MicroRNA 218 Mediates the Effects of Tbx5a Over-
Expression on Zebrafish Heart Development
Elena Chiavacci1, Luca Dolfi1, Lorena Verduci1, Francesco Meghini1, Gaia Gestri2, Alberto Mercatanti
Monica Evangelista, Stephen W. Wilson2, Federico Cremisi3, Letizia Pitto1*
1Institute of Clinical Physiology, CNR, Pisa, Italy, 2Department of Cell and Developmental Biology, University College London, London, United Kingdom, 3Scuola Normale
Superiore, Pisa, Italy
, a member of the T-box gene family, encodes one of the key transcription factors mediating vertebrate heart
development. Tbx5 function in heart development appears to be exquisitely sensitive to gene dosage, since both
haploinsufficiency and gene duplication generate the cardiac abnormalities associated with Holt2Oram syndrome (HOS),
a highly penetrant autosomal dominant disease characterized by congenital heart defects of varying severity and upper
limb malformation. It is suggested that tight integration of microRNAs and transcription factors into the cardiac genetic
circuitry provides a rich and robust array of regulatory interactions to control cardiac gene expression. Based on these
considerations, we performed an in silico screening to identify microRNAs embedded in genes highly sensitive to Tbx5
dosage. Among the identified microRNAs, we focused our attention on miR-218-1 that, together with its host gene, slit2, is
involved in heart development. We found correlated expression of tbx5 and miR-218 during cardiomyocyte differentiation of
mouse P19CL6 cells. In zebrafish embryos, we show that both Tbx5 and miR-218 dysregulation have a severe impact on
heart development, affecting early heart morphogenesis. Interestingly, down-regulation of miR-218 is able to rescue the
heart defects generated by tbx5 over-expression supporting the notion that miR-218 is a crucial mediator of Tbx5 in heart
development and suggesting its possible involvement in the onset of heart malformations.
Citation: Chiavacci E, Dolfi L, Verduci L, Meghini F, Gestri G, et al. (2012) MicroRNA 218 Mediates the Effects of Tbx5a Over-Expression on Zebrafish Heart
Development. PLoS ONE 7(11): e50536. doi:10.1371/journal.pone.0050536
Editor: Xiaolei Xu, Mayo Clinic, United States of America
Received January 23, 2012; Accepted October 26, 2012; Published November 30, 2012
Copyright: ? 2012 Chiavacci et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research project was funded by Tuscany Region (n.4177) and the Medical Research Council (to GG and SW). All the funders gave the financial
support and they had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
The formation of the mature vertebrate heart with separated
chambers and valves involves a complex orchestration of gene
expression. Numerous genes are critical for cardiac morphogen-
esis, although their exact functions and their integration with other
cardiac regulators is poorly understood .
The T-box gene tbx5 encodes a key transcription factor for
vertebrate heart development [2,3]. Tbx5 function in the heart is
gene dosage sensitive, as both haploinsufficiency and gene
duplication give rise to Holt2Oram syndrome (HOS). HOS is
a highly penetrant autosomal dominant disease characterized by
congenital malformations of the heart and upper limbs, which are
two sites of Tbx5 expression [4–7]. Nonetheless, the molecular
mechanisms accounting for gene dosage sensitivity are not known.
Mice heterozygous for mutations in Tbx5 display many of the
phenotypic abnormalities of individuals with HOS [8,9]. Compa-
rable defects are seen in the zebrafish Tbx5 mutant heartstrings,
suggesting that Tbx5 expression and function have been conserved
throughout vertebrate evolution [10,11]. In the murine model of
HOS, gene expression profiling of a tbx5 allelic series demonstrat-
ed that Tbx5 could regulate hundreds of downstream genes .
Examination of the expression dynamics of mouse genes regulated
by Tbx5 indicates that Tbx5 can act via an array of independent
mechanisms, some of which include direct DNA binding as has
been shown for Gja5 , and others via indirect mechanisms that
may involve complex regulatory networks.
