transcriptional regulation in the early ectodermal lineage of ascidian embryos

Original Article

Transcriptional regulation in the early ectodermal lineageof ascidian embryos

Yosuke Horikawa, Haruka Matsumoto, Fumika Yamaguchi,† Satomi Ishida andShigeki Fujiwara*

Department of Applied Science, Kochi University, 2-5-1 Akebono-cho, Kochi-shi, Kochi, 780-8520, Japan

In ascidian embryos, ectodermal tissues derive from blastomeres in the animal hemisphere. The animal hemi-sphere-specific gene expression is observed as early as the 16-cell stage. Here, we characterized animal hemi-sphere-specific enhancers of three genes, Ci-ephrin-Ad, Ci-TGFb-NA1 and Ci-Fz4. Deletion analyses identifiedminimal essential elements. Although these elements contained multiple GATA sequences, electrophoreticmobility shift assays revealed that only some of them were strong binding sites for the transcription factorCi-GATAa. On the other hand, the motif-searching software MEME identified an octamer, GA (T/G) AAGGG,shared by these enhancers. In Ci-ephrin-Ad and Ci-TGFb-NA1, the octamer was GATAAGGG, which stronglybound Ci-GATAa. The 397-bp upstream region of Ci-ephrin-Ad contained two strong Ci-GATAa-binding sites,one of which was the octamer motif. Mutation in the octamer motif, but not the other Ci-GATAa-binding site,severely affected the enhancer activity. The 204-bp upstream region of Ci-TGFb-NA1 contained four strongCi-GATAa-binding sites, including the octamer motif. Mutation only in the octamer motif, leaving the otherthree Ci-GATAa-binding sites intact, abolished the enhancer activity. These results suggest a crucial role for theoctamer motif.

Key words: ascidian, ectoderm, enhancer, GATA, transcriptional activation.

Introduction

The ectoderm arises from the animal hemisphere in

chordates, including amphibians and ascidians (Lem-

aire et al. 2008). It is subsequently subdivided into the

epidermis and central nervous system. The mecha-nism of neural induction has been extensively studied

(for reviews see Stern 2005; Schmidt et al. 2013). Fac-

tors promoting epidermal specification and suppress-

ing neural fate have also been identified, which are

activated by bone morphogenetic protein (e.g. Qiao

et al. 2012; Tr�ıbulo et al. 2012). However, few genes

have been identified that were expressed in the

uncommitted general ectoderm. Our knowledge about

the mechanisms of specification of the epidermis and

general ectoderm is fragmentary.

The third cleavage of ascidian embryos divides the

embryo into four animal and four vegetal blastomeres.

The animal blastomeres mainly give rise to the epider-

mis and central nervous system (Conklin 1905; Nishida1987). Nishikata et al. (2001) reported that mRNAs of

a few genes were predominantly localized in the ani-

mal blastomeres of the 8-cell embryo, although it is

not clear whether this localization is achieved by spe-

cific zygotic expression or asymmetrical distribution of

maternal mRNA. The mRNAs localized to the animal

blastomeres include those encoding a WD repeat-con-

taining protein, an F-actin-capping protein, a calcineurininhibitor, and a putative GTP-binding protein (Nishikata

et al. 2001). It is unclear whether these proteins are

involved in the ectodermal genetic regulatory cascade.

At the 16-cell stage, many genes are expressed

exclusively in the animal hemisphere. These include

the genes encoding transcription factors; Ci-SoxF

(Imai et al. 2004) and Ci-fog (Rothb€acher et al. 2007).

Components of cell–cell signaling pathways are alsoexpressed in the animal hemisphere; Ci-ephrin-Aa,

Ci-ephrin-Ad, Ci-TGFb-NA1, Ci-Smad1/5 and Ci-Fz4

*Author to whom all correspondence should be addressed.Email: [email protected]†Present address: Laboratory of Molecular Biology, Medical

Research Center, Kochi Medical School, Kochi, 783-8505,Japan.Received 3 September 2012; revised 9 October 2013;

accepted 10 October 2013.© 2013 The AuthorsDevelopment, Growth & Differentiation © 2013 Japanese

Society of Developmental Biologists

Develop. Growth Differ. (2013) 55, 776–785 doi: 10.1111/dgd.12100

The Japanese Society of Developmental Biologists

(Imai et al. 2004; Hamaguchi et al. 2007; Picco et al.

