Foxd4l1.1 negatively regulates transcription of neural repressor ventx1.1 during neuroectoderm formation in Xenopus embryos

Neuroectoderm formation is the first step in development of a proper nervous system for vertebrates. The developmental decision to form a non-neural ectoderm versus a neural one involves the regulation of BMP signaling, first reported many decades ago. However, the precise regulatory mechanism by which this is accomplished has not been fully elucidated, particularly for transcriptional regulation of certain key transcription factors. BMP4 inhibition is a required step in eliciting neuroectoderm from ectoderm and Foxd4l1.1 is one of the earliest neural genes highly expressed in the neuroectoderm and conserved across vertebrates, including humans. In this work, we focused on how Foxd4l1.1 downregulates the neural repressive pathway. Foxd4l1.1 inhibited BMP4/Smad1 signaling and triggered neuroectoderm formation in animal cap explants of Xenopus embryos. Foxd4l1.1 directly bound within the promoter of endogenous neural repressor ventx1.1 and inhibited ventx1.1 transcription. Foxd4l1.1 also physically interacted with Xbra in the nucleus and inhibited Xbra-induced ventx1.1 transcription. In addition, Foxd4l1.1 also reduced nuclear localization of Smad1 to inhibit Smad1-mediated ventx1.1 transcription. Foxd4l1.1 reduced the direct binding of Xbra and Smad1 on ventx1.1 promoter regions to block Xbra/Smad1-induced synergistic activation of ventx1.1 transcription. Collectively, Foxd4l1.1 negatively regulates transcription of a neural repressor ventx1.1 by multiple mechanisms in its exclusively occupied territory of neuroectoderm, and thus leading to primary neurogenesis. In conjunction with the results of our previous findings that ventx1.1 directly represses foxd4l1.1, the reciprocal repression of ventx1.1 and foxd4l1.1 is significant in at least in part specifying the mechanism for the non-neural versus neural ectoderm fate determination in Xenopus embryos.

Once the neural ectoderm is formed and neural transcription factors (nTFs) are expressed, some of the earliest expressed nTFs need to prevent cells from reverting to a non-neural fate [25][26][27] . The primary neuroectoderm expresses various transcription factors that may inhibit it from becoming the epidermis 25,28 . Foxd4l1.1 of the forkhead box (fox) family of transcription factors (also known as Xenopus fork-head expressed in the dorsal lip, foxd4l1.1, with other designations being xflip, foxd4-like1, foxd4l1, xfd-12 and foxd5b) is reported as one of the earliest neural genes and is evolutionally conserved and expressed in neuroectoderm across vertebrates including humans [29][30][31] . Foxd4l1.1 is known to actively participate in various developmental events, such as immature neuroectoderm fate maintenance, neural plate formation and neural differentiation. It has been shown that the ectopic expression of DNBR stimulates foxd4l1.1 mRNA expression level in animal cap explants 18,[32][33][34] . Further, transition of ectoderm-neuroectoderm is regulated by fine-tuning of several transcription factors such as foxd4l1.1, zic2/3, sox1-3/d and xiro1-3 25,35 . Foxd4l1.1 has been documented to increase the expression of neural specific genes, including sox2/3, geminin, n-tubulin, ncam, and neuroD in a dose-dependent manner, leading to neural differentiation 36 . Also, ectopic expression of foxd4l1.1 inhibits BMP4/Smad1 signaling and leads to neural differentiation and neuroectoderm formation 34 .
We have previously shown that foxd4l1.1 inhibits the promoter activity of ventx1.1 and promotes primary neurogenesis in Xenopus embryos 15 . A study documented that foxd4l1.1 and its engrailed (repressor) conjugated construct (EnRfoxd4l1.1) induce the expression of neural markers such as xngnr1, n-tubulin, geminin and xnr3 in animal cap explants 37 . However, the molecular details for foxd4l1.1 in ectoderm-neuroectodem specification and foxd4l1.1-mediated BMP4/Smad1 inhibition, leading to neuroectoderm formation, remain to be elaborated.