Several genes regulated by mouse Tbx5 encode transcription
factors (TFs), or are involved in transcriptional regulation
suggesting that HOS might in part be the consequence of
‘‘misregulation of regulators’’. Besides TFs, microRNAs (miRNAs)
play key roles in heart development and cardiac diseases [12–14].
TFs and miRNAs comprise two major layers of gene regulatory
networks with strictly interconnected activities: TFs control
miRNA expression and many miRNA targets are TFs. There is
increasing evidence that TFs and miRNAs can work cooperatively
through mutual cross-regulation . Starting from these con-
siderations, we performed an in silico screening to identify miRNAs
embedded in genes highly sensitive to Tbx5 dosage. We focused
our attention on miR-218-1 that, together with its host gene slit2, is
involved in heart development . We confirmed a correlation
between tbx5 and miR-218 expression and showed that alterations
of miR-218 expression have a significant impact on zebrafish heart
development. Interestingly, down-regulation of miR-218 is able to
rescue most of the defects generated by Tbx5 over-expression,
demonstrating the pivotal role of miR-218 in mediating the effects
of Tbx5 dosage on heart development. These data support the
idea that a miRNAs/Tbx5 regulatory circuit is crucial in cardiac
PLOS ONE | www.plosone.org1November 2012 | Volume 7 | Issue 11 | e50536
Identification of Tbx5-modulated miRNAs
To identify miRNAs modulated by Tbx5, we developed
a bioinformatic tool to search for miRNAs within introns of
Tbx5-controlled genes (Fig. S1). As a source of Tbx5 targets, we
used genes identified by microarray analysis of a conditional
mouse allelic series of tbx5  and of a mouse 1H cell line infected
with adenovirus expressing tbx5 . Four miRNAs were
identified: miR-218-1, miR-678, mir-719 and miR-335 (Table 1).
We focused our attention on miR-218 since: i) it is conserved from
human to zebrafish (www.mirbase.org); ii) the miR-218-1 host
gene, slit2, is highly sensitive to Tbx5 mis-expression ; iii) the
secreted Slit ligands, together with their Robo receptors,
contribute to the control of oriented cell tissue growth during
chamber morphogenesis of the mammalian heart ; iv) Slit/
miR-218/Robo are part of a regulatory loop required during heart
tube formation in zebrafish .
tbx5 and miR-218 are Co-expressed in Mouse Tissues and
in Cardiomyocyte Differentiation of P19CL6 Cells
We compared tbx5, slit2 and miR-218 expression in newborn
mouse lung, liver, brain, aorta, skeletal muscle and heart. slit3
expression was also analyzed since its host miRNA, miR-218-2,
cannot be separately quantified because it is identical to miR-218-
1. In agreement with the literature , we observed tight co-
expression of slit2 and miR-218, and a general correlation among
tbx5, slit2, slit3 and miR-218 expression (Fig. S2).
To assess whether there are functional regulatory interactions
among Tbx5, Slit2 and miR-218, we first examined these genes in
an in vitro model for cardiomyocyte differentiation. P19CL6 cells
proliferate in growth medium (GM) and differentiate into beating
cardiomyocytes in differentiation medium (DM) [20,21]. P19CL6
increased the expression of cardiac differentiation markers such as
GATA4, a-MHC, CX40 and decreased the expression of the
marker of pluripotency Oct4 after a few days of culture in DM,
compared to cells maintained in GM (Fig. 1A). A progressive
increase in tbx5 expression was also observed (Fig. 1B), which was
paralleled by an increase in slit2, slit3 and miR-218 transcripts. To
show that the slit/miR-218 increase was at least partially dependent
on Tbx5, tbx5 was up- or down-regulated by transfecting P19CL6
cells with a tbx5-carrying expression vector (CMV-Tbx5), or with
a siRNA mix directed against tbx5, respectively. Tbx5 over-
expression tripled slit2 expression, almost doubled miR-218
expression and had no effect on slit3 expression (Fig. 1C). On
the other hand, tbx5 silencing, the effect of which was highest
2 days after silencing (6th day in culture, see Methods), caused
significant reduction of slit2 and miR-218 expression 4 days after
transfection (8th day in culture, Fig. 1D). Pre-miRNA 218-1
expression paralleled the increase in miR-218 level during
cardiomyocyte differentiation (Fig. S3A) and after Tbx5 modula-
tion (Fig. S3B). Moreover, the transfection of a siRNA mix against
Slit2 cut the level of Slit2 by half without affecting miR-218
expression, supporting the idea that miR-218 expression depends
on the regulation of Slit2 transcription rather than on its
translation. Overall these results suggest that the expression of
Slit2 and its embedded microRNA miR-218-1 are modulated by
Tbx5 during cardiomyocyte differentiation.
