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35
Development 136, 35-39 (2009) doi:10.1242/dev.030981
brachyury null mutant-induced defects in juvenile ascidian
endodermal organs
Shota Chiba1, Di Jiang1,*, Noriyuki Satoh2 and William C. Smith1,†
We report the isolation of a recessive ENU-induced short-tailed mutant in the ascidian Ciona intestinalis that is the product of a
premature stop in the brachyury gene. Notochord differentiation and morphogenesis are severely disrupted in the mutant line. At
the larval stage, variable degrees of ectopic endoderm staining were observed in the homozygous mutants, indicating that loss of
brachyury results in stochastic fate transformation. In post-metamorphosis mutants, a uniform defect in tail resorption was
observed, together with variable defects in digestive tract development. Some cells misdirected from the notochord lineage were
found to be incorporated into definitive endodermal structures, such as stomach and intestine.
KEY WORDS: Notochord, Endoderm, Metamorphosis, Ascidian, Ciona intestinalis
1
Department of Molecular, Cell and Developmental Biology, University of California
Santa Barbara, Santa Barbara, CA 93106, USA. 2Department of Zoology, Graduate
School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
*Present address: Sars International Centre for Marine Molecular Biology, NO-5008
Bergen, Norway
†
Author for correspondence (e-mail: [email protected])
Accepted 28 October 2008
MATERIALS AND METHODS
Animals
Adult C. intestinalis were collected at the Santa Barbara Yacht Harbor. ENU
treatment and culturing of animals was as described (Hendrickson et al.,
2004; Moody et al., 1999).
SNP linkage mapping
Larvae from crossed heterozygous mutant 411 adults were segregated by
phenotype and then pooled in groups of 50 to 200. Genomic DNA was
isolated from pooled larvae as described (Hendrickson et al., 2004). Fortysix PCR primer sets that amplify loci on the various chromosome arms were
designed based on the C. intestinalis genome sequence. The full sequences
of the panel of primers are available on request.
Sequence of mutant brachyury allele
Genomic DNA for dideoxy sequencing of the brachyury gene from mutant
411 was amplified by specific primers that covered the entire ORF: 5⬘ATGACGTCATCAGATAGTAAGTTAGC-3⬘ (bra1F) and 5⬘-TCACAAAGAAGGTGGCGT-3⬘ (bra1R); or bra1F and 5⬘-GGTTCGTATTTATGCAGAGAGT-3⬘ (bra4R).
Microarray analysis
Microarray analysis was performed using the C. intestinalis Oligoarray ver.1
(Yamada et al., 2005). Two hundred nanograms of Trizol-isolated
(Invitrogen) total RNA from each sample were used. Replicates included
swapping of the fluorescent dyes between the samples.
Immunohistochemical staining and whole-mount in situ
hybridization
Embryos were stained with BODIPY-FL phallacidin (Invitrogen), DAPI
(Sigma) and rabbit anti-GFP (Invitrogen). Alexa 488 (Invitrogen) was used
as a secondary antibody for the anti-GFP antibody. Immunostaining was as
described previously (Veeman et al., 2008). Alkaline phosphatase (AP)
activity was detected as described (Whittaker and Meedel, 1989). AP
staining reactions were allowed to proceed for 40-60 minutes at room
temperature.
For whole-mount in situ hybridization, antisense RNA probes were
transcribed from cDNA clones in the C. intestinalis gene collection release
1 (Satou et al., 2002). In situ hybridization was as described (Satou et al.,
1995).
Single tadpole PCR
Single larvae were genotyped for the brachyury locus after AP staining or
in situ hybridization. Genomic DNA was isolated for PCR from single larvae
as described previously (Veeman et al., 2008). Four μl samples of the
digested larvae or negative controls (from 11 μl total) were PCR-amplified
for 40 cycles using the bra1F and bra4R primers (see above).
