Xenopus Nodal Related, Xnr3; dorsal lip

Xenopus Nodal Related, Xnr3, nucleic acid

Pictures and text from Richard Harland and Bill Smith.

The pictures show the mutually exclusive regions of expression of Xnr3 (blue) and Xwnt-8 (brown) in the late blastula and early gastrula. Xnr3 was visualized with a fluorescein probe, an alkaline phosphatase coupled anti-fluorescein antibody which was developed with NBT/BCIP. The Xwnt-8 was visualized with a digoxigenin probe, alkaline phosphatase coupled anti-digoxigenin and vectastain II.

Xnr3 is expressed exclusively in the superficial layer of the organizer (see Smith and Harland, 1995 for more pictures). Note the rapid change from stage 9 to stage 10-. We assume that at these stages the cells expressing Xnr3 are fairly constant (but have not proven this). At later times Xnr3 turns off as cells go around the lip. If the cells expressing Xnr3 are constant at this time, then this region is converging to the dorsal midline.
Xwnt-8 expression changes rapidly when vizualized by this method. At the onset of expression the regions of Xnr3 and Xwnt-8 abut one another, and Xwnt-8 expression is seen throughout the vegetal region (see also Lemaire P; Gurdon JB. A role for cytoplasmic determinants in mesoderm patterning: cell-autonomous activation of the goosecoid and Xwnt-8 genes along the dorsoventral axis of early Xenopus embryos. Development, 1994 May, 120(5):1191-9). As the embryo approaches gastrulation, staining is no longer apparent in the in the region next to the organizer and the vegetal pole. Lateral mesoderm is converging to the dorsal midline at this stage, so the absence of Xwnt-8 staining must reflect extinction of expression, presumably as a result of dorsalizing signals from the organizer. Staining appears punctate because transcripts are most concentrated in the nuclei at these stages.

For other pictures of Xwnt-8 expression see Christian, J. L., McMahon, J. A., McMahon, A. P., and Moon, R. T. (1991). XWnt-8 a Xenopus Wnt-1/int-1-related gene responsive to mesoderm-inducing growth factors, may play a role in ventral mesodermal patterning during embryogenesis. Development 111, 1045-1055. or the xwnt 8 page.

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Reference; Smith, W.C., McKendry, RM, Ribisi, S. and Harland, R.M. (1995) A nodal-related gene defines a physical and functional domain within the Spemann organizer. Cell, 82, 37-46
Plasmid name; pDor3 (download restriction map)
xnr3 cDNA in pGEM5Zf(-)
download sequnce
Genbank accession number; U25993
For mRNA cut with various enzymes, e.g. BamHI and transcribe with SP6
For in situ probe cut with EcoRI and transcribe with T7
Available from Bill Smith (plasmid is subject to materials transfer agreement) or Richard Harland
Technical notes:The whole mount method does not usually reflect the expression of genes in the vegetal mass accurately (see Bolce ME; Hemmati-Brivanlou A; Harland RM. XFKH2, a Xenopus HNF-3 alpha homologue, exhibits both activin-inducible and autonomous phases of expression in early embryos Developmental Biology, 1993 Dec, 160(2):413-23; Frank D; Harland RM. Localized expression of a Xenopus POU gene depends on cell-autonomous transcriptional activation and induction-dependent inactivation. Development, 1992 Jun, 115(2):439-48) However, in this experiment this combination of probes and stain clearly worked at stage 9. In general this combination has not worked as well as shown here. Any suggestions for ways to make this more reliable would be gratefully received. The method used here was similar to that used in Knecht, A.K., Good, P.J., Dawid, I.B., and Harland, R.M. (1995) Dorsal-Ventral Patterning and Differentiation of Noggin-Induced Neural Tissue in the Absence of Mesoderm Development 121, 1927-1936

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