In the present study, we focused on the mechanisms of reciprocally exclusive germ-layer (ectoderm, mesoderm and neuroectoderm) specification in early vertebrate embryogenesis. In the mesoderm region, both BMP/ Smad1 and FGF/Xbra synergistically upregulate transcription of neural repressor ventx1.1 and inhibit neuroectoderm formation in Xenopus embryos 15,18 . On the other hand, the neuroectoderm region may require a neuroectoderm specific repressor in order to inhibit ventx1.1 expression. In this work, we found that foxd4l1.1 and engrailed-foxd4l1.1 (EnRfoxd4l1.1) negatively regulated BMP4/Smad1 signaling and inhibited ventx1.1 expression. It is also shown that Foxd4l1.1 directly binds to the proximal region of endogenous ventx1.1 promoter and inhibits ventx1.1 transcription during neuroectoderm formation. Foxd4l1.1 also inhibits the direct binding of Xbra-Smad1 to block the synergistic activation of ventx1.1 transcription. This study suggests that neuroectoderm specific repressor Foxd4l1.1 inhibits expression of the neural repressive transcription factor ventx1.1 to commit and maintain the neuroectoderm fate, obviating mesoderm commitment during germ-layer specification of Xenopus embryos.

Foxd4l1.1 abolishes Smad1-induced transcription activation of ventx1.1. EnRfoxd4l1.1 inhib-
ited expression of BMP4 and its target genes, resulting in the neuroectoderm formation in the animal cap explants ( Fig. 1a,b). A previous study demonstrated that ventrally injected foxd4l1.1 mRNA reduces the number of pSmad1/5/8 positive cells in the ventral epidermis region of embryos 34 and also inhibits expression of BMP4-targeted genes, ap2 and epi-keratin, while it induces the expression of chordin-stabilizing factor, sizzled (szl) 34 . In Xenopus, the expression domains of szl and bmp4 overlap and it was shown that szl expression is strongly dependent on BMP4 35 . Therefore, we examined whether foxd4l1.1 indeed inhibits Smad1 activity and Smad1-induced transcription activation of ventx1.1. To examine the Smad1 activity, 3BRE (triple-repeat of BMP4 response elements of activated Smad1 binding sites, 3XCAG ACA 16 ) reporter gene construct was injected with and without EnRfoxd4l1.1 at the one-cell stage and collected the injected embryos at stage 11 for reporter gene assays. EnRfoxd4l1.1 significantly decreased the relative reporter activity of 3BRE construct compared to that of 3BRE alone (Fig. 4a). Smad1 activity is reportedly positively dependent on its C-terminal phosphorylation and negatively on its linker region phosphorylation 21 . Since the cytoplasmic retention of Smad1 is mediated either by inhibition of its C-terminal phosphorylation (pSer-463/465) (BMP4 inhibition dependent) or the increase of MAPK-mediated linker region phosphorylation (pSer-206) (FGF dependent) 21,22,39,40 , we examined the phosphorylation status of Smad1 to test on which pathway mediated ventx1.1 repression by foxd4l1.1. Our results indicated that foxd4l1.1 not only reduced C-terminal phosphorylation of endogenous Smad1 (Fig. 4b, first line, pSmad1(463/465)), but it also increased linker region phosphorylation in Xenopus embryos (Fig. 4b, 2nd line, pSmad1(206)). We also examined phosphorylation changes for overexpressed Flag-Smad1 and changes in phospho-MAPK levels with and without EnRfoxd4l1.1 and foxd4l1.1 expression (Fig. 4c). Flag-Smad1 phosphorylation and phospho-MAPK levels were enhanced in foxd4l1.1 and EnRfoxd4l1.1 injected embryos (Fig. 4c). We then examined the localization of Smad1 in presence and absence of Foxd4l1.1 using immunofluorescence staining