DEVELOPMENT
INTRODUCTION
The T-box transcription factor brachyury has an essential role in
chordate mesoderm development (Herrmann and Kispert, 1994;
Showell et al., 2004). The structure and function of brachyury has
been extensively characterized in a number of chordates, including
the ascidian (Satoh, 2003; Showell et al., 2004). In ascidians,
brachyury is expressed in the notochord precursor and the notochord
(Corbo et al., 1997; Imai et al., 2000; Yasuo and Satoh, 1993), and
experimental manipulations have confirmed its role in notochord
development (Di Gregorio et al., 2002; Satou et al., 2001; Yamada
et al., 2003; Yasuo and Satoh, 1998). Both the molecular pathway
inducing brachyury expression in the ascidian notochord precursor,
and direct and indirect downstream targets of brachyury have been
well characterized (Hotta et al., 2000; Hotta et al., 2008; Imai et al.,
2006; Nishida, 2005; Takahashi et al., 1999; Yagi et al., 2004).
Functional study of brachyury in ascidians has relied largely on
the misexpression of DNA constructs, the downregulation of
brachyury expression by targeting upstream factors, and morpholino
knockdown (Di Gregorio et al., 2002; Satou et al., 2001; Takahashi
et al., 1999; Yamada et al., 2003). Although these approaches have
been fruitful, they frequently result in mosaic and/or incomplete
knockdown. Here, we report an N-ethyl-N-nitrosourea (ENU)induced null mutant allele of brachyury in Ciona intestinalis.
Several new and surprising results come from the study of this
mutant. Significantly, despite the lack of brachyury function, the
homozygous mutants show a highly variable misexpression of
endoderm markers in the transfated notochord lineage. The mutant
line has allowed us to follow the consequences of loss of brachyury
through metamorphosis, where we find that re-directed cells of the
notochord lineage are able to contribute to the definitive endoderm
of the juvenile.
36
RESEARCH REPORT
Development 136 (1)
Reverse transcription-PCR (RT-PCR)
RNA was isolated with Trizol at the following stages: 110-cell, early tailbud,
larva, and stage 3 juvenile (Chiba et al., 2004). The SMART RACE cDNA
amplification kit (Clontech) was used for cDNA synthesis. The cDNA
samples were amplified with primers bra1F and bra1R for brachyury, or
with 5⬘-CGTTTTCCCATCCATCGTAG-3⬘ and 5⬘-CCAGCAGATTCCATACCAAG-3⬘ for cytoplasmic actin.
Tail ablation
RESULTS AND DISCUSSION
Isolation of an ENU-induced Ciona intestinalis
brachyury mutant
Five hundred and twenty-nine F1 adults were screened and 14 mutants
in nine complementation groups were isolated. Several of the mutants
had short tail phenotypes, including mutant 411 shown in Fig. 1A. At
a gross level, the head and trunk of the mutant 411 larvae appeared
largely normal, while the tail appeared to lack vacuolated notochord
cells. Single nucleotide polymorphism (SNP)-based linkage analysis
using 46 sets of PCR primers targeting the various chromosome arms
was used to link mutant 411 to chromosome arm 12p (Fig. 1B).
Chromosome arm 12p contains at least three notochord genes (data
not shown), including brachyury (Shoguchi et al., 2008). The
brachyury gene from homozygous 411 mutants was found to have six
nucleotide substitutions in exons compared with the published C.
intestinalis cDNA sequence (NM_001078478). The most significant
of these substitutions was a cytosine for thymine in exon 2 that
changed amino acid 49 from an arginine to a stop codon (Fig. 1C). The
predicted protein from this mutant gene would be truncated at the
beginning of the DNA-binding T-box (Fig. 1C). The fact that the
mutant 411 phenotype is similar to previous reports for Ciona
embryos with downregulated or knocked-down brachyury (Di
Gregorio et al., 2002; Satou et al., 2001; Yamada et al., 2003) allowed
us to confidently conclude that the mutant phenotype was due to a
loss-of-function brachyury allele. In addition, we observed that four
genes previously shown to be downstream of brachyury [noto4,
noto8, prickle (pk) and tropomyosin (Hotta et al., 2000)] and laminin
alpha 3/4/5 were not expressed in homozygous mutant 411 embryos
(data not shown). Because the predicted protein is so severely
truncated, the mutation is likely to be a null. In the remainder of this
manuscript we will refer to this mutant as brachyury– (bra–).
brachyury mutant at tailbud stages
To investigate the morphology and fates of the notochord lineage in
the homozygous mutants (bra–/–), the line was crossed to a stable line
expressing GFP under the control of the C. intestinalis brachyury 5⬘
regulatory region (Joly et al., 2007). At the early tailbud stage (Fig.
