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First published online 26 November 2008
doi: 10.1242/dev.029157
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Research Report |
1 Department of Molecular and Cell Biology, Center for Integrative Genomics,
University of California, Berkeley, 142 LSA#3200, Berkeley, CA 94720,
USA.
2 Center for Cell Dynamics, Friday Harbor Laboratory, University of Washington,
620 University Road, Friday Harbor, WA 98250, USA.
* Author for correspondence (e-mail: wshi{at}berkeley.edu)
Accepted 27 October 2008
SUMMARY
Convergent extension (CE) is the narrowing and lengthening of an embryonic field along a defined axis. It underlies a variety of complex morphogenetic movements, such as mesoderm elongation and neural tube closure in vertebrate embryos. Convergent extension relies on the same intracellular molecular machinery that directs planar cell polarity (PCP) in epithelial tissues, including non-canonical Wnt signaling components. However, it is not known what signals coordinate CE movements across cell fields. In the simple chordate Ciona intestinalis, the notochord plate consists of just 40 cells, which undergo mediolateral convergence (intercalation) to form a single cell row. Here we present evidence that a localized source of FGF3 in the developing nerve cord directs notochord intercalation through non-MAPK signaling. A dominant-negative form of the Ciona FGF receptor suppresses the formation of polarized actin-rich protrusions in notochord cells, resulting in defective notochord intercalation. Inhibition of Ciona FGF3 activity results in similar defects, even though it is expressed in an adjacent tissue: the floor plate of the nerve cord. In Xenopus mesoderm explants, inhibiting FGF signaling perturbs CE and disrupts membrane localization of Dishevelled (Dsh), a key regulator of PCP and CE. We propose that FGF signaling coordinates CE movements by regulating PCP pathway components such as Dsh.
Key words: Ciona, FGF, Ascidian, Convergent extension, Notochord, Planar cell polarity
INTRODUCTION
In vertebrates and ascidians, the formation of the primary embryonic body
axis depends on the lengthening of the axial mesoderm by convergent extension
(CE), a process in which cells intercalate medially along the anteroposterior
(AP) axis (Keller, 2002
;
Wallingford et al., 2002).
Members of the planar cell polarity (PCP) pathway, including Dishevelled
(Dsh), Prickle and Van Gogh/Strabismus, are recruited to specific regions on
the cell membrane upon activation of the non-canonical Wnt pathway
(Lawrence et al., 2004;
Jones and Chen, 2007;
Seifert and Mlodzik, 2007) and
transduce the Wnt/PCP signal into changes in actin dynamics and cellular
motility. Disrupting these membrane protein complexes causes severe defects in
tail extension and neural tube closure
(Wallingford, 2006),
suggesting a central role for these genes in chordate embryogenesis.
Tissue-level CE typically begins with coordinated polarization and movement
of individual cells. During Xenopus mesoderm formation, cells across
the dorsal marginal zone (DMZ) form actin protrusions and lamellopodia
preferentially along the mediolateral (ML) axis, events that are controlled by
Dsh and other PCP genes (Wallingford et
al., 2000). However, most of the known PCP components are
intracellular molecules that function cell-autonomously to regulate
cytoskeletal rearrangements. The putative extracellular signals that might
establish the initial polarization of a field of cells have yet to be
identified (Barrow, 2006;
Casal et al., 2006;
Lawrence et al., 2007).
Here, we present evidence for a non-autonomous mechanism for establishing ML polarity in the Ciona notochord. A localized source of FGF3 in the ventral midline of the neural tube signals to the underlying notochord plate to direct ML intercalation. Hence, FGF signaling could serve as the instructive signal for coordinating cell behavior over long distances and establishing the direction of CE in chordates.
MATERIALS AND METHODS
Ciona
Adult Ciona intestinalis were obtained and maintained as
previously described (Corbo et al.,
1997).
In situ hybridization and immunohistochemistry
In situ hybridization, diphosphorylated (dp) Erk staining and double
antibody/in situ hybridization were performed according to Davidson et al.
(Davidson et al., 2006).
Phalloidin staining and lamellopodia counting were performed according to
Munro et al. (Munro et al., 2002a).