of animal cap explants. Results showed that Foxd4l1.1 led to cytoplasmic retention of Smad1 with complete exclusion from the nucleus for Smad1 in animal cap explants (Fig. 4d). Figure 4b,c indicated that foxd4l1.1 increased the linker region phosphorylation of Smad1. We then attempted to address    (Fig. 4e). We confirmed whether presence of fgf8b affected the linker region phosphorylation of Smad1 and Erk (p44/42 MAPK) phosphorylation in our system (Fig. 4f). We observed the reduction of 3BRE activity by EnRfoxd4l1.1. (Fig. 4a). Similarly, the 3BRE activity was reduced in presence of fgf8b and the reduction was recovered by the MEK inhibitor, U0126 (Fig. 4g). These results indicated that foxd4l1.1 not only inhibits Smad1 activation, but may also lead to cytoplasmic retention of Smad1 by activating FGF signaling. Since FGF signaling induces expression of xbra, which is a well-known inducer of mesoderm and is neural inhibitory in Xenopus 15,24 , we further examined xbra and fgf4 (efgf) expression with and without foxd4l1.1 in gastrula embryos and animal cap explants. Foxd4l1.1 strongly inhibited xbra expression in whole embryos (Fig. 4h) and xbra and fgf4 were not expressed in foxd4l1.1 injected animal caps (Fig. 4i), indicating that Foxd4l1.1 activates FGF/MAPK signaling for Smad1 retention in the cytoplasm (Fig. 4c,d), but inhibits efgf and xbra expression (Fig. 4h). We then examined whether Foxd4l1.1 inhibits the direct binding of Smad1 within the proximal region of endogenous ventx1.1 promoter. The ChIP-PCR results indicated that ectopic expression of HA-Foxd4l1.1 reduced the direct binding of Smad1 within the proximal region of ventx1.1 promoter (Fig. 4j (ChIP-PCR), 4 k (ChIP-qPCR)). In this study, we did not conclusively show that phosphorylation of Smad1 was likely to be initially inhibited by Foxd4l1.1; however, the results collectively indicated that (1)

Discussion
In the present study, we wanted to uncover a potential mechanism for non-neural ectoderm exclusion seen in neural ectoderm areas as part of an established transcriptional gene regulatory network (GRN). Here, we focused on elaborating the repressive activity of the neural specific transcription factor (TF), foxd4l1.1. Foxd4l1.1 is one of a number of evolutionally conserved earliest repressor TFs produced in neuroectoderm territory, post inhibition of BMP signaling in the dorsal ectoderm region. We found that foxd4l1.1, as a neural ectoderm specific TF, repressed ventx1.1, a neural repressor TF. Ventx1.1 as a neural repressor gene is an immediate early zygotic repressor TF, which is a direct target of BMP/Smad1 and FGF/Xbra signaling in the ventral ectoderm and mesoderm regions 15,16 . Together, our results suggest that repressive non-neural and neural TFs are mutually antagonistic in specifying the non-neural versus neural ectoderm activation areas in the nucleus. The implication and significance of this study is discussed below from the view point of the factors involved in neural induction. The role of extracellular levels of BMP in specifying naive ectoderm cells either to become epidermal or neural ectodermal has been understood since the mid 1990s [5][6][7][8] , and that the vertebrate neuroectoderm develops through the inductive signals emanating from the dorsal mesoderm of Spemann organizer has been a finding since 1924 41 . However, only recently, the discovery that the organizer is an antagonist center has led to a shift in thinking of neuroectoderm specification being a default neurogenesis process rather than an active neural induction one 11,42 .
