An Evolutionarily Conserved Mesodermal Enhancer in Vertebrate Zic3

Zic3 encodes a zinc finger protein essential for the development of meso-ectodermal tissues. In mammals, Zic3 has important roles in the development of neural tube, axial skeletons, left-right body axis, and in maintaining pluripotency of ES cells. Here we characterized cis-regulatory elements required for Zic3 expression. Enhancer activities of human-chicken-conserved noncoding sequences around Zic1 and Zic3 were screened using chick whole-embryo electroporation. We identified enhancers for meso-ectodermal tissues. Among them, a mesodermal enhancer (Zic3-ME) in distant 3′ flanking showed robust enhancement of reporter gene expression in the mesodermal tissue of chicken and mouse embryos, and was required for mesodermal Zic3 expression in mice. Zic3-ME minimal core region is included in the DNase hypersensitive region of ES cells, mesoderm, and neural progenitors, and was bound by T (Brachyury), Eomes, Lef1, Nanog, Oct4, and Zic2. Zic3-ME is derived from an ancestral sequence shared with a sequence encoding a mitochondrial enzyme. These results indicate that Zic3-ME is an integrated cis-regulatory element essential for the proper expression of Zic3 in vertebrates, serving as a hub for a gene regulatory network including Zic3.

Enhancer screening in chicken embryos. We carried out embryo electroporation 36,37 to identify enhancers of chicken Zic1 and Zic3. The analysis focused on evolutionary conserved noncoding element (CNE) between human and chicken genome. Twenty-five and twenty-eight CNEs within 150 kb flanking regions were selected for Zic1 and Zic3, respectively ( Fig. 2A). The selection of distal CNEs (more than 50 kb apart) was based on the conservation between human and teleost fish sequences. The CNEs were placed upstream of the herpes simplex virus thymidine kinase (TK) promoter-driven EGFP reporter vector. TK promoter was shown to be neutral in terms of the expression in chick embryos in previous studies 36 . The CNE-TK-EGFP reporters were co-electroporated with a control vector (EF-LacZ) that directs ubiquitous expression. Electroporation was carried out at HH4, and the reporter gene expression profiles were monitored until HH11.
As a result, we observed clear enhancer activities for two CNEs for Zic1 and three CNEs for Zic3 (Fig. 2). These CNEs showed spatially restricted enhancer activities. In the neural tissue, Zic1-NE (neural enhancer), Zic3-NE1 (neural enhancer 1), Zic3-NE2 (neural enhancer 2) directed the reporter gene expression (Fig. 2B-D). As to the anterior-posterior (A-P) extent of expression in the HH11 neural tube (Fig. 2B,D), Zic1-NE enhancer activity was observed all along the A-P axis with enhancement in the hindbrain and the spinal cord region. Zic3-NE1 and Zic3-NE2 activities were observed in the hindbrain and the anterior spinal cord region. Along the dorsal-ventral (D-V) axis, Zic1-NE directed wide expression at the HH7 neural plate, but the expression was dorsally restricted at HH16 neural tube as determined by electroporation of the reporter constructs into neural tube at HH11 (Supplementary Fig. S1). The change in expression mimicked that of Zic proteins in the chicken neural tube development 24 . Zic3-NE1 and Zic3-NE2 directed wide expression along D-V axis in the neural tube at all the stages (Supplementary Fig. S1). In the mesodermal derivatives, Zic1-ME (mesodermal enhancer) reporter expression was observed weakly at somites, whereas Zic3-ME reporter expression was strong at both unsegmented paraxial mesoderm and somites ( Fig. 2B-D, Supplementary Fig. S1), mimicking distribution of Zic1 and Zic3 transcripts (Fig. 1). The spatially restricted activities of all five enhancers can be traced back to 7 hours after transfection (HH6) when the enhancer activity became evident, and it continued until 24 hours post-transfection (HH11) ( Supplementary Fig. S2, Fig. 2B).