Morpholinos, PCR and enhancer constructs
C. intestinalis FGF3 (Ci-FGF3) splice donor
(5'-CCGATGTTTGACTTACTTTGCGGCG-3') and splice acceptor
(5'-CAATCTCAGCTGTGAAAATAGAAAT-3') morpholinos were co-injected
with Brachyury::GFP (10 ng/µl) at a total concentration of 1.5 mM. qRT-PCR
was performed with total RNA extracted from 
15 embryos using SYBR
chemistry on an ABI 3700 real time PCR system with the following primers:
Ci-FGF3 (5'-ATAACAAGTCGCCGCAAACT-3',
3'-TTGCTGTGTCGGTTCATAGC-5') and Cytoplasmic actin 7 (internal
control, 5'-CTCCATCATGAAGTGCGATGTT-3',
3'-CATTCTGTCGGCGATTCCA-5'). A nerve cord-specific enhancer for the
FGF3 homolog in Ciona savignyi (Cs-FGF3) was cloned
by fusing the first intron (6.2 kb) to a 5 kb genomic DNA fragment from
the 5' flanking region. The nerve cord enhancer 00124
(5'-TATATATACTGTTGTGCCAG-3',
3'-CATCTTGGTTAAAACTGATTC-5'), notochord enhancer Noto1
(5'-GGCTTGGTCAGTTGAATC-3',
3'-CGTAAACAACTTCATAATTTTG-5'), muscle enhancer MyoD
(5'-GGCTTACGCATCTCGAGCGAACC-3',
3'-CTCTTGAGAGATACACGTCATCG-5') and endodermal strand enhancer
00794 (5'-CATTCTGCGCTGCTGTTG-3',
3'-CGGTTTTGCTTTCACAACTTT-5') were cloned using the indicated
primers.
Time-lapse movies
For the time-lapse movie examining notochord intercalation, the dorsal
anterior quadrants from wild-type and mutant embryos were isolated at the
mid-gastrula stage and recorded over a period of 5 hours every 15 seconds. For
notochord cell protrusive activity, electroporated embryos were developed to
the mid-neurula stage, dissociated in Ca2+/Mg2+-free
medium with trypsin (Christiaen et al.,
2008) and recovered for 15 minutes before mounting on a
poly-L-lysine-coated slide. Movies were taken every 6 seconds over
a period of 15 minutes.
Xenopus explants
Xenopus embryos were obtained, reared and injected as described
(Sive et al., 2000). Embryos
were injected in the animal hemisphere at the 2-cell stage for isolation of
animal caps and equatorially into two dorsal blastomeres at the 4-cell stage
for isolation of DMZ. Capped mRNAs were synthesized using the mMessage Machine
Kit (Ambion) and injected as follows: Xdsh-GFP
(Rothbacher et al., 2000) 120
pg; PKC-
-YFP (Kinoshita et al.,
2003) 100 pg; membrane RFP 200 pg. Animal caps (stage 9) were
removed and cultured in 3/4x NAM
(Sive et al., 2000) for 30
minutes before fixation and immunohistochemistry. The DMZ above the blastopore
lip was excised from stage 10+ embryos in 1x Steinberg's medium. DMZs
were allowed to heal until whole-embryo siblings reached stage 10.5, then
transferred to 1x Steinberg's medium containing 120 µM SU5402 or DMSO
and cultured at 15°C until stage 12. Embryos and explants were fixed in
MEMFA (Sive et al., 2000),
dehydrated in methanol, then rehydrated and processed for in situ
hybridization or bleached in 1% hydrogen peroxide solution prior to
immunohistochemistry. The following antibodies were used: chicken anti-GFP
1:500 (Abcam); rabbit anti-RFP 1:500 (Molecular Probes); Tor70 (notochord,
mouse IgM) (Bolce et al.,
1992); and 12/101 (muscle, mouse IgG)
(Kintner and Brockes,
1984).
RESULTS AND DISCUSSION
Non-MAPK FGFR signaling is required for notochord convergent extension
In tadpoles of Ciona intestinalis, the notochord is composed of a
single row of 40 cells that forms through ML intercalation, a common mode of
CE (Miyamoto and Crowther,
1985). To identify potential regulators of notochord
intercalation, we searched for genes that are specifically expressed in the
notochord plate and found that the Ciona FGFR gene is strongly
expressed in the notochord before and after intercalation
(Fig. 1A,B). This expression is
distinct from the role of FGF signaling in notochord induction at the 32-cell
stage of embryogenesis (Kim et al.,
2000; Kim and Nishida,
2001), which depends on MAPK signaling through maternal FGFR and
the transcriptional activation of Ciona Brachyury (Ci-Bra).
