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Xnot-2 is a homeobox gene expressed in Spemann's organizer. Here we present evidence that microinjection of synthetic Xnot-2 mRNA leads to the formation of notochord. Microinjection into the dorsal side of the Xenopus embryo results in greatly expanded notochords. Nearby somitic and prechordal mesoderm becomes recruited into these enlarged notochords, which also affect CNS patterning. Two early genes expressed in the developing notochord, chd and XFKH-1, are activated by Xnot-2. We conclude that gain-of-function of Xnot-2 promotes notochord formation.
FIG. 1. The Xnot-2 gene induces notochordal tissue. (A) One of the very few secondary axes resulting after ventral injection of pSPXnot-2 mRNA showing a second notochord. (B and C) Embryos injected dorsally into two adjacent blastomeres at the 4-cell stage with pSPXnot-2 mRNA and stained for notochord with MZ15. Note the enlarged notochords; some extend to the anterior end of the embryo. A control embryo (injected with prolactin mRNA) is shown on top of C. (D) Embryo injected ventrally at the 4-cell stage with pSPXnot-2 showing a stretch of ectopic secondary (27) notochord. (E and F) Reduced muscletissue (arrow) is observed on one side of an embryo injected with 200 pg/embryo of pSPXnot-2 mRNA (F), but not in uninjected controls (E). These embryos are from the same experiment shown in B and C. (G and H) Sagittal histological sections of control (prolactin mRNA injected) (G) or of an embryo with expanded notochord (H). Notochord was stained with MZ15 before sectioning and staining with light green. Injection of pSPXnot-2 mRNA causes a loss of the prechordal mesoderm and its replacement by notochord. After injection (H), the cephalic flexure is missing, a single ventricular cavity is present, and the morphology of the CNS is altered; pcm, prechordal mesoderm; pe, pharyngeal endoderm; fb, forebrain; hb, hindbrain; ad, aqueduct connecting the third and fourth ventricles. (I) N-CAM staining of pSPXnot-2 mRNA injected embryo (bottom) and of a control embryo (top) analyzed at stage 39. Note that the brain has a single ventricular cavity and that N-CAM staining is more intense in the CNS of the injected embryo. In this extreme case eyes are missing. (J) pSPXnot-2 and lacZ mRNAs coinjected into UV-treated embryos and analyzed at control stage 30. The rescued notochord is stained in brown by MZ15 and b-galactosidase activity (lineage) is in light blue. Note that the injected cells (arrowheads) populate the notochord, particularly in its posterior region that is still MZ15 negative; no, notochord. (K) Lineage tracing (blue) indicates that in wild-type embryos Xnot-2 mRNA injection leads to the adoption of notochordal cell fate. When sectioned (L), the lineage label was found principally in the expanded notochord of this ventrally injected embryo. (L) Cells injected with Xnot-2 and lacZ mRNAs contribute to the expanded notochord after ventral injection into wild-type embryos. The lineage label can be seen in the enlarged notochord (no) and in the ventral spinal cord (sc). (M and N) Chordin mRNA expression (analyzed by in situ hybridization) is induced in the early neurula stage embryo ventralized by UV treatment after diagonal injection of pSPXnot-2 mRNA at the 8-cell stage (N). The two lines of expression suggest that cells undergo convergence and extension movements after pSPXnot-2 mRNA injection. Chordin expression is absent in the UV-treated control embryo (M). (O and P) XFKH-1 mRNA expression analyzed by in situ hybridization is expanded in early neurula stage embryos after injection of pSPXnot-2 mRNA into the 4-cell stage embryos (P). Compare this with the narrow wild-type XFKH-1 expression in midline structures (O).