2007). Cell adhesion molecules (Ci-d1-protocadherin-like and Ci-d-protocadherin-4) are also specifically

expressed (Noda & Satoh 2008).

Transcription of Ci-fog in the animal hemisphere is

activated by the transcription factor Ci-GATAa

(Rothb€acher et al. 2007). A Ci-GATAa-specific mor-

pholino oligo suppresses the enhancer activity of

Ci-fog (Rothb€acher et al. 2007). Transcriptional activa-tion of Ci-otx in the animal hemisphere also requires

Ci-GATAa (Bertrand et al. 2003). The enhancer regions

of these genes contain multiple GATA sequences (Ber-

trand et al. 2003; Rothb€acher et al. 2007), although

binding of the Ci-GATAa protein to these sequences

was not demonstrated. In addition, it is not yet clear

whether only Ci-GATAa is responsible for activation of

animal hemisphere-specific genes.In the present study, we identified animal hemisphere-

specific enhancer elements within the 5′ flanking regions

of three genes (Ci-ephrin-Ad, Ci-TGFb-NA1 and Ci-Fz4).

These genes were chosen because their specific

expression was clear and strong (Imai et al. 2004;

Picco et al. 2007). We found multiple GATA

sequences in the upstream regions of these genes.

Electrophoretic mobility shift assays revealed thatsome, but not all, of them strongly bind the

Ci-GATAa protein. However, disruption of only one of

them, whose GATA sequence was followed by AGGG,

severely affected the enhancer activity. Based on these

results, the possible involvement of transcription factor

(s) other than Ci-GATAa is discussed.

Materials and methods

Animals

Juvenile adults of Ciona intestinalis were purchased

from the National Bio-Resource Project of MEXT,

Japan. The animals were cultured in Tosa Bay near

the Usa Marine Biological Institute of Kochi University.

Eggs and sperm were obtained from the gonoducts ofmature adults. Eggs were inseminated with non-self

sperm. Fertilized eggs were dechorionated with 0.05%

actinase E (Kaken Pharmaceutical) and 1% sodium

thioglycolate. Embryos were reared in artificial seawa-

ter Super Marine Art SF1 (Tomita Pharmaceutical).

Construction of plasmids

Genomic DNA fragments corresponding to the pro-

moter region of Ci-ephrin-Ad, Ci-TGFb-NA1 and Ci-

Fz4 were amplified by polymerase chain reaction

(PCR), using the primers ephrin1.7F and ephrin1.7R

for Ci-ephrin-Ad, tgfna3.0F and tgfna3.0R for

Ci-TGFb-NA1, and fz2.0F and fz2.0R for Ci-Fz4 (TableS1). The PCR products were inserted into pGEM-T

(Promega). The Ci-ephrin-Ad fragment was excised

with PstI and BamHI from pGEM-T, while the

Ci-TGFb-NA1 and Ci-Fz4 fragments were excised with

XhoI and BamHI. These fragments were inserted into

pSP72-1.27 (Corbo et al. 1997). The resultant plas-

mids were named Ephrin1648Z, Tgfna2940Z and

Fz1964Z, respectively.For construction of Ephrin545Z, Ephrin497Z, Eph-

rin447Z, Ephrin397Z and Ephrin333Z, PCR was

carried out using a lacZ-specific primer (gal-A2) and a

Ci-ephrin-Ad-specific primer (ephrin545F, ephrin497F,

ephrin447F, ephrin397F and ephrin333F, respectively;

Table S1). Ephrin1648Z was used as a template. For

construction of Tgfna799Z, Tgfna599Z, Tgfna399Z,

Tgfna204Z and Tgfna66Z, PCR was carried out usingtgfna3.0R and one of the following primers (tgfna799F,

tgfna599F, tgfna399F, tgfna204F and tgfna66F,

respectively; Table S1). Tgfna2940Z was used as a

template. For construction of Fz1425Z, Fz1294Z,

Fz975Z, Fz784Z and Fz387Z, PCR was carried out

using fz2.0R and one of the following primers

(fz1425F, fz1294F, fz975F, fz784F and fz387F, respec-

tively; Table S1). Fz1964Z was used as a template.PCR products were once inserted into pGEM-T,

excised with XhoI and BamHI, and inserted into

pSP72-1.27.

