© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 1062-1072 doi:10.1242/dev.119685
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
teashirt is required for head-versus-tail regeneration polarity in
planarians
ABSTRACT
Regeneration requires that the identities of new cells are properly
specified to replace missing tissues. The Wnt signaling pathway
serves a central role in specifying posterior cell fates during planarian
regeneration. We identified a gene encoding a homolog of the
Teashirt family of zinc-finger proteins in the planarian Schmidtea
mediterranea to be a target of Wnt signaling in intact animals and
at posterior-facing wounds. Inhibition of Smed-teashirt (teashirt) by
RNA interference (RNAi) resulted in the regeneration of heads in
place of tails, a phenotype previously observed with RNAi of the
Wnt pathway genes β-catenin-1, wnt1, Dvl-1/2 or wntless. teashirt
was required for β-catenin-1-dependent activation of posterior
genes during regeneration. These findings identify teashirt as a
transcriptional target of Wnt signaling required for Wnt-mediated
specification of posterior blastemas.
KEY WORDS: Wnt, Neoblasts, Patterning, Planarians, Regeneration,
Teashirt
INTRODUCTION
A central issue in animal regeneration is how positional information
is maintained and regenerated for proper specification of cell types,
organs and tissue patterns (Wolpert, 1969; French et al., 1976;
Reddien, 2011). The planarian Schmidtea mediterranea is a
flatworm with nearly unlimited capacity to regenerate, and is an
emerging system for the study of positional information in
regeneration. Planarian regeneration requires a population of
proliferative cells called neoblasts, which includes pluripotent
stem cells (Wagner et al., 2011). Neoblasts can specialize to produce
lineage-specific progenitors for the regeneration of missing target
tissues (Lapan and Reddien, 2011; Scimone et al., 2011, 2014a;
Wenemoser et al., 2012; Cowles et al., 2013; Currie and Pearson,
2013; Reddien, 2013; van Wolfswinkel et al., 2014), and must
receive appropriate instructions during regeneration in order
to replace missing tissues correctly. The choice of whether to
regenerate a head or a tail at transverse amputation planes,
sometimes called regeneration polarity, is a classic problem in
regeneration and a paradigm for molecular dissection of the
positional information that guides regeneration (Morgan, 1898;
Reddien, 2011). In a fragment lacking both a head and a tail, anterior
cells must receive instructions to produce head tissues, whereas
posterior cells must be specified to form tail tissues. This
1
Howard Hughes Medical Institute, MIT Biology and Whitehead Institute for
Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA.
2
Department of Molecular Biosciences, Northwestern University, 2205 Tech Drive,
Hogan Hall Room 2-144, Evanston, IL 60208, USA.
*These authors contributed equally to this work
‡
Author for correspondence ([email protected])
Received 9 November 2014; Accepted 15 January 2015
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head-versus-tail regeneration decision occurs at transverse wounds
at essentially any position along the head-to-tail axis (Randolph,
1897; Morgan, 1898).
Wnt signaling regulates tissue identity in a wide array of
processes across many investigated metazoan phyla (Peifer et al.,
1991; Harland and Gerhart, 1997; Hobmayer et al., 2000;
Eisenmann, 2005; Stoick-Cooper et al., 2007; Petersen and
Reddien, 2009a). Wnts are expressed in the posterior during early
development and have a central role in patterning the primary axis in
many organisms (Petersen and Reddien, 2009a; Niehrs, 2010). In
planarian regeneration, Wnt signaling inhibits head and promotes
tail regeneration at posterior-facing wounds. RNAi of the Wnt
pathway components Smed-β-catenin-1 (β-catenin-1), Smed-wnt1
(wnt1, formerly wntP-1) and Smed-wntless (wntless) results in the
regeneration of heads at posterior-facing wounds (Gurley et al.,
2008; Iglesias et al., 2008; Petersen and Reddien, 2008, 2009b;
Adell et al., 2009). wnt1 expression in intact animals is restricted to a
set of cells near the tip of the tail referred to as the posterior pole
(Petersen and Reddien, 2008). Following injury, wnt1 transcription
is generically induced, such that wnt1 is expressed at essentially all
wound sites (Petersen and Reddien, 2009b). RNAi of the gene
Smed-notum (notum) results in the regeneration of tails in place of
heads, a phenotype opposite to that observed in wnt1 or β-catenin-1
RNAi animals (Petersen and Reddien, 2011). notum is expressed in
a cluster of cells at head tips referred to as the anterior pole (Petersen
and Reddien, 2011). Following injury, notum expression is
generically induced, but its expression is more robust at anteriorfacing wounds than at posterior-facing wounds (Petersen and
Reddien, 2011). For example, a flank incision that is allowed to seal
will induce notum transcription, but preferentially on the posterior
side of the incision (anterior facing). notum is a negative regulator of
Wnt signaling, inhibiting the pathway at anterior-facing wounds and
allowing head regeneration to occur at suitable wounds (Petersen
and Reddien, 2011). Whereas these early steps of the planarian
head-tail regeneration choice have been identified, little is known
about how the output of the Wnt pathway is modulated or what
downstream genes are targeted to mediate regeneration polarity.
Teashirt proteins are found across the Bilateria and can interact
with Wnt signaling in several developmental contexts. teashirt was
first identified in Drosophila and collaborates with homeotic genes
to control prothoracic segment specification (Fasano et al., 1991;
Roder et al., 1992; Andrew et al., 1994; de Zulueta et al., 1994;
McCormick et al., 1995). Drosophila teashirt mutants can have
segment polarity defects similar to those caused by disrupted Wnt
signaling, and these defects can be suppressed by ectopic stabilized
β-catenin (Fasano et al., 1991; Peifer et al., 1991; Gallet et al.,
1998). In Xenopus, a Teashirt homolog (XTsh3) is expressed at the
presumptive dorsal embryo side near the site of gastrulation. XTsh3
inhibition causes ventralization, a phenotype also seen following
perturbation of β-catenin (Onai et al., 2007). XTsh3 can enhance
Wnt signaling activity in a β-catenin-dependent manner (Onai et al.,
DEVELOPMENT
Jared H. Owen1, *, Daniel E. Wagner1,*, Chun-Chieh Chen1, Christian P. Petersen1,2 and Peter W. Reddien1,‡
RESEARCH ARTICLE
Development (2015) 142, 1062-1072 doi:10.1242/dev.119685
2007). In both flies and frogs, Teashirt can localize to the nucleus
and bind β-catenin, but its exact molecular function remains unclear
(Gallet et al., 1999; Onai et al., 2007). In other vertebrates, Teashirt
proteins have been implicated in myogenesis and in the
development of the nervous system, particularly the specification
of hindbrain structures; whether Teashirt functions with the Wnt
pathway is unknown in several of these contexts (Koebernick et al.,
2006; Caubit et al., 2010; Erickson et al., 2011).
