Use of an oocyte expression assay to reconstitute inductive signaling.
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.
Lustig
K D
KD
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Kirschner
M W
MW
D028341:Activins; D000818:Animals; D002352:Carrier Proteins; D015816:Cell Adhesion Molecules, Neuronal / Q000096:biosynthesis; D003001:Cloning, Molecular; D017931:DNA Primers; D004625:Embryo, Nonmammalian / Q000502:physiology*; D005260:Female; D016332:Fetal Tissue Transplantation / Q000502:physiology*; D005775:Gastrula / Q000502:physiology; D015870:Gene Expression; D006133:Growth Substances / Q000096:biosynthesis; D007265:Inhibins / Q000096:biosynthesis*; D008648:Mesoderm / Q000502:physiology*; D008845:Microinjections; D009865:Oocytes / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011506:Proteins; D012313:RNA / Q000008:administration & dosage / Q000378:metabolism; D015398:Signal Transduction; D014982:Xenopus laevis; D000099176:Noggin Protein
D028341:Activins; D002352:Carrier Proteins; D015816:Cell Adhesion Molecules, Neuronal; D017931:DNA Primers; D006133:Growth Substances; D007265:Inhibins; D011506:Proteins; D012313:RNA; D000099176:Noggin Protein