Site-directed mutagenesis was performed according

to the protocol described by Fujiwara et al. (1998). To

create point mutations within the Ci-ephrin-Ad enhan-

cer, the XhoI-BamHI fragment of Ephrin397Z was

inserted into pBluescript II SK+ (Stratagene). Uracil-containing single-stranded DNA was prepared using

the Escherichia coli strain CJ236 (Takara). Mutagenic

oligonucleotides used were E-S1 m, E-S2 m, E-G1 m,

E-G2 m, E-S1G1 m and E-Mm (Table S1). For point

mutations in the Ci-TGFb-NA1 enhancer, we trans-

formed CJ236 with pGEM-T containing the PCR prod-

uct using primers tgfna204F and tgfna3.0R (described

above). Mutagenic oligonucleotides used were T-Gmand T-Mm (Table S1).

Electroporation and detection of transgene expression

Plasmid DNA was prepared using QIAGEN tip-100

(Qiagen). Transgenes were introduced into dechorion-

ated embryos by electroporation, according to the pro-

tocol described by Corbo et al. (1997). Expression oflacZ was detected by in situ hybridization as described

by Kanda et al. (2009). In each experiment, 100–400electroporated embryos were observed under an

AZ100 microscope (Nikon). Embryos expressing lacZ

in one or more blastomeres were counted as positive.

ª 2013 The Authors

Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists

Early ectodermal enhancers in ascidians 777

The percentage of positive embryos among morpho-logically normal embryos was calculated.

Electrophoretic mobility shift assay

The entire translated region of Ci-GATAa was amplified

by PCR using the cDNA clone “cien229100” as a tem-

plate (Tassy et al. 2010; http://www.aniseed.cnrs.fr/

index.php). The PCR was carried out using primersGATA-F and GATA-Nhe-R (Table S1), and Tks Gflex

DNA polymerase (Takara), according to the protocol

supplied by the manufacturer. The product of the PCR

was briefly treated with Taq DNA polymerase (Ampli-

qon) to create the 3′ overhang of a single adenine

nucleotide, and inserted into pGEM-T. The Ci-GATAa

cDNA was again amplified using Tks Gflex DNA poly-

merase and the primers GATA-F and M13-Forward-40(5′-GTTTTCCCAGTCACGAC-3′). The product of the

PCR was excised with NheI. The pCMX-hRARa plas-

mid (Umesono et al. 1991) was digested with MscI

and NheI to remove the cDNA encoding human reti-

noic acid receptor a (hRARa). The Ci-GATAa cDNA

was inserted into the pCMX vector. The Ci-GATAa

protein was produced using the TNT coupled reticulo-

cyte lysate system (Promega). Double-stranded DNA

probes were labeled with [c-32P] adenosine triphos-phate (Perkin Elmer Japan), according to the protocol

described by Kanda et al. (2009). Electrophoretic

mobility shift assays (EMSAs) were carried out using a

Gel-shift assay system (Promega). Autoradiograms

were obtained using BAS2500 (Fuji Film).

Results

Identification of early animal-hemisphere enhancers

A fragment of the C. intestinalis genomic DNA was

obtained, which contained the promoter region of Ci-

ephrin-Ad, including putative transcription and transla-

tion initiation sites (Fig. 1a). The fragment was fused

in-frame with lacZ and introduced into fertilized eggs

by electroporation (Fig. 1b). The resultant transgene,named Ephrin1648Z, contained 1648 bp of the 5′flanking region, according to the gene model

KH.C3.716.v1.A.SL1-1 in the C. intestinalis genome

database (Satou et al. 2005; http://ghost.zool.kyoto-u.

ac.jp/SearchGenomekh.html). Ephrin1648Z was intro-

duced into one-cell embryos. The embryos were fixed

at the 16-cell stage and the lacZ mRNA was detected

by in situ hybridization. Ephrin1648Z was expressed in

(a)

(b)

(c) (d)

(e) (f)

(g) (h)

Fig. 1. The animal hemisphere enhancer of Ci-ephrin-Ad. (a) The genomic structure of the Ci-ephrin-Ad gene, predicted in the Ciona

intestinalis genome database (Satou et al. 2005; http://ghost.zool.kyoto-u.ac.jp/SearchGenomekh.html). Exons are indicated by boxes.

