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FIGURE 1 The BTB/POZ zinc- finger protein Zbtb14 promotes induction of posterior neural tissue and suppresses epidermal differentiation. (a) Structure of Xenopus laevis, Xenopus tropicalis and human Zbtb14 proteins. BTB/POZ domain, Broad- complex, Tramtrack and Bric- a- brac/Poxvirus and Zinc- finger domain. X. laevis zbtb14 encodes a 452- amino- acid protein containing a BTB/POZ domain with 98% and 97% amino acid identity to the X. tropicalis and human domains, respectively, and five C2H2- type zinc- finger domains with over 93% and 86% amino acid identity to the X. tropicalis and human domains, respectively. (b) Overexpression of Zbtb14 promotes neural induction and reduces epidermal differentiation in ectodermal explants. zbtb14-GR mRNA (125 pg and 250 pg) was injected into the animal region of 4- cell- stage embryos. Ectodermal explants were isolated from 12 embryos at the blastula stage, treated with DEX to activate Zbtb14, and cultured until late neurula stage (stage 20). Expression of marker genes was determined by semi- quantitative RT- PCR. Emb and –RT indicate whole- embryo RNA processed in, respectively, the presence or absence of reverse transcriptase. histone H4 was used as a loading control. (c–j) Zbtb14 expands the expression region of the neural plate markers sox2 and ncam (arrows) at the expense of epidermal keratin expression (arrowheads). Together with β-galactosidase (β-gal) mRNA, zbtb14-GR (125 pg) mRNA was injected unilaterally into both dorsal and ventral animal blastomeres of 8- cell- stage embryos. The injected side of the embryo is indicated by brackets. Expression of sox2, ncam and epidermal keratin analyzed by whole- mount in situ hybridization is shown in purple and β- Gal is stained in red. The top six panels (c–h) show dorsal views with posterior to the top. The bottom panels (i and j) show diagonal views with posterior to the right. (k–r) Zbtb14 expands the expression of the posterior marker cad2 (cdx2) and the spinal cord marker hoxb9 (m and p, respectively; black arrows), and the anterior limits of hoxb9 expression shift anteriorly with respect to the uninjected side of the embryo (r; white arrowheads). By contrast, Zbtb14 severely reduces the expression region of both the fore- and midbrain marker otx2 and the mid- and hindbrain marker en2 on the injected side (k and l, respectively; black arrowheads). Interestingly, the hindbrain marker krox20 shows both reduced and anteriorly shifted expression with respect to the uninjected side of the embryo (black and orange arrowheads in o and n/q, respectively). (q) and (r) show enlarged views of n and p, respectively. Dotted lines in (q) and (r) indicate the median line of embryos.
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FIGURE 2 Expression patterns of zbtb14 during early Xenopus embryogenesis. (a) Temporal expression profile of zbtb14 analyzed by semi- quantitative RT- PCR during X. laevis embryogenesis. –RT indicates whole- embryo RNA (stage 1) processed in the absence of reverse transcriptase. histone H4 was used as a loading control. (b–m) Expression pattern of zbtb14 analyzed by whole- mount in situ hybridization during the early development of X. tropicalis. Expression of zbtb14 is observed in the dorsal region (arrowheads) at the early gastrula stage (b, c and c’). Sagittal half embryos cut through the blastopore region (arrows) at the gastrula stage are shown in (c) and (d). (c’) indicates a magnified view of (c). Significant expression of zbtb14 is observed in the neural plate at early neurula stages (e–g). Cross- sections of early neurula- stage embryos (i and j), showing localized expression of zbtb14 mainly in the ectoderm (i). At late neurula stages, expression of zbtb14 is detected in the neural tube and eye region (k and l), and expression is stronger in the anterior than in the posterior region. In (d), (h), (j) and (m), hybridization with a sense probe did not show significant signals in these regions, except for non- specific staining (shown as asterisks in i, j, l, and m). Images in (b), (c), and (d) show lateral side views of gastrula- stage embryos with the dorsal lip (arrows) to the right. Images in (e), (h), and (k) show dorsal side views of embryos with posterior to the top. (f) shows the lateral side view of the embryo in (e) with posterior to the top and dorsal to the left. (g) shows the posterior side view of the embryo in (e) with dorsal to the top. Images in (l) and (m) show the frontal view with dorsal to the top. The dotted line in each panel of (i) and (j) indicates the boundary between the ectoderm and mesoderm. (n and o) X. tropicalis gastrula embryos dissected into dorsal and ventral halves were analyzed by both semi- quantitative RT- PCR (n) and qPCR (o). zbtb14 was predominantly expressed in the dorsal region at the early gastrula stage.
