lower panels: explants were detached from the oocytes when sibling embryos reached the early tailbud stage, and then assayed by RT-PCR for expression of the mesodermal marker muscle actin or the neural marker NCAM. Each sample consisted of a pooled set of six explants, and each PCR reaction contained the cDNA equivalent of one-tenth of an explant.
	Fig. 7. Induction of muscle by Xnr1. Ectodermal explants from early blastula or early gastrula embryos were grafted onto uninjected oocytes or onto oocytes injected with 1 ng Xnr1 RNA. When sibling embryos reached the early tailbud stage, the explants were detached from the oocytes, sectioned and immunostained with the muscle-specific 12/101 antibody. Differential interference contrast (DIC) and corresponding fluorescence micrographs are shown.
	Fig. 8. Induction of brachyury and goosecoid by Xnr1. Ectodermal explants from late blastula stage embryos were grafted onto uninjected oocytes or onto oocytes injected with the indicated amount of Xnr1 RNA. Explants were detached from the oocytes when sibling embryos reached the late gastrula stage, and then assayed by RT-PCR for expression of brachyury or goosecoid. Each sample consisted of a pooled set of six explants, and each PCR reaction contained the cDNA equivalent of one-tenth of an explant. EF1-a was assayed to show that comparable amounts of RNA were isolated from each sample.