The open reading frame is in green. The 1.6-kb upstream region examined in the present study is indicated. (b) Diagrams showing the

different 5´ flanking regions used for the analysis in (c–h). The E1 enhancer is indicated by a red box. Percentage of embryos expressing

lacZ among normally developing embryos is indicated. (c–h) The expression pattern of transgenes shown in (b). All panels show the

animal side of the 16-cell embryos. The anterior side is up. Names of the blastomeres are indicated in (c).

ª 2013 The Authors


778 Y. Horikawa et al.

all of the blastomeres (a5.3, a5.4, b5.3 and b5.4 blas-tomere pairs) in the animal hemisphere (Fig. 1c).

Transgenes containing 545, 497, 447, or 397 bp of

the upstream sequence gave essentially similar pat-

terns of expression, although some embryos did not

express lacZ in all of the eight blastomeres (Fig. 1d–g).No embryo carrying Ephrin333Z expressed lacZ in any

blastomere (Fig. 1h). These results indicate that 397

bp of the 5′ flanking region of Ci-ephrin-Ad is suffi-cient, and the region between �397 and �334,

named E1, is necessary for transcriptional activation in

the animal hemisphere.

We also isolated the promoter regions of Ci-TGFb-NA1and Ci-Fz4, and placed them upstream of lacZ. (Figs 2

and 3). A transgene named Tgfna2940Z contained

2940 bp of the 5′ flanking region of Ci-TGFb-NA1,according to the gene model KH.C4.547.v1.A.nonSL1-1(Fig. 2a,b). Tgfna2940Z was expressed in all of the

eight blastomeres in the animal hemisphere of the

16-cell embryo (Fig. 2c). Deletion analyses revealed

that 204 bp of the upstream sequence of Ci-TGFb-NA1 was sufficient, and the region between �204 and

�66, named T1, was necessary for transcriptional acti-

vation in the animal hemisphere (Fig. 2d–h). Fz1964Z

contained 1964 bp of the 5′ flanking region of Ci-Fz4,according to the gene model KH.C6.162.v2.A.

SL2-1 (Fig. 3a,b). Fz1964Z recapitulated the animal

hemisphere-specific expression of Ci-Fz4 (Fig. 3c).Deletion analyses revealed that 975 bp of the upstream

sequence of Ci-Fz4 was sufficient, and the region

between �975 and �784, named F1, was necessary

for transcriptional activation in the animal hemisphere

(Fig. 3d–h). The region between �783 and �67 was

deleted from Fz975Z. The resultant transgene was

not expressed, suggesting that the proximal region

(downstream to �784) also contains enhancer element(s) necessary for transcriptional activation (data not

shown).

Possible binding sites for transcription factors

We searched for sequence motifs shared by Ci-eph-

rin-Ad, Ci-TGFb-NA1 and Ci-Fz4, using MEME (Bailey

& Elkan 1994; http://meme.sdsc.edu/meme/intro.html).MEME identified an octamer sequence, GA (T/G)

AAGGG. The E1 and T1 enhancers contained a single

octamer motif each (GATAAGGG), named E-M1 and

T-M1, respectively (Fig. 4). MEME did not find this

motif within the 975-bp proximal region of Ci-Fz4.

However, we found a similar sequence, GAAAAGGG,

at nucleotide position �73 of Ci-Fz4 (data not shown).

We also searched for putative binding sites for knowntranscription factors. Ci-GATAa is necessary for activa-

tion of genes in the animal hemisphere of the Ciona

(a)

(b)

(c) (d)

(e) (f)

(g) (h)

Fig. 2. The animal hemisphere enhancer of Ci-TGFb-NA1. (a) The genomic structure of the Ci-TGFb-NA1 gene, predicted in the Ciona

intestinalis genome database. Exons are indicated by boxes. The open reading frame is in green. The 2.94-kb upstream region exam-

ined in the present study is indicated. (b) Diagrams showing the different 5´ flanking regions used for the analysis in (c–h). The T1 enhan-

cer is indicated by a red box. Percentage of the embryos expressing lacZ among normally developing embryos is indicated. (c–h) The

expression pattern of transgenes shown in (b). All panels show the animal side of the 16-cell embryos. The anterior side is up.