Here, we present the characterization of a teashirt gene in
planarians. teashirt was previously unstudied in regeneration and in
non-Ecdysozoan protostomes. We present evidence that planarian
Smed-teashirt (teashirt) is an early target of Wnt signaling and is
necessary for the output of Wnt signaling in planarian regeneration,
promoting tail and inhibiting head regeneration.
The planarian Schmidtea mediterranea genome encodes one
Teashirt-family zinc-finger protein. Smed-teashirt encodes a
predicted 1107 amino acid protein, with 23.9% sequence
identity to Drosophila Teashirt and 23.5% sequence identity to
Xenopus XTsh3. The sequence contains two Cx2Cx12HMx4Htype zinc-finger motifs, sharing unique domain structure with
other Teashirt family proteins (supplementary material Fig. S1).
The presence of multiple copies of this atypical zinc-finger motif
is a hallmark of Teashirt family proteins (Manfroid et al., 2004).
Smed-Teashirt lacks the basic motif, the homeodomain and the
second C-terminal zinc finger found in vertebrate orthologs,
which are also lacking in Drosophila Teashirt and the Drosophila
Teashirt paralog Tiptop (Onai et al., 2007). The three mouse
teashirt genes can rescue the Drosophila teashirt loss-of-function
phenotype, suggesting that Teashirt proteins have similar
biochemical activities in highly divergent animals (Manfroid
et al., 2004). We also identified an 11 amino acid domain
(NPLSXLQSVM) in planarian Teashirt that is present in all
known vertebrate orthologs (82% identity) but absent from
Ecdysozoan orthologs (supplementary material Fig. S1).
Teashirt and regeneration polarity
RNAi of teashirt resulted in a highly penetrant polarity phenotype
with heads being regenerated at posterior-facing wounds (Fig. 1A;
supplementary material Fig. S2; n=36/49 trunks). teashirt RNAi
with head fragments (see Materials and Methods, 4-day RNAi
protocol) also yielded a two-headed phenotype (19/47 two-headed
and 18/47 abnormal tail blastemas; 38/38 controls were normal).
In situ hybridization to detect the neuronal marker choline
acetyltransferase (Smed-chat) showed that teashirt(RNAi)
animals regenerated a symmetrical nervous system with brains at
both ends of the primary axis, similar to the β-catenin-1(RNAi)
phenotype (Fig. 1B; supplementary material Fig. S3). The ectopic
presence of anterior marker expression (Smed-sFRP-1) and the
absence of posterior marker expression (Smed-wntP-2, also known
as wnt11-5) in teashirt(RNAi) posterior blastemas confirmed that
animals regenerated heads in place of tails (Fig. 1C,D). wntP-2/
wnt11-5 orthology is difficult to assign, making it unclear whether
this gene encodes a Wnt11-family member (Gurley et al., 2010);
we refer to the gene here as wntP-2 or wntP-2/wnt11-5. Less
stringent RNAi resulted in additional posterior defects, including
lack of a blastema, a misshapen blastema or partial head
regeneration (supplementary material Fig. S2). Similar posterior
blastema defects have also been described for wnt1(RNAi) animals
(Adell et al., 2009; Petersen and Reddien, 2009b). teashirt RNAi
Fig. 1. teashirt RNAi causes posterior blastema polarity reversal. (A) Trunk
fragments 12 days after amputation. Control RNAi trunks regenerated posterior
tails (n=56/56), whereas teashirt(RNAi) trunks regenerated posterior heads
with eyes (white arrowheads) (n=36/49). (B) teashirt(RNAi) animals
regenerated cephalic ganglia in anterior and posterior blastemas, shown by
in situ hybridization with chat. (C,D) teashirt(RNAi) animals expressed the
anterior marker sFRP-1 at both ends (C) and did not express the posterior
marker wntP-2 (D) (100% of double-headed animals; n>4, each marker).
(A-D) Animals viewed dorsally; anterior is leftwards; dotted lines indicate
approximate blastema/old tissue boundary. (E) Weak individual wnt1 and
teashirt RNAi using dsRNA diluted to one-third of the working concentration
with control (C. elegans unc-22) dsRNA failed to cause regeneration of twoheaded planarians (n=8, each). By contrast, combining the diluted dsRNA for
each gene (one-third wnt1, one-third teashirt, one-third unc-22) caused
regeneration of two-headed animals (n=4/8 with another animal displaying
failed posterior regeneration). Posterior is downwards. (F,G) Fluorescence
in situ hybridization for cintillo (Oviedo et al., 2003) showed a decrease in
putative mechanosensory cells in teashirt(RNAi) and β-catenin-1(RNAi) 7-day
anterior blastemas (Hoechst marks nuclei in white). (H) Fluorescence in situ
hybridization for the pigment cup marker tph showed mis-shaped eyes in both
teashirt (n=5/14) and β-catenin-1 (n=4/6) RNAi 7-day anterior blastemas. Scale
bars: 200 µm in A-D; 500 µm in E; 50 µm in F,H. Error bars indicate s.d.
combined with weak RNAi of wnt1 (under conditions that do not
cause a two-headed regeneration phenotype for RNAi of either
gene alone) caused enhancement of the phenotype (two-headed
animals were observed), demonstrating partial Wnt signaling
perturbation can cause synthetic defects when combined with
partial teashirt inhibition (Fig. 1E).
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DEVELOPMENT
RESULTS
Planarian Teashirt
Previously, inhibition of genes encoding core components of the
Wnt secretion and signal transduction pathway (β-catenin-1, wnt1,
Dvl-1/2 and wntless) was shown to result in the regeneration of
posterior heads (Gurley et al., 2008; Petersen and Reddien, 2008,
2009b; Adell et al., 2009). The similarity of the teashirt(RNAi)
phenotype to the phenotype caused by inhibition of these core Wnt
pathway components therefore raises the possibility that teashirt has
an essential role in the outcome of Wnt signaling in planarian
regeneration. In Xenopus, a Teashirt protein can act with β-catenin
to establish the dorsoventral (DV) axis, and Drosophila teashirt
mutants mis-specify certain tissues along the anteroposterior (AP)
axis (Fasano et al., 1991; Onai et al., 2007); however, teashirt genes
were not previously known to be required for establishment of the
polarity of an AP axis, and this raises the possibility that, like Wnt
signaling (Petersen and Reddien, 2009a), Teashirt proteins will be
involved in AP axis polarization in many metazoans.
