Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
* Author for correspondence (e-mail: j61056{at}hpc.cmc.osaka-u.ac.jp)
Accepted 2 August 2005
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SUMMARY |
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Key words: ZFHX1, Sip1, Lens, Foxe3, Smad8
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Introduction |
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Sip1 and EF1 are very similar in structure and share DNA-binding
sequence specificity to the E2-box-like motif CACCT(G) in vitro; their
activity as a transcriptional repressor has been demonstrated using several
reporter constructs (Comijn et al.,
2001
; Kamachi et al.,
1995
; Postigo and Dean,
1997
; Remacle et al.,
1999
; Sekido et al.,
1994
; Sekido et al.,
1997
; van Grunsven et al.,
2001
), suggesting a shared regulatory function. However,
EF1 lacks the SBD sequence and does not bind Smad proteins in vitro
(van Grunsven et al., 2003
),
suggesting a unique Smad-dependent regulation exerted by Sip1. Expression
patterns in the mouse embryo partly overlap but are generally diversified
between two protein genes, e.g. only Sip1 is expressed in the lens and null
mutant mouse phenotypes produced by targeted gene inactivation are distinct
(Takagi et al., 1998
;
Van de Putte et al.,
2003
).
Although the augmentation of the Sip1 binding of Smads by Alk
receptor-mediated phosphorylation through their MH2 domain has been
demonstrated (Verschueren et al.,
1999), whether or how the binding of a Smad protein affects
transcriptional regulation by Sip1 has not been elucidated. Although all
experiments using full-length Sip1 and CACCT(G)-containing target sequences
indicate a repression activity, supported by its binding to the co-repressor
CtBP (van Grunsven et al.,
2003
) as for
EF1
(Furusawa et al., 1999
), it
may still be only one of the functions of this multi-faceted protein.
During the embryonic development of mice, early Sip1 expression in
gastrula (e.g. E8.0) is observed primarily in the neural plate, neural crest
and paraxial mesoderm (Van de Putte et
al., 2003); however, late Sip1 expression occurs in
various tissues (T. Miyoshi, M. Maruhashi, T. Van de Putte, H.K., D.
Huylebroeck and Y.H., unpublished). The Sip1-null knockout mouse
embryos die around E9.5 after heart dysfunction and embryo turning failure
(Van de Putte et al., 2003
).
Thus, the floxed (flanked by loxP sites) Sip1 allele was generated
(Higashi et al., 2002
), which
enables cell-lineage-specific Sip1 inactivation.
As demonstrated in this study, in the lens lineage, Sip1 is
expressed after lens placode induction. The lens is a simple tissue and is one
of the best characterized in terms of transcriptional regulation
(Kondoh, 1999;
Kondoh, 2002
). The
lens-lineage-specific ablation of the floxed Sip1 gene can be
achieved by using the lens enhancer of Pax6
(Kammandel et al., 1999
;
Williams et al., 1998
) in
controlling Cre recombinase. Therefore, the consequence of lens-specific
Sip1 inactivation was investigated, and two steps in lens development
dependent on Sip1 activity were characterized: (1) the separation of the lens
epithelium and surface ectoderm by the removal of the connecting lens stalk;
and (2) the progression of lens fiber precursors in the bow region into
-crystallin-expressing mature fiber cells. In this study Foxe3
activation, which is involved in the first step, was demonstrated to be
dependent on Sip1 activity. The Foxe3 promoter was activated by Sip1
and this activation was augmented by the specific interaction of Sip1 with
Smad8 in a transfection assay. This study is the first clear demonstration
that Smad-Sip1 interactions are significant in transcriptional regulation.
Given evidence of the involvement of Sip1 in many important processes of
embryogenesis, not limited to the lens
(Eisaki et al., 2000;
Papin et al., 2002
;
Sheng et al., 2003
;
Van de Putte et al., 2003
;
van Grunsven et al., 2000
),
this study provides new insight into the regulatory functions of this
interesting transcription factor.