Among the five CNEs, Zic1-NE homologous region in mouse genome was included in the cochlear nucleus and dorsal spinal cord enhancer region within the 2.9 kb region of Zic1 transcription start site ( Fig. 2A) that was characterized in a previous study 27 . The other CNEs were thought to be novel. Zic1-ME was located in an intron of Zic4, which is placed 13 kb upstream of Zic1 in a head-to-head manner ( Fig. 2A). Zic3-NE1, Zic3-NE2, and Zic3-ME were located in the 3′ flanking region of Zic3 ( Fig. 2A). In chicken, they are 45, 50, and 65 kb apart from Enhancer activities of the five CNEs in mouse embryos. To characterize the five CNEs, we tested if the homologous region in the mouse genome shows enhancer activities in mouse embryos by a transgenic reporter  Top indicates the scales in kilobase, centered at the transcription initiation of Zic1 or Zic3 the + (plus) direction is that of transcription. Blue thin vertical lines below gene names indicate the protein coding exons. Clustered green vertical lines indicate the presence of sequence similarity with the human sequence at each position of the sequences of animals indicated at the left side (mouse, chicken, Xenopus, and zebrafish). Extent of conservation is indicated as the height of bars. The extent of conservation among the 100 vertebrate species selected at UCSC genome browser is indicated by 100 vert. Black vertical lines in the CNE line indicate the position of tested CNEs in this study. Ol-a-d and Dr-e1, known regulatory elements identified in teleost fishes 30,31  assay. We prepared transgenes in which the mouse CNE homologues were placed upstream of the Heat shock protein 68 (Hsp68) promoter driven-LacZ. The transgene expression profiles were examined by β-galactosidase activities in the primary transgene recipient mice at embryonic day (E) 10.5 or 11.5.
In the mouse neural tissue, Zic1-NE gave the most robust expression in spinal cord and frequently at dorsal forebrain and midbrain (Fig. 3A,B,L). Zic3-NE2 also showed frequent expression in hindbrain and spinal cord, and Zic3-NE1 transgenes showed frequent expression in the spinal cord (Fig. 3C,D,L). All three CNEs directed the reporter gene expression in spinal cord. Although Zic1-NE transgene showed consistent activity in the dorsal spinal cord where the Zic1 was expressed, Zic3-NE1 and Zic3-NE2 were not consistent with the endogenous Zic3 expression 17,38 . In the mouse mesoderm derivatives, Zic3-ME frequently showed expression in the axial mesoderm including the unsegmented mesoderm, newly generated somites, and the dermomyotome of the anteriorly located mature somites (Fig. 3I,J,L), and less frequently at external layer of brain (Fig. 3I,K,L). Zic1-ME directed expression in the somite less frequently and weakly (Fig. 3G,H,L).
Thus, the expression patterns of the mouse Zic1 and Zic3 CNE-driven reporters were similar to those in chicken embryos, indicating that the enhancer functions are evolutionary conserved between mouse and chicken. However, the expression may not always be limited to the region of mouse Zic1 and Zic3 expression at the corresponding stage (Fig. 3M,N). In this regard, Zic3-ME-driven reporter appears to reliably mimic the Zic3 expression in chicken as well as mouse 17,39 . We therefore focused on the analysis of Zic3-ME in the following experiments.
Zic3-ME was required for the Zic3 expression in the mesodermal tissue. We next addressed the significance of Zic3-ME by generating Zic3-ME knockout (KO) mice. The mice were generated by replacing entire Zic3-ME by a loxP sequence using homologous recombination in ES cells ( Supplementary Fig. S3). We examined Zic3 expression in the E6.5 and E8.5 in Zic3-ME-deficient (Zic3-ME −/Y ) and wild type (Zic3-ME +/Y ) mice. By in situ hybridization, we found loss of Zic3 transcript in the mesoderm of E6.5 Zic3-ME-deficient embryos (5/5), and an intact expression in the epiblast (Fig. 4B,D,F,J, Supplementary Fig. S4). At E8.5, the Zic3 expression at somites was lost (4/4) ( Fig. 4N,P, Supplementary Fig. S4). These results indicate that the Zic3-ME contains cis-regulatory elements required for the proper Zic3 expression at mesoderm and the somites in mouse embryos at the corresponding stages 38,40 .