Teashirt and other Wnt-dependent processes
Teashirt proteins can have roles in many developmental contexts,
including Drosophila eye, wing disk and gut development, as well
as hindbrain patterning in zebrafish and myogenesis in mouse (Pan
and Rubin, 1998; Erkner et al., 1999; Waltzer et al., 2001; Singh
et al., 2002; Wu and Cohen, 2002; Erickson et al., 2011; Faralli
et al., 2011). We therefore examined tissue markers in regenerating
teashirt(RNAi) animals for additional defects. Both teashirt and
β-catenin-1 RNAi animals showed significant decreases in the
number of head rim candidate mechanosensory cells (cintillo+)
(Fig. 1F,G). In addition, both teashirt and β-catenin-1 RNAi
resulted in minor morphology defects in regenerated eyes (Fig. 1H).
Drosophila teashirt, together with wingless, dpp, eyeless and
homothorax, is known to control eye morphogenesis (Singh et al.,
2002; Bessa and Casares, 2005). Overall brain morphology and the
presence of other neuronal lineages (e.g. cholinergic and
serotonergic), however, were largely unaffected by teashirt or
β-catenin-1 RNAi (supplementary material Fig. S3). These data
indicate that in addition to polarity phenotypes, RNAi of Smedteashirt and β-catenin-1 can also result in similar nervous system
defects.
teashirt affects the expression of anterior and posterior
position control genes during tail regeneration
The teashirt(RNAi) polarity defects suggest Teashirt might regulate
expression of genes at posterior-facing wounds that are involved in
the specification of head and tail tissue identity. There are numerous
genes expressed regionally and constitutively in adult planarians
that are candidates to specify positional information to tissues and
are referred to as position control genes (PCGs) (Reddien, 2011;
Witchley et al., 2013). PCGs display regional expression and are
implicated in patterning, either because PCG RNAi causes a
patterning phenotype or because the PCG encodes a predicted
component of a pathway associated with planarian patterning
(Reddien, 2011; Witchley et al., 2013). PCGs are expressed in
planarian body wall muscle cells and define a candidate ‘GPS’-like
coordinate system of positional information (Witchley et al., 2013).
We examined the expression of PCGs that normally display
anterior- or posterior-restricted expression during regeneration in
teashirt(RNAi) animals.
wnt1 is wound induced as the first known step in posterior
regeneration (Petersen and Reddien, 2009b). notum is expressed
preferentially at anterior-facing wounds by 6 h of regeneration and
is the earliest known anterior regeneration-specific step (Petersen
and Reddien, 2011). Polarized notum expression is β-catenin
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Development (2015) 142, 1062-1072 doi:10.1242/dev.119685
dependent (Petersen and Reddien, 2011). Wound-induced wnt1 and
notum expression was similar in teashirt and control RNAi trunk
fragments, suggesting that the teashirt(RNAi) phenotype is not
explained by a defect in regulation of this phase of the head-versustail regeneration decision (Fig. 2A,B). Both wnt1 and wntP-2
promote posterior fate in regeneration, and wnt1 promotes wntP-2
expression (Petersen and Reddien, 2009b). teashirt RNAi resulted
in decreased wntP-2 expression at posterior-facing wounds 48 h
after amputation (Fig. 2C). Overall, we did not detect a requirement
for teashirt in the β-catenin-1-dependent expression of notum early
at anterior-facing wounds but did detect a requirement for teashirt in
early expression of a posterior-specific PCG during regeneration
(Fig. 2C).
We next used qRT-PCR to examine the expression of multiple
PCGs during regeneration. nou darake (ndk), ndl-4, sFRP-1,
notum and prep were selected as anterior PCGs (Cebrià et al.,
2002; Gurley et al., 2008; Petersen and Reddien, 2008, 2011; Rink
et al., 2009; Felix and Aboobaker, 2010); wnt2 and ndl-3 were
selected as prepharyngeal PCGs (Petersen and Reddien, 2008;
Rink et al., 2009); and wntless, wntP-2, frizzled-4 ( fz-4), axinB
and wnt1 were selected as posterior PCGs (Gurley et al., 2008;
Petersen and Reddien, 2008; Adell et al., 2009; Iglesias et al.,
2011). We used a highly parallel microfluidic qRT-PCR platform
(Fluidigm) to assess gene expression in wild-type, control, βcatenin-1 and teashirt RNAi animals (supplementary material
Fig. S4). At 72 h of regeneration, a timepoint that allowed robust
detection of regenerative PCG expression by qRT-PCR, teashirt
and β-catenin-1 RNAi animals deviated from the control in a
similar manner (Fig. 2D). Specifically, in head fragments
regenerating a tail, many anterior PCGs were expressed more
highly in teashirt and β-catenin-1 RNAi animals than in the
control, whereas some posterior PCGs were expressed at lower
levels in teashirt and β-catenin-1 RNAi animals than in the control
(Fig. 2D). In general, the effect of β-catenin-1 RNAi on PCG gene
expression during regeneration was more robust than teashirt
RNAi, which is expected, given the incomplete penetrance of the
teashirt RNAi phenotype. Whereas most genes behaved similarly
in teashirt and β-catenin-1 RNAi animals, the effects of RNAi of
these two genes on ndl-3 expression were opposite. ndl-3
expression differs from the other PCGs in that its expression is
largely prepharyngeal and extends from the anterior into the
midbody (Rink et al., 2009); these results indicate that regulation
of this gene might differ from the other PCGs. Possibly because of
its low levels of expression at posterior wounds, notum levels were
variable between biological replicates. However, notum behaved
similarly to posterior (rather than anterior) PCGs. Whereas notum
expression during regeneration is preferentially at anterior-facing
wounds at early (6-18 h) timepoints, it is also expressed at low
levels during posterior regeneration (Petersen and Reddien, 2011).
These results suggest that expression of notum during posterior
regeneration is Wnt signaling dependent.
We next used principal component analysis (PCA) to assess
underlying transcriptional signatures in teashirt(RNAi) regenerating
tissues. PCA was performed using transcript levels for 12 PCGs and
compared 72 h anterior- or posterior-facing wound sites from wildtype, control RNAi, β-catenin-1 RNAi and teashirt RNAi animals.