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Materials and methods |
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Immunohistology
Anti-crystallin antibodies were raised in rabbit by injecting the following
peptides ligated to keyhole limpet hemocyanin. Anti--crystallins
(recognizing both
A and
B), CVSREEKPSSAPSS;
anti-ßA-crystallins without cross-reaction to the ßB class,
CHAQTSQIQSIRRIQQ; and anti-
-crystallins, GKITFYEDRSFQGRC. The embryos
were fixed with 4% paraformaldehyde in phosphate-buffered saline at 4°C
overnight, dehydrated, embedded in paraffin and cut into 6 µm serial
sections. The sections were treated with anti-crystallin primary antibodies
and Alexafluor568-conjugated anti-rabbit IgG (Molecular Probe) antibodies,
stained for nuclei with DAPI and mounted in Permafluor anti-fade reagent
(Immunotech).
In situ hybridization
Embryo sections were hybridized with specific probes as described
previously (Uchikawa et al.,
1999). The full-length Sip1 cDNA probe
(Van de Putte et al., 2003
),
Pax6 3' UTR probe (Xu et
al., 1999
), Sox1 3' UTR probe
(XhoI-StuI fragment), and probes for Foxe3, Maf,
-crystallins and Pdgfra
(Yamada et al., 2003
) were
used.
TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling) assay
Apoptotic cells in histological sections were detected by the TUNEL
technique using an Apo-Alert DNA Fragmentation Assay kit (Clontech).
TUNEL-positive nuclei among DAPI (4',6-diamidino-2-phenylindole)-stained
nuclei in the anterior and posterior lens halves were counted in meridian
sections through lenses, and data of individual embryo specimens were
combined.
Transfection
Lens epithelial cells were prepared from E14 chicken embryos and cultured
for transfection as previously described
(Muta et al., 2002).
Collagen-coated 24-well plates were inoculated with one-fifth of the
epithelial cells derived from one lens per well. Similarly, E10 gizzard cells
were inoculated at 4x104 cells per well. A 1.5 µg
plasmid-DNA mixture for transfection, typically containing 100-200 ng of a
Foxe3 promoter-ligated GL3 firefly luciferase gene (Promega), 1-500
ng of effector plasmids and 10 ng of phRG-TK Renilla luciferase expression
plasmid, was transfected into cells in a well using 3 µg of Fugene6
(Roche). Luciferase activity was measured after 48 hours using a Dual
Luciferase Reporter Assay kit (Promega) and an LB940 Mithras Multilabel Reader
(Berthold Technologies), normalizing firefly luciferase activity using Renilla
luciferase. Transfection was carried out at least in triplicate. The activity
of Smad expression vectors (provided by Drs M. Kawabata and K. Miyazono)
driven by the elongation factor I enhancer/promoter was confirmed by the
activation of p3GC2-Lux (Smad1, 5 and 8) or p3TP-Lux (Smad 2 and 3)
(Ishida et al., 2000
) in lens
epithelial cells.
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Results |
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Lens-specific inactivation of Sip1 gene
To clarify the intrinsic functions of Sip1 in lens cells, the Sip1
gene was ablated in a lens-lineage-specific fashion, using embryos homozygous
for the floxed Sip1 allele, in which the action of Cre recombinase
causes the loss of detectable Sip1 protein
(Higashi et al., 2002)
(Fig. 2A).
|
When these Cre transgenes were introduced into homozygous floxed Sip1 embryos, they developed defective eyes; otherwise, they exhibited normal growth and fertility. In the homozygous floxed Sip1 mouse population, the Cre transgenes were transmitted according to the Mendelian ratio. The lens defect was identical between the two Cre transgenic lines, Pax6(LP)-Cre and Pax6(Lens)-Cre; therefore, data using these two Cre lines were combined and used in the following analysis.
Two major defects of lens development in the absence of Sip1 activity
The consequence of the loss of Sip1 activity in the lens lineage was
investigated at the histological level using various markers. Expression of
Cre did not interfere with lens placode development (E9.5) (data not shown) or
invagination (E10.5) (Fig.
