Nanog safeguards early embryogenesis against global activation of maternal β-catenin activity by interfering with TCF factors

Maternal β-catenin activity is essential and critical for dorsal induction and its dorsal activation has been thoroughly studied. However, how the maternal β-catenin activity is suppressed in the nondorsal cells remains poorly understood. Nanog is known to play a central role for maintenance of the pluripotency and maternal -zygotic transition (MZT). Here, we reveal a novel role of Nanog as a strong repressor of maternal β-catenin signaling to safeguard the embryo against hyperactivation of maternal β-catenin activity and hyperdorsalization. In zebrafish, knockdown of nanog at different levels led to either posteriorization or dorsalization, mimicking zygotic or maternal activation of Wnt/β-catenin activities, and the maternal zygotic mutant of nanog (MZnanog) showed strong activation of maternal β-catenin activity and hyperdorsalization. Although a constitutive activator-type Nanog (Vp16-Nanog, lacking the N terminal) perfectly rescued the MZT defects of MZnanog, it did not rescue the phenotypes resulting from β-catenin signaling activation. Mechanistically, the N terminal of Nanog directly interacts with T-cell factor (TCF) and interferes with the binding of β-catenin to TCF, thereby attenuating the transcriptional activity of β-catenin. Therefore, our study establishes a novel role for Nanog in repressing maternal β-catenin activity and demonstrates a transcriptional switch between β-catenin/TCF and Nanog/TCF complexes, which safeguards the embryo from global activation of maternal β-catenin activity.


Introduction
and it is crucial for embryonic architecture formation and cell 122 survival [42]. 123 In the present study, we independently generated two MZnanog alleles with 124 zebrafish, and demonstrated that maternal Nanog interacts with maternally 125 deposited TCF, thereby safeguards the embryo against formation of β-126 catenin/TCF transcriptional activation complex which may induce hyper-127 dorsalization of the embryo. Our study thus uncovers a negative regulation 128 system of maternal β-catenin activity, by revealing Nanog as a novel 129 transcriptional switch between Nanog/TCF and β-catenin/TCF complexes. observe that the nuclear β-catenin localized not only at the dorsal-most 139 blastomeres with high amount but also in some ventral and lateral cells with low 140 amount in early zebrafish embryos at 3.5 hour-post-fertilization (hpf) (Fig. 1a), 141 just like that in Xenopus, lower levels of nuclear β-catenin also presents 142 throughout the embryo at blastulae stage [48,49]. Furthermore, we showed that overexpression of β-catenin at non-dorsal cells could induce ectopic 144 expression of maternal β-catenin targets, boz and chd (Fig. S1), suggesting 145 that the low level of ventrally located endogenous nuclear β-catenin activities 146 should be repressed in early embryo. 147 Therefore, we were curious about how the ventrally distributed nuclear   At mid-gastrulation, the moderate-dose morphants showed strong expansion 204 of otx2 (dorsal ectoderm marker) (Fig. 2i, j) and ventral shrinkage of foxil 205 (ventral ectoderm marker) (Fig. 2k, l) of nanog MO (160pg per embryo), wnt8a mRNA (0.1pg per embryo) and tcf7l1a 220 MO (800pg per embryo) to obtain normal brain patterning in the injected 221 embryos. However, when nanog MO was co-injected with wnt8a mRNA or tcfl1a 222 MO, a majority of embryos developed with the headless phenotype (Fig. 3b), 223 suggesting the genetic interaction between nanog and zygotic Wnt signaling.

224
To further investigate the crosstalk between Nanog and Wnt/β-catenin signaling, 225 we performed genetic interaction experiments. When compared with WT, the 226 expression of zygotic Wnt target gene sp5l was expanded to the animal pole 227 and ventral ectoderm, and the expression of Wnt antagonist dkk1b and frzb 228 was decreased in the nanog morphants (Fig. 3c, d). Injection of wnt8a MO 229 largely restored the expression of those genes in nanog morphants (Fig. 3c, d).
which were absent in the low-dose nanog MO injected embryos, were 232 recovered after wnt8a knockdown (Fig. 3c,d). All these data indicate that 233 Nanog negatively regulates Wnt/β-catenin signaling. 234 We then investigated the repressive role of Nanog on Wnt/β-catenin 235 signaling activity by in vivo and in vitro TOPflash assay. Knockdown of nanog 236 with low and moderate dosages of nanog MO resulted in dose-dependent up-237 regulation of Wnt signaling activity in embryos (Fig. 3e). In 293T cells, the 238 TOPflash activity was significantly increased after transfection of WT β-catenin.