Various studies indicate that across vertebrates, including for zebrafish, frog, and mouse embryos, input from BMP4 is required for ectoderm formation and that inhibition of BMP signaling has a conserved role for anterior neuroectoderm formation 27 . BMP4 is among more than 30 known BMP proteins that are mainly involved in epidermal induction and neural inhibition 8,11,13 . BMP signals express target genes that include gata, vent and msx families of TFs. Direct target TFs of BMP signaling that include gata1b, msx1 and ventx1.1 indeed inhibit neuroectoderm and represent the inhibitory aspect of BMP signals. This indicates that certain TFs in epidermis actively protect against becoming neuroectodermal via a transcriptional repression function of certain direct target TFs of the BMP pathway [12][13][14][15] . Albeit which BMP target gene(s) is essential for epidermis specification or the details of the relevant GRNs among various non-neural TFs still remain to be addressed. In this study, we selected ventx1.1 as a target TF to examine Foxd4l1.1 function in preventing cells from reverting to a non-neural fate. The reasons on why we focused on ventx1.1 are the following: First, ventx1.1 is expressed throughout the embryonic ectoderm in blastula to gastrula embryos such as with msx1 and ventx2. www.nature.com/scientificreports/ Second, ventx1.1 induces the epidermis and inhibits the formation of the dorsal mesoderm and neural tissue 14,17 . Third, ventx1.1 is a neural repressor, being a direct target TF of BMP4 16,18 . Fourth, inhibition of ventx1.1 induces neural ectoderm together with FGF in animal cap explants, similar to DNBR treated animal caps 8,24 . Fifth, the reporter activity of ventx1.1 promoter construct is reduced by foxd4l1.1 15 . Ventx1.1 transcript is found in the ventral ectoderm and mesoderm, but is completely absent in the organizer and the neuroectoderm region in early gastrula embryos 43 . Although Ventx1.1 has yet to be proven as an essential master TF among the direct target BMP TFs in epidermal specification, our previous study that ventx1.1 directly represses foxd4l1.1 led to examine the reciprocal repression between them in specifying the non-neural versus neural ectoderm fates at the transcription level. For a given ectodermal region, among exclusively expressed neuroectoderm or ectoderm genes, certain TFs may function as repressors in excluding expression of genes of alternate fates. Such a proposal has been put forward by Sasai 25 . Once, the neural ectoderm is formed and neural transcription factors (nTFs) are expressed, some of the earliest expressed nTFs are needed to prevent cells from reverting to a non-neural fate 25,26 . The primary neuroectoderm expresses various transcription factors that may block it from becoming an epidermis [25][26][27][28] . Foxd4l1.1 (xfd-12, xflip, foxd5a, b) transcript is exclusively expressed in the superficial layer, namely neuroectoderm, of cells above the dorsal lip of the Spemann organizer territory at the early gastrula 31,43 . Both Foxd4l1.1 and Ventx1.1 contain a strong repressive domain 17,18,37,44 with mutually exclusive expression in the ectoderm/ ventral mesoderm and the neuroectoderm/organizer, respectively, at the time of the ectoderm/neuroectoderm commitment for early gastrula. We hypothesized that BMP signal modulation leads to expression of non-neural versus neural TFs, with at least one being essential in preventing cells from reverting to alternate fates. We thus examined foxd4l1.1 as one of the earliest expressed nTFs, which block neuroectoderm from becoming epidermis. Exclusive presence of a repressive gene in a given territory would at least be a strategy to exclude essential gene expression involved in alternate germ layer commitment. For example, ectopic overexpression of organizer genes including gsc, chordin and noggin in ventral region of 4-cell stage embryos leads to two-axis formation in whole embryos. On the other hand, overexpression of ventral specific genes including vent1.1, vent1.2 and bmp4 in dorsal region of 4-cell stage embryos leads to headless embryos. As such, overexpression studies in developing embryos suggest that ectopic signaling or presence of certain TF(s) in competent cells convert their GRNs.