3A,D); however, the first marked defect was observed at E11.5
(Fig. 3B,E) when the lens
vesicle was normally separated from the surface ectoderm by tissue
reorganization and local apoptosis involving the connecting lens stalk
(van Raamsdonk and Tilghman,
2000). The Cre recombinase action in the lens lineage was
confirmed using R26R mouse background. In the Sip1-defective lens, a
thick stalk connecting the vesicle and ectoderm was persistent, and the
vesicle was smaller (Fig. 3E).
The lens vesicle that developed in the floxed Sip1 embryo lacked
Sip1 expression in the surface ectoderm, stalk and anterior region of
the vesicle, but had some residual Sip1 expression in the posterior
half, as determined by in situ hybridization
(Fig. 3F). This is in contrast
to the uniform expression of Sip1 in the lens vesicle in the normal
embryo (Fig. 3C). Thus, in the
absence of Sip1 activity, the cells positioned in the lens stalk
persist.
At E14.5 and even at newborn (P0) stages the lens stalk still remains when Sip1 is inactivated in the lens lineage (Fig. 3I,J), and a defect in lens mass development is evident. A stalk-persistent lens produced in dyl/dyl (Foxe3 mutant) lens is shown in Fig. 3K for comparison.
When apoptotic cell distribution was measured using the TUNEL method from E10.5 to E12.5, apoptotic cells were mainly distributed in the anterior half of both normal and Sip1-defective lenses. In Sip1-defective lenses, the apoptotic cell population increased significantly, but the increment in apoptosis rate relative to that observed in the normal lens was comparable between the anterior and posterior halves at E10.5 and E11.5 without any specific suppression of apoptosis in the stalk region, and higher in the anterior half at E12.5 (Fig. 3L,M). Therefore, the increased apoptosis rate in Sip1-defective lenses accounts for the smaller lens size, but does not appear to contribute to either the persistence of lens stalk, or the specific loss of mature lens fibers in the posterior lens to be described below.
To clarify the cellular and molecular bases of lens development defects,
various lens markers were examined at the histological level. At E12.5,
anti-A/B-crystallin antibodies stained all cells of normal and
Sip1-defective lenses (Fig.
4A,F). ßA-crystallins are expressed at a low level in
immature fiber cells in the bow region where the Sip1 expression
level is high (Fig. 1E), and at
a high level in mature lens cells (Fig.
4B). In Sip1-defective lenses, only a low
ßA-crystallin expression level was observed in the posterior-most side of
the lens vesicle, which may correspond to the bow region of the normal lens.
Mature lens fiber cells are marked by
-crystallins in normal lenses
(Fig. 4C). The most distinct
characteristic of a Sip1-defective lens is the total absence of
-crystallin expression (Fig.
4H). This absence of
-crystallin expression was confirmed
using in situ hybridization (data not shown).
|
-Crystallin genes are regulated by Sox1 and Maf, which
synergistically function in the activation of their lens-specific promoters
(Kamachi et al., 1995
;
Kawauchi et al., 1999
;
Kim et al., 1999
;
Nishiguchi et al., 1998
;
Ring et al., 2000
), but the
inactivation of Sip1 does not affect expression of these
transcription factor genes, as indicated by in situ hybridization
(Fig. 4D,E,I,J).
Thus, two major defects were observed in the Sip1-defective lens: (1) persistent lens stalk; and (2) the arrest of lens fiber cell maturation at the bow-region stage.
Loss of Foxe3 activation in Sip1-defective lens
A persistent lens stalk is called Peter's anomaly in human congenital
diseases, and accompanies the inactivation of the transcription factor Foxe3
(Blixt et al., 2000;
Brownell et al., 2000
).
Homozygous dyl (dysgenetic lens) mice with a mutation in the
Foxe3 gene (Blixt et al.,
2000
; Brownell et al.,
2000
) or a low Pax6 activity affecting Foxe3 expression
(Brownell et al., 2000
;
Dimanlig et al., 2001
) are
documented examples. This prompted us to examine the possibility that
Foxe3 activity is affected in Sip1-defective lenses
(Fig. 5). In addition to
Foxe3 (Fig. 5A,E),
Pdgfra gene under Foxe3 regulation
(Blixt et al., 2000
)
(Fig. 5B,F), and an upstream
gene Pax6 required for Foxe3 expression
(Brownell et al., 2000
)
(Fig. 5C,G) were analyzed for
their expression using in situ hybridization.
|
Pax6 expression was basically unaffected in the
Sip1-defective lens (Fig.