1D,E), the GFP-fluorescing cells in bra–/– embryos were organized
into a mass of tissue that was nearly as long and narrow as the wildtype notochord, and the overall anterior/posterior (A/P) length of the
embryo was similar to wild type. Despite this, the convergent
extension (C/E) of the notochord lineage was severely disrupted. In
embryos double stained with DAPI and phallacidin (Fig. 1F,G), the
disrupted morphology of the notochord lineage was evident and, most
noticeably, the cells of the notochord lineage failed to intercalate into
a single column, unlike in wild-type embryos (Fig. 1G, arrow).
Fig. 1. An ENU-induced short tailed mutant. (A) Sibling larvae,
either wild type (top) or homozygous for the 411 mutation (bottom).
Scale bar: 100 μm. (B) SNP-based linkage analysis. Examples of unlinked
(chromosome 01q, left panel) and linked (chromosome 12p, right
panel) loci are shown. Panels show sequence traces from pooled
mutant (411–/–) or heterozygous larvae and homozygous wild-type
mixed siblings (411+/– + 411+/+). The red arrows indicate SNPs. (C) The
mutant 411 has a premature stop codon in the brachyury gene. (Top)
The gene model of brachyury. (Middle) Partial sequence of brachyury
on exon 2. The point mutation is shown in red. (Bottom) Predicted
models of wild-type and mutant brachyury protein. (D-G) Disrupted
notochord morphology in bra–/– embryos. (D,E) Maximum intensity
projections of confocal stacks through wild-type (D) and bra–/– (E) early
tailbud stage embryos (dorsal view). Notochords were visualized by
staining embryos carrying a stable brachyury promoter:GFP transgene
with an anti-GFP antibody. The smaller cells in the background are
mesenchyme (Me). (F,G) Lateral view of wild-type (F) and bra–/– (G) early
tailbud stage embryos stained with phallacidin (green) and DAPI (blue).
Notochord lineage cells are indicated by arrows. Scale bars: 50 μm.
brachyury occupies a key position in the gene regulatory network
that specifies notochord fate (Showell et al., 2004). Approximately
forty candidate brachyury targets have been identified in C.
intestinalis (Hotta et al., 2000; Takahashi et al., 1999). The null
DEVELOPMENT
The larvae (3 to 6 hours post-hatching) were bisected at the neck using
tungsten needles. The resulting tailless trunks were cultured at 18°C in
seawater with 50 mg/l streptomycin that was changed daily. Four days after
attachment (equivalent to juvenile stage 5), the juveniles were fed a microalgae
mixture to label the digestive tract. On the following day, the animals were
observed to determine whether a complete digestive tract was present, as
would be indicated by the presence of food in the lumen and feces.
Ascidian brachyury null mutant
RESEARCH REPORT
37
Fig. 2. Notochord cells express
endoderm markers in bra–/– mutant.
(A-C) Alkaline phosphatase (AP) activity
in wild-type (A) and bra–/– (B,C) larvae.
The two bra–/– larvae are representative
of the range of ectopic AP activity
observed. (D-F) Expression of the
endoderm/endodermal strand marker
Kyotograil2005.572.7.1. in wild-type
(D) and bra–/– (E,F) larvae. The black
arrowhead indicates ectopic expression
in the mutant larva. Genotypes were
confirmed by PCR. The red arrowheads
in the lower panels indicate the
mutation that results in a premature
stop codon in the bra– mutant.
(G,H) Ectopic AP activity in the
notochord lineage of wild-type and
mutant larvae. The notochord lineage is
indicated by GFP fluorescence.
(G) Merged image for a wild-type larva.
No colocalization of AP and GFP
expression was found in anterior
(A-line) notochord cells. (H) Merged
image for a bra–/– larva. Ectopic AP
activity and GFP fluorescence are
colocalized (arrow) in the mutant.
Scale bars: 100 μm.
Notochord cell fate transformation in brachyury
mutants
The manipulation of factors upstream of notochord specification,
including bFGF, β-catenin, ZicN and FoxD, demonstrates the
potential for presumptive notochord cells to assume neural and
endodermal fates in ascidians (Imai et al., 2000; Imai et al., 2002a;
Kumano et al., 2006; Minokawa et al., 2001). However, morpholino
knockdown of brachyury in Ciona savignyi resulted in a short tail
phenotype, but with no reported ectopic endoderm (Satou et al., 2001).