ª 2013 The Authors



embryo (Rothb€acher et al. 2007). The binding

sequence for Ci-GATAa is 5′-GATA-3′, and it had no

strong constraint on surrounding sequences (K. Nitta

& P. Lemaire, pers. comm., 2012). Ci-SoxF is the only

transcription factor whose mRNA is exclusively

detected in the animal hemisphere at the 16-cell stage(Imai et al. 2004). The core recognition sequence for

the Sox group transcription factors is AACAAT (Mertin

et al. 1999). In vitro binding experiments revealed that

the consensus sequence for C. intestinalis Sox pro-

teins is (A/G) (A/C) CAA (T/A) (K. Nitta & P. Lemaire,

pers. comm., 2012). We found six GATA-binding sites

and two Sox-binding sites within the 397-bp proximal

region of Ci-ephrin-Ad (Fig. 4a). The E1 enhancer con-tained two GATA-binding sites (named E-G1 and E-

G2) and one Sox-binding site (named E-S1; Fig. 4a,b).

The 204-bp upstream region of Ci-TGFb-NA1 con-

tained no Sox-binding site, but four GATA-binding

sites (Fig. 4a). The T1 enhancer contained three of

them (named T-G1–T-G3; Fig. 4c). E-M1 and T-M1

included the GATA sequence (E-G2 and T-G3, respec-

tively, Fig. 4).

The octamer motif is necessary for enhancer activity

Since the E1 enhancer is necessary for transcriptional

activation in the animal hemisphere, we tested the

requirement of putative binding sites for transcription

factors by site-directed mutagenesis. First, a point

mutation was generated at the Sox-binding site within

the E1 enhancer (E-S1; Fig. 5a,b). The mutant trans-

gene, named Ephrin (S1 m) Z, was expressed normally

in the animal hemisphere at the 16-cell stage (Fig. 5b).Another possible Sox-binding sequence was found

downstream of nucleotide position �333 (E-S2;

Fig. 5a). Disruption of E-S2, or a double mutation in

E-S1 and E-S2, did not affect the expression in the

animal hemisphere (Fig. 5c,d). One of the two GATA-

binding sites within the E1 enhancer (E-G1) was

mutagenized (Fig. 5a,e). The mutant transgene, named

Ephrin (G1 m) Z, was expressed in the animal hemi-sphere (Fig. 5e). In contrast, a mutation in the other

GATA sequence (E-G2) caused complete silencing of

the reporter gene (Fig. 5f). Note that E-G2 overlaps

with E-M1 (GATAAGGG) (Figs. 4b and 5a), and that a

point mutation in E-G2 completely inactivated the

enhancer that still contained five additional GATA

sequences (see Fig. 4a). We then introduced a point

mutation into E-M1 without disrupting E-G2 (GAT-AAGGG to GATActtt). The resultant transgene, named

Ephrin (M1 m) Z, was not expressed in any blastomere

(Fig. 5g). About 83% of the embryos carrying Eph-

rin397Z expressed lacZ, while only 14% of the

embryos carrying Ephrin (M1 m) Z expressed it.

(a)

(b)

(c) (d)

(e) (f)

(g) (h)

Fig. 3. The animal hemisphere enhancer of Ci-Fz4. (a) The genomic structure of the Ci-Fz4 gene, predicted in the Ciona intestinalis

genome database. Exons are indicated by boxes. The open reading frame is in green. The 1.96-kb upstream region examined in the

present study is indicated. (b) Diagrams showing the different 5´ flanking regions used for the analysis in (c–h). The F1 enhancer is indi-

cated by a red box. Percentage of the embryos expressing lacZ among normally developing embryos is indicated. (c–h) The expression

pattern of transgenes shown in (b). All panels show the animal side of the 16-cell embryos. The anterior side is up.