Contributions of each of the 12 PCGs to the first two principal
components are illustrated in a loadings plot (Fig. 2E, left). With the
exception of ndl-3, which is expressed prepharyngeally, and notum,
for which expression is β-catenin-1 dependent, anterior and
posterior-specific PCGs are oppositely weighted in the first
principal component (PC1). PC1 explains the majority (>65%) of
DEVELOPMENT
RESEARCH ARTICLE
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Development (2015) 142, 1062-1072 doi:10.1242/dev.119685
the total variance in the dataset, and distinguishes anterior- from
posterior-facing wounds for wild-type and control RNAi specimens
(Fig. 2E, right panel). Posterior-facing wounds from teashirt and
β-catenin-1 RNAi animals did not cluster with posterior-facing
wounds in control animals. Instead, these samples more closely
resembled the expression signature of anterior-facing wounds
(Fig. 2E). We conclude that teashirt and β-catenin-1 are similarly
required for the differential expression of anterior and posterior
PCGs during tail regeneration.
teashirt is expressed in the posterior
In uninjured adults, teashirt mRNA was present in a broad gradient
along the AP axis with the signal strongest in the tail (Fig. 3A).
teashirt was expressed more broadly than previously known PCGs,
including in the central nervous system (cephalic ganglia and nerve
cords), in the pharynx (and strongly just anterior to the pharynx), in
the dorsal head rim and in the epidermis (Fig. 3A,B). Posterior
teashirt expression was present in subepidermal cells, which
included but was not restricted to muscle cells marked by
collagen expression (Fig. 3C). The posterior-to-anterior graded
expression and strong expression at the anterior end of the pharynx
is similar to the wntP-2-expression pattern (Petersen and Reddien,
2008) (Fig. 1D), and these two genes were co-expressed in cells at
the anterior end of the pharynx compartment (Fig. 3D).
We next assessed teashirt expression during regeneration.
Consistent with its elevated posterior expression in uninjured
animals, teashirt levels became elevated at posterior-facing wounds
by 16-24 h post-amputation (Fig. 4A). In amputated tail fragments,
in which teashirt is initially broadly expressed, the posterior teashirt
expression domain retracted towards the posterior pole over time
(Fig. 4A). This process of gene expression retraction to the
posterior, and away from anterior-facing wounds, likely reflects the
rescaling of existing tissues to a smaller total body size in a
regenerative process called morphallaxis (Reddien and Sánchez
Alvarado, 2004). Irradiation can ablate all proliferating cells
(neoblasts) in adult planarians and blocks regeneration (Dubois,
1949; Reddien et al., 2005). Dynamic expression of several PCGs
still occurs in amputated animals exposed to irradiation (Petersen
and Reddien, 2009b; Gurley et al., 2010; Witchley et al., 2013),
demonstrating that some PCG expression domain changes can occur
in existing differentiated tissue after injury. Dynamic teashirt
expression features during regeneration (at posterior-facing wounds
and rescaling in tail fragments) were also observed in amputated
irradiated fragments (Fig. 4B,C).
We next used qRT-PCR to compare the expression of teashirt
during regeneration with the expression of several anterior and
posterior-specific PCGs during regeneration. Time course analysis
of PCG expression at anterior- and posterior-facing wounds
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DEVELOPMENT
Fig. 2. teashirt is required for normal PCG
expression during tail regeneration.
(A) teashirt(RNAi) trunk fragments properly
expressed wnt1 12 h after amputation (teashirt RNAi:
6/6 anterior- and posterior-facing wounds; control:
7/7 anterior-facing and 6/7 posterior-facing
wounds). Anterior is upwards. Scale bars: 200 µm.
(B) Irradiated teashirt(RNAi) trunks properly
expressed notum 14 h after amputation (n=11/12).
Anterior is leftwards. Scale bars: 200 µm.
(C) teashirt RNAi resulted in decreased wntP-2
expression at posterior wounds in irradiated head
fragments after 48 h of regeneration (n=16/31, three
experiments). Anterior is upwards. Scale bars:
200 µm. (D) Quantitative RT-PCR (qRT-PCR) of
position control genes (PCG) following teashirt or
β-catenin-1 RNAi. Cartoon displays PCG
expression domains. RNA was harvested from
dissected posterior-facing wound sites of
dsRNA-injected head fragments 72 h
post-amputation. Biological triplicates consisted of
pooled wound sites from six to nine individual
animals. Data depict mean log2-fold changes in
transcript levels between teashirt or β-catenin-1
RNAi specimens and control RNAi specimens.
Error bars indicate s.d. Individual replicates are
indicated by dots. Asterisks indicate significance
determined by t-tests between sets of biological
triplicate delta-Ct values (*P<0.05) and with
standard Bonferroni adjustment (**P<0.0042).
(E) Principal component analysis (PCA) of PCG
transcriptional signatures in 72 h post-amputation
wound sites. Left: PCA loadings indicate relative
contributions of each gene to principal components
1 and 2. Right: PCA plot. Each dot represents a
pooled wound site sample. Colors and shapes
indicate anterior versus posterior wound orientation
or RNAi treatment. Percentages of total dataset
variance explained by each principal component
are shown. Plot regions corresponding to anterior or
posterior-specific transcriptional states are
indicated by shaded ellipses.
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Development (2015) 142, 1062-1072 doi:10.1242/dev.119685
hybridization some teashirt expression was apparent at 12 h or 24 h at
all wound sites, including at anterior-facing wounds and side incisions
(Fig. 4E; supplementary material Fig. S5B,C). This expression was not
strong enough to be detected above the basal overall level of teashirt
expression in all tissues of wound-site fragments used in qRT-PCR
experiments. These data indicate that teashirt expression is also
moderately wound induced. Over time, after this wound-induced
phase teashirt expression became strong at posterior-facing wounds
and was largely restricted to clusters of apparent regenerating cephalic
ganglia at anterior-facing wounds (Fig. 4E).
The teashirt expression pattern has three key characteristics that are
consistent with its role as a factor involved in positional identity
specification during regeneration: regionalized expression in uninjured
animals, polarized expression during regeneration of heads and tails,
and expression during early regeneration that is independent of new
cell production. Few genes (including notum, wnt1, wntP-2, wntless
and bmp4) are known to possess these expression characteristics and an
axial tissue-identity RNAi phenotype in regeneration (Molina et al.,
2007; Orii and Watanabe, 2007; Reddien et al., 2007; Adell et al.,
2009; Petersen and Reddien, 2009b, 2011; Gurley et al., 2010).