5C,G). In contrast to Pax6 expression in the normal lens,
which is downregulated in the posterior region, Pax6 expression
prevails throughout the lens in Sip1-defective lens, but this
difference is accounted for by the lack of a mature fiber compartment in the
latter. Thus, Sip1 and Pax6 are both assigned as upstream regulator of
Foxe3. As Pax6-null embryos develop no lens structure
(Hill et al., 1991;
Hogan et al., 1986
), it was
not determined whether Sip1 itself is regulated by Pax6.
Activation of Foxe3 promoter by Sip1 thorough interaction with Smad8
The 6.2 kb 5' flanking region upstream of the SmaI site has
promoter activity sufficient for controlling the expression of a lacZ
transgene in the mouse lens (Brownell et
al., 2000). Whether the Foxe3 promoter is regulated by
Sip1 and whether this regulation depends on interaction with Smads were
investigated. The 6.2-kb promoter sequence was ligated to a luciferase
reporter gene (Fig. 6A), then
transfected with a Sip1 expression vector into primary-cultured lens
epithelial and gizzard (smooth muscle) cells of chicken embryos. Results using
these cells were similar and data for the lens epithelial cells are shown in
Fig. 6.
|
Under the same transfection conditions, various Smad proteins were
expressed by co-transfection (Fig.
6C). Smad1, Smad5 and Smad8, mediating BMP signals, and Smad2 and
Smad3, mediating TGFß signals, bind Sip1 in vitro through the SBD
(Verschueren et al., 1999). Of
these Smads, only Smad8 caused a significant Sip1-dependent activation of the
Foxe3 promoter, threefold more activation than Sip1 alone, while
Smad8 by itself had no effect on the Foxe3 promoter
(Fig. 6C). However, using
SBD-deleted Sip1, exogenous Smad8 did not augment Sip1-dependent activation
(Fig. 6C). Under the same
transfection condition, Smad1, Smad5 and Smad8 exhibited comparable levels of
transactivation of the 3GC2-luciferase reporter gene
(Fig. 6D). It was rather
unexpected that Smad1 or Smad5 had no significant effect under the
transfection condition, although they are generally considered to act
analogously to Smad8 (ten Dijke and Hill,
2004
). When an inhibitory Smad, Smad6 or Smad7, was co-expressed
with Smad8, the effect of Smad8 was cancelled and only the activation level
attainable by Sip1 alone remains (Fig.
6E). The effect of Smad8 was saturated at approximately threefold
the activation level attained by Sip1 alone, regardless of whether the
original activation level by Sip1 alone was sevenfold
(Fig. 6F) or nearly 20-fold
(Fig. 6F).
|
The 6.2 kb promoter region was divided into four blocks, A to D, from the proximal side, and the block responsible for activation by Sip1 and Smad8 was then investigated. The removal of the upstream blocks B to D did not greatly affect the activation of the Foxe3 promoter by Sip1 or by Sip1 plus Smad8, and the 1.26 kb block A promoter region is sufficient (Fig. 6G). As shown below using transgenic mouse embryos, block A is not involved in the lens-specific regulation of the Foxe3 promoter. The Sip1-dependent activation of the Foxe3 promoter and its augmentation by Smad8 is also observed in gizzard cells (see Fig. S1 in the supplementary material), confirming their tissue non-specific effect.
The shortening of the promoter sequence to 287 bp maintains the capacity
for Sip1-dependent activation and further augmentation by Smad8
(Fig. 6G). Further shortening
of the promoter to 127 bp resulted in the loss of response to Sip1. The
unrelated -crystallin promoter was not affected by either Sip1 or Sip1
plus Smad8 (Fig. 6G). This
observation suggests that the activation of the Foxe3 promoter by
Sip1 and Smad8 involves the proximal region.
