© 2013. Published by The Company of Biologists Ltd | Development (2013) 140, 4703-4708 doi:10.1242/dev.100339
RESEARCH REPORT
A time delay gene circuit is required for palp formation in the
ascidian embryo
Tatsuro Ikeda, Terumi Matsuoka and Yutaka Satou*
KEY WORDS: Ciona intestinalis, Gene circuit, Palp, Brain, Placode
INTRODUCTION
The ascidian Ciona intestinalis is a simple chordate belonging to the
tunicate group. The anterior animal cells (a-line) of the eight-cell
Ciona embryo give rise to the trunk epidermis, brain and palps,
adhesive organs containing sensory neurons (Nishida and Satoh,
1983) (Fig. 1A). FGF9/16/20, which is expressed in vegetal cells
from the 16-cell to gastrula stage, then induces expression of the
neural markers Otx (Hudson and Lemaire, 2001; Bertrand et al.,
2003; Hudson et al., 2003) and DMRT1 (Tresser et al., 2010) in the
future brain/palp (a7.9 and a7.10) and brain/epidermis (a7.13) cells
at the 32- and 64-cell stages. At the gastrula stage, bi-potential
brain/palp progenitor cells in the anterior part of the neural plate
become further fate restricted, separating into distinct palp
progenitors and brain progenitors (Nicol and Meinertzhagen, 1988)
(Fig. 1B). After this fate restriction, only the brain progenitors
express ZicL (Imai et al., 2004) in response to FGF9/16/20
signaling, because the brain progenitors, but not the palp
progenitors, abut on FGF9/16/20-expressing vegetal cells (Wagner
and Levine, 2012). Thus, even though FGF9/16/20 signals are
present as early as the 16-cell stage, the brain progenitors respond
to these signals to express ZicL only after the developmental fates
Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku,
Kyoto, 606-8502, Japan.
*Author for correspondence ([email protected])
Received 20 June 2013; Accepted 4 September 2013
of the brain and palps diverge. The mechanism by which this timing
is coordinated is still unknown.
RESULTS AND DISCUSSION
Identification of novel zinc finger genes expressed at the
16-cell stage
During a microarray screen for zygotically activated genes in early
Ciona embryos (Matsuoka et al., 2013), we identified a gene that is
expressed in all anterior animal cells (a-line cells) at the 16-cell
stage (Fig. 1C). Prior to the 64-cell stage, expression of this gene
diminishes in all but one descendant, a7.13, and then is lost in a7.13
by the early gastrula stage. This gene is also transiently expressed
in b6.5 at the 32-cell stage, and in a pair of posterior vegetal cells
between the 32-cell and early gastrula stages.
The gene we identified encodes a C2H2-type zinc-finger protein
with sequence similarity to Blimp-1/PRDM1 in other animals.
However, it lacks the PR/SET-domain common to Blimp-1 in other
animals. Whereas Blimp-1 proteins in other animals have four or
five zinc fingers, this has only three. A molecular phylogenetic
analysis failed to give strong support (data not shown). Therefore,
we named this gene Blimp-1-like Zinc finger gene 1 [BZ1; DNA
Data Bank of Japan (DDBJ) accession number AB819270],
although it is possible that this gene is a divergent Blimp-1. Another
gene encoding a protein with 67% overall amino acid identity to
BZ1 and an identical zinc finger domain was found 15 kb upstream
of BZ1, and we named this BZ2 (supplementary material Fig. S1;
DDBJ accession number AB819271).
The expression pattern of BZ2 was almost identical to that of BZ1,
except that BZ2 expression at the 16-cell stage was undetectable and
that BZ1 and BZ2 were expressed in non-overlapping tissues of
tailbud embryos (Fig. 1D).