By contrast, the later zygotic expression of FGFR in the notochord
plate and definitive notochord was not associated with MAPK activity
(Fig. 1G,H). Moreover,
inhibition of MAPK activation with the pharmacological MEK inhibitor U0126 did
not affect notochord intercalation when applied after the the notochord fate
is specified (Fig. 1I).
FGFR signaling acts through three major downstream pathways: Ras/MAPK,
PLC
/Ca2+ and PI3K/Akt
(Bottcher and Niehrs, 2005). To
determine whether an alternative FGF signaling pathway might be essential for
notochord development, a dominant-negative form of the Ciona FGFR
(dnFGFR) (Davidson et al.,
2006) was specifically expressed in the notochord lineage after
the late gastrula stage using the Ci-Noto1 enhancer
(Takahashi et al., 1999). The
transgene caused defects in notochord intercalation
(Fig. 1C-F) but did not affect
notochord cell fate specification (see Fig. S2 in the supplementary material).
Many of the defective notochord cells failed to undergo appropriate shape
changes (Fig. 1E') and
rearrangement. The resulting embryos had shorter and wider tails owing to the
failure in CE (Fig. 1, compare
F with D).
To better visualize notochord morphogenesis, we performed time-lapse
microscopy on dorsal-anterior notochord/neural tube explants (DA explants)
(Munro and Odell, 2002b)
isolated from wild-type and dnFGFR Ciona embryos. These DA explants
preserve the normal spatial relationship between notochord and neural plate.
In wild-type DA explants, notochord cells polarize, form dynamic membrane
protrusions and intercalate along a distinct axis to produce an elongated
structure (see Movie 1 in the supplementary material). By contrast, in DA
explants from dnFGFR mutant embryos, notochord cells had diminished
membrane protrusions and failed to intercalate, resulting in a wide notochord
plate resembling that seen in wild-type embryos before CE (see Movie 2 in the
supplementary material).
In Drosophila wing bristle development, Frizzled mutants
exhibit both cell-autonomous and non-cell-autonomous PCP defects
(Klein and Mlodzik, 2005). The
Ciona FGFR seems to function strictly in a cell-autonomous manner. In
mosaically transformed embryos, only those notochord cells that express the
dnFGFR transgene displayed a rounded shape and failed to intercalate
(Fig. 1E'). By contrast,
neighboring wild-type notochord cells lacking the transgene underwent normal
intercalation to form a single row of cells. Together, these data suggest that
FGF signaling - independent of the MAPK pathway - is essential for notochord
intercalation in the Ciona tadpole.
FGFR signaling controls polarized actin organization in notochord cells
Lamellopodia-like protrusions form at the ML edges of notochord precursor
cells within the Ciona notochord plate
(Munro and Odell, 2002a;
Jiang et al., 2005) and are
presumed to be the driving force for directional intercalation. To determine
whether the dnFGFR transgene interferes with the formation of
polarized lamellopodia, phalloidin staining and confocal microscopy were used
to reconstruct the three-dimensional organization of actin protrusions in the
notochord (Fig. 1J). In
wild-type embryos at the late neurula stage, an average of 0.42 actin
protrusions per internal edge was observed in the notochord plate, comparable
with previous results from another ascidian species, Boltenia villosa
(Munro and Odell, 2002a). Most
of these protrusions were positioned within 45° of the ML axis as compared
with the AP axis in the notochord plate, which is consistent with the ML
direction of intercalation. When the dnFGFR transgene was expressed
in the notochord, there was a 50% reduction in the number of actin
protrusions at internal notochord plate cell boundaries
(Fig. 1J). Moreover, the
remaining protrusions displayed a randomized orientation instead of the
2:1 ML:AP ratio seen in wild-type notochord. This combined reduction in
actin protrusions and polarity might underlie the defective notochord
intercalation in dnFGFR embryos.
FGF signaling is not only necessary for the formation of oriented actin
protrusions in the notochord, but is also sufficient to induce ectopic cell
protrusive activity. Dissociated wild-type notochord cells form random
protrusions at low frequency in culture
(Jiang et al., 2005) (see
Movie 3 in the supplementary material). Incubating notochord cells with
recombinant human basic FGF (bFGF) protein causes the cells to form multiple
and larger protrusions at much higher frequency (see Movie 4 in the
supplementary material), whereas cells expressing the dnFGFR
transgene failed to respond to the ectopic FGF signal (see Fig. S5 and Movie 5
in the supplementary material). Thus, FGF signaling might regulate
Ciona notochord intercalation by inducing locally oriented,
actin-based protrusions.
