Glinka A et al. (1996), Combinatorial signalling by Xwnt-11 and Xnr3 in...

XB-ART-17319

Mech Dev 1996 Dec 01;602:221-31.

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Combinatorial signalling by Xwnt-11 and Xnr3 in the organizer epithelium.

Glinka A , Delius H , Blumenstock C , Niehrs C .

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The epithelium of the Spemann organizer plays an important role in embryonic axis formation and transplantation experiments have shown that epithelial organizer cells have potent axis-inducing potential. Known axis-inducing molecules like noggin and chordin are not expressed in the epithelium and cannot account for its inductive properties. Xwnt-11 is expressed in the epithelium but has only poor dorsalizing activity. In an expression screen for genes that are able to functionally cooperate with Xwnt-11 we have identified a cDNA encoding Xenopus nodal-related 3 (XNR3), a member of the TGF-beta family, coexpressed with Xwnt-11 in the organizer epithelium. Xwnt-11 and Xnr3 act highly cooperatively in inducing secondary embryonic axes and dorsalizing ventral mesoderm. Xwnt-11/Xnr3 interfere with BMP signalling without themselves inducing chordin or noggin. The results indicate that induction by the organizer epithelium may result from the combinatorial action of instructive Xnr3 and permissive Xwnt-11 signalling.

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Species referenced: Xenopus
Genes referenced: chrd h4c4 nodal nodal1 nodal3.1 nodal3.2 nog tgfb1 wnt11b

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	Fig. 1. Xwnt-ll and Xnr3 are specifically coexpressed in the organizer epithelium. (A) In situ hybridization of sagitally cut stage 10.5 gastrulae with Xnr3 and Xwnt-ll. Dorsal is to the right and animal on top. Note expression of both genes in the epithelial layer of the dorsal blastopore lip. (B) Dorsal blastopore lips of early Xenopus gastrulae (one batch) were dissected and deep cells (deep) were separated from the epithelial layer (epith.). RNA was prepared from both tissues and analyzed by RT-PCR for expression of the genes indicatedH4, Histone H4 was used for normalization
	Fig. 2. (A-D) Xwnt-ll and Xnr3 act combinatorially in inducing secondary embryonic axes. (A--C) Phenotype of embryos microinjected at the fourcell stage into two ventral blastomeres each with 25 pg Xwnt-I 1 and 5 pg preprolactin (PPL) (A), 5 pg Xnr3 and 25 pg PPL (B), or 25 pg Xwnt-I 1 and 5 pg Xnr3 mRNA ((2). (D) Two ventral blastomercs of four-cell stage embryos wen~ injectexl each with increasing mRNA doses of Xnr3 alone (Xnr3), Xwnt-ll alone (Xwnt-11) or coinjeeted with increasing doses of Xnr3 and 25 pg XWnt-ll (Xnr3 + 25 pg Xwnt-ll). The secondary axes formed as a function of injected mRNA is shown in %. For every data point an average of 62 embryos was microinjected in 5 embryo batches.
	Fig. 3. (A) Xwnt-11/Xnr3 mRNA-injected cells behave like organizer cells. Eight-cell stage embryos were coinjeeted with a mixture of 20 pg Xnr3, 100 pg Xwnt-ll and 50 ng Fluorescein dextran (FDA) into one blastomere as well as 50 ng Rhodamin dextran (RDA) into the neighboring blastomere (schematically shown in the upper dgh0. Embryos displaying secondary embryonic axes (14 of 23, one batch) were analyzed histologically. A transverse section with the dorsal side up shows RDA-containing cells recruited in muscle (mu) and neural tube (nt) of the secondary axis. The notochord of the primary axis (no) is seen on the left. (B) Xwnt-lllXnr3 mRNA-injected cells can give rise to immature notochord. 32-cell stage embryos were eoinjected into C4 blastomeres with a mixture of 5 pg Xnr3, 25 pg Xwnt-ll mRNA and colloidal gold. Embryos (9/9 secondary axes, one batch) were processed by silver staining. Labelled cells can be seen ventral of the somites (so) in a thin rod of intercalating cells, most likely representing immature notochord in prevaeuolation stage.
	Fig. 4. Transplantation of epithelial cells expressing Xwnt-11/Xnr3 mRNA induces secondary embryonic axes. The epithelial layer of stage 10.5 gastrulae was transplanted to the ventral marginal zone of host stage 10.5 gastrulae. The epithelial layer was derived from ventral marginal zones (A, 0% secondary axes, n = 5), dorsal marginal zones (B, 45% secondary axes, n = 11) or ventral marginal zones from embryos microinjected with 250 pg Xwnt-11 and 50 pg Xnr3 mRNA per blastomere at the four-cell stage (C, 43% secondary axes, n = 14) (two batches of embryos analyzed). White arrow points to secondary axis in (B).
	Fig. 5. (A) Microinjection of Xwnt-lllXnr3 mRNA in animal caps induces neural but not mesodermal markers. Four-cell stage embryos were injected in the animal part of each blastomere with 25 pg Xwnt-ll and 5 pg PPL (Xwnt-ll), 5 pg Xnr3 and 25 pg PPL (Xnr3), or 25 pg Xwnt-ll and 5 pg Xnr3 mRNA (Xnr3 + Xwnt-11). At the late blastula stage injected and uninjected animal caps (Co) were dissected and cultivated until sibling embryos reached stage 10.5 or stage 28. RNA was prepared and analyzed by RT-PCR for the expression of marker genes indicated (3 embryo batches), embryo, RNA from whole embryos; -RT, sample without reverse transcriptase. (B) Microinjection of Xwnt-lllXnr3 mRNA dorsalizes ventral mesoderm. Embryos were microinjected at the four-cell stage into two ventral blastomeres each with 25 pg Xwnt-ll and 5 pg PPL (Xwnt-11), 5 pg Xnr3 and 25 pg PPL (Xnr3), or 25 pg Xwnt-ll and 5 pg Xnr3 mRNA (Xnr3 + Xwnt-11). At the early gastrula stage injected and uninjected 60 fragments of ventral (VMZ) or uninjected dorsal marginal zones (DMZ) were dissected and cultivated until sibling embryos reached stage 10.5 or stage 20. RNA was prepared and analyzed by RT-PCR for the expression of marker genes indicated (4 embryo batches). All RT-PCR assays in which mRNA was injected were carried out with VMZ explants.