BZ1 and BZ2 are required for proper differentiation of the
brain and palp lineages
We knocked down BZ1 and BZ2 by injecting specific morpholino
oligonucleotides (MOs) into fertilized eggs. All BZ1 morphant
larvae show clear brain defects, with 6% also losing palps (n=51),
whereas BZ2 morphant larvae appear normal (n=7) (supplementary
material Fig. S2A,B). However, when both MOs are simultaneously
injected, the brain and palps are severely disrupted compared with
normal larvae (n=34, 100%; Fig. 2A,B), indicating functional
redundancy between BZ1 and BZ2. Indeed, the palp markers islet
and ZF220 are absent in BZ1 and BZ2 double morphants (BZ1/2
morphants) at the tailbud stage (Fig. 2C-F). EphrinA-d, one of the
earliest markers for the palp lineage (Imai et al., 2004; Wagner and
Levine, 2012), is lost at the late gastrula stage in BZ1/2 morphants,
whereas FoxC, another earliest marker for the palp lineage, is not
affected (Fig. 2G-J). Six3/6, which is normally expressed in the
anterior row of the two rows of the brain progenitor cells at the late
gastrula state (Imai et al., 2004; Moret et al., 2005), is ectopically
expressed in the palp progenitor cells in BZ1/2 morphants, whereas
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DEVELOPMENT
ABSTRACT
The ascidian larval brain and palps (a putative rudimentary placode)
are specified by two transcription factor genes, ZicL and FoxC,
respectively. FGF9/16/20 induces ZicL expression soon after the bipotential ancestral cells divide into the brain and palp precursors at
the early gastrula stage. FGF9/16/20 begins to be expressed at the
16-cell stage, and induces several target genes, including Otx, before
the gastrula stage. Here, we show that ZicL expression in the brain
lineage is transcriptionally repressed by Hes-a and two Blimp-1-like
zinc finger proteins, BZ1 and BZ2, in the bi-potential ancestral cells.
ZicL is precociously expressed in the bi-potential cells in embryos in
which these repressors are knocked down. This precocious ZicL
expression produces extra brain cells at the expense of palp cells.
The expression of BZ1 and BZ2 is turned off by a negative autofeedback loop. This auto-repression acts as a delay circuit that
prevents ZicL from being expressed precociously before the brain
and palp fates split, thereby making room within the neural plate for
the palps to be specified. Addition of the BZ1/2 delay timer circuit to
the gene regulatory network responsible for brain formation might
represent a key event in the acquisition of the primitive
palps/placodes in an ancestral animal.
RESEARCH REPORT
Development (2013) doi:10.1242/dev.100339
MYTF, which is normally expressed in the posterior row, is not
affected (Fig. 2K-N). ZicL, which is normally expressed in the brain
lineage at the early gastrula stage, is ectopically expressed in the
palp lineage in BZ1/2 morphants (Fig. 2O,P). Thus, BZ1 and BZ2
are necessary for proper specification of the brain and palp lineages.
Previous studies revealed essential roles for ZicL and FoxC in
specification of the brain and palps, and that ZicL regulates Six3/6,
but not FoxC and MYTF (Imai et al., 2006; Wagner and Levine,
2012). EphrinA-d is also regulated by ZicL, because EphrinA-d
expression is expanded to the brain lineage in ZicL morphants
(supplementary material Fig. S3A). Next, we made a construct in
which the ZicL coding sequence was linked to the DMRT1 upstream
sequence. With this construct, ZicL is expected to be expressed in
the brain/palp lineage from the 64-cell to late gastrula stages. When
this construct is injected into fertilized eggs, EphrinA-d, islet and
ZF220 are lost, and Six3/6 is ectopically expressed in the palp
lineage (supplementary material Fig. S3B,C; Fig. 2Q,R). Thus,
overexpression of ZicL generates a similar phenotype to knockdown
of BZ1 and BZ2, suggesting that repression of ZicL might be the
primary function of BZ1 and BZ2.
BZ1 and BZ2 temporally control ZicL expression
To understand how BZ1 and BZ2 regulate ZicL expression, we made
constructs in which either of the BZ1 or BZ2 coding sequences was
linked to the DMRT1 upstream sequence. Following injection of these
constructs into an anterior animal cell at the eight-cell stage, ZicL
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expression is completely or partially lost in the injected octant
(Fig. 3A,B). Thus, BZ1 and BZ2 repress ZicL expression in the
brain/palp lineage. In BZ1/2 morphants, ZicL is precociously
expressed at the 64-cell stage in the bi-potential progenitors a7.9 and
a7.10 (Fig. 3C,D). FGF signaling is required for this precocious
expression, because ectopic ZicL expression is rarely observed in
BZ1/2 morphants treated with the MEK inhibitor U0126 at the 44-cell
stage (Fig. 3E). In addition, BZ1/2 morphant embryos treated with
recombinant human basic fibroblast growth factor (bFGF) at the 44cell stage ectopically express ZicL in a7.9/a7.10 and in neighboring
epidermal/brain progenitor cells (a7.13/a7.14), whereas similarly
treated uninjected embryos do not show ectopic ZicL expression
(Fig. 3F,G). Therefore, FGF signaling acts positively on a7.9/a7.10 to
induce ZicL expression at the 64-cell stage, and BZ1 and BZ2 repress
the precocious expression of ZicL in normal embryos at this stage.