Ciona FGF3 is expressed in the floor plate above the converging notochord
The preceding results suggest an instructive role for FGF signaling in
notochord intercalation. To identify the putative ligand, we examined the
expression patterns of all six FGF genes in the Ciona genome. Only
one of these, Ci-FGF3/7/10/22 (Ci-FGF3), is expressed in
close proximity to the developing notochord
(Imai et al., 2002). The
Ciona dorsal nerve cord consists of four rows of ependymal cells in
the tail (Crowther and Whittaker,
1992). Ci-FGF3 is expressed in the ventral-most row of
cells, which constitutes the rudimentary floor plate of the nerve cord in
tailbud stage embryos (Fig. 2A)
and is located immediately above the midline of the notochord.
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; Davidson et al.,
2005). Indeed, an 11-kb Cs-FGF3 regulatory DNA fully
recapitulated the normal FGF3 expression pattern in C.
intestinalis when attached to a lacZ reporter gene, including
staining in a single row of cells comprising the floor plate at tailbud stages
(Fig. 2, compare C with A). At
earlier stages, when the notochord plate starts to undergo intercalation, the
Cs-FGF3 regulatory DNA drove reporter expression in the two rows of
floor plate progenitors, which are in direct contact with the midline of the
notochord plate (Fig. 2D,E).
Later in development, the floor plate progenitors intercalate at the midline
during neural closure to form the floor plate
(Fig. 2F,G). Thus, it appears
that Ci-FGF3 is expressed at the right time and place to serve as a
signal for notochord intercalation.
Ci-FGF3 is required for actin polarization and notochord convergent extension
To address the function of Ci-FGF3 in Ciona notochord
development, we designed two antisense morpholino oligos (MOs) targeting the
splice donor/acceptor sites flanking the first intron of Ci-FGF3.
qRT-PCR assays indicated that injection of both MOs caused a substantial
reduction in the steady-state levels of Ci-FGF3 transcripts (see Fig.
S3 in the supplementary material). Ci-FGF3 morphant embryos displayed
a variety of CE defects, including noticeably shortened tails
(Fig. 3A-C). Confocal imaging
revealed several classes of mutant phenotypes with varying severity. In the
most extreme cases, the mutant notochord consisted of two or three rows of
cells instead of one, resulting in an extremely reduced tail that was about
the same length as the trunk region (Fig.
3A'-C'). To determine whether Ci-FGF3
regulates actin organization in the notochord, we performed phalloidin
staining and confocal reconstruction of actin structures in Ci-FGF3
morphants. As seen for the dnFGFR transgene, the FGF3 MOs
caused both a reduction in the number of actin protrusions and a randomization
of protrusion orientation (Fig.
3E). The FGF3 morphant phenotype was stronger than that
of dnFGFR mutants because the dominant-negative form of the receptor
is not fully penetrant in the notochord.
Ci-FGF3 is expressed in a spatially localized pattern
that corresponds to the ML polarity of actin protrusions. To determine whether
FGF3 provides a positional cue triggering CE, we asked whether
misexpressing FGF3 in neighboring tissues could affect notochord
intercalation. We used four different tissue-specific enhancers to direct
Ci-FGF3 expression in the nerve cord, notochord, tail muscles and
endodermal strand. Such misexpression resulted in varying degrees of
intercalation defects in the notochord
(Fig. 3F-I'). The most
severe defects were obtained upon misexpression of Ci-FGF3 in the
notochord (Fig. 3F,F'),
which emphasizes the need for the instructive cue to be expressed in a
neighboring tissue. Relatively mild defects were obtained when
Ci-FGF3 was misexpressed in the tail muscles
(Fig. 3H,H'), which is
consistent with the possibility that the muscles serve as a secondary source
of intercalary signals, as seen in other ascidians
(Munro and Odell, 2002b).
Finally, overexpression of Ci-FGF3 in the nerve cord resulted in
severe intercalary defects (Fig.
3G,G'), suggesting that both the levels and location of the
Ci-FGF3 signal are important for proper notochord intercalation.
Defects in notochord intercalary behavior did not result from changes in
notochord fates (see Fig. S4 in the supplementary material).
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in Xenopus). We asked whether FGF signaling is required to promote
membrane localization of the Dsh protein. This is hard to address in
Ciona owing to the lack of Ci-Dsh antibodies and the variable
penetrance of the dnFGFR transgene in the Ciona notochord.
