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The vertebrate body is organized along the dorsal-ventral (DV) and anterior-posterior (AP) axes by the BMP and Wnt pathways, respectively. We previously reported that Xenopus Zbtb14 promotes dorsalization (neural induction) of ectoderm by inhibiting BMP signaling and also posteriorizes neural tissue by activating Wnt signaling, thereby coordinating the patterning of the DV and AP axes during early development. Although it has been reported that human ZBTB21 binds to ZBTB14 and is involved in gene expression in cultured mammalian cells, the function of Zbtb21 in early embryonic development remains unknown. Here, we show that Xenopus Zbtb21 plays an essential role in AP axis formation in the early Xenopus embryo. zbtb21 and zbtb14 are co-expressed in the dorsal region of embryos during gastrulation. Simultaneous overexpression of Zbtb21 and Zbtb14 in ectodermal explants enhances the neural-inducing activity of Zbtb14. Moreover, knockdown experiments showed that Zbtb21 is required for the formation of posterior neural tissue and the suppression of anterior neural development. Collectively, these results suggest that in cooperation with Zbtb14, Zbtb21 has a crucial function in AP patterning during early Xenopus embryogenesis.
Fig. 1. Comparison of Xenopus and human Zbtb21 protein sequences.
GenBank accession numbers for X. tropicalis Zbtb21 and H. sapiens ZBTB21 are NP_001090699.1 and NP_001091872.1, respectively. BTB/POZ and zinc finger domains are indicated by green and red boxes, respectively. Amino acids conserved between the two sequences are shaded in black. Amino acid number is shown on the right. Multiple protein sequence alignment was performed using the Clustal W method. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2. Expression of X. tropicalis zbtb21 and zbtb14 during early development.
(A) RT-PCR analysis of the temporal expression of zbtb21 and zbtb14. “-RT” indicates RNA of whole embryos (stage 1) examined in the absence of reverse transcriptase. (B–S) Spatial expression of zbtb21 analyzed by WISH in developing embryos at stages 4, 6, 9, 11, 11.5, 15, 16, 22, and 27. The expression of zbtb21 is shown in blue/purple (B–J); hybridization with a sense probe did not show significant signals (K–S), except for non-specific staining (shown as asterisks in J and S). Expression of zbtb21 is observed in the dorsal ectoderm (arrowheads in E and F) and the neural plate (arrowheads in G), at the gastrula and early neurula stages, respectively. At the late neurula stage, zbtb21 is expressed in the neural tube and eye region (H and I), and expression is detected in the brain, eyes, otic vesicle, branchial arches, and kidney (J) at the tailbud stage. (B-D, and K-M) Animal view. (E and N) Vegetal view with dorsal to the top. (F and O) Sagittal view with dorsal to the right of a dissected gastrulaembryo cut through the blastopore region (arrows). (G, I, P, and R) Dorsal view with anterior to the left. (H, J, Q, and S) Lateral side view with the anterior to the left. (T) X. tropicalis gastrula embryos (stage 10.5) dissected into dorsal and ventral halves were analyzed by qPCR analysis. The data are presented as means ± SE. ∗∗∗P < 0.001 vs. dorsal, Student's t-test. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. Zbtb21 and Zbtb14 cooperate to induce posterior neural tissue in ectodermal explants.
zbtb14-GR (125 pg) mRNA alone or with zbtb21 mRNA (500 pg and 1000 pg) was injected into the animal hemisphere of 4-cell stage embryos. Ectodermal explants were isolated at the blastula stage, cultured until the neurula stage (stage 19/20), and analyzed by both semi-quantitative RT-PCR (A) and qPCR (B). “Emb” and “-RT” indicate whole embryos examined in the presence or absence of reverse transcriptase, respectively. The qPCR data are presented as means ± SE. NS, not significant. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. Zbtb14 alone, Student's t-test with Bonferroni correction. (C) Xt Zbtb21 interacts with Xt Zbtb14. COS cells were transfected with the indicated combination of expression constructs, cultured for 2 days, and then treated with MG132 for 6 h before harvest. Flag-tagged Zbtb21 (Xt Zbtb21-Flag) was immunoprecipitated (IP) from cell lysates with an anti-Flag antibody, and the precipitates were immunoblotted (IB) with an anti-Myc antibody (top). Input cell lysates were immunoblotted with anti-Myc (middle) or anti-Flag (bottom) antibodies. The interaction between Xt Zbtb21 and Xt Zbtb14 was consistently observed in at least three independent experiments.
Fig. 4. Zbtb21 is required for the formation of posterior neural tissue and the suppression of anterior neural development.
Thirty-five ng of Control MO (Cont MO; A, E), Zbtb21 MO (B, F), or Zbtb21 MO with 750 pg of Xt zbtb21 mut mRNA (C, G) was injected unilaterally into 2-cell stage X. laevis embryos. Expression of hoxb9 (A–C) and pax6 (E–G) analyzed by WISH are shown in blue/purple. The injected side (indicated by brackets) was identified by the fluorescence signal. Expression of hoxb9 on the Zbtb21 MO-injected side was severely reduced (yellow arrowheads in B) and the anterior limits of hoxb9 expression shifted posteriorly with respect to the uninjected side of the embryo (white arrowhead compared to black one in B), and Xt zbtb21 mut mRNA partially but significantly rescued the reduction of hoxb9 expression caused by Zbtb21 MO (white arrowhead in C). All panels show dorsal views with posterior to the top. (D) A summary of the phenotypes in repeat experiments of (A–C). The degree of reduction in the hoxb9 expression domains was scored and categorized into four types: ■, Severe; Image 1, Moderate; Image 2, Weak; □, No reduction. Numbers of embryos per experimental group are indicated above the bars. Expression of pax6 on the Zbtb21 MO-injected side was expanded (yellow arrowheads in F), and Xt zbtb21 mut mRNA partially rescued the expansion of pax6 expression caused by Zbtb21 MO (G). All panels show frontal views with dorsal to the top. (H) A summary of the phenotypes in repeat experiments of (E–G). The degree of expansion of pax6 expression in the anterior region was scored and categorized into four types: ■, Severe; Image 1, Moderate; Image 2, Weak; □, No expansion. Numbers of embryos per experimental group are indicated above the bars. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