The brain/palp progenitors at the 64-cell stage are derived from a
pair of anterior animal cells (a6.5) in the 32-cell embryo. Although
no precocious ZicL expression was detected in BZ1/2 morphants at
the 32-cell stage, we found ectopic and precocious ZicL expression
in the anterior animal cells of BZ1, BZ2 and Hes-a triple morphants
(Fig. 3H-J). Hes-a transcriptional repressor gene is normally
expressed in all of the blastomeres except for the posterior-most pair
of cells at the 16-cell stage, and the expression in the animal
hemisphere diminishes by the 32-cell stage (Imai et al., 2004). By
contrast, in embryos injected with only the Hes-a MO, ectopic
expression is observed only weakly in a small number of cells
DEVELOPMENT
Fig. 1. BZ1 and BZ2 are expressed in the
progenitors of the neural plate. (A) Cell lineages of
the anterior animal cells. Cells fated to give rise to
the brain and palps are shown in blue and red,
respectively. Blue and green arrows indicate the
onset of Otx and DMRT1 expression. (B) Schematics
of the brain and palp lineages of cells in the early and
late gastrulae. The brain and palp progenitors are
shown in blue and red, respectively. The names of
individual cells, indicated at the left half of each
illustration, are prefixed with the IDs shown next to
the illustration. Vertical bars indicate sister
relationships between blastomeres. (C,D) In situ
hybridization for BZ1 (C) and BZ2 (D) from the 16cell to early tailbud stages. All embryos except the
tailbud embryos are shown in animal view. The
tailbud embryos are in lateral view.
RESEARCH REPORT
Development (2013) doi:10.1242/dev.100339
Fig. 2. BZ1 and BZ2 are required for proper
brain and palp formation. (A-B′) Tadpole
larvae developed from eggs injected with a lacZ
MO (A,A′), and BZ1 and BZ2 MOs (B,B′). Palps
and the brain region are shown by red and blue
arrowheads. A′ and B′ show higher
magnification views of the trunk region of larvae
(different individuals from those shown in A and
B). (C-P) The expression of islet (C,D), ZF220
(E,F), EphrinA-d (G,H), FoxC (I,J), Six3/6 (K,L),
MYTF (M,N) and ZicL (O,P) in control
(C,E,G,I,K,M,O) and BZ1/2 morphant
(D,F,H,J,L,N,P) embryos at the tailbud stage
(C-F), at the late gastrula stage (G-N) and at the
early gastrula stage (O,P). (G-P) The
expression is illustrated schematically in the
lower right by red (palps) and blue (brain).
(Q,R) The expression of islet (Q) and ZF220 (R)
in embryos expressing ZicL under the DMRT1
upstream sequence at the tailbud stage. The
number of embryos examined and the
proportion of embryos that each panel
represents are shown within the panels.
BZ1 and BZ2 expression is transient, owing to a negativefeedback loop
The results described above indicate that transient expression of BZ1
and BZ2 is important for ZicL expression in the brain lineage. This
transient expression is controlled mainly by BZ1, because BZ1 and
BZ2 expression is strongly upregulated in BZ1 morphants (Fig. 4AD), but weakly in BZ2 morphants (Fig. 4E,F).
We injected synthetic BZ1 and BZ2 mRNAs into embryos.
Because the synthetic BZ1 and BZ2 mRNAs lack the endogenous
3′-UTRs, we were able to measure the amount of the endogenous
mRNAs of BZ1 and BZ2 by RT-qPCR with primers designed to their
3′-UTRs. Whereas levels of the maternal control mRNA macho-1
are unchanged by injection of BZ1 and BZ2 mRNAs, BZ1 and BZ2
expression levels are greatly reduced by injection of synthetic BZ1
mRNA (Fig. 4G). By contrast, injection of BZ2 mRNA rarely
reduces BZ1 and BZ2 expression. Therefore, feedback repression of
BZ1 and BZ2 expression by BZ1 results in transient expression of
these two repressor genes.