Sharing a relatively rare set of expression characteristics with this
exclusive group points to teashirt as an important regulator of early
tissue identity specification in regeneration.
Fig. 3. teashirt is expressed in the planarian posterior. (A) Whole-mount
in situ hybridization for teashirt showed graded expression highest at the
posterior end. teashirt was also expressed anterior to the pharynx, and in the
brain and nerve cords (ventral view). Arrowheads indicate the signal in folds of
epidermis. Anterior is upwards. Scale bars: 200 µm. (B) Single confocal planes
following fluorescence in situ hybridization show teashirt expression in the
brain, dorsal head rim cells, anterior to and in the pharynx, and in the
epidermis. Anterior is leftwards. Scale bars: 50 µm. (C) Double fluorescence
in situ hybridization showed teashirt and collagen co-expression in some
(yellow arrowheads), but not all (white arrowheads), teashirt+ subepidermal
cells (single confocal planes). DAPI-labeled nuclei are in gray. Scale bars:
10 µm. (D) Double fluorescence in situ hybridization showed that teashirt and
wntP-2 were co-expressed in cells anterior to the pharynx. Scale bar: 20 µm.
confirmed anterior regeneration-biased elevation of sFRP-1, ndk,
wnt2, ndl-3 and ndl-4, and posterior-biased elevation of wntless,
wnt1, axinB, fz4, wntP-2 and teashirt (Fig. 4D). Consistent with
previous observations (Adell et al., 2009; Petersen and Reddien,
2009b), transient early (by 6 h post-injury) wound-induced
expression of wntless and wnt1 at anterior-facing wounds was
also apparent. Most expression change trends were similar in
irradiated animals, confirming that dynamic expression features of
many PCGs at wounds are largely independent of new tissue
formation. Hierarchical clustering revealed that teashirt expression
patterns were highly similar to those of other posterior PCGs
(Fig. 4D). Posterior teashirt expression occurred after the woundinduced phase of wnt1 expression (∼6-12 h), but before wnt1
expression becomes polarized to be present only in posterior-facing
blastemas (∼48 h) in regenerating posterior poles (Petersen and
Reddien, 2009b). Posterior teashirt expression also occurred at a
similar time or before the detected expression of other known
posterior genes (Fig. 4D; supplementary material Fig. S5A), such as
wntP-2 (Petersen and Reddien, 2009b). During head regeneration,
two small symmetrical teashirt expression domains appeared in
anterior-facing blastemas, probably representing expression in the
developing brain (Fig. 4E).
By qRT-PCR teashirt showed a robust expression increase at
posterior- but not anterior-facing wounds. However, by in situ
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Constitutive expression of tissue patterning factors could provide
a system for maintaining proper regional tissue identity during
cell turnover and for maintaining body proportions during growth
and degrowth (Reddien, 2011). Consistent with this hypothesis,
β-catenin-1 RNAi causes gradual loss of AP polarity in uninjured
animals, eventually resulting in hypercephalized animals (Gurley
et al., 2008; Iglesias et al., 2008; Petersen and Reddien, 2008).
This suggests that Wnt signaling maintains positional information
along the AP axis during homeostatic tissue turnover. The
posterior teashirt expression domain was substantially reduced 4
days after RNAi of β-catenin-1 in uninjured animals (Fig. 5A).
This rapid change occurred prior to the loss of posterior cell types
[assessed by continued expression of Smed-fibrillin ( fibrillin) or
Smed-mag-1 (mag-1)], and before detectable anteriorization
occurred (assessed with sFRP-1 expression) (Fig. 5A). Other
posterior PCG expression (wnt11-1 and wnt1) was not robustly
decreased at this early time point (4 days) following β-catenin-1
RNAi (supplementary material Fig. S6). Together, these data
indicate that the loss of posterior teashirt expression in β-catenin1(RNAi) animals is the consequence of reduction in Wnt
signaling, rather than an indirect consequence of a gradual loss
of posterior tissue. teashirt inhibition in uninjured animals
resulted in an enlarged pharynx, occurring at low penetrance
(n=4/52) (supplementary material Fig. S7), and we did not detect
expression abnormalities for posterior markers (wntP-2, frizzled-4
and plox4) in uninjured teashirt(RNAi) animals (supplementary
material Fig. S7). However, it is possible that the lack of effect in
tissue turnover is the consequence of incomplete inhibition by
RNAi.
APC is a negative regulator of β-catenin protein stability in the
Wnt signaling pathway (Stamos and Weis, 2013). Inhibition of
β-catenin-1 and APC cause opposite phenotypes in planarians, with
APC RNAi causing regeneration of tails in place of heads (Gurley
et al., 2008). Inhibition of β-catenin-1 and APC in uninjured animals
resulted in opposite changes in many PCGs by 10 days of RNAi
(Fig. 5B). β-catenin-1 RNAi resulted in general upregulation of
anterior PCGs and downregulation of posterior PCGs, whereas
DEVELOPMENT
Posterior teashirt expression requires Wnt signaling
RESEARCH ARTICLE
Development (2015) 142, 1062-1072 doi:10.1242/dev.119685
APC(RNAi) caused the opposite effect. teashirt was downregulated
in β-catenin-1(RNAi) animals similar to the case for other posterior
PCGs (Fig. 5B). In APC(RNAi) animals, four different primer pairs
for teashirt consistently showed moderate upregulation of teashirt
but the effect was modest and not significant for all primer sets
(Fig. 5B). In general, APC RNAi caused weaker PCG expression
changes than did β-catenin-1 RNAi in these experiments in
uninjured animals.
Whereas the posterior-specific teashirt expression domain was
dependent upon β-catenin-1, expression of teashirt in the nervous
system did not decrease following β-catenin-1 RNAi (Fig. 5A). In
β-catenin-1(RNAi) head fragments, teashirt was expressed in newly
regenerated posterior brains and maintained in pre-existing anterior
brains, suggesting that Wnt-independent regulation of teashirt
transcription in the nervous system exists (Fig. 5C). In Drosophila,
teashirt expression in the trunk is initially Wnt dependent, but
becomes Wnt independent as development progresses, and
transcription factors such as Antennapedia and Eyeless can
positively regulate its expression (Roder et al., 1992; Gallet et al.,
1998; Pan and Rubin, 1998).