The mutant line described here offers a stable, non-mosaic
background in which to study the effects of loss of brachyury function.
On examination of the pan-neural gene ETR-1 and muscle actin, there
was no indication of notochord cells transfating to neural or
endodermal tissues in bra–/– larvae (data not shown). By contrast, the
examination of alkaline phosphatase (AP) activity, as a marker of
endoderm, revealed definitive, but highly variable, transfating of
notochord to endoderm-like cells in the bra–/– larvae. Approximately
one-half of the bra–/– larvae had ectopic AP staining in the core of the
tail (Fig. 2B,C). Because of the variability in AP staining, the
genotypes of individual larvae were determined by genomic PCR and
dideoxy sequencing (Fig. 2B,C, lower panels). Identical results were
obtained by in situ hybridization using an endoderm-specific marker,
kyotograil2005.572.7.1 (Fig. 2E,F). Finally, AP activity colocalized
with brachyury promoter-driven GFP fluorescence in bra–/– larvae,
but not in wild-type larvae, indicating that the notochord cells were
transfated to endoderm in the homozygous mutant (Fig. 2G,H).
The fate of cells in the notochord lineage of bra–/– embryos not
expressing ectopic endoderm markers is unknown, although by
morphology and gene expression they do not become notochord.
Previous reports show that ascidian Zic can prevent notochord cells
from becoming endoderm, but Zic is not expressed in the notochord
until after gastrulation (Imai et al., 2002b; Kumano et al., 2006).
Thus, one possibility is that transfating of notochord cells in bra–/–
embryos is repressed late in the process of determination, and that
only a fraction of the embryos are able to overcome the repression
and express ectopic endoderm markers.
Although genes required for tail elongation, including pk and
laminin alpha 3/4/5 are not expressed in bra–/– mutants, tail
elongation was not completely disrupted, as was observed in
aimless/chongmague double homozygous mutants in C. savignyi
(Veeman et al., 2008). In fact, the A/P axis in bra–/– mutants was
similar to that of wild-type embryos at early tailbud stage. One
possibility is that the transfated notochord cells are following the
morphogenetic pathway of endoderm in bra–/– mutants. Cells of
the endodermal strand do form a single-file row underlying the
notochord in the wild type, although the morphogenetic
mechanisms are unknown. Alternatively, there may be an intrinsic
and cell fate-independent program in the notochord lineage to
undergo at least the initial steps of C/E.
Transfated notochord lineage contributes to
definitive endoderm
Following the larval stage, ascidians undergo metamorphosis. In wildtype ascidians, the tail is largely reabsorbed and most adult organs are
present by juvenile stage 4 (Fig. 3A,D) (Chiba et al., 2004). bra–/–
larvae also undergo metamorphosis, but the resulting juveniles are
highly abnormal (Fig. 3B,C). As with larvae, bra–/– juveniles were
variable (Table 1), with some showing a more moderate phenotype
DEVELOPMENT
mutation reported here provides a new tool for investigating
brachyury function. Microarray analysis was used to identify three
new notochord genes of unknown function that are downregulated
in the homozygous bra–/– mutant larvae (see Fig. S1 in the
supplementary material).
38
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Development 136 (1)
Fig. 3. bra–/– juveniles have defects in tail
resorption and digestive tract
development. (A) Stage 4 wild-type juvenile.
A small amount of tail debris is still evident
(arrow). (B,C) Stage 4 bra–/– juveniles showing
the range of defects observed, from moderate
(B) to severe (C). Note the extensive tail debris
remaining in B (arrow). (D) Higher
magnification image of digestive tract of
juvenile A. (E) Higher magnification image of
digestive tract of juvenile B. Although the
esophagus and the stomach can be
distinguished in the bra–/– juvenile, they are
malformed. E, esophagus; ST, stomach. Scale
bars: 100 μm. (F-J) Fate of notochord lineage
as indicated by GFP fluorescence during
metamorphosis. At stage 3 of metamorphosis
the GFP-expressing cells are found displaced
further towards the stalk in bra–/– (G) than in
wild-type (F) juveniles. By stage 4, the
remaining notochord debris is greatly reduced
in wild-type (H), but not in bra–/– (I,J) juveniles.