Conclusions
FGF9/16/20 is expressed as early as the 16-cell stage, and is used
repeatedly to induce different genes in different cells at different
times by functioning in combination with different transcription
factors (Imai et al., 2002; Bertrand et al., 2003; Imai et al., 2003;
Yagi et al., 2004; Imai et al., 2006; Kumano et al., 2006; Lamy et
al., 2006; Hudson et al., 2007; Picco et al., 2007; Shi and Levine,
2008; Hashimoto et al., 2011). The present study shows that the
same signaling pathway can evoke differential outputs (ZicL
expression or not) in a single lineage of cells at different times by
functioning in combination with Hes-a and BZ1/BZ2 repressors
(supplementary material Fig. S4). Without these repressors, the
brain/palp progenitors precociously express ZicL, and therefore the
palps are not properly specified. After the brain/palp progenitors
divide, the palp precursor cells do not abut on FGF9/16/20expressing cells (Wagner and Levine, 2012), and therefore ZicL is
not induced in the palp lineage.
The duration of this repression is probably determined by a
negative-feedback circuit of BZ1/2, the kinetics of which are likely
to depend on mRNA and protein synthesis and turnover rates. Our
results suggest that this duration coincides with cell division of the
brain/palp progenitors, and thus that the feedback loop acts as a
‘delay timer’ to temporally control ZicL expression in the brain
lineage without regulating the cell division directly.
This situation contrasts with instances in which cell cycle genes are
directly controlled by regulatory gene circuits. In vertebrates, the key
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DEVELOPMENT
(Fig. 3K). Therefore, at the 32-cell stage, ZicL is repressed by Hesa, BZ1 and BZ2.
Ectopic expression of ZicL in BZ1/2/Hes-a morphants at the 32cell stage is dependent on FGF signaling, because this ectopic
expression is lost in BZ1/2/Hes-a morphants treated with U0126
(Fig. 3L) and because bFGF treatment evokes ectopic expression in
all anterior ectodermal cells of BZ1/2/Hes-a morphants (Fig. 3M,N).
RESEARCH REPORT
Development (2013) doi:10.1242/dev.100339
Fig. 3. Precocious expression of
ZicL is repressed by BZ1, BZ2 and
Hes-a. (A,B) ZicL expression at the
early gastrula stage in embryos
expressing BZ1 (A) and BZ2 (B) under
the DMRT1 upstream sequence. The
overexpression constructs were
injected into one anterior animal cell of
eight-cell embryos. Descendants of
the blastomere injected with the
constructs are encircled by pink
dashed lines. The expression is
illustrated schematically at the bottom
right in blue (brain). (C-G) ZicL
expression at the 64-cell stage in
control (C,F) and BZ1/2 morphant
(D,E,G) embryos (shown in anterior
views; animal pole up). The embryo
shown in E is treated with U0126, and
the embryos in F and G are treated
with bFGF. (H-N) ZicL expression at
the 32-cell stage in control (H,M),
BZ1/2 morphant (I), BZ1/2/Hes-a
morphant (J,L,N) and Hes-a morphant
(K) embryos (shown in animal views).
The embryo shown in L is treated with
U0126, and the embryos in M and N
are treated with bFGF. Ectopic
expression is shown by yellow
arrowheads. Black arrowheads
indicate the expression of ZicL.
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MATERIALS AND METHODS
Animals
Ciona intestinalis adults were obtained from the National Bio-Resource
Project of MEXT. An inhibitor of MEK signaling (U0126, Sigma) (Hudson
et al., 2003) and human recombinant bFGF (Sigma) were used at 10 μM,
and 100 ng/ml, respectively.
Microarray screen
We manually isolated individual blastomeres from 16-cell embryos, and
obtained gene expression profiles at single-cell resolution with microarrays
(Matsuoka et al., 2013). We injected synthetic mRNAs for genes that were
activated in specific cells at the 16-cell stage, and found that injection of BZ1
mRNA severely disrupted morphology. We therefore examined the function
of BZ1 in the present study. Data from the microarray screen will be
available in Gene Expression Omnibus under accession number GSE45575.
Knockdown and overexpression studies
We used the same MOs (Gene Tools) for Hes-a [E(spl)/Hairy-a] and ZicL
that we used in a previous study (Imai et al., 2006). We designed two MOs
for BZ1 (BZ1a: 5′-CAGAAGATCGTAATTACCTCGATGT-3′; and BZ1b:
5′-CGTAACTTTCGCGGTGATTCCTCAT-3′) and one MO for BZ2 (5′GTCTGAACACACATGATTCCGACAT-3′). Both combinations of
BZ1a/BZ2 MOs and BZ1b/BZ2 MOs gave the same morphological
phenotype and elicited precocious expression of ZicL in the neural lineage.
Although we could not design additional MOs for BZ2 because of its short
5′-UTR, the BZ2 MO gave specific phenotypes only when it was injected
with the BZ1a or BZ1b MO, strongly suggesting specificity of this BZ2
MO. We describe results obtained from injection of the BZ1a and BZ2
MOs.