In the adult Zic3-ME-deficient mice, we did not observe the kinky-tail-like abnormality that is observed in Zic3-deficient mice 41,42 . Zic2 expression in the mesoderm and the epiblast (Fig. 4H) and Brachyury expression in the primitive streak at E6.5 (Fig. 4L, Supplementary Fig. S4), the notochord, and the caudal mesoderm at E8.5 (Fig. 4R,T) remained intact in the Zic3-ME-deficient embryos. These results indicate that the altered Zic3 expression is not due to the changes in embryonic architecture, but reflects the direct regulation Zic3 gene expression by Zic3-ME.
Delineation and characterization of the core region in Zic3-ME. Since our findings revealed the biological significance of Zic3-ME, we defined the core region of Zic3-ME. For the deletion mutants of chicken Zic3-ME (Fig. 5), the enhancer activity was assayed using whole-embryo electroporation. Sequential deletions from both ends revealed two critical segments within a 633 bp region, which is highly conserved among vertebrates (Figs 5 and 6). We named the 633 bp region as Zic3-ME core. Zic3-ME core sequence was then queried against public DNase I hypersensitive site (DNase-HS) and chromatin-immunoprecipitation sequencing (ChIP-seq) databases.
We found that, Zic3-ME core was included in the DNase I-HS in ES cells, mesodermal cells, and developing neural cells of both human and mouse ( Fig. 6A-C). In the mature tissues, brain, retina, and spinal cord cells showed Zic3-ME core in DNase-HS, but not in the heart, skeletal muscle, fat, blood, vascular cells, gut, lung, or liver (ENCODE database) 42 .
The binding sites for Nanog, TCF-LEF, Zic2, Twist, and Myc were clustered at the most strongly conserved region where the highest DNase-HS peak lies (Figs 6 and 7). By contrast, TF binding sites for two T-box factors (T and Eomes) were located outside of the peak and included in the segment 2 ( Fig. 6B,C), the deletion of which abolishes the somite expression directing activity. The segment 2 sequence is conserved in vertebrates, but not in the teleost fishes (Medaka and Zebrafish, Fig. 6). Although another region, which is necessary for the somite expression in the chicken embryos (segment 1), contained the predicted binding sites for Oct4 and Twist, the sites are not clearly conserved in mammalian species. indicated above the pictures. Numbers indicate those of tested embryos. (C) Reporter GFP signals at HH9 (12 h after electroporation). Expression patterns at additional time points are shown in Supplementary Fig. S2. Scale bar, 1 mm. (D) Spatial distribution of enhancer activity in a summary.
Positions of ChIP-seq peaks matched to those of the binding motifs ( Fig. 6B,C). However, Ep300, Chd7, Hdac2, Rad21, and Usf1 showed their ChIP-seq peaks irrespective of the binding sequences in agreement with their known roles as cofactor or in chromatin remodeling. Tcf12 (a basic helix-loop-helix protein, also known as Heb) showed the presence of additional peaks besides the peak on the predicted binding sites. Presence of the binding-site-independent-peak depended on the Nodal signal, raising a potential controlling mechanism of Zic3-ME (see Discussion, Figs 6C and 8D, and Supplementary Figs S5 and S7). Interestingly, positions of the DNase-HS peaks with Zic3-ME are different between ES cells and fetal brain tissue both in human and mouse (Fig. 6C). Brain DNA-HS peaks are placed at the distal end of the Zic3-ME core region, overlapping the Eomes and T ChIP-seq peaks. ES DNase-HS peaks coincide with the peaks of Nanog/Oct4/Zic2/Tcf3. The difference of DNase-HS suggest that the Zic3-ME higher order chromatin structure dynamically changes during the differentiation from ES cells into immature neural cells.