Reciprocal transcriptional repression of ventral and dorsal opposing homeobox genes gsc and ventx1/2 has also been proposed to in part mediate dorsoventral patterning to ensure robust and reproducible embryonic development through triple depletion of gsc, ventx1, and ventx2 45 . Various TFs have been proposed and examined for being reciprocal means of repression in early vertebrate embryogenesis 46,47 . Involvement of foxd4l1.1 and ventx1.1 in neuroectoderm versus ectoderm specification cannot be ruled out in the present study and require www.nature.com/scientificreports/ further study. However, in this work, we mainly focused on the maintenance/protective role of foxd4l1.1 in the expressed cells of neuroectoderm for fate reversion. From our previous work, inhibition of BMP signaling induces foxd4l1.1 expression, which is directly repressed by Ventx1.1 18 . In the present study, foxd4l1.1 induced neuroectoderm formation which was also inhibited by co-injection of ventx1.1 (Fig. 1b), indicating that ventx1.1 needs to be repressed for proper neuroectoderm formation in both cases of foxd4l1.1 induced neuroectoderm and DNBR induced foxd4l1.1 expression. In this study, we show that foxd4l1.1 inhibited ventx1.1 expression. We examined whether the inhibition was direct or indirect (or both). It was shown that Foxd4l1.1 directly binds to one of the cis-acting FREs of ventx1.1 (FRE1; ATA AAA , − 82 to − 76 region of the promoter) to inhibit ventx1.1 transcription (Fig. 2e-g). We also found an indirect means of foxd4l1.1 mediated ventx1.1 repression, separately occurring through Xbra and Smad1. For Xbra inhibition, Foxd4l1.1 bound to Xbra protein to diminish its affinity to the XbRE element (ATCA CAC TT, − 107 to − 99 region) of ventx1.1 promoter (Fig. 3b-f). In addition, foxd4l1.1 inhibited xbra expression (Fig. 4h), although we did not exactly elaborate on how foxd4l1.1 inhibits xbra expression. We speculate that it is from a direct inhibition of xbra expression though Foxd4l1.1 binding to the 5′-flanking/promoter region of xbra. A genome-wide ChIP-Seq analysis of Foxd4l1.1 implicated such a pathway with Foxd4l1.1 binding to the 5′-flanking region of xbra in Xenopus embryos (data not shown). However, the exact mechanism remains to be demonstrated.
For foxd4l1.1 mediated Smad1 inhibition, it has been documented that ventrally injected foxd4l1.1 reduces the number of phosphorylated Smad1 (activated Smad1) positive cells and inhibits BMP4/Smad1 downstream targets epi-keratin and ap2 in Xenopus embryos 34 . Furthermore, foxd4l1.1 increases expression of szl, suggesting that foxd4l1.1 reduces nuclear localization of Smad1 either by inhibiting bmp4 expression or by Sizzled-mediated inhibition of BMP4 in a Chordin-dependent manner 34 . In the present study, we found that foxd4l1.1 reduced bmp4 expression (Fig. 1a, lane2, 4 vs lane5). Currently, we do not know exactly how foxd4l1.1 downregulated bmp4 expression. We and others have elaborated on the possibility of BMP expression being regulated by a positive feedback loop 48,49 . Foxd4l1.1 mediated reduction of bmp4 expression may be through inhibiting activation of Smad1 since activated Smad1 may be involved in zygotic bmp4 expression during mid-blastula transition 50 . We confirmed Smad1 inactivation using a Smad1 specific reporter (3BRE) (Fig. 4a). Interestingly, we found that foxd4l1.1 increased linker region phosphorylation of Smad1 (pSer-206) (Fig. 4b,c). Since Smad1 is negatively regulated by FGF/MEK/Erk-mediated phosphorylation of Smad1 linker region 21,22 , we elaborated on FGF signal involvement. We observed that both foxd4l1.1 and EnRfoxd4l1.1 increased fgf8a/b expression (Fig. 4e). These suggested that foxd4l1.1 increased Smad1 linker region phosphorylation could be mediated by FGF8/MAPK signaling. Our confocal image results showed that foxd4l1.1 led to a cytoplasmic retention of Smad1 and completely excluded nuclear localization of Smad1 in animal cap explants (Fig. 4d). These observations are basically the same as reported by Yan et al. (2009) 34 . We used animal cap explants, while Yan's experiments used the ventral epidermal part in whole embryos. A noted difference was the detection of endogenous phospho-Smad1/5/8 in Yan's report, while, we overexpressed tagged Smad1 and detected the ectopically expressed Smad1 using confocal microscopy. Foxd4l1.1 increased fgf8a/b expression in animal cap explants (Fig. 4e). The question of whether other fgfs in addition to fgf8a/b are also involved could be raised. We examined the expression of fgf4 (efgf) and xbra with both foxd4l1.1 and EnRfoxd4l1.1 injected embryos. Neither injection induced xbra or fgf4 in the animal cap explants, suggesting that FGF4/MAP-kinase/xbra loop is not involved in foxd4l1.1 mediated Smad1 linker phosphorylation. Furthermore, whether fgf8a/b induction occurs through Foxd4l1.1 activator function or indirectly through Foxd4l1.1 repressor function would be interesting to explore. Both foxd4l1.1 mRNAs induced fgf8a/b, implying that fgf8a/b induction may occur indirectly through Foxd4l1.1 repressor function and Xbra/ FGF positive feedback loop was not the reason for Smad1 inactivation. Similar to Foxd4l1.1, Zbtb14 reduces the levels of phosphorylated Smad1/5/8 in Xenopus 51 . Overexpression of zbtb14 promotes neural induction similar to that of foxd4l1.1. While BMP inhibition and overexpression of foxd4l1.1 induces anterior neural tissue, zbtb14 promotes posterior neural tissue and suppresses anterior neural tissue. Presently, we cannot delineate the connection between Foxd4l1.1 and Zbtb14 in neural induction and Smad1 inactivation, and details of foxd4l1.1 mediated BMP/Smad1 signal attenuation and fgf8 involvement during neuroectoderm formation await more elaboration.