miR-218a Over-expression Affects Zebrafish Heart
To analyze the role of miR-218 in heart development, we
decided to use zebrafish since this model is particularly informative
for studying cardiac early patterning networks due to its relatively
simple two-chambered heart coupled with its ability to develop
even in the absence of a functioning heart. Moreover, various data
derived from Tbx5 knock-down experiments in zebrafish [10,11]
have revealed developmental defects of the heart and limbs
comparable to the defects observed in human with Tbx5
mutations [4,22] or in Tbx5 knock-down mice [9,23], suggesting
that the functional role of this crucial transcription factor is
In zebrafish, as in mammals, two isoforms of miR-218, miR-
218a-1 and miR-218a-2, are embedded in slit3 and slit2 genes,
respectively. A third genomic copy of this miRNA, miR-218b, is
present in fish. However miR-218b, an intergenic miRNA, has very
low expression, suggesting that its contribution to the global miR-
218 level might be irrelevant . miR-218a1/2, slit2 and slit3 are
highly expressed in zebrafish neural tissue (Fig. 2A, [24,25]) and
Fig. 2B [26–28]. In cardiac tissue, slit2 and slit3 are clearly
detectable at 48 hpf (Fig. 2B), while the expression of miR-218a1/2
is barely detectable by in situ hybridization (ISH) up to 48 hpf (not
shown) but is visible around 72 hpf (Fig. 2A).
To over-express miR-218a1/2, we injected double stranded
RNA oligonucleotide with a miR-218a sequence (miR-218a mimic)
in Tg(cmlc2:eGFP) embryos. Injection of 260, 135 and 35 pg of miR-
218a mimic generated embryos with cardiac defects in a dose-
dependent way (Fig. 2C). At the highest dose (2 ng in Fig. 2C), we
observed a slight decrease in embryos with cardiac defects, which
was balanced by the increased number of dead embryos and, to
a lesser extent, of embryos with different morphological defects. As
specificity controls, 2 ng of either miR-492, a control miRNA, that
is not annotated in zebrafish, or miR-214, which is not heart-
specific, were injected without generating embryos with cardiac
defects in significant percentages (Fig. 2C). Conversely, in miR-
218a over-expressing embryos we found different cardiac defects
accounting for the ratios shown in Fig. 2C: hearts failing to
complete looping, ventricles showing very irregular walls, and atria
that were strongly reduced and sometimes stretched to a thin
‘‘string-like’’ morphology (Fig. 2F,G). To down regulate miR-218a,
we injected either a morpholino targeting the mature form of miR-
218a (MOM-218), or a longer morpholino also targeting the
Drosha cleavage site of pre-miR-218a (MOD-218, see Methods and
). The knockdown efficiency of these morpholinos was
Table 1. miRNAs identified by bioinformatic approach within introns of Tbx5-modulated genes.
MicroarrayGene symbolmiRNA Tbx5 controlProposed biological function
Mori/PlagemanMest miR-335positive Trabeculation, mouse embryonic cardiac expression 
MoriNupl 1miR-719negative Nuclear pore complex
MoriSlit 2miR-218-1positive Secreted negative regulator of axonal extension
MoriHrmtl 1 miR-678positiveS-adenosylmethionine-dependent methyltransferase activity
MiR-218 is a Tbx5 Effector in Heart Development
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confirmed by their ability to down-regulate mature miR-218 (Fig.