Sequence similar to Zic3-ME core exists in the 3′ flanking region of Zic2. In bioinformatics analysis of Zic3-ME, we noticed the presence of Zic3-ME-related sequences in vertebrate genomes. A BLAST homology search against human genome identified a sequence similar to Zic3-ME, which resides 270 kb downstream of Zic2 (named as Zic3-ME-like, Fig. 7A,C). The sequence alignment showed that the Zic3-ME-like is similar  to the most strongly conserved region of Zic3-ME core where the predicted binding sites for Myc and LEF1 are present (Fig. 7A). Surprisingly, a 59 bp region downstream of the Zic3-ME-like included the protein coding exon of propionyl-CoA carboxylase alpha subunit (PCCA) that is transcribed from the transcription start site located 100 kb downstream of Zic2 (Fig. 7C). The human genome also contained a paralogue of PCCA, known as methylcrotonoyl-CoA carboxylase 1 (MCCC1). MCCC1 was found to be located 38 Mb downstream of Zic1 (Fig. 7C). As the PCCA amino acid sequence encoded in Zic3-ME-like is highly conserved in MCCC1 protein (Fig. 7B), a traceable homology existed between Zic3-ME and Zic3-ME-like in the MCCC1 region. The juxtaposition of Zic2-PCCA is conserved widely in the vertebrate species including teleost fish (zebrafish) and cartilaginous fish (elephant shark), and the tandem array of Zic1-MCCC1 was partially conserved in amniotes, including at least cow and chicken (UCSC genome browser, https://genome.ucsc.edu/). However, we did not find any traits of the enhancers (DNase-HS and histone modification) in the ENCODE database. Based on these results, we inferred the evolutionary history of Zic3-ME (See Discussion and Fig. 8E).

Discussion
This study revealed enhancers critical for Zic1 and Zic3 expression in chicken and mouse embryos. The five enhancers, Zic1-NE, Zic1-ME, Zic3-NE1, Zic3-NE2, and Zic3-ME directed the spatially restricted expression in neural or mesodermal tissue. We hypothesize that they are coordinately involved in the regulation of Zic1 and Zic3 expression during embryonic development. However, they may be only a few pieces of enhancers necessary for the proper expression of Zic1 and Zic3. This is because we targeted only the evolutionary conserved region between human and chicken genomes. In this regard, studies on Sox2 enhancer are supportive. Full scanning of upstream and downstream 100 kb of chicken Sox2 regions identified 26 enhancers, of which most (25 out of 26) corresponded to a fraction of conserved sequence blocks between chicken and mammalian genomes 36,47 . The exceptional one enhancer was proposed to be unique to the chicken 47 . Therefore, the conserved sequence-targeted enhancer screening as in this study would be effective to identify developmentally critical enhancers if the comprehensiveness is not absolutely needed.
Cis-regulatory elements for Zic1 have been investigated in regions 2.9 kb upstream of mouse Zic1 transcription start site 27 , 5.0 kb upstream of Xenopus Zic1, 73 kb upstream and 45 kb downstream of zebrafish Zic1 48 , and 106 kb upstream and 36 kb downstream of medaka fish (Oryzias latipes) Zic1 gene 31 . These studies identified dorsal spinal cord and cochlear nucleus enhancer (Zic1-NE) and core promoter in mouse 27 , BMP inhibitor-responsive element in Xenopus 28 , and neural tube and mesodermal enhancer 31 . We showed that Zic1-NE enhancer activities are conserved in chicken and mouse. Whether or not the enhancer activities are conserved in vertebrates awaits further investigation. However, it is noteworthy that that major mesodermal enhancers in teleost fish reside distantly (11-60 kb) in the 5′ flanking region of Zic1 and they contain the four blocks of sequences conserved among  Red thick bar indicates Zic3 ME core, From top to bottom, histone modification (H3K4Me1), DNase HS signal, and each animal species (mouse, zebra finch, chicken, X. tropicalis, medaka, and zebrafish). At the bottom, location of the indicated TF binding sites defined by the prediction algorithm and ChIP-seq peaks. ChIPseq peaks of P300, Rad21, Sin3a, Hdac2, and Chd7 were identified at Zic3-ME in ES cells without predicted binding sites. Asterisks at TF indicate the ChIP-seq peaks identified in both human and mouse whereas no asterisks indicate the peaks identified in either human or mouse ChIP-seq studies. Double asterisks indicate the presence of the binding motifs only in the chicken sequence. Bold letters indicate the verification by both ChIPseq peaks and TF binding site prediction, and non-bold letters indicate verification by either of the methods.