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FIGURE 3 Zbtb14 is required for formation of posterior neural tissue and suppression of anterior neural development. (a–d) With or without β-gal mRNA, 35 ng of Control MO (a; Cont MO), 6mis Zbtb14 MOa (b), Zbtb14 MOa (c) or Zbtb14 MOa with 500 pg of 5′ del-zbtb14 mRNA (d) was injected unilaterally into 2- cell- stage embryos. Expression of ncam analyzed by whole- mount in situ hybridization is shown in purple and β- Gal is stained in red. The injected side was identified by either β- Gal staining or fluorescence signal (See Materials and Methods for details). In embryos injected with Zbtb14 MOa, expression of ncam on the injected side was severely reduced (arrowheads in c) and 5′ del-zbtb14 significantly rescued the reduction of ncam expression induced by Zbtb14 MOa (arrows in d). The injected side of the embryo is indicated by brackets. All panels show dorsal views with posterior to the top. (e) Summary of phenotypes obtained from several repeats of the experiments shown in (a–d). The degree of recovery in the ncam expression domains was scored and categorized into three types: ■, Severely; , Weakly; □, unchanged. Numbers of embryos per experimental group are indicated above the bars. (f–u) Together with β-gal mRNA, either Cont MO (35 ng, left columns) or Zbtb14 MOa (35 ng, right columns) was injected unilaterally into 2- cell- stage embryos. Expression of sox2 (early neural marker, f and g), ncam (late neural marker, h and i), n-tubulin (differentiated neuronal marker, j and k), epidermal keratin (E. keratin) (epidermal marker, l and m), myod (paraxial mesoderm marker, n and o), pax6 (neural plate and eye field marker, p and q), krox20 (hindbrain marker)/rx2a (eye field marker, r and s), and hoxb9 (spinal cord marker, t and u) genes analyzed by whole- mount in situ hybridization are shown in purple and β- Gal is stained in red. In embryos injected with Zbtb14 MOa, expression of ncam and n-tubulin on the injected side is reduced; however, expression of sox2 is not significantly reduced. The expression pattern of myod is not significantly affected by Zbtb14 MOa. Expression of rx2a on the injected side is expanded, whereas that of krox20 is reduced (s). Expression of hoxb9 is also decreased and shifted posteriorly (horizontal lines mark approximate anterior limits of hoxb9 expression; u). In accordance with these observations, the spinal cord expression of pax6 is suppressed (arrowheads), while the anterior expression domains of pax6 and rx2a are expanded toward the posterior and lateral sides (arrows) (q and s). The injected side of the embryo is indicated by brackets. All panels show dorsal views with posterior to the top, except for pax6 and krox20/rx2a, which show anterior (front) views with dorsal to the top.