Lens-specific regulation of Foxe3 promoter
When the 6.2 kb Foxe3 promoter was ligated to a lacZ
transgene construct and primary transgenic mouse embryos were produced, lens-
and brain-restricted lacZ expression was observed at E12.5
(Fig. 7A), confirming a
previous report (Brownell et al.,
2000). To investigate tissue-specific regulation, the effect of
deleting various blocks was examined (Fig.
7A). The removal of the most distal block D, resulting in the
promoter blocks A+B+C, did not have any appreciable effect, but further
deletion of block C leaving promoter blocks A+B caused a large decrease in the
expression level in the lens and the loss of expression in the brain. When
block B was removed from A+B+C blocks, leaving A+C blocks of the promoter
region, transgene expression in the lens and brain was indistinguishable from
that using the full 6.2 kb sequence. By contrast, with only the most proximal
block A, transgene expression was not observed. These results indicate that
block C includes the major lens-specific element and a brain element, and
block B contains a minor lens element, and that the combination of the
activity of these blocks with the Sip1-dependent, cell type nonspecific
activity of block A elicits Foxe3 expression in embryonic lenses, as
summarized in Fig. 7B.
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Discussion |
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In lens development, Sip1 is first activated in the lens placode, then after the lens vesicle is formed, Sip1 expression is confined to the vesicle without detectable expression in the surface ectoderm. After mature lens fibers develop, strong Sip1 expression is confined to the lens epithelium and bow region, and the expression is very low in the mature lens fibers (Fig. 1). The consequence of the lens-lineage-specific ablation of Sip1 revealed two major Sip1-dependent steps in lens development (Fig. 3), consistent with the Sip1 expression pattern.
The first significant defect is a persistent stalk connecting the surface
ectoderm and lens epithelium (Fig.
3). This defect, called Peter's anomaly as a congenital disease in
humans, is shared by defects in the transcription factors Pax6 or Foxe3, and a
common denominator is the loss of functional Foxe3
(Blixt et al., 2000;
Brownell et al., 2000
;
Dimanlig et al., 2001
). A Sip1
defect also causes the downregulation of Foxe3 in the lens epithelium
(Fig. 5). This observation
indicates that the Foxe3 gene is downstream of Sip1 in the
regulatory pathway.
The second defect of the Sip1-defective lens is lack of mature
lens fibers expressing -crystallins (Figs
3,
4). Sip1 is strongly
expressed in the bow region in the normal lens where ßA-crystallins are
already expressed, signifying the initiation step of fiber differentiation.
The bow region has been thought of as merely a zone of transition between the
epithelial fiber precursor and mature lens fibers. However, the arrest of lens
fiber differentiation in the ßA-crystallin-positive
-crystallin-negative bow region state strongly suggests that
Sip1 expression in immature fiber cells promotes lens fiber
maturation.
Regulation of Foxe3 promoter
The possible involvement of the Foxe3 promoter in the
Sip1-dependent activation of Foxe3 was examined, using cell
transfection. Up to 20-fold activation of the 6.2 kb Foxe3 promoter
was observed from exogenous Sip1 (Fig.
6A,B). For this activation, a 1.2 kb promoter sequence was
sufficient (Fig. 6F).
The activation of the Foxe3 promoter by exogenous Sip1 allowed
examination of the effect of exogenous Smads on its regulation. Of the Smads
that bind Sip1 in vitro (Verschueren et
al., 1999), only Smad8 further augmented Sip1-mediated
Foxe3 promoter activation by threefold
(Fig. 6). However, this Smad8
effect was not observed using SBD (Smad-binding domain)-deleted Sip1,
demonstrating the involvement of a direct Sip1-Smad8 interaction. Amino acid
sequence comparison of Smad8, Smad1 and Smad5 indicates that Smad8 has a
considerably shorter and diversified linker sequence between MH1 and MH2
domains than the other two (see Fig. S2 in the supplementary material).