ª 2013 The Authors



The T1 enhancer contains three GATA sequences,one of which (T-G3) overlaps with T-M1 (GATAAGGG)

(Figs 4c and 6a). Disruption of T-G3 (GATAAGGG to

GtaAAGGG) completely silenced the expression of

lacZ (Fig. 6b). A point mutation of T-M1 that did not

disrupt T-G3 (GATAAGGG to GATActtt) also caused

severe inactivation of the enhancer (Fig. 6c). About

81% of the embryos carrying Tgfna204Z expressed

lacZ, while 3.6% of the embryos carrying Tgfna(M1 m) Z did so.

Binding of the Ci-GATAa protein to wild-type and

mutagenized DNA sequences

Electrophoretic mobility shift assays were carried out

using a full-length Ci-GAGAa protein synthesized in

vitro. An oligonucleotide probe containing the E-M1(GATAAGGG) and its flanking sequences formed a

DNA/protein complex (Fig. 7a, lane 2). We prepared amutant probe (E-M1 m), in which AGGG of E-M1 was

disrupted without affecting GATA. E-M1m did not form

a complex with Ci-GATAa (Fig. 7a, lane 3). A probe

containing E-G1 formed a complex with Ci-GATAa

(Fig. 7a, lane 4). E-G3, E-G5 and E-G6 formed a weak

band (Fig. 7a, lanes 5, 7, 8). No specific shift was

observed with E-G4 (Fig. 7a, lanes 6). A probe con-

taining T-M1 (overlapping with T-G3) and T-G2 formeda complex with Ci-GATAa (Fig. 7b, lane 2). The shifted

band became weak when mutation was created in

either T-G2 or T-G3 (Fig. 7b, lanes 3, 5). When the

AGGG moiety of T-M1 was disrupted without affecting

GATA, the complex’s formation was also affected

(Fig. 7b, lane 4). No shifted band was observed when

both T-G2 and T-G3 were mutated (Fig. 7b, lane 6).

T-G1 and T-G4 also strongly formed a complex withCi-GATAa (Fig. 7b, lanes 7, 8).

Discussion

Binding of Ci-GATAa is strongly influenced by flanking

sequences

Direct binding assays of the ascidian GATA proteinwere first reported in the present study. Our EMSA

revealed cases in which the affinity of the Ci-GATAa

binding to DNA was strongly affected by the

sequences surrounding the core GATA sequence. The

397-bp upstream region of Ci-ephrin-Ad contains six

GATA sequences. However, only two of them (E-G1

and E-G2) can strongly bind Ci-GATAa. These results

seem inconsistent with unpublished results obtainedby K. Nitta and P. Lemaire (pers. comm., 2012).

They searched for a consensus Ci-GATAa-binding

sequence by the systematic evolution of ligands using

exponential enrichment (SELEX). They did not see a

strong preference outside of the GATA sequence,

although TGATAA was a little better (K. Nitta & P.

Lemaire, pers. comm., 2012). In the present study,

Ci-GATAa seemed to prefer GATAAGGG. The under-lined nucleotides may not have been recognized by

SELEX, possibly because only two or three of the

underlined nucleotides are conserved in many cases.

Bertrand et al. (2003) and Rothb€acher et al. (2007)

carried out mutagenesis of many GATA sequences

within the enhancer region of Ci-otx and Ci-fog, and

reported strong suppression of reporter genes. Similar

results were obtained by antisense-mediated knock-down of Ci-GATAa (Bertrand et al. 2003; Rothb€acher

et al. 2007). Two critical GATA sequences within the

Ci-fog enhancer are GATAAAGT and GATACTTT

(Rothb€acher et al. 2007). The former is similar to the

octamer motif, but the latter looks like the mutant

(a)

(b)

(c)

Fig. 4. Putative transcription factor-binding sites in the E1 and

T1 enhancers (a) Schematic diagrams showing the 5´ flanking

regions of Ci-ephrin-Ad and Ci-TGFb-NA1. Putative binding

sites for the GATA and Sox transcription factors are indicated

by blue and yellow boxes, respectively. The octamer motif

(GATAAGGG) is indicated by red boxes. (b) Nucleotide

sequence of the E1 enhancer. (c) Nucleotide sequence of the

T1 enhancer.

