Lustig KD and Kirschner MW (1995), Use of an oocyte expression assay to reconstitu...

XB-ART-19517

Proc Natl Acad Sci U S A 1995 Jul 03;9214:6234-8.

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Use of an oocyte expression assay to reconstitute inductive signaling.

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We have developed a paracrine signaling assay capable of mimicking inductive events in the early vertebrate embryo. RNA encoding one or more secreted proteins is microinjected into a Xenopus laevis oocyte. After a brief incubation to allow translation, a piece of embryonic tissue competent to respond to the signaling protein is grafted onto the oocyte. The secreted protein's effect on the grafted explant is then scored by assaying expression of tissue-specific markers. Explants of ectodermal tissue from blastula or gastrula stage embryos were grafted onto oocytes that had been injected with RNA encoding activin or noggin. We found that the paracrine assay faithfully reconstitutes mesoderm induction by activin and neural induction by noggin. Blastula-stage explants grafted onto activin-expressing oocytes expressed the mesodermal marker genes brachyury, goosecoid, and muscle actin. Gastrula-stage explants grafted onto noggin-expressing oocytes expressed neural cell adhesion molecule (NCAM) and formed cement gland. By injecting pools of RNA synthesized from a cDNA expression library into the oocyte, we also used the assay to screen for secreted neural-inducing proteins. We assayed 20,000 independent transformants of a library constructed from LiCl-dorsalized Xenopus laevis embryos, and we identified two cDNAs that induced neural tissue in ectodermal explants from gastrula-stage embryos. Both cDNAs encode noggin. These results suggest that the paracrine assay will be useful for the cloning of novel signaling proteins as well as for the analysis of known factors.

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Species referenced: Xenopus laevis
Genes referenced: actl6a gsc nog tbxt

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References [+] :

Cho, Molecular nature of Spemann's organizer: the role of the Xenopus homeobox gene goosecoid. 1991, Pubmed, Xenbase

	FIG. 1. Principle of the paracrine signaling assay. Synthetic RNA encoding one or more secreted signaling proteins is microinjected into a Xenopus laevis oocyte. After a brief incubation period to allow RNA translation, a piece ofX laevis embryonic tissue competent to respond to the signaling protein (e.g., an ectodermal explant) is grafted onto the oocyte. The signaling properties of the expressed protein are tested by analyzing gene expression in the grafted tissue.
	FIG. 2. Mesoderm induction by activin. Ectodermal explants from blastula stage embryos were grafted onto uninjected control oocytes (for A) or onto oocytes injected with 50 pg of activin RNA (for B) or the indicated amount of activin RNA (for C). For A and B, the oocyte-explant conjugates were photographed when sibling embryos reached the early tailbud stage. (x23.) For C, explants were detached from the oocytes when sibling embryos reached the indicated stage, and then assayed by RT-PCR for expression of the indicated genes.
	FIG. 3. Neural induction by noggin. Ectodermal explants from early gastrula-stage embryos were grafted onto uninjected control oocytes or oocytes injected with the indicated dose of nogginA5' or activin RNA. For A, explants were detached from the oocytes when sibling embryos reached the early tailbud stage, and then assayed by RT-PCR for expression of the indicated genes. For B and C, the oocyte-explant conjugates were photographed when sibling embryos reached the early tailbud stage. (Scale bar = 200 p,m.)