S4A), and to rescue the phenotype caused by miR-218a over-
expression (Fig. S4B). Nonetheless, injection of either MOD-218 or
MOM-218 (not shown) generated no cardiac edema or cardiac
defects even at very high dose (12 ng) in any of the genetic
backgrounds we tested (Fig. 2D,H). This is consistent with the
extremely low level of miR-218a during early stages of de-
velopment (data not shown and [16,29]).
Injection of miR-218a mimic or MOs-218 in Tg(flk1:eGFP),
which express GFP driven by the endothelial-specific enhancer of
flk1, allowed a direct visualization of vascular integrity. As
previously reported , MO-218 microinjection did not cause
gross alteration in vascular structures (Fig. S5C) nor the
hemorrhagic events described in mice after miR-218a knock down
. miR-218a over-expression did not affect vessel morphology
either (Fig. S5B), supporting the idea that in zebrafish miR-218a
does not overtly influence the organization of blood vessels during
Finally, we analyzed the expression of some cardiac markers in
embryos over-expressing miR-218a. Despite the strong cardiac
morphological alterations, miR-218a over-expressing embryos
showed normal expression of the ventricular myosin heavy chain
and the atrial myosin heavy chain (Fig. S6), as previously described
in Tbx5 (hst) mutants . It has been shown that Tbx5 activity is
crucial for the specification of the AV boundary and valve
formation . To verify whether miR-218 over-expression can
affect cardiac valve development, we analyzed the expression of
the tie-2 gene, a member of the Tie family of tyrosine kinase
receptors, which is expressed mainly in endothelial cells  and is
up regulated during atrio-ventricular canal differentiation [32,33].
In hemizygous Tg(tie-2:GFP) embryos, the transgene has low
expression  and it is easy detectable only in the atrio-
ventricular canal thus allowing to visualize the valve tissues
(Fig. 3A, a,a9). Hemizygous Tg(tie-2:GFP) embryos injected with
miR-218a showed an increase in Tie-2 expressing cells in the
ventricle and atrium (Fig. 3A, b,b9 and Fig. 3B,C). A similar Tie-2
dysregulation was observed in hemizygous Tg(tie-2:GFP) embryos
after Tbx5 misexpression (Fig. 3A, c,c9,d,d9 and Fig. 3B).
All together these data indicate that correct expression of miR-
218 is crucial for proper cardiac development.
miR-218 Over-expression Decreases the Migration of
Recent data have shown that, in zebrafish, delayed heart field
migration was caused by either miR-218a reduction or by silencing
of Robo1, an established target of miR-218a [16,19]. In line with
data showing robo1 as target of miR-218a, over-expression of miR-
218a significantly reduces the translational rate of a reporter
construct (sensor), carrying GFP coding sequence upstream of
robo1 39UTR (Fig. S7). Therefore, we decided to verify whether
miR-218a over-expression might modify the migration of bilateral
heart field cells to the midline. miR-218a mimic was injected in
Figure 1. tbx5 and miR-218 are co-expressed in cardiomyocyte differentiation of P19CL6 cells. A, qRT-PCR analysis of cardiac (a-MHC,
Cx40, GATA4), muscle (Myosin), neural (Pax6, b-3-tubulin) and pluripotency (Oct4) markers in P19CL6 differentiating cells (8,10 days) compared to
P19CL6 in growth medium (GM). B, qRT-PCR analysis of tbx5, slit2, slit3 and miR-218 relative expression in either expanding (GM) or differentiating
(8,10,12 days) P19CL6 cells. (C-D), P19CL6 differentiating cells, 48 h after CMV-Tbx5 transfection compared to cells transfected with empty vector (C)
or different times after Tbx5-siRNA transfection compared with cells transfected with siRNA-Ct (D). The timing course of the silencing experiment in
(D) is described in the ‘‘Cell culture and transfection’’ section of methods. Results are standardized against GAPDH for genes, and against U6 for
MiR-218 is a Tbx5 Effector in Heart Development
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Tg(cmlc2:eGFP) embryos and the migration of the GFP-expressing
cells was followed by confocal analysis. Myocardial cells migrated
more slowly in miR-218a than in miR-Ct injected embryos (Fig. 4).