Previously, our studies show that Smad1 and Xbra physically interact and synergistically cooperate to increase ventx1.1 transcription in Xenopus embryos 15 . Foxd4l1.1 reduced Smad1 and Xbra physical interaction possibly, first, due to reduced C-terminal phosphorylation of Smad1 via reduced BMP expression, and second, via competition for Xbra since both Foxd4l1.1 and Smad1 bind to Xbra protein (Figs. 5b and s4). However, the latter reason is less essential since both Foxd4l1.1 and EnRFoxd4l1.1 (which does not contain Xbra binding C-terminal domain) reduced Smad1 and Xbra physical interaction (Fig. 5b, Fig. s4a,b). C-terminal phosphorylation of Smad1 is crucially required for the interaction with the N-terminal domain of Xbra 52 . However, this explanation is not enough to address more effective Foxd4l1.1 mediated reduction of Smad1 binding on the ventx1.1 promoter with presence of Xbra (Fig. 5c,d (lane 5 vs lane6)) when compared with absence of Xbra (Fig. 4j,f). At this moment, we only speculate that the difference may be dependent on the difference of involved FGF characters and additional work is necessary to explain how Xbra contribute positively and negatively for Smad1 binding on context dependent manner.
EnRfoxd4l1.1 inhibited the relative promoter activity for ventx1.1 (− 2481) promoter construct by up to eightfold while it reduced that of ventx1.1 (− 157) construct by up to 0.5-fold. This may be due to ventx1.1 (− 2481) promoter containing more than one functional FRE for Foxd4l1.1. The ventx1.1 (− 2481) promoter region contains an additional six putative cis-acting FREs for Foxd4l1.1 (Fig. s3, putative FREs; highlighted in red). A point mutation within the confirmed FRE1 in ventx1.1 (− 2481, − 233 and -233 mBRE) promoter constructs indicated that the 5′-flanking region of ventx1.1 contains more than one consensus FRE, actively participating in Foxd4l1.1mediated negative regulation of ventx1.1 transcription (Fig. 5f). EnRFoxd4l1.1 more efficiently inhibited BREmutated ventx1.1 (− 233) mBRE promoter constructs (Fig. 5e,f, bar 9 vs bar 10). Surprisingly, EnRFoxd4l1.1 increased the relative promoter activity of doubly-mutated ventx1. www.nature.com/scientificreports/ ( Fig. 5f bar 11 vs bar12). This induction may be caused by inhibition of endogenous goosecoid (gsc) expression by EnRFoxd4l1.1. Our study demonstrates that gsc inhibited ventx1.1 expression as well as the relative activity of ventx1.1 promoter construct 16 . Ventx1.1 (− 233) promoter contained the direct binding response element for Gsc (GRE: ATT TGC , − 195 to − 190 region of the promoter; highlighted in blue in Fig. s3, unpublished data), which was experimentally identified. In summary, we propose a model for Foxd4l1.1 inhibiting the transcription of ventx1.1 in its exclusively occupied region of neuroectoderm via the mechanisms elaborated in this work and leading to primary neurogenesis in Xenopus embryos ( Fig. 6; right panel; highlighted as red and blue lines for the evidence shown in this paper). In this paper, we provide evidence on how Foxd4l1.1 represses ventx1.1 transcription in neuroectoderm. This work provides an insight on how Foxd4l1.1 negatively regulates the neural repressive BMP-Smad1-ventx axis, specifically at the transcription level for the neural repressor ventx1.1 and the Foxd4l1.1 exclusively occupied territory of neuroectoderm. In the "neuroectoderm" areas, we propose that the dominant repressory role of Foxd4l1.1 on ventx1.1 transcription is via the FRE-domain areas bound by Foxd4l1.1 as the BMP/Smad1 levels are relatively low already. In Fig. 6, we depict the direct or indirect regulatory