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The Notch signaling pathway plays an important role in many cell-fate decisions during development. Here we investigate the regulation and function of the conserved gene XNAP, which is a member of the Delta-Notch synexpression group in Xenopus. XNAP encodes a small protein with two C-terminal tandem ankyrin repeats which is expressed in the neurectoderm and in the presomitic mesoderm in a pattern that resembles that of other component of the Notch pathway. When a myc-tag form of XNAP is overexpressed in Xenopus or Hela cells, XNAP protein is detected both in the nucleus and the cytoplasm. In embryos and in animal cap assays, XNAP expression is activated, perhaps directly, by the Notch pathway and this activation appears to be Su(H) dependent. Overexpression of XNAP in embryos decreases Notch signaling, which leads to an increase in the number of primary neurons that form within the domains of the neural plate where neurogenesis normally occurs. In culture Hela cells, XNAP overexpression interferes with ICD activation of a Notch regulated reporter gene. Together, these data indicate that XNAP is a novel target of the Notch pathway that may, in a feedback loop, modulate its activity.
Fig. 3. XNAP mRNA expression during embryogenesis and in adult organs. (A,B) XNAP mRNA expression during embryogenesis and in adult tissues analyzed by RNAase protection. FGFr or EF-α are used as internal controls. (C–N) Whole-mount in situ analysis of XNAP expression during Xenopus early embryogenesis. Nieuwkoop–Faber stages of embryogenesis are indicated. All embryos are shown with anterior to the right. (C–F) Dorsal view; (G–I) dorso-anterior view; (J,K) lateral view. (L–N) Transversal section at the level of the otic vesicles of embryos stained with XNAP, ESR9 or X-Delta-1. The stripes of XNAP expression in the posterior neural plate are indicated by asterisks in (E) and are labeled l (lateral), i (intermediate) and m (medial) in (F,G). Abbreviations: ANR, anterior neural ridge; BC, branchial crest segment; NT, neural tube; OV, otic vesicle; PSM, presomitic mesoderm; TBD, tailbud domain; VFM, ventral fore and midbrain.
Fig. 4. XNAP expression is positively regulated by proneural genes and by the Su(H) Notch pathway. (A) Whole mount in situ hybridization analysis of XNAP expression in embryos injected into one cell at the two cell stage with the mRNA as indicated. Each panel shows views of neurula stage injected embryos with anterior to the right. Overexpression of the proneural X-NGNR-1 and Xash3 genes as well as of the active forms of Notch (Notch ICD) and Su(H) (Su(H)Ank) induces XNAP expression. In contrast, overexpression of the dominant negative form of X-delta-1(X-Delta-1Stu) inhibits XNAP expression. Due to the short time of staining performed to visualize the strong ectopic XNAP expression induced by ICD or Su(H)Ank, the endogenous XNAP expression on the uninjected sides of these embryos cannot be seen. (B) RNAase protection analysis of the expression of XNAP and ESR1 in animal caps derived from embryos injected at the four cell stage with the indicated mRNA. When indicated, Noggin mRNA was added to neuralize the cells. FGFr RNA expression is used as a loading control. Ectoderm was removed from embryos at blastula stages and assayed at neurula stage. Note that the XNAP induction by ICD overexpression is higher in neuralized than in non-neuralized cells (left panel). (C) RNAase protection analysis performed as described in (B) showing that the induction of XNAP by Notch ICD can be effectively blocked by inclusion of a the dominant negative mutant of Su(H), Su(H)DBM, while that by X-NGNR-1 is only partially affected (right panel).
Fig. 6. Effects of XNAP on primary neurogenesis. Xenopus embryos at the two cell-stage were injected into one cell with the indicated RNA along with LacZ mRNA. Injected embryos were fixed at neurula stage, stained with X-gal and hybridized with the indicated probes. Each panel shows dorsal views of neurula stage injected embryos, with anterior to the right. Views (C,D) and (G,H) show both sides of the same injected embryo. (A,C–F) The injected side of XNAP overexpressing embryos show increased number of primary neurons. This phenotype is very similar to that observed in X-Deltastu injected embryos. (B) XNAPank reduces the number of N-tubulin positive cells; (G,H) XNAP slightly increases X-Delta expression; (I,J) XNAP reduces ESR1 and ESR10 expression in early neurula stage embryos.