SCIeNtIfIC REPORTS | (2018) 8:14954 | DOI:10.1038/s41598-018-33235-y teleost fishes and mammals 31 (Ol-a-d in Fig. 2A). Insertion of a large transposon between the teleost mesodermal enhancers and Zic1 results in mesoderm-specific loss of Zic1 expression 31 . If the Ol-a-d enhancer activities are conserved in chicken or in mouse, Zic1-ME may control the mesodermal expression cooperatively with them.
Two cis-regulatory elements for have been reported in the distant region upstream of zebrafish Zic3 30 . These enhancers are distinct from Zic3-NE1, Zic3-NE2, and Zic3-ME analyzed in this study. Zic3-E1 (16 kb upstream of zebrafish Zic3, Dr-e1 in Fig. 2A) directs reporter gene expression at the neural plate border, and E2 (53 kb upstream) enhancer activity is present in the neural plate border, anterior neural plate, and posterior paraxial mesoderm at an early stage (12 hours post fertilization), anterior dorsal neural tube, and posterior paraxial mesoderm at a later stage (24 hours post fertilization). E1, but not E2, shows clear enhancer signatures in terms of an epigenetic modification peak, summed TF-binding peak, and DNase-HS peaks in human or mouse genomes ( Fig. 6 and Supplementary Fig. S5). Zic3-ME and Zic3-E1 may associate with the transcriptional regulatory complex on the Zic3 coding region in mammalian embryos (Fig. 8A), because humans and/or mouse Zic3-E1, Zic3 coding region, and Zic3-ME are all in the open chromatin structure as evidenced by DNase-HS peaks (Supplementary Fig. S5) and are all associated with the active and primed enhancer signature (H3K4Me1) 49 (ENCODE database). Furthermore, all three regions are bound by proteins associated with chromatin remodeling factors (Chd7 and Hdac2) or the factor defining higher order chromatin architecture (Rad21) 50,51 in ES cells ( Fig. 6 and Supplementary Fig. S5).
In the differentiation processes of mesoderm and its derivatives, Zic3-ME binding of TFs related to the mesodermal differentiation may play important roles (Fig. 8B). These include the LEF-TCF TFs that mediate Wnt-β-catenin signaling, which have well known roles in mesodermal differentiation [52][53][54] . Brachyury (T), a direct target of Wnt-β-catenin signaling 55 , is a T-box type mesodermal TF that directly activates the mesodermal Zic3 expression in Xenopus embryos 26,56 . Another T-box factor, Eomes, is essential for mesoderm formation and the recruitment of prospective mesodermal cells to the primitive streak 57 . Zic2, another member of the Zic family, has been shown to increase the paraxial mesoderm progenitors at the primitive streak cooperatively with Zic3 38 . In addition, Zic2 and Zic3 are also required for precise somitogenesis 38 . These results indicate that Zic3-ME acts as a highly integrated hub to control mesodermal development.
In previous studies, Zic3 overexpression suppresses Xenopus Brachyury expression and Wnt-β-catenin signaling in mesoderm, resulting in an impaired notochord development 58 , whereas Brachyury upregulates Zic3 mesodermal expression 26 . On the other hand, both mouse Zic2 and Zic3 are required to increase the paraxial mesodermal cells in mice 38 . The results are consistent if we assume that the role of Zic3 is temporally limited to enhancing mesoderm generation, and not in the generation of the notochord at a later stage. This idea is also supported by the expression profile of the chicken Zic3 that is expressed in the notochord progenitor but not in the notochord itself. Collectively, the Zic3-Brachyury (T) regulatory loop (Fig. 8C) may be critically involved in controlling mesodermal development.
Zic3-ME also contains the brain enhancer activity (Fig. 3I,K). The expression of Zic3 in the brain was detected at E10.5 (Fig. 3N, and ref. 17 ), and Eomes (Tbr2) is expressed in the brain beginning around E10.0 59 . Together with the shift of a ChIP-seq peak to the Eomes binding site during neural differentiation (Fig. 6C), we speculate that Eomes could be involved in the brain enhancer activity of Zic3-ME (Fig. 8B).
