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FIG. 1. Xnr4 is a member of the TGFb superfamily that is expressed in the Spemann organizer. (A) The amino acid sequence of Xnr4.
A hydrophobic region at the N-terminus is underlined and the predicted proteolytic cleavage site (RRXRR) is indicated by a box. Cysteine
residues in the mature region are shown in bold typeface. (B) A lineup of the amino acids of mature Xnr4 with those of mouse nodal and
the other Xenopus nodal-related factors (beginning with the first conserved cysteine of the mature region). The cDNA sequence for Xnr4
has been submitted to GenBank under accession No. U79162.
FIG. 2. (A) Whole-mount in situ hybridizations of gastrula and neurula stage embryos with antisense probe recognizing Xnr4 (panels on
the left) and with control Xnr4 sense probe (panels on the right). The gastrula (stage 10) embryo is shown as a vegetal view with the dorsal
blastopore lip at the top of the panel and the neurula (stage 19) embryo is shown as a dorsal view with the posterior end at the bottom
of the panel. (B) An in situ hybridization of a sagittal section of a gastrula stage embryo showing the expression of Xnr4 in the deep cells
of the dorsal blastopore lip. (C) An in situ hybridization of a transverse section of a late neurula stage embryo showing expression of Xnr4
in the notochord and the ventral neural tube.
FIG. 3. Xnr4 mRNA injection partially rescues a dorsal axis in a UV-ventralized embryo. (Top) A sibling stage control embryo, DAI
5 (n 18). (Middle) A UV-ventralized embryo, DAI 0 (n 26). (Bottom) A UV-ventralized embryo injected in the marginal zone region
of one cell at the four-cell stage with 50 pg of Xnr4 mRNA (DAI 2). A partial axial rescue of DAI 1 or 2 occurred in 77% of the
embryos injected with 50 pg of Xnr4 mRNA (n 17/22), with 23% having a DAI 2 (n 5/22). Injection of 5 pg of Xnr4 mRNA did
not efficiently rescue a dorsal axis (n 7/19, DAI 1; and n 0/19, DAI 2) and injection of 500 pg of Xnr4mRNAled to hyperdorsalization
of embryos (n 21). Control UV-ventralized embryos injected with 50 pg of b-galactosidase mRNA had a DAI 0 (n 19).
FIG. 4. Xnr4 has the ability to dorsalize and induce mesoderm.
(A) RT-PCR analysis of marginal zones injected with 2 ng of a
linearized Xnr4-XEX plasmid shows that zygotic expression of
Xnr4 leads to expression of muscle actin and a lack of aT1-globin
expression in ventral marginal zone explants. b-Galactosidase
mRNA-injected marginal zones, uninjected marginal zones, and
whole embryos were analyzed as controls. (B) Animal cap assay
with Xnr4 mRNA. Caps were scored at stage 11 for early mesodermal markers. Xbra and Xwnt8 are expressed in caps injected
with 1 ng of Xnr4 mRNA. Gsc, noggin, and siamois are not
expressed in these injected caps. (C) Animal caps injected with
1 ng of Xnr4 mRNA, and scored at stage 28, express muscle
actin, but do not express aT1-globin. In all cases, EF1-a is used
as an RT-PCR control for RNA recovery.