However, given the demonstration of similar activities of Smad1, Smad5 and
Smad8 in various assays (Moustakas et al.,
2001
; ten Dijke and Hill,
2004
), it is possible that Smad1 and Smad5 also contribute to
Sip1-dependent gene regulation in different contexts. In any case, this is the
first clear demonstration that Smad interaction affects the regulatory
potential of the Sip1 protein.
|
The block C sequence has a region strongly conserved between mouse Foxe3 and human FOXE3, and with the aid of this sequence conservation, Grainger's group has independently identified the corresponding region in Xenopus as the lens element of the Foxe3 promoter (H. Ogino and R. Grainger, personal communication).
Gene activation involving Sip1 activity
Gene activation by the action of Sip1 shown in this study expands the
horizon of gene regulation involving ZFHX1 family transcription factors. Sip1
and EF1 bind almost identical sets of sequences, owing to their highly
conserved Krueppel-type zinc finger sequences
(Funahashi et al., 1993
;
Verschueren et al., 1999
). In
addition, the bipartite zinc-finger clusters each bind to a similar set of
sequences with a consensus of CACCT(G)
(Remacle et al., 1999
;
Sekido et al., 1997
), it has
been postulated that ZFHX1 proteins bind a pair of CACCT(G) sequences in a
two-footed fashion (Remacle et al.,
1999
). Under such conditions full-length Sip1 or
EF1
clearly exhibited the repression of gene transcription
(Comijn et al., 2001
;
Funahashi et al., 1993
;
Kamachi and Kondoh, 1993
;
Papin et al., 2002
;
Remacle et al., 1999
;
Sekido et al., 1994
;
Sekido et al., 1997
).
However, several lines of evidence support the view that the gene repression thorough a CACCT(G) pair is just one of many modes of regulatory function associated with ZFHX1 proteins.
(1) With a knockout allele of EF1 lacking C-terminal zinc fingers,
only a minor nonlethal phenotype develops in homozygous mouse
(Higashi et al., 1997
), in
contrast to more severe lethal defects with a null allele
(Takagi et al., 1998
). This
indicates that N-terminal zinc fingers are sufficient for DNA binding and
exerting a regulatory function.
(2) The binding consensus CACCT(G) of N- and C-terminal zinc fingers was
determined using oligonucleotide sequence pools preferentially binding to
respective zinc finger clusters (Sekido et
al., 1997). Re-examination of these sequences indicated that
N-terminal zinc fingers bind DNA with a more relaxed specificity, including,
for example, CACANNT.
(3) The 6.2 kb promoter sequence of Foxe3 contains frequent recurrent CACCT sequences, many located in blocks B, C and D, but the removal of these upstream blocks did not significantly affect the response to exogenous Sip1 (Fig. 6G).
It has not been determined whether the Sip1 protein directly binds to the Foxe3 promoter DNA, but further analysis of Foxe3 promoter activation will reveal how Sip1 is involved in gene activation and how interaction with Smad affects its regulatory potential.
Smad-Sip1 interaction in transcriptional regulation
Since the discovery of Sip1 as a Smad-binding protein, Smads have been
implicated in Sip1-mediated transcriptional regulation
(Verschueren et al., 1999),
but this study provides the first definitive evidence that Smad-Sip1
interaction has an impact on Sip1-dependent gene regulation.
The mechanism of augmenting the Sip1-dependent activation of the Foxe3 promoter from interaction with Smad8 is not clear, but this interaction is not required for basal activation by Sip1 (up to 20-fold activation of the Foxe3 promoter), as the same activation level is achieved using SBD-deleted Sip1 (Fig. 6B). A possible model would be that Sip1 itself possesses an activation domain that is exposed upon binding to a proper sequence, and Smad8 bound to Sip1 provides an additional activation domain.
|
Sip1 gene activity is implicated in many steps of embryogenesis:
gastrulation in chickens (Sheng et al.,
2003), mesodermal development in Xenopus
(Papin et al., 2002
) and
neural crest development in mice (Van de
Putte et al., 2003
). This study revealed important aspects of gene
regulation mediated by Sip1 leading to gene activation. Most importantly,
interaction with Smad proteins, at least with Smad8, modifies the
transcriptional regulation mediated by Sip1. These new features of
Sip1-mediated transcriptional regulation should help understanding of
processes involving Sip1.
![]() |
ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/20/4437/DC1
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