1067
DEVELOPMENT
Fig. 4. teashirt is expressed in the posterior during regeneration. (A) In regenerating fragments viewed dorsally, new teashirt expression occurred at posterior
wounds by 16 h (n=21/27). By 48 h, teashirt expression in tail fragments retracted towards the posterior. (B) teashirt expression at posterior-facing wounds also
occurred in irradiated animals (n=45/56), and expression also retracted towards the tail tip at 48 h. (C) Top, log2-fold changes in teashirt levels for each timepoint
relative to time zero. Error bars indicate s.d. Asterisks denote significance determined by t-tests between each timepoint and time zero using sets of biological
triplicate delta-Ct values. Colors indicate wound orientation. Bottom, four independent primer sets for teashirt display similar trends. (D) Heatmap of gene
expression changes during anterior or posterior regeneration, determined by qRT-PCR. Total RNA was harvested from wound sites at specified times postamputation. Some specimens were γ-irradiated (6000 rads) 4 days prior to amputation, as indicated. Heatmap displays mean log2-fold changes in transcript
levels between each timepoint and time zero. Dendrogram reflects hierarchical clustering of genes based on Euclidean distance. Anterior and posterior-specific
PCGs are indicated in magenta and green, respectively. (E) Whole-mount in situ hybridization showing teashirt wound-site expression at 0, 12 and 72 h postamputation (hpa). Short development times were used to allow detection of wound-site expression without other expression locations obscuring signal.
Background level was determined with a teashirt ‘sense’ probe (see supplementary material Fig. S5B). Above-background teashirt expression at wounds was
evident at 12 and 72 hpa. Anterior expression of teashirt at 72 hpa was confined to presumptive brain regions. Scale bars: 200 μm.
RESEARCH ARTICLE
Development (2015) 142, 1062-1072 doi:10.1242/dev.119685
catenin-1(RNAi) animals than in the control, with the effect most
pronounced at posterior-facing wounds (Fig. 6B). teashirt RNAi did
not affect β-catenin-1 transcript levels (supplementary material
Fig. S8). Conversely, RNAi of APC caused increased teashirt
expression at anterior-facing wounds compared with the control
(Fig. 6B). We next assessed the effects of β-catenin-1 and APC
RNAi on both the posterior induction and the anterior rescaling of
teashirt by directly comparing transcript levels at 72 h with those at
0 h post-amputation. teashirt levels failed to change from its already
low levels during regeneration in β-catenin-1(RNAi) animals, and
were elevated with time at both wound types in APC(RNAi) animals
(Fig. 6C; supplementary material Fig. S8). Together, these findings
demonstrate that teashirt transcriptional regulation at wounds in
planarian regeneration is positively influenced by Wnt signaling.
Fig. 5. Posterior teashirt expression is dependent on Wnt signaling.
(A) Posterior teashirt expression was reduced by 4 days after RNAi of
β-catenin-1, whereas brain expression persisted (n=12/15). This occurred
before loss of posterior cell types, indicated by in situ hybridization for fibrillin
(n=10/10) and mag-1 (n=12/12). Anterior-restricted sFRP-1 expression was
not altered, indicating that major polarity defects have not yet occurred (n=7/7).
(B) qRT-PCR analysis of position control gene (PCG) transcriptional changes
following β-catenin-1 or APC RNAi in intact uninjured animals. RNA was
harvested from uninjured animals after 10 days of RNAi. Biological triplicates
consisted of four individual animals each. teashirt transcriptional changes were
measured by four independent primer sets (I-IV). Error bars indicate s.d.
Individual replicates are indicated by dots. Asterisks indicate significance
determined by t-tests between sets of biological triplicate delta-Ct values
(*P<0.05) and with standard Bonferroni adjustment (**P<0.0031). (C) teashirt
was expressed in both anterior and posterior brains of head fragments
subjected to β-catenin-1 RNAi (n=8/9). (A,C) Anterior is leftwards. Scale bars:
200 µm.
Wnt signaling promotes teashirt expression during tail
regeneration
We next tested whether teashirt expression during regeneration
was dependent upon Wnt signaling. First, we assessed teashirt
expression in irradiated head fragments 48 h after amputation. Both
β-catenin-1(RNAi) and wnt1(RNAi) head fragments showed
decreased or absent teashirt expression (Fig. 6A). We next
assessed teashirt expression at anterior-facing and posterior-facing
wounds (from tail and head fragments, respectively) by qRT-PCR.
At 72 h after injury, teashirt displayed lower expression in β1068
The choice made at transverse amputation planes to regenerate a
head or a tail in planarians has emerged as a paradigm for studying
the molecular logic of how wounds initiate suitable regeneration
programs (Reddien, 2011). This process requires Wnt signaling in
planarians, with homeotic phenotypes (replacing heads with tails or
vice versa) caused by activating or inhibiting the Wnt pathway.
Here, we describe the identification of an additional gene, teashirt,
that is required for this head-tail regeneration decision. The teashirt
RNAi phenotype is highly similar to that caused by Wnt pathway
inhibition in planarians at the molecular and regeneration outcome
levels. Furthermore, teashirt transcriptional activation during
posterior regeneration requires Wnt signaling. The preferential
expression of teashirt during posterior regeneration occurred after
initial wound-induced activation of wnt1. teashirt was required for
the subsequent activation of posterior-specific position control
genes, such as wntP-2. Therefore, teashirt is regulated by Wnt
signaling and is required for the output of Wnt signaling in
regeneration (Fig. 7). We propose a model for the initial phases of
planarian head-versus-tail regeneration in which teashirt is required
for proper PCG expression (Fig. 7). In this model, wounding
initiates wnt1 expression 3-6 h following injury. Notum inhibits
Wnt signaling at anterior-facing wounds to promote head
regeneration. At posterior-facing wounds that will regenerate a
tail, Wnt signaling activates a program of gene expression involving
posterior PCGs, rather than anterior PCGs. teashirt is required for
this Wnt-dependent gene expression program.
Anterior and posterior poles are small clusters of cells at the head
and tail tip, respectively, and are required for normal planarian head
and tail regeneration and PCG expression (Felix and Aboobaker,
2010; Hayashi et al., 2011; Blassberg et al., 2013; Chen et al., 2013;
Scimone et al., 2014b; Vásquez-Doorman and Petersen, 2014;
Vogg et al., 2014). However, in contrast to teashirt RNAi, inhibition
of either anterior or posterior pole formation does not cause a
reversal of the head-tail regeneration choice. Unlike inhibition of
pole formation, inhibition of β-catenin-1 or teashirt causes a gene
expression signature switch: posterior-facing wounds not only fail
to express tail PCGs but inappropriately express head PCGs. These
findings suggest that Wnt signaling and teashirt regulate a broad
program of gene expression events – giving wounds anterior or
posterior character – and that pole formation is just one component
of this program.