ª 2013 The Authors



sequences that did not bind Ci-GATAa in the present

study. None of the seven GATA sequences within the

Ci-otx enhancer look similar to the strong binding sites

(Bertrand et al. 2003). Considering the present results,

direct binding assays will be necessary to confirm that

these GATA sequences are genuine Ci-GATAa-binding

sites.

Possible role of the octamer motif shared by the

animal hemisphere enhancers

The MEME software identified the octamer motif

shared by the E1 and T1 enhancers. Point mutations

of the motif resulted in severe reduction of enhancer

activity. There are at least three possible explanations

of how the octamer motif contributes to transcriptionalactivation. First, Ci-GATAa binds to the motif and acti-

vates transcription. The affinity of the GATA sequence

for Ci-GATAa is affected by the adjacent sequence,

where AGGG facilitates the binding of Ci-GATAa to the

GATA sequence. Second, the octamer motif is a bind-

ing site for a transcription factor other than Ci-GATAa.

Although E-M1 and T-M1 can bind Ci-GATAa in vitro,

these sequences may be occupied in vivo by another

transcription factor that recognizes the octamer motif

as a whole. Third, Ci-GATAa binds to the GATA part

of the octamer motif, while another transcription factorsynergistically binds to the AGGG part. The combina-

torial binding of two transcription factors may acceler-

ate transcriptional activation.

Mutations in E-M1 and T-M1 (GtaAAGGG or GAT-

Acttt) indeed affected the binding of Ci-GATAa, sup-

porting the first possibility. However, this does not

exclude the second and third explanations. The 397-bp

upstream region of Ci-ephrin-Ad contains two strongCi-GATAa-binding sites (E-G1 and E-G2). However,

only E-G2 is necessary, while E-G1 is dispensable for

transcriptional activation. Disruption of E-G1 revealed

that only a single strong Ci-GATAa-binding site (and a

few weak sites?) was sufficient for transcriptional

(a)

(b)

(c)

(d)

(e)

(f)

(g)

Fig. 5. Effect of point mutations in putative transcription factor-binding sites in the 5´ flanking region of Ci-ephrin-Ad. Diagrams of

transgenes and their pattern of expression are shown side-by-side. Percentage of the embryos expressing lacZ among normally devel-

oping embryos is also indicated. (a) Ephrin397Z, carrying no mutation. (b) Ephrin (S1 m) Z, carrying a mutation in the Sox-binding site E-

S1. (c) Ephrin (S2 m) Z, carrying a mutation in the other Sox-binding site E-S2. (d) Ephrin (S1S2 m) Z, carrying mutations in both E-S1

and E-S2. (e) Ephrin (G1 m) Z, carrying a mutation in the GATA sequence E-G1. (f) Ephrin (G2 m) Z, carrying a mutation in the GATA

sequence E-G2, which overlaps with the octamer motif E-M1. (g) Ephrin (M1 m) Z, carrying a mutation in E-M1. The mutation was

created to convert GATAAGGG into GATATCCC, not to disrupt E-G2.

ª 2013 The Authors



activation. In contrast, the Ci-TGFb-NA1 upstreamregion contained four binding sites for Ci-GATAa. Dis-

ruption of only one of them (T-G3 or T-M1) abolished

the enhancer activity, although three strong binding

sites remained. These observations suggest that theoctamer motif is not just one of many Ci-GATAa-bind-

ing sites. In vitro binding of Ci-GATAa to the octamer

sequence does not necessarily mean that the octamer

is also legitimately occupied in vivo by Ci-GATAa.

Maternal Ci-GATAa mRNA is ubiquitous, and seems to

be translated in both animal and vegetal blastomeres

(Bertrand et al. 2003). Disruption of the b-cateninfunction caused ectopic expression of the Ci-fog repor-ter gene in the vegetal hemisphere, suggesting that

b-catenin suppresses Ci-GATAa at the post-transla-

tional level (Rothb€acher et al. 2007). As described

above, one of the critical GATA sequences within the

Ci-fog enhancer is similar to the octamer motif. It is

therefore possible that the octamer motif is also involved

in the animal-specific and b-catenin-sensitive transcrip-

tional activation. Extensive binding analyses usingnuclear extracts and identification of binding proteins will

help elucidate what binds to the octamer motif in vivo.