However, FGF signaling has also been implicated in CE of the mesoderm in
Xenopus laevis (Sivak et al.,
2005). Therefore, we used Xenopus DMZ explants to assess
the relationship between FGF signaling and the subcellular localization of a
Xenopus Dsh (Xdsh)-GFP fusion protein. As a control, we also assayed
the localization of PKC--YFP, which is a known downstream target of the
FGF pathway that becomes membrane-localized upon FGFR activation
(Kinoshita et al., 2003;
Sivak et al., 2005). Because
FGF signaling is required for specification of the mesoderm (including
notochord) in Xenopus (see Fig. S6 in the supplementary material), we
manipulated FGF signaling by treatment with the FGFR pharmacological inhibitor
SU5402 after mesoderm induction (from stage 10.5 onwards). This treatment
results in short embryos owing to defects in CE (see Fig. S7 in the
supplementary material).
In wild-type Xenopus DMZ explants undergoing CE, Xdsh-GFP and
PKC--YFP were localized primarily to the cell membrane
(Fig. 4A,B). Upon SU5402
treatment, Xdsh-GFP was excluded from the membrane and was instead distributed
throughout the cytoplasm (Fig.
4C), suggesting that FGFR signaling is required for Xdsh membrane
localization. PKC--YFP was exclusively localized to the cell membrane
in wild-type DMZ explants, whereas the majority of the protein was present in
the cytoplasm after treatment with SU5402
(Fig. 4D). As expected, DMZ
explants did not elongate after drug treatment. Together, these data suggest
that FGF signaling is required for Dsh membrane localization in the
Xenopus dorsal mesoderm during CE. Since Dsh is also required for CE
in Ciona (Keys et al.,
2002), similar mechanisms are likely to operate in Ciona
notochord intercalation.
Directional movement of individual cells in embryogenesis can be achieved
via diverse mechanisms, including chemotaxis, differential protrusive activity
and differential adhesion. A key feature of CE movements is the coordination
of uniform asymmetric cell behavior across large fields of cells. We have
presented evidence that during Ciona notochord formation, FGF3 is
released from the developing floor plate of the neural tube to coordinate ML
polarity and CE movements of the notochord plate. This is consistent with the
previous observation that ascidian DA explants can form elongated notochord
rudiments in culture, whereas isolated notochord precursors alone do not
(Munro and Odell, 2002b). Our
results show that the instructive signal emanating from the neural plate to
direct notochord CE in Ciona is a member of the FGF signaling pathway
that works through the non-MAPK pathway downstream of FGFR. One candidate is
the NRH receptor that functions downstream of FGFR to promote DMZ protrusive
activity (Chung et al., 2005),
although Ciona lacks a clear NRH homolog.
FGF signaling might play similar roles in coordinating complex
morphogenetic processes in vertebrate development. As in Ciona, an
early phase of MAPK-mediated FGF signaling is required to induce mesoderm fate
in the frog embryo through the activation of genes such as Brachyury.
After mesoderm specification, non-MAPK FGF signaling has been implicated in
axial elongation in the Xenopus neurula
(Sivak et al., 2005), but the
details of this mechanism remain unclear. The complex and often overlapping
expression patterns of 23 FGFs and four FGFRs in the early Xenopus
embryo (S.P., unpublished) prevent the unambiguous identification of a
ligand-receptor relationship underlying CE, in contrast to Ciona.
However, it is possible that a similar tissue-tissue interaction is involved
in establishing the polarity of Xenopus DMZ cells.
Cells undergoing CE need to transduce the extrinsic polarity cues (such as
FGF) into coordinated (planar) cell behavior
(Lawrence et al., 2002;
Lawrence et al., 2004). It is
likely that FGF signaling achieves this effect through interacting with the
non-canonical Wnt/PCP pathway. It has been shown previously that FGFR is
required for membrane localization of PKC-, which physically interacts
with Dsh (Kinoshita et al.,
2003; Sivak et al.,
2005). The Ciona genome encodes ten Wnts, but none of
these is expressed in the notochord, raising the possibility they might play
permissive roles in the notochord, as seen in other systems. We propose that a
localized FGF signal released by a developing tissue functions as an
extracellular positional cue to directionally activate the intracellular PCP
pathway in cells of an adjacent tissue (see Fig. S1 in the supplementary
material). This coordination in cell polarity is the first step towards the
orchestrated cell movements that underlie CE.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/1/23/DC1
Footnotes
We thank Dr Naoto Ueno for the Xenopus PKC--YFP construct.
The work was supported by
NIH grants to M.L.,
Richard Harland (for S.M.P.) and E.M. Deposited in PMC for release after 12
months.
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