The Teashirt family and Wnt signaling
teashirt encodes a broadly conserved zinc-finger protein, with
orthologs found throughout the animal kingdom. In other
DEVELOPMENT
DISCUSSION
teashirt and the head-versus-tail regeneration decision
RESEARCH ARTICLE
Development (2015) 142, 1062-1072 doi:10.1242/dev.119685
organisms, it has been suggested that Teashirt can function to
increase the relative strength of the cellular response to Wnt
(Gallet et al., 1998; Onai et al., 2007). We found that teashirt in
planarians is required for the outcome of β-catenin-dependent
processes during regeneration. Planarians are members of the
superphylum the Lophotrochozoa. Xenopus is a chordate and part
of the deuterostomes, and Drosophila is an arthropod and a
representative from the ecdysozoans. Thus, whereas teashirt is still
understudied, connections between Teashirt proteins and the
outcome of Wnt signaling now exist for all three major branches of
the Bilateria. This raises the possibility that Teashirt proteins will
be involved in many Wnt-dependent processes throughout the
Metazoa, a possibility that can be explored by study of Wnt
signaling and Teashirt in distinct tissue and organism contexts.
In the case of Xenopus, Xtsh3 can affect DV axis development
through its role in β-catenin activity for Spemann’s organizer
Fig. 7. Model for the role of teashirt in planarian head-tail regeneration.
A developmental pathway for the initiation of head versus tail regeneration.
wnt1 is activated by wounding and, through Wnt signaling, activates teashirt.
During anterior regeneration, notum inhibits Wnt signaling (later notum roles
are not the focus of this model). During initiation of posterior regeneration,
teashirt helps promote the expression of posterior position control genes
(PCGs) (such as wntP-2) and inhibit the expression of anterior PCGs to give a
tail regeneration signature. It is unknown whether these effects (depicted by
black arrows) reflect direct action. Gray arrows indicate that it is unknown
whether all action of β-catenin-1 will involve teashirt.
formation, which coordinates early embryonic patterning (Onai
et al., 2007). After gastrulation in Xenopus, Wnt signaling promotes
AP axis polarization; whether a Teashirt protein has a role in this AP
axis polarization process remains unknown. Wnt signaling
promotes AP axis polarization in many animals throughout the
animal kingdom (Petersen and Reddien, 2009a). Data from
planarians therefore raise the possibility that Teashirt proteins will
have a broad role, together with Wnt signaling, in AP axis
development in the Metazoa. Wnt signaling controls a large array of
important processes in addition to AP axis development, including
stem cell proliferation, wound healing, polarization of the animalvegetal axis of early embryos in many species and posterior growth.
Because of this diversity of roles, Wnt signaling components have
been the subject of intensive investigation, and have been targeted
for the development of chemical therapeutic approaches. An
important direction will therefore be to determine the diversity of
contexts in which Teashirt activity is important for Wnt-signaling
outcomes, such as in the context of human diseases.
We conclude that Teashirt is a key factor in regulating the
response to Wnt signaling in the polarization of the planarian
primary body axis during regeneration. These findings in planarians
predict that Teashirt proteins will prove to be involved in the
development of the primary axis in many other animals.
Furthermore, these findings support the possibility that Teashirt
will prove to be an important but presently understudied
determinant of the outcome of Wnt signaling activity in many of
the diverse and important biological processes controlled by Wnt
signaling throughout the Metazoa.
MATERIALS AND METHODS
Animal culture and radiation treatment
Asexual S. mediterranea (strain CIW4) animals were starved 7-14 days
prior to experiments. Animals were exposed to 6000 rads using a dual
Gammacell-40 cesium-137 source and amputated 4-6 days after irradiation.
1069
DEVELOPMENT
Fig. 6. Wnt signaling regulates teashirt expression during
regeneration. (A) teashirt was expressed at posterior wounds in
irradiated head fragments at 48 h. Less expression (see
individual blue dots, which represent cells) was observed at
head fragment posterior-facing wounds following RNAi of wnt1
(n=16/32) and β-catenin-1 (n=16/16). Animals viewed ventrally;
anterior is leftwards. Scale bars: 200 µm. (B,C) qRT-PCR
analysis of teashirt levels in β-catenin-1 and APC RNAi animals.
RNA was harvested from anterior- or posterior-facing wound
sites (0 h or 72 h post-amputation) cut from animals after
10 days of β-catenin-1, APC or control RNAi. Biological
triplicates consisted of five to eight wound sites per sample.
(B) Data are mean log2-fold changes in teashirt levels at 72 h
wound sites between β-catenin-1 or APC RNAi and control RNAi
samples. (C) Data are mean log2-fold changes in teashirt levels
between 72 h and 0 h wound sites for each RNAi condition. Error
bars indicate s.d. Individual replicates are indicated by dots.
Asterisks denote significance determined by t-tests between
sets of biological triplicate delta-Ct values. See also
supplementary material Fig. S8.
RESEARCH ARTICLE
Smed-teashirt and RNAi
The teashirt sequence was determined using RACE (Ambion). teashirt was
amplified (5′-CAACGAAATCCGGAGGAAA-3′ and 5′-TTTGGACTCTTGGACGGTTC-3′) from cDNA. β-catenin-1, wnt1, wntP-2, APC, notum
and control (C. elegans unc-22) constructs have been previously described
(Petersen and Reddien, 2008, 2009b, 2011). RNAi experiments used
dsRNA-expressing bacteria mixed with liver as food or dsRNA injection
(Petersen and Reddien, 2009b). All RNAi involved matched control RNAi
animals that had undergone the same procedure. Injection protocol: animals
underwent amputation and injection on day 0; animals were then injected on
day 1; wound sites were removed and animals were injected on day 3;
animals were then injected on day 4; wound sites were removed and animals
were injected on day 6; animals were then injected on day 7. Wound site
removal removed ∼200 μm of pre-existing tissue next to the injury. This
protocol was performed with trunk fragments (lacking heads and tails;
Fig. 1A-H and Fig. 2A,B) or head fragments (lacking trunk and tail;
Fig. 2C-E and Fig. 6A). Final wound site removal (experimental
amputation, day 6) in head fragments used for gene expression analyses
amputated tissue that would include most wound-induced gene expression
(≥250 μm). A short ‘4-day RNAi protocol’ with head fragments (day 0,
amputate and inject; day 1, inject; day 3, amputate and inject; day 4, inject)
also generated a teashirt phenotype. For Fig. 5A, β-catenin-1 and control
dsRNA-expressing bacteria were fed and animals were fixed 4 days later.