Even after injection of high miR-218 doses, cardia bifida was never
observed, suggesting that miR-218 over-expression slows down but
does not arrest the migration of cardiomyocytes to the midline. On
the contrary, MOD-218 injection did not affect cmlc2-positive cells
migration (Fig. 4C), further confirming that reducing the level of
this miRNA during the first hours of development has no overt
effects on heart development.
These data show that the cardiac defects observed in miR-218a
over-expressing embryos might be at least in part related to defects
in heart field migration.
Tbx5 Over-expression can be Rescued by Down-
regulation of miR-218
To further investigate the functional interaction between Tbx5
and miR-218a, we assessed whether the morphological alterations
generated by Tbx5 misexpression might be compensated for the
Figure 2. miR-218 over-expression affects cardiac development. A, miR-218a ISH of 72 hpf embryos. B, slit2 and slit3 ISH of 48 hpf embryos.
nt, neural tube; ht, heart. C,D, phenotypes induced at 72 hpf by increasing doses of miR-218 mimic (C) or MOM/MOD-218 (D) injection. The percentage
of embryos with the indicated defects was averaged across multiple independent experiments carried out in double blind. The total numbers of
embryos analyzed were as follows: Ct miRNA (1 ng) n=293; miR-214 mimic (1 ng) n=104; miR-492 mimic (1 ng) n=103; miR-218 mimic (35 pg)
n=107; miR-218 mimic (135 pg) n=180; miR-218 mimic (260 pg) n=318; miR-218 mimic (2 ng) n=180; MO-Ct (8 ng) n=207; MOD-218 (12 ng)
n=323; MOM-218 (2 ng) n=112; MOM-218 (4 ng) n=165; MOM-218 (8 ng) n=182. E-H, phenotypic analysis of miR-218a misregulation in
Tg(cmlc2:eGFP) embryos. Confocal images of representative transgenic embryos showing the presence or the absence of pericardial edema (e;top)
and heart morphology (bottom). F,G examples of heart defects with different degrees of severity. a, atrium, v, ventricle, e, cardiac edema. Dotted lines
encircle ventricle (white) or atrium (red). Red arrow in G (bottom panel) indicates a shrunken, elongated ventricle typical of the heartstring
phenotype. Scale bars: white or black 100 mm, red 25 mm.
MiR-218 is a Tbx5 Effector in Heart Development
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modulation of miR-218a. We reasoned that if the phenotype
induced by Tbx5 over-expression was due, at least in part, to
increased expression of miR-218, the co-injection of MO-218
should rescue the Tbx5 gain of function phenotype. To test this
hypothesis, Tbx5a was over-expressed by injecting its mRNA into
the cytoplasm of one–cell-stage embryos. Tbx5 over-expression
affects heart development in humans [35,36],mice  and chicks
, while no reports describe the effect of Tbx5 over-expression in
zebrafish. Over-expression of tbx5a mRNA induced a range of
cardiac defects, from cardiac edema in 25% of embryos injected
with low doses of mRNA (35 pg), to mild cases of looping defects
or absence of looping and alteration of chamber morphology in
60% of embryos injected with higher doses (100 pg, Fig. 5A and
C, panels b9–d9). Pectoral fins were occasionally asymmetric in
embryos injected with 200 pg of tbx5a transcript (Fig. 5C, panel d).
