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Wnt signaling pathways are involved during various stages in the development of many species. In Xenopus, the accumulation of beta-catenin on the dorsal side of embryo is required for induction of the organizer, while the head structure formation requires inhibition of Wnt signaling. Here, we report a role for xIdax, a negative regulator of Wnt signaling. XIdax is expressed in neural tissues at the neurula stage, and in the restricted region of the tadpolebrain. Ectopic expression of xIdax inhibits the target gene expression, suggesting that xIdax can inhibit canonical Wnt signaling. To examine the function of xIdax, a morpholino oligo for xIdax (xIdaxMO) was designed. An injection into an animal pole cell caused a loss of forebrain. The anterior neural marker expression is decreased in xIdaxMO-injected embryo, suggesting that xIdax is required for anterior neural development. Moreover, a negative regulator that acts downstream of xIdax rescued this defect. We propose that Idax functions are dependent on the canonical Wnt pathway and are crucial for the anterior neural development.
Figure 2. Temporal and spatial distribution of the xIdax gene transcript. A: Temporal expression patterns of xIdax (row 1), Cerberus (Cer, row 2), chordin (Chd, row 3), and ODC (row 4) were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Row 5 shows the negative control using templates without RT. Upper numbers indicate the Nieuwkoop stage. UF indicates an unfertilized egg. B-M: Spatial expression patterns were examined by in situ hybridization. Experiments were done with xIdax antisense (B,E,H,I), xIdax sense (C,F), Dvl activity antisense (D,G), Sox2 antsisense (J,K), and N-CAM (L,M). A, Anterior; P, Posterior. B: At the mid-neurula stage, very weak expression of xIdax was detected in the presumptive neural region. E: At the late neurula stage, xIdax was expressed in the neural plate. D,G: Dvl activity expression in the neurula is similar with that of xIdax. H,I: At the 2-day tadpole stage, xIdax is expressed in the inner cells of the head, probably in the ventral boundary of the brain hemisphere (arrow). I, K, and M are magnified figures of H, J, and L, respectively. eye, eye vesicle; fb, forebrain; hb; hindbrain; mb; midbrain; ov, otic vesicle.
Figure 5. XIdaxMO injection causes anterior neural development. A: Observation of marker gene expression in XIdaxMO-injected embryo. XIdaxMO (lanes 3, 5) or standard controlMO (lanes 2, 4) were injected into animal pole (AP) cells, then cultured and harvested at stage 18 (lanes 1-3) or stage 34 (lanes 4-5). In an IdaxMO-injected embryo, expressions of both forebrain marker genes BF-1 (row 1) and Rx1 (row 2), forebrain and midbrain marker Otx2 (row 3), and mid-hindbrain boundary marker En2 (row 4) were all reduced. On the other hand, posterior neural marker HoxB9 (row 5) was expressed normally in the XIdaxMO-injected embryo. Expressions of N-CAM is also reduced (row 6). B-E: In situ hybridization with Krox20, En2, and BF-1 as probes. C: In a XIdaxMO-injected embryo, BF-1 expression is obviously reduced. D,E: Similar reduction can be observed in a Dvl-injected embryo (D), whereas a GSK-3 beta injection causes expansion of the BF-1 expression to the posterior region (E). Other neural-specific markers expression in XIdaxMO. XIdaxMO and lacZ were injected into one side of the AP cells. E: Red-gal staining marked the injected side. At the region injected with XIdaxMO, Sox2 expression was attenuated.