DEVELOPMENT
transcription factors MyoD and Myf5 control cell cycle withdrawal
and induction of differentiation in skeletal muscle cells (Kitzmann et
al., 1998). This type of regulation is likely to be more robust.
Nevertheless, evolutionary processes might favor delay timers in early
embryos, probably because gene regulatory networks in early animal
embryos typically have a cascade-like structure (Davidson et al.,
2002; Levine and Davidson, 2005; Imai et al., 2006).
Although we do not have solid evidence that BZ1 and BZ2 are
Blimp-1 orthologs, it should be noted that Blimp-1 is required for
proper anterior neural tissues in a variety of animals including
amphioxus, zebrafish and Xenopus (de Souza et al., 1999; Roy and
Ng, 2004; Onai et al., 2010). In zebrafish, blimp-1 (prdm1a –
Zebrafish Information Network) is expressed at the boundary of the
neural plate and non-neural ectoderm. Loss of blimp-1 activity
inhibits specification of neural crest cells and the primary sensory
neurons, whereas misexpression of blimp-1 generates
supernumerary primary sensory neurons. It has been suggested that
the ascidian palps represent a rudimentary placode and might be
derived from an ancestral structure that evolved into separate
anterior placodes in the vertebrate lineage (Manni et al., 2004;
Mazet et al., 2005; Wagner and Levine, 2012). Addition of the
BZ1/2 delay timer circuit to the gene regulatory network responsible
for brain formation might represent a key event in the acquisition of
the primitive palps/placodes in an ancestral animal, because this
circuit makes room within the neural plate for the palps to be
specified by a FoxC-dependent mechanism.
RESEARCH REPORT
Development (2013) doi:10.1242/dev.100339
Fig. 4. Negative-feedback loops of BZ1 and BZ2 expression. (A-F) Expression of BZ1 (A,C,C′,E) and BZ2 (B,D,D′,F) at the early gastrula stage in normal
embryos (A,B), BZ1 morphants (C-D′) and BZ2 morphants (E,F). Embryos in A-F are shown in lateral views (animal pole right). C′ and D′ show animal views of
the embryos in C and D, respectively. Arrowheads in C-F indicate upregulated expression. (G) The amount of endogenous BZ1 and BZ2 mRNAs is measured
by qPCR in embryos injected with GFP (control), BZ1 and BZ2 mRNAs. The relative amount of mRNA in the experimental embryos compared with control
embryos is shown. A maternal mRNA, macho-1, is used as a control. Error bars indicate standard errors between two biological duplicates.
Whole-mount in situ hybridization
The detailed procedure for whole-mount in situ hybridization was described
previously (Imai et al., 2004). To discriminate between BZ1 and BZ2
transcripts, we used 437-bp and 658-bp portions of the 5′-regions of cDNAs
for BZ1 and BZ2 cDNAs.
RT-qPCR
For RT-qPCR, we extracted RNA from wild-type embryos and embryos
injected with BZ1, BZ2 and Gfp mRNAs. The RNA was reverse-transcribed
with an oligo-dT primer. The obtained cDNA samples were then analyzed
by quantitative PCR with the SYBR green method. For each qPCR, the
amount of cDNA used was equivalent to two-thirds of an embryo. Primers
used were: BZ1, 5′-GAATTGGACATGAGAATAAAACAACTACA-3′ and
5′-TGCTTTGAAGATGCGCATAAA-3′; BZ2, 5′-AAAAACACGTCAGAAGTAGCTCTCTAAA-3′ and 5′-CCCAGGCACTTGAAAGTTAACAGT3′; Macho-1, 5′-CCCAGTATGCACCAAATTCAGA-3′ and 5′-TGGTGAGAAAACGGGTGAAAC-3′.
Acknowledgements
We thank Reiko Yoshida and Chikako Imaizumi and all staff members of the
Maizuru Fisheries Research Station of Kyoto University for collecting and cultivating
Ciona intestinalis under the National Bio-Resource Project (NBRP) of MEXT, Japan.
Competing interests
The authors declare no competing financial interests.
Author contributions
T.I. designed and performed the experiments. T.M. performed the experiment to identify
BZ1 and BZ2 genes. Y.S. designed and supervised the study, and wrote the paper.
Funding
This research was supported by Grants-in-Aid from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) and the Japan Society for the
Promotion of Science (JSPS) [21671004 to Y.S.].
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.100339/-/DC1
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