tbx5a mRNA injection also caused eye morphology malformations
ranging from asymmetrically positioned eyes, fusion of eyes or
reduction/absence of eyes (Fig. 5C, panels b,c). None of these
defects was observed at significant percentages in hundreds of
embryos injected with comparable doses of GFP mRNA (not
shown). Increased miR-218a expression was observed as a conse-
quence of Tbx5 up regulation (Fig. 5B). Co-injection of 8 ng of
either MOD-218a or MOM-218a along with 100 pg of tbx5a
mRNA restored normal heart morphology and looping in a high
percentage of injected embryos and reduced the number of
embryos with eye defects as compared to control-injected embryos
(Fig. 6A and B, panels b,b9). Co-injection of MO-218 was also able
to counteract the tie-2 expansion observed in Tbx5 dysregulated
embryos (Fig. 6 C,D). This result strengthened our hypothesis that
Figure 3. miR-218a over-expression leads to the expansion of tie-2 expression. A, confocal images of 72 hpf Tg(tie-2:GFP) embryos injected
with 260 ng of control miRNA (a,a9), 260 ng of miR-218a mimic (b,b9), 2 ng of MO-Tbx5a (c,c9) or 100 pg of mRNA Tbx5a (d,d9). A, magnification of the
control valve is shown in the inset in panel a9. Labels: A, atrium, V, ventricle. B, FACS analysis of cells dissociated from 72 hpf Tg(tie-2:GFP-cmlc2:eRFP)
embryos injected as described in A. C, confocal images of 72 hpf Tg(tie-2:GFP-cmlc2:eRFP) embryos injected with 260 ng of miR-Ct (top) or with miR-
218a mimic (bottom). The control valve is magnified in the inset in panel a9. White scale bars: 100 mm, red scale bars 25 mm.
MiR-218 is a Tbx5 Effector in Heart Development
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the effect of Tbx5 over-expression on heart development might, at
least in part, be be mediated by miR-218.
The apparent lack of phenotype that we observed after MO-
218a injection (Fig. 2D,I) and the very low expression of miR-218
at early developmental stages, suggest that decreased miR-218a
should not contribute to the phenotype generated by Tbx5
knockdown. To verify this assumption we downregulated Tbx5
using a morpholino directed against the tbx5a translational start
site (MO-Tbx5a). The effectiveness of this morpholino was
confirmed by the injection of GFP-carrying a chimeric target
(Fig. S8A,B) and its specificity was confirmed by rescuing the MO-
Tbx5a phenotype co-injecting in vitro transcribed Tbx5a-mRNA
(not shown). Tg(cmlc2:GFP) embryos injected with MO-Tbx5a
showed the heart and limb defects characteristic of the well
described heartstrings phenotype (Fig. S8E,G and [10,11]). In
addition to tbx5a, a second tbx5 isoform has been recently
identified in zebrafish, tbx5b, which has lost the characteristic
forelimb/pectoral fin expression of tbx5 genes . Consequently,
we also designed a morpholino against this tbx5 isoform, MO-
Tbx5b (Fig. S8C,D). Similarly to MO-Tbx5a injection, MO-
Tbx5b injection caused looping defects and stretched cardiac
chambers, but it did not induce Tie-2 mis-expression and lack of
fins (Fig. S8F). For these reasons and since the penetrance of the
MO-Tbx5b phenotype was much lower compared to MO-Tbx5a
(compare Fig. S8G,H) we chose MO-Tbx5a for further analysis.
Co-injection of mimic miR-218a did not rescue the cardiac edema
and looping defects generated by MO-Tbx5a (Fig. S9A). Instead,
the co-injection increased both the frequency and the severity of
the mutant phenotype, doubling the number of heartstring-like
embryos (Fig. S9A) and the number of embryos that showed
a particularly extended edema at 48 hpf (Fig. S9B). This synergism
was further demonstrated by MO-Tbx5a and miR-218a mimic co-
injection in sub-phenotypic doses. In fact, zebrafish embryos
tolerated the injection of either 35 pg of miR-218a or 0,5 ng of
MO-Tbx5a well and only rarely revealed heart looping defects as
compared to un-injected siblings (Fig. 2A and Fig. S9C,D).
However, the injection of both MO-Tbx5a and miR-218a mimic at
these doses dramatically increased the number of embryos with
looping defects (Fig. S9C,E).
As a whole, these data support a functional interaction between
Tbx5 and miR-218a in heart morphogenesis.
