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The transcription factor Zic1 plays important roles in patterning the neural plate in early vertebrate development. However, few genes that are regulated by Zic1 are known. We have identified a new direct downstream target gene of Zic1 that we have named Xfeb. Xfeb is a member of the pathogenesis-related (PR) protein superfamily and contains five tandem SCP domains. The sequence of Xfeb suggests that it may possess serine protease activity. Xfeb is expressed in the presumptive hindbrain region during neurula stages and in somite tissues later in development. Xfeb represses the hindbrain gene hoxB1 and the anterior neural gene otx2, suggesting that Xfeb is involved in regionalizing the neural plate, possibly by ensuring a posterior expression limit for otx2.
Figure 3. Temporal and spatial expression of Xfeb. A: A developmental profile of Xfeb expression was generated by quantitative RT-PCR using RNA from staged Xenopus embryos (top). EF-1 alpha and ODC served as loading controls (bottom). Y axes represent the relative abundance of transcripts in log scale and developmental stages are listed along the bottom. Xfeb expression begins at early gastrula stage 10 and continues throughout neurula stages. B-I: Characterization of Xfeb expression by whole mount in situ hybridization. Xfeb is expressed in hindbrain regions at stage 13 (B) and stage 17 (C). Xfeb expression overlaps extensively with zic1 expression (D; stage 17). At stage 20, Xfeb is expressed in a broad area in the midbrain and hindbrain and lateral to the neural tube (E). At stage 23, Xfeb is expressed in the midbrain and hindbrain region (F, arrowheads). The enlarged region (equivalent to box in F) shows Xfeb expression in a chevron pattern in the trunk (G). Double in situ hybridizations of stage 17 embryos with probes for Xfeb (brown) and en-2 (orange, arrowheads in H) and with probes for Xfeb (brownish purple) and wnt1 (purple, arrowheads in I) show that Xfeb is expressed in the hindbrain region up to the midbrain/hindbrain boundary, as defined by wnt1 staining. J: To confirm the relative staining patterns of en-2 and wnt1 in Xenopus, double in situ hybridization with wnt1 (purple) and en-2 (turquoise) was performed. All embryos are shown as dorsal views with the anterior ends up, except F and G, which are side views with dorsal sides up.
Figure 4. Zic1 regulates Xfeb expression in whole embryos. A,B: Zic1 induces ectopic Xfeb expression. Two-cell albino embryos were injected into one cell with 100 pg zic1 Delta C RNA and 25 pg lacZ RNA tracer. Embryos were fixed at stage 17 and stained for beta -galactosidase (blue) and Xfeb (brown). Ectopic Xfeb expression (white arrowheads) is induced by zic1 Delta C in the neural plate (A) and in non-neural tissues (B). C: A dominant interfering construct of Zic1 (dnzic1; Merzdorf and Sive, ) inhibits endogenous Xfeb expression; 100 pg dnzic1 (together with 25 pg lacZ RNA tracer) inhibited endogenous Xfeb expression on the injected side in stage-14 embryos. Asterisks mark the posterior ends of the embryos. In all embryos, the injected sides are on the left. Black arrowheads mark the endogenous Xfeb expression domains.
Figure 5. Xfeb represses otx2 and hoxB1 expression. A: Xfeb represses otx2 expression in animal cap assays. Two-cell embryos were injected with 1 pg noggin RNA (lane 2), 500 pg Xfeb RNA (lane 3), or their combination (lane 4). Animal caps were dissected at stage 9 and cultured to equivalent of stage 23. RT-PCR analysis demonstrated that otx2 was induced by Noggin (lane 2), but repressed by co-expression of Xfeb (lane 4). Uninjected animal caps were used as controls (lane 1). Muscle actin primers verified the absence of mesoderm; -RT samples control for genomic DNA. B, C: Xfeb represses the expression of otx2 and hoxB1 in whole embryos. Two-cell albino embryos were co-injected into one cell with 250 pg Xfeb RNA and 25 pg lacZ RNA. Embryos were stained for beta -galactosidase (blue) and by in situ hybridization for otx2 (B, stage 17) and hoxB1 (C, stage 15). otx2 and hoxB1 expression was significantly reduced on the injected (left) sides. The embryos are shown as anterior views.