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FIGURE 4 Zbtb14 regulates BMP and Wnt signaling. (a) Zbtb14 decreases endogenous levels of phosphorylated Smad1/5/8 in Xenopus ectodermal cells. zbtb14-GR mRNA (250 pg) was injected into the animal region of 4- to 8- cell- stage embryos. Embryos were treated with DEX, and ectodermal explants were isolated at the blastula stage and cultured until the early gastrula stage (stage 10.5). Whole- cell lysates from explants were immunoblotted with anti- phospho Smad1/5/8 (pSmad1/5/8), anti- Smad1 (MO5), anti- HA, and anti- Tubulin antibodies, respectively. Zbtb14- GR was tagged at the C- terminus with the HA epitope. Tubulin was used as a loading control. (b) Zbtb14 decreases levels of pSmad1/5/8 induced by the constitutive active BMP receptor HA- CA- Alk6 in cultured HeLa cells. The effect of Zbtb14 depends on the proteasomal degradation pathway, which is inhibited by the proteasome inhibitor MG132. HeLa cells were transfected with the indicated combination of expression constructs, and then treated with or without MG132 for 6 hr before cell harvest. Whole- cell extracts were immunoblotted with the indicated antibodies. Tubulin was used as a loading control. Graph (right) shows quantification of the pSmad1/5/8 immunoblot which was normalized to Tubulin (left). (c) Zbtb14 decreases protein levels of BMP- regulated Smads (Smad1 and Smad8), Co- Smad (Smad4), TGF- β- regulated Smad (Smad2), and I- Smads (Smad6 and Smad7). COS cells were transfected with the indicated combination of expression constructs. Whole- cell extracts were immunoblotted with the indicated antibodies. Tubulin was used as a loading control. (d) Zbtb14 interacts with the R- Smad Smad3 and I- Smads (Smad6 and Smad7). COS cells were transfected with the indicated combination of expression constructs, and then treated with MG132 for 6 hr before harvest. Flag- tagged Smads (F- Smads) were immunoprecipitated (IP) from cells lysates with anti- Flag antibodies, and the precipitates were immunoblotted (IB) with anti- Myc antibodies (top). Input cells lysates were immunoblotted with anti- Myc (middle) or anti- Flag (bottom) antibodies. Interactions of Zbtb14 with Smad3, Smad6, and Smad7 were repeatedly observed in independent experiments (e and g; data not shown). (e) Zbtb14 interacts with Smurf1 and Smurf2. COS cells were transfected with the indicated combination of expression constructs, and then treated with MG132 for 6 hr before harvest. Flag- tagged Smads were immunoprecipitated from transfected cells with anti- Flag antibodies, and the precipitates were immunoblotted with anti- Myc antibodies (top). Input cells lysates were immunoblotted with anti- Myc (middle) or anti- Flag (bottom) antibodies. (f) Zbtb14 stabilizes cytosolic β- Catenin, which transduces canonical Wnt signaling. COS cells were transfected with Myc- tagged Zbtb14 and cell lysates were incubated with concanavalin A (Con A) beads to enrich free cytosolic β- Catenin by removing cadherin- bound β- Catenin. Input extracts were immunoblotted with anti- Myc (middle) or anti- Tubulin as a loading control (bottom). Graph (bottom) shows quantification of the immunoblot (top) which was normalized to Tubulin. (g) Zbtb14 interacts with β- TrCP as well as with Smad7. COS cells were transfected with the indicated combination of expression constructs, and then treated with MG132 for 6 hr before harvest. Flag- tagged constructs were immunoprecipitated from cells lysates with anti- Flag antibodies, and the precipitates were immunoblotted with anti- Myc antibodies (top panel). Input extracts were immunoblotted with anti- Myc antibodies (second panel) or anti- Flag antibodies (bottom panel). Extracts were also immunoprecipitated with anti- Flag and then immunoblotted with anti- Flag antibody (third panel), in order to detect β- TrCP, which is expressed at low levels.
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FIGURE5 Zbtb14 coordinates the establishment of DV and AP axes in Xenopus. (a) By the early neurula stage, neural tissue is formed on the dorsal side of the embryonic ectoderm by BMP antagonists, and then regionalized into the future fore- , mid- and hindbrain and the spinal cord along the AP axis by posteriorizing signals such as Wnt. Zbtb14 promotes neural induction and inhibits epidermal differentiation by inhibiting BMP signaling, thereby regulating DV patterning. In addition, Zbtb14 promotes the formation of posterior neural tissues and suppresses anterior neural development by modulating Wnt signaling. (b) Mechanistically, Zbtb14 reduces the level of both total and phosphorylated Smad1/5/8 proteins to suppress BMP signaling and also induces an accumulation of β- Catenin to promote Wnt signaling by associating with Smurfs/I- Smads (Smad6 and Smad7) and β- TrCP, respectively. Zbtb14 plays a crucial role in the formation of DV and AP axes by regulating both the BMP and Wnt pathways during early Xenopus embryogenesis.
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