Although this was not directly assayed in this study, Zic3-ME may be involved in controlling pluripotent stem cells. Because many DNase-HS studies indicate that Zic3-ME is in the open chromatin structure in human and mouse ES/iPS cells (Fig. 6, Supplementary Fig. S5, ENCODE database), and Zic3-ME is bound by pluripotency-associated TFs (Nanog and Oct4) (Fig. 6, Supplementary Fig. S5). In ES cells, knockdown of Oct4, but not of Nanog, suppresses Zic3 expression 60,61 . Oct4 interacts with Nanog and associates with multiple transcriptional repression complexes including Sin3A complex 62 . Zic3-ME is likely to be targeted by the Sin3A/ deacetylase complex, which has been shown to cooperate functionally with Nanog to promote pluripotency 63 . Therefore, we speculated that Nanog was involved in fine-tuning Zic3-ME function.
On the other hand, Zic3 protein binds to a promoter region of Nanog and directly upregulate Nanog expression in ES cells 64 (Fig. 8C). Moreover, Zic3 and Nanog prevent endodermal lineage specification, and Zic3 expression was required for ES cell pluripotency 6 . Thus, the Zic3 and Nanog feedback loop as a whole would be associated with the establishment of pluripotency (Fig. 8C). Further investigation on the regulation of Zic3-ME in ES cells would be beneficial for better understanding of the mechanism underlying pluripotency.
Additionally, the ChIP-seq results clarified the role of Zic3-ME regulation in the mesoderm-definitive endoderm bifurcation (Fig. 8D). Tcf12 (also called Heb, a basic helix-loop-helix [bHLH] E protein that forms a heterodimer with another bHLH TF) binds Zic3-ME in a partially Nodal signaling-dependent manner (Fig. 6C,  Supplementary Fig. S5) 65 . Depletion of Tcf12 in mesendodermal cell results in mesodermal differentiation 65 . Tcf12 has been proposed to be a Smad2/3 cofactor 66 that link Nodal signaling 65 . T also interacts and collaborates with Smad2/3, but mediates mesoderm formation 67 . Tcf12 and T could alternatively associate with Zic3-ME, since we found ChIP-seq peaks for Smad2/3, Tcf12, and T at the same site in Zic3-ME (Fig. 6C, Supplementary  Fig. S5). Furthermore, Tcf12 knockdown increases Zic3 expression in mesendodermal cells ( Supplementary  Fig. S7). Combining the fact that Zic3 knockdown in ES cells results in the endodermal differentiation 6 , Zic3-ME may be involved in the switching between mesodermal and endodermal differentiation of ES cells (Fig. 8D).
(C) Representative DNase-seq and ChIP-seq results of human and mouse ZIC3/Zic3-ME. Box with dotted lines indicate the entire Zic3-ME regions and boxes with solid lines indicate Zic3-ME core regions. Tcf12 (ESmesendoderm) and Tcf12 (ES-m.e.(mesendoderm) + Nodal block) indicate the embryoid body treated for 2 days with 100 ng/ml Activin and 100 ng/ml Activin plus 10 μM SB431542 (inhibitor of Smad2/3 phosphorylation) respectively 65 . The overall profiles including other known Zic3 enhancers and their derivations are indicated in Supplementary Fig. S6 and Table S1 respectively.
SCIeNtIfIC REPORTS | (2018) 8:14954 | DOI:10.1038/s41598-018-33235-y Finally, based on the presence of additional Zic3-ME-related sequences, we hypothesize the origin of Zic3-ME as follows. Ancestral Zic3-ME may have existed in the vertebrate ancestor after the tandem head-to-head duplication 5 (Fig. 8E). At this point, prototypal ME may have coexisted with a protein coding exon of common ancestor gene for PCCA/MCCC1 in the 3′ flanking region of Zic1/2/3 common ancestor. The whole genome quadruplication and subsequent loss of one copy 5 may have generated two additional sets of the "head-to-head tandem Zic genes", PCCA/MCCC1/ME. Thereafter, diversification of PCCA/MCCC1/ME may have occurred where Zic3-ME kept and presumably acquired additional TF binding sites sequences, PCCA exon retained the protein coding sequence with remnant TF binding sequences, and MCCC1 retained the protein coding information, but lost all of the TF binding sequences. We think Zic3-ME may provide us an intriguing model of enhancer evolution, awaiting further validation by experimental and computational molecular phylogenetic analyses.  Plasmid construction. We used pd2EGFP-1 in which a herpes simplex virus thymidine kinase promoter from pRL-TK (Promega) was inserted at its 5′ multiple cloning site. CNEs were PCR cloned from chicken BAC clones and inserted upstream of the TK-d2EGFP unit. Control EF-LacZ vector was generated by inserting LacZ cassette from pMC1871 (Amersham) and oligonucleotide containing an initiation methionine into pEF-BOS vector 68 . For the transgenic assay in mouse, CNEs were inserted into an Hsp promoter-driven β-galactosidase reporter cassette 69 . In situ hybridization. In situ hybridization (ISH) was performed essentially as described 38,58 .