Our data show that miR-218 is part of a regulatory circuit
through which Tbx5 controls heart morphogenesis. Previous
studies in mice identified slit2, which encodes miR-218-1 within
one of its introns, as one of the genes highly sensitive to Tbx5
dosage . Moreover, the coordinate expression of miR-218-1 and
its host genes has been largely documented both in physiological
(mouse development ) and pathological (cancer progression
[39,40]) conditions. We showed a functional relation between
Tbx5, Slit2 and miR-218 in P19CL6 cells in which a progressive
increase of Tbx5, Slit2 and miR-218 expression was observed
during cardiomyocyte differentiation. Moreover, we showed that
Tbx5 deregulation affects miR-218 expression. Tbx5 might
regulate slit2 expression directly or indirectly. However the
presence of T-box consensus sequences upstream of both mouse
and fish slit2 transcription start site, as identified by the Transfac
program (http://www.biobase-international.com; not shown),
supports the hypothesis that Tbx5 might directly bind to and
activate the slit2 promoter. To demonstrate that the Tbx5/miR-
218 regulatory circuit is also functional during development, we
used the zebrafish as a model system. Through functional assays in
zebrafish, we showed that either over-expression or down-
regulation of Tbx5 affects heart morphogenesis. In line with the
hypothesis that miR-218 might be a Tbx5 effector, we demon-
strated that miR-218a deregulation generates cardiac defects
(Fig. 2A). As in zebrafish the expression level of miR-218a is
Figure 4. miR-218 over-expression causes a delay in early heart
field migration. A,B, images of Tg(cmlc2:eGFP) embryos injected with
260 pg of miR-Ct (A) or with 260 pg of miR-218a mimic (B) at different
times of development. Dorsal view, anterior at the bottom. After
confocal analysis, embryos were left to develop until 72 hpf when they
were screened for the presence of edema. White scale bars: 150 mm. C,
migration velocities of myocardial Tg(cmlc2:eGFP) cells as quantified
from time-lapse images. Five embryos for each experiment were
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Figure 5. tbx5 over-expression causes eye, cardiac and fin defects. A, phenotypes generated by increasing doses of tbx5a mRNA. The
percentage of embryos with the indicated defects was averaged across multiple independent experiments. The total number of embryos analyzed
was as follows: mRNA-Tbx5a (35 pg) n=48; mRNA-Tbx5a (100 pg) n=199; mRNA-Tbx5a (200 pg) n=131;. B, qRT-PCR analysis of miR-218a relative
expression in 24 and 34 hpf embryos injected with 100 pg of tbx5a mRNA compared with embryos injected with 100 pg of GFP mRNA. C, phase-
contrast and confocal images of representative transgenic Tg(cmlc2:eGFP) embryos at 72 hpf showing eye, heart and fin morphological defects
induced by the injection of 100 pg (b,b9–c-c9) or of 200 pg (d,d9) of tbx5a mRNA. Arrowheads indicate eye alteration, arrows show fin absence. Labels:
A, atrium, V, ventricle. Dotted lines encircle ventricle (white) and atrium (red). Scale bars: black 100 mm, red 25 mm.
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extremely low during the first stages of development (our
observation is supported also by the data of Fish et al.  and
by data regarding miRNA microarray of Thatcher et al. , the
introduction of very low amounts of miR-218a mimic into embryos
at the one- or two-cell stage generated a severe cardiac phenotype.
miR-218a over-expressing hearts were not able to complete the
looping process, showed marked alteration of the cardiac chamber
morphology and mis-expression of a marker of valve cardiac
tissues. A large pericardial edema was visible after 48 hpf. The
frequency and severity of all these cardiac phenotypes were dose-
dependent (Figs. 2 and 3). Our data suggest that at least part of
these morphological anomalies may be due to a reduced migration
rate of myocardial cells (Fig. 4). Conversely, miR-218a down
regulation, even through injection of high doses of two different
morpholinos, did not affect heart development.