Other transcription factors

In sea urchin embryos, maternally provided SoxB1 is

responsible for gene expression in the presumptive

ectodermal region in the animal hemisphere (Kennyet al. 1999). Sox transcription factors are expressed

mainly in the central nervous system in vertebrates

(a)

(b)

(c)

Fig. 6. Effect of point mutations in putative transcription factor-

binding sites in the 5´ flanking region of Ci-TGFb-NA1. Diagrams

of trangenes containing mutations and their pattern of expression

are shown side-by-side. Percentage of the embryos expressing

lacZ among normally developing embryos is indicated. (a)

Tgfna204Z, carrying no mutation. (b) Tgfna (G3 m) Z, carrying a

mutation in the GATA sequence T-G3, which overlaps with the

octamer motif T-M1. (c) Tgfna (M1 m) Z, carrying a mutation in

T-M1. The mutation was created to convert GATAAGGG into

GATATCCC, not to disrupt T-G3.

(a) (b)

Fig. 7. Electrophoretic mobility shift

assay. Double-stranded radioactive

probes were incubated with in vitro

translated Ci-GATAa protein. For lane

1, TC14-3 was used as a control pro-

tein. TC14-3 is a calcium-dependent

galactose-binding lectin, whose cDNA

was obtained from the budding ascid-

ian Polyandrocarpa misakiensis (Mat-

sumoto et al. 2001). DNA/protein

complexes were fractionated on a poly-

acrylamide gel. The black arrowhead

indicates specific signals of the com-

plex’s formation. The open arrowhead

indicates non-specific signals. Probes

used are indicated below the gel

image. Lower case letters (in red) indi-

cate mutagenized nucleotides. (a) The

GATA sequences within the Ci-ephrin-

Ad upstream region were examined. (b)

The GATA sequences within the Ci-

TGFb-NA1 upstream region were

examined.

ª 2013 The Authors



(Pevny & Lovell-Badge 1997). In the Ciona embryo,SoxF is specifically expressed in the animal hemi-

sphere from the 16-cell stage (Imai et al. 2004). SoxB1

is also expressed in all of the eight animal blasto-

meres, although it is expressed in anterior vegetal

blastomeres (Imai et al. 2004). Therefore, these Sox

factors may play a role in the gene regulatory network

in the animal hemisphere. In the present study, we

found two putative Sox-binding sequences in the 5′flanking region of Ci-ephrin-Ad. However, these sites

were unnecessary for transcriptional activation, at least

at the 16-cell stage. Although animal hemisphere-spe-

cific expression of many transcription factors, signaling

proteins and receptors is observed at the 16-cell

stage, these proteins require a time-lag to exhibit their

function.

Since zygotic expression at the 16-cell stage is theearliest, maternal transcription factors likely play impor-

tant roles. A large amount of expressed sequence tag

(EST) and in situ data have been accumulated for

maternal transcripts (e.g. Nishikata et al. 2001). In con-

trast, information about maternal proteins is still limited

(Nomura et al. 2009; Endo et al. 2011). In addition to

analyzing enhancers, it is important to characterize

maternal proteins involved in activation of the gene reg-ulatory network at the earliest stage of embryogenesis.

Acknowledgments

This work was supported in part by Grants-in-aid from

the Japan Society for the Promotion of Science

(#22570214 and #25440192). We thank Kazuko Hiray-

ama, Chikako Imaizumi, Shota Chiba, Nori Satoh, Yu-taka Satou and the Maizuru Fisheries Research Station

of Kyoto University for providing us with animals

through the National Bio-Resource Project, MEXT,

Japan. We are particularly grateful to the late Ms. Ka-

zuko Hirayama who helped us set up the culture sys-

tem in Kochi University. We also thank Kaz Nitta for

sharing his unpublished data.

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Supporting Information

Additional supporting information may be found in the

online version of this article at the publisher’s web-site:

Table S1. Primers used for construction of plasmids.

ª 2013 The Authors



transcriptional regulation in the early ectodermal lineage of ascidian embryos

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