Chicken embryo electroporation and enhancer assay. For the description of the chicken developmental stages, Hamburger and Hamilton stages were used 35 . Chicken embryo electroporation and whole embryo culture was carried out essentially as described 36,37 . Fertilized chicken eggs were purchased from Inoue Egg Farm (Kanagawa, Japan). Briefly, fertilized eggs were incubated at 38 °C for 26 hours. The chicken embryos at HH4 were excised, and attached to a sterilized paper filter with hole. The yolks attached on embryos were rinsed with Hank's balanced salt. The embryos were placed upside down on a 2 × 2 mm platinum plate electrode (cathode) on a dish (CUY700P, Nepagene). After one microliter of transfection cocktail (2 μg/μl CNE-TK-d2EGFP, 1 μg/μl of pEF-LacZ, 0.5 μg/μl fast green was injected into the space between the blastoderm and vitelline membrane using a glass pipette, electric pulses (10 V, 50 ms, 100 ms intervals, five times) were delivered using the electroporator (CUY21, Nepagene). The transfected embryos were placed on the agarose-albumin plate 36 , covered with yolk supernatant diluted in Hank's solution and incubated at 38 °C.
Generation and analysis of transgenic and Zic3-ME KO mice. Transgenic mice were generated at Nihon SLC (Shizuoka, Japan). A BAC clone containing Zic3 was purchased from the BACPAC Resources of the Children's Hospital, Oakland Research Institute. Zic3-ME targeting vector was constructed to replace Zic3-ME with a neomycin resistance gene cassette flanked by a loxP sequence (Neo). Homologous genomic DNA with the Neo cassette was joined with a diphtheria toxin A cassette for negative selection. Linearized targeting vectors were electroporated into C57BL/6J ES cells (EmbryoMax, Millipore) and homologous recombinants were isolated by G418 selection. The ES clones were screened by Southern blot analysis ( Supplementary Fig. S3). Correctly targeted ES clones were injected into blastocysts of C57BL/6J mice, which were then used to produce chimeric mice. After confirmation of germ line transmission, the Neo cassette was removed by crossing mice that had germ line transmission with transgenic mice expressing Cre recombinase in germ cells 70 . The Cre recombination was confirmed by PCR and Southern blot analyses. Mutant animals were genotyped by PCR using DNA and the following primers: Forward primer F1 (5′-CTATGCTCATCGCTTTCGCCATCTAA-3′) and Reverse primer R1 (5′-ATTTTCACGGC CAGCAGTGTTGATAG-3′) for the knockout (KO) allele; and Forward primer F2 (5′-TCTGTGAGGGG ATGTTGGAT-3′), and Reverse primer R2 (5′-CCCTGCAGCATGGAGATAAG-3′) for the wild type (WT) allele.
formation, and subsequent high levels of Nodal signaling induces differentiation into definitive endoderm.
Binding of Tcf12 to the T-binding site in Zic3-ME is dependent on Nodal signal and suppresses Zic3 expression. (E) It has been proposed that Zic paralogues appeared because of a tandem gene duplication and a subsequent quadruplication in the early vertebrate. This study predicts the presence of a common ancestral gene for PCCA, MCCC1, and Zic3-ME next to the two Zic genes as indicated in the second line. After quadruplication, PCCA, MCCC1, and Zic-ME may have diverged respectively. Ur indicates the common ancestor of the following genes.
Red indicates the enhancer property, and black indicates the protein-coding property.