axes, as supported by our data. With the role of extracellular BMP/BMPR in fate determination being in the literature for many years, the intracellular details in transcription regulation of neural/non-neural TFs have been lacking and that this model serves to fill in some of the current gaps in the literature. Homologues of Xenopus foxd4l1.1 are highly conserved across vertebrates that include zebrafish, mouse and also humans. All the homologues of foxd4l1.1 are similarly expressed in the neural ectoderm of embryos [53][54][55] . Recently, Sherman et al. reported that foxd4 in mouse is required for transition of a pluripotent ES cell to a neuroectodermal stem cell, suggesting that mouse foxd4 has a similar function to its Xenopus orthologue 56 . Similarly, our study may provide an additional insight on neuroectoderm differentiation in early embryogenesis across vertebrates.

Materials and methods
Ethics statement. This  Ventx1.1 promoter constructs. The 2.5 kbs of 5′-flanking region of positive clone was subcloned into the pGL-2 basic plasmid (Promega, Madison, WI) and was designated as the − 2481 bps construct. Serially-deleted ventx1.1 promoter mutants and triple-repeated BMP4-response element (BRE) were generated from − 2481 bps construct and subcloned into a pGL-2 basic plasmid by PCR amplification 16 . Numbering of the reporter construct was for the distance 5′-upstream of the translation start site (ATG). RNA isolation and RT-PCR. EnRfoxd4l1.1 (280 pg/embryos) and other mRNA (Myc-xbra (1 ng/embryo), Flag-smad1 (1 ng/embryo) and HA-foxd4l1.1 (3 ng/embryo)) was injected into the animal pole of one-cell stage embryos that were then cultured in 30% MMR solution until stage 8 and also for control non-injected embryos. Animal caps were then dissected from the injected and non-injected embryos and incubated until stage 11 and 24 in L-15 medium. Total RNA was isolated from whole embryos or animal caps using RNA-bee reagent following the manufacturer's instructions (TEL-TEST, Friendwood, TX) as described by Kumar et al. (2018) 15 . PCR was performed by using oligonucleotides according to the following conditions as described in Table 1.

Embryo injection and explants culture. Xenopus laevis
Quantitative RT-PCR (qPCR). The qPCR reactions were performed by using an Applied Biosystems Ste-pOnePlus Real-Time PCR System with KAPA SYBER FAST qPCR Master Mix. All the real-time values were averaged and compared using the threshold cycle (CT) method, in which the amount of target RNA (2 − ΔΔCT) was normalized against the endogenous expression of ODC (ornithine decarboxylase) (ΔCT). The qPCR reactions were performed when RT-PCR reaction results need to be quantified (Fig. s1) www.nature.com/scientificreports/ elsewhere published data (vent1.1) 57 was not repeated. All qPCR reactions were repeated three time using independent samples to present data with standard deviations and statistical significance.
Luciferase assays. Levels of relative luciferase activity were measured as described by Kumar et al. (2018) 15 .

Identification of binding sites of transcription factors and site-directed mutagenesis.
The binding sites of transcription factors including Foxd4l1.1 (FRE1) and Smad1 (BRE) were identified using serially-deleted reporter gene constructs (Table 2) and site-directed mutagenesis. Site-directed mutagenesis of FRE1 and BRE within ventx1.1 promoter constructs were performed by a site-directed mutagenesis kit (Muta-Direct, iNtRON Biotechnology, Seongnam, Korea) by using the oligonucleotides listed in Table 3. Site-directed mutagenesis of XbRE has been described by Kumar et al. 15 .