239
However, when the cells were co-transfected with different amounts of Nanog, 240 the TopFlash activity showed significantly reduction in a dose-dependent 241 manner (Fig. 3f). We then performed a similar TopFlash assay in the cells 242 transfected with ∆N-β-catenin, which can sustainably enter the nucleus to 243 active the transcriptional activity [29]. To our surprise, co-transfection of Nanog 244 could still significantly repress the Wnt activity resulting from overexpression of 245 "activated form" of β-catenin (∆N-β-catenin) in a dose dependent manner (Fig. 246 3g). These results indicate that the Nanog could effectively repress the 247 transcriptional activity of nucleus-located β-catenin. In order to fully characterize the role of nanog in early embryonic 252 development, we generate nanog mutants using TALEN mediated mutagenesis as described in our previous study [54]. We identified two mutant lines -one 254 with 2-bp deletion (-2) and the other one with 1-bp insertion (+1) (Fig. S2a). The   To verify that the Nanog protein was completely absent in MZnanog, we 271 detected the protein level of Nanog by western blot. In WT embryos, Nanog 272 protein could be detected from 64-cell stage and decreased rapidly during 273 gastrulation. After the 75% epiboly stage, Nanog protein was no longer detected, 274 whereas the Nanog protein could not be detected in both types of MZnanog embryos (Fig. 4a). Immunostaining analysis of Nanog further confirmed that the 276 Nanog is mainly localized in the cell nucleus and there is no Nanog expression 277 in the MZnanog (Fig. 4b). These data suggested that the Nanog was completed 278 disrupted in MZnanog mutant. 279 We also compared the transcript level of nanog in MZnanog ihb97 and 280 MZnanog ihb98 . The maternal nanog mRNAs were absent in the MZnanog ihb97 . and chd in WT embryos (Fig. 4c, d). TopFlash assay further supported that the 295 Wnt activity was elevated in MZnanog embryos, and this effect could be 296 suppressed by overexpression of gsk3b or ck1a (Fig. 4f). WISH analysis of chd at sphere stage further confirmed the rescue effect by gsk3b or ck1a (Fig. 4g). 298 More interestingly, by overexpression of gsk3b or ck1a, the hyper-dorsalization 299 defects of MZnanog at gastrula stage could be partially rescued (Fig. 4h). 300 These results illustrate that loss of nanog function leads to over-activation of 301 maternal Wnt/β-catenin activity.   It is well-known that the activation of canonical Wnt pathway leads to 316 translocation of β-catenin into the cell nucleus [13,55]. We then aimed to test 317 whether the over-activation of Wnt activity in MZnanog is caused by increased 318 accumulation of β-catenin in the nucleus. Firstly, we examined the level of nuclear β-catenin in MZnanog at 4 hpf by western blot by using wnt8a-320 overexpressed embryo as positive control. Unlike that the ratio of active β-321 catenin/total β-catenin was remarkably increased in wnt8a-overexpressed 322 embryos, there was no significant difference between MZnanog and WT 323 embryos (Fig. 5d, e). We further checked the nuclear β-catenin accumulation 324 in MZnanog by immunostaining at 3 hpf, and found that the nuclear localized 325 β-catenin in MZnanog appeared comparable to that in WT (Fig. 5f). Taken 326 together, we demonstrate that the hyper-activation of maternal β-catenin 327 activity is not due to the increased nuclear translocation of β-catenin. of Nanog is required for its suppression activity on Wnt/β-catenin signaling.

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Nanog interferes with the binding of β-catenin to TCF7 in vitro 375 Since Nanog dose not regulate the nuclear β-catenin level and its 376 suppression of β-catenin transcriptional activity relies on its N-terminal, we 377 inquired whether Nanog physically interacts with β-catenin or its nuclear 378 partners, such as TCF/Lef. To start with, we firstly identified that Tcf7 and Tcf4 379 are both activator-type TCF, since localized injection of tcf7 or tcf4 mRNA alone, 380 or co-injection with β-catenin mRNA, tcf7 and tcf4 could efficiently induce the 381 ectopic expression of maternal β-catenin targets, boz and chd (Fig. S4), 382 supporting the previous report that Tcf7 acts as β-catenin-dependent trans-383 activators with Lef1 [59][60][61].

393
Next, we aimed to understand that which part of Tcf7 is responsible for its 394 interaction with Nanog. We generated different forms of Tcf7 including full- Since Nanog and β-catenin could both bind to Tcf7, there is a possibility 411 that Nanog may interfere the interaction between β-catenin and TCF. We then 412 performed a competitive binding assay in which the input of β-catenin and TCF 413 was consistent in each sample and the input of Nanog was gradually increased. The above data showed that Nanog binds to the Tcf7 and interferes the     Competing interest statement 550 The authors declare no competing interests.

553
Zebrafish maintenance 554 All the zebrafish used in this study were maintained and raised as previously               W T + n a n o g _ F L + n a n o g _ C T + v p 1 6 -n a n o g