In the stage 4 bra–/– juveniles, the GFPexpressing cells have incorporated into
definitive endodermal structures, such as the
stomach (arrow in I) or intestine (arrow in J).
(K) RT-PCR shows the absence of brachyury
transcript in larvae and in stage 3 juveniles.
110, 110-cell stage; eTB, early tailbud stage;
SL, larva; Juv, stage 3 juvenile.
Table 1. Tail ablation
Digestive tract development*
Swimming larvae
WT with tail
WT without tail
bra–/– with tail
bra–/– without tail
Good
Moderate
Severe
195 (86%)
80 (68%)
2 (1%)†
30 (11%)
0 (0%)
1 (1%)
174 (70%)
167 (60%)
31 (14%)
36 (31%)
74 (30%)
86 (30%)
*Good means the juvenile had a complete digestive tract. Moderate means the
juvenile had a recognizable, but incomplete, digestive tract. Severe means the juvenile
had no recognizable digestive tract. Typical phenotypes of these are shown in Fig. 3.
†
This juvenile completed tail resorption.
Despite the fact that the brachyury gene is not transcribed past the
tailbud stage, the perdurance of GFP allows us to follow the notochord
lineage well into the juvenile stage (Fig. 3F-J) (Deschet et al., 2003).
As has been described previously (Cloney, 1978), the remnants of the
notochord are coiled at one end of the developing juvenile at stage 3
(Fig. 3F). By stage 4, only a small clump of GFP-positive cells is
found between the esophagus and the stomach (Fig. 3H). In contrast
to wild type, most GFP-positive cells in the mutant were found more
centrally located in the trunk region at stage 3 (Fig. 3G). Most
significantly, at stage 4 there was a much greater persistence of GFPpositive cells, many of which had been incorporated into definitive
endodermal organs, such as the stomach and the intestine (Fig. 3I,J,
arrows), suggesting that the cells were not following the apoptotic
pathway of normal notochord cells but rather were transfated.
Rescue of metamorphosis by tail ablation
The above observations that transfated notochord cells in bra–/–
animals could contribute to definitive digestive organs suggested
that gut development was abnormal due to an excess of cells. As a
possible mechanism to correct this defect, the tails of larvae were
removed and the remaining trunks were allowed to settle and
undergo metamorphosis. Tail ablation resulted in an increase from
1% to 11% of bra–/– animals that had well-formed digestive tracts,
indicating that the persistence of the tail remnants in the juvenile
disrupts normal development (Table 1).
DEVELOPMENT
with well-developed adult organs, such as endostyle and
protostigmata (e.g. Fig. 3B), while others were more severely
disrupted (e.g. Fig. 3C). Even in the less severe examples the digestive
tracts were abnormal and did not appear to make a complete tract (e.g.
Fig. 3E). Nearly all bra–/– juveniles arrested at this stage, presumably
because of these defects in the digestive system.
The failure of the bra–/– juveniles to make a functional digestive
tract could have several underlying causes. One possibility is that
brachyury is needed in the endoderm lineage at metamorphosis for
its proper development. However, brachyury transcript could not be
detected by RT-PCR in wild-type larvae and stage 3 juveniles,
making this unlikely (Fig. 3K).
In conclusion, the mutant line reported here will provide a useful
tool for future studies on notochord specification and brachyury
function. Among the unresolved issues is the variability in the
expression of endoderm markers in bra–/– mutants. Investigation in
this area may provide an insight into the quantitative effects of other
loci and mechanisms of embryonic robustness.
This work was supported by grants from the NIH (HD38701 and GM075049) to
W.C.S., and a Grant-in-aid for Scientific Research from MGXT to N.S.
(No.17018018). We thank Dr Yasunori Sasakura, Maizuru Fisheries Research
Station of Kyoto University, Mrs Yasuyo Kasuga, Dr Michael T. Veeman, Erin L.
Mulholland and the Santa Barbara Ascidian Stock Center for their help with this
project. Deposited in PMC for release after 12 months.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/1/35/DC1
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DEVELOPMENT
Ascidian brachyury null mutant