For Figs 5 and 6, β-catenin-1, APC-1 and control dsRNA-expressing
bacteria were fed to animals on day 0, 2, 4 and 7; on day 10, RNA was
collected for Fig. 5B and amputation occurred for Fig. 6B,C. For long-term
teashirt inhibition experiments (supplementary material Fig. S7), animals
were fed dsRNA-containing food every 4 days for 8 weeks.
Development (2015) 142, 1062-1072 doi:10.1242/dev.119685
Analysis software using the Linear (Derivative) Baseline Correction Method
and the Auto (Global) Ct Threshold Method. For each target transcript, melt
curve analysis was used to eliminate Ct measurements associated with
abnormally high or low Tm values.
Normalization and analysis of qRT-PCR Data
Processing and normalization of qRT-PCR data was performed using the R
statistical computing environment. For each chip, technical replicates were
averaged and fold changes calculated using the ‘Delta Delta Ct’ (ΔΔCt)
method. For each of three biological replicates, samples were first normalized
to the average Ct value of four housekeeping transcripts: g6pd, clathrin, luc7
and ubiquilin (ΔCt). ΔCt values were then subtracted between samples and a
reference (e.g. control RNAi or time zero) to generate ΔΔCt values reflecting
log2-fold changes. Significance was determined by t-tests (unpaired, equal
variance) between sets of biological triplicate ΔCt values. Bar plots of qRTPCR data represent mean log2-fold changes (±s.d.). Dots represent individual
ΔΔCt values. Heatmaps were generated using the heatmap.2 function in R, and
dendrograms reflect hierarchical clustering based on Euclidean distance. For
principal component analysis (PCA), each sample was normalized to average
housekeeping levels as above. Transcript levels were then converted to
z-scores, and PCA was performed on scaled, centered data using the prcomp
function. PCA plots and ‘PC loading’ plots were generated using ggplot2.
Note added in proof
Analysis of the role of teashirt in planarians has also been described by
Reuter et al. (2015), published during final publication preparations of this
manuscript.
Acknowledgements
Whole-mount in situ hybridization and fluorescence in situ hybridization
were performed as described previously (Pearson et al., 2009).
Quantitative reverse-transcriptase PCR (qRT-PCR)
Samples were processed as in supplementary material Fig. S4 and analyzed on
the Fluidigm Biomark platform using 96.96 Dynamic Array IFC chips.
Dissected wound sites or whole animals were collected and dissociated in
0.75 ml Trizol (Life Technologies) by gentle trituration with a P1000 tip (for
∼1-2 min, room temperature) and then stored (−80°C). The RNA from the
samples was purified according to manufacturer guidelines and resuspended in
dH2O. RNA concentrations were determined by Qubit using the RNA HS
Assay kit (Life Technologies) and normalized to 100 ng/μl in dH2O. Each
sample (500 ng) was dispensed into a 96-well plate and treated with 1 U of
amplification-grade DNAse I (Life Technologies) for 15 min (at room
temperature). DNAse was heat-inactivated for 10 min (at 65°C) in the presence
of ∼2.5 mM EDTA. Samples were diluted 1:100 in dH2O and stored (−80°C).
Multiplex reverse-transcription (50°C for 20 min) followed by 15 PCR
amplification cycles were performed using pooled (outer) primers at 50 nM
each for 28 target transcripts (see supplementary material Table S1) and a
Superscript III/Platinum Taq enzyme mix (Life Technologies). Outer primers
were digested with ExoI (15 U/well, New England Biolabs) at 37°C (30 min),
followed by enzyme inactivation (80°C for 15 min). Samples were diluted 1:5
in TE and 1 μl of each was used to evaluate g6pd levels by conventional qRTPCR (7500 Fast PCR System, Applied Biosystems). Samples were loaded
onto a 96.96 Dynamic Array IFC chip and transcript levels assessed using a
separate inner primer set (see supplementary material Table S1) for each
transcript. Amplified cDNA (3.3 μl/sample) was mixed with 2.7 μl of
EvaGreen Taq Supermix (Bio-Rad) supplemented with 20× DNA-Binding
Mix (Fluidigm). For each inner primer pair, 10 μM of forward and reverse
primer mixes were diluted 1:1 with 2× Assay Loading Reagent (Fluidigm). Six
technical replicates for each of four housekeeping transcripts (g6pd, clathrin,
luc7 and ubiquilin) and three technical replicates for the remaining 24
transcripts were prepared for each chip. Primer mixes and samples were loaded
onto the left and right sides of the IFC chip, respectively; priming and loading
of the IFCs and operation of the Biomark instrument were performed
according to the manufacturer’s instructions. Temperature cycling used a
thermal mixing step, 44 fast PCR cycles (96°C for 5 s; 60°C for 20 s) and
melting (60-95°C). Data were analyzed using Fluidigm Real-Time PCR
1070
We thank Sylvain Lapan for the cintillo probe, and Lucila Scimone, Katrina
Longenecker, Irving Wang and the Reddien lab for manuscript comments.
Competing interests
The authors declare no competing or financial interests.
Author contributions
J.H.O., D.E.W., C.-C.C., C.P.P. and P.W.R. designed the experiments. D.E.W.
and C.P.P. identified teashirt as expressed at posterior wounds. J.H.O. identified
and characterized the teashirt RNAi phenotype. Experiments leading to: Fig. 1
were carried out by J.H.O. and C.-C.C.; Fig. 2, D.E.W., J.H.O., C.-C.C. and
P.W.R.; Fig. 3, D.E.W. and J.H.O.; Fig. 4, D.E.W., J.H.O. and C.-C.C.; Fig. 5,
J.H.O. and D.E.W.; Fig. 6, D.E.W. and J.H.O.; supplementary material Fig. S1,
J.H.O.; supplementary material Fig. S2 J.H.O. and C.-C.C.; supplementary
material Fig. S3, C.-C.C.; supplementary material Fig. S4, D.E.W.;
supplementary material Fig. S5, D.E.W. and C.-C.C.; supplementary material
Fig. S6, C.-C.C., supplementary material Fig. S7, J.H.O.; and supplementary
material Fig. S8, D.E.W. and P.W.R.
Funding
P.W.R. is an Investigator of the Howard Hughes Medical Institute and an associate
member of the Broad Institute of Harvard and MIT. We acknowledge support from
the National Institutes of Health (NIH) [R01GM080639] and from the Keck
Foundation. Deposited in PMC for release after 6 months.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.119685/-/DC1
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