Since Tbx5 and Slit2 are both expressed in myocardial cells
 we hypothesized that early Tbx5 over-expression might cause
heart malformation through early activation of miR-218a and
silencing of the target genes of this miRNA. This hypothesis is
supported by the observation that miR-218 down-regulation by
MO-218a injection rescues the effects of Tbx5 over-expression
(Fig. 6). robo1 has been identified as a target of miR-218 in many
different organs and tissues [19,39,41]. Fish et al  showed that
robo1 is a miR-218a direct target and that early robo1 down-
regulation by morpholino injection in zebrafish embryos induces
severe pericardial edema and heart defects caused by reduced
migration rate of endocardial cells. Thus, robo1 is a candidate gene
through which Tbx5 and miR-218a early over-expression affects
heart development. Fish et al reported that miR-218a down
regulation, performed by the microinjection of one of the two
morpholinos that we used in our study, causes severe cardiac
defects and cardiac edema through reduced migration of
endocardial and myocardial cells. This is inconsistent with both
our data and with the results of Fish et al.  who observed that
Robo1 knock-down generates the same phenotype, because the
same authors also showed that Robo1 is targeted by miR-218. In
fact, an effect of miR-218a on the migration of endocardial and
myocardial cells is not expected, as miR-218a seems not to be
substantially expressed before 24 hpf [16,29], when the fusion of
migrating cardiac cells is about to be completed. We speculate that
the timing of miR-218a upregulation during heart development is
crucial for heart morphogenesis. miR-218 and Robo1 are supposed
to be upregulated and downregulated, respectively, when myo-
cardial cell migration is about to end. In this view, Tbx5 mis-
expression by mRNA microinjection at the one-cell stage might
speed up the up-regulation of miR-218 and reduce the migration of
myocardial cells precociously, which in turn might affect heart
morphogenesis by impairing the correct interaction between
myocardial and endocardial cells.
A negative role of miR-218 on cell migration has been
highlighted in different biological contexts. In mice, it has been
shown that miR-218 regulates vascular patterning by modulating
Slit-Robo signaling . The authors showed that miR-218-driven
repression of Robo1/2 and of heparan sulfate proteoglycans
(HSPGs), which are proteins essential for Slit/Robo signaling,
negatively affects endothelial cell (EC) migration. miR-218 has also
been shown to affect cancer progression by inhibiting tumor cell
migration and metastasis via the repression of the Slit2/Robo1
pathway in gastric  and in nasopharyngeal  tumors,
respectively. Therefore our data showing a migration delay of
cmlc2-positive cardiac precursors in embryos over-expressing miR-
218 (Fig. 4), but not in embryos in which miR-218 was down-
regulated by MO-218 injection (Fig. 4C), are in line with these
In the human heart, Tbx5 is expressed not only in the
myocardium, but also throughout the embryonic epicardium and
coronary vasculature. Using chick development as a model system,
Hatcher et al.  showed that, in vivo, over-expression of
biologically active Tbx5 inhibits proepicardial cell migration.
Although we do not know whether the slit2 is expressed in
proepicardial cells, it is tempting to speculate that the up
regulation of the Tbx5-miR-218 circuit might also impact the
proepicardial cell migration by targeting robo1 or other cell
migration regulators such as Semaphorins, some members of this
large class of molecules being predicted targets of miR-218 (not
Tbx5 over-expression affects heart development in different
organisms. In humans, tbx5 gene duplication produces cardiac
abnormalities [35,36]; in mice, persistent cardiac Tbx5 over-
expression results in heart looping defects and abnormalities of
early chamber development . In chicks, Tbx5 over-expression
determines a significant decrease in heart size and a marked
decrease in ventricular trabeculation . We also observed
looping and cardiac chamber alterations in zebrafish after
injection of tbx5a mRNA in embryos at one-cell stage. Un-
expectedly, the severity of cardiac morphology defects was
paralleled by the severity of eye defects (Fig. 5). Tbx5 is expressed
in optic primordia from zebrafish to humans [43,44] and its mis-
expression has been shown to affect eye morphogenesis and the
visual projection in chicks . Moreover, ophthalmological
examination of HOS patients revealed alteration of dorso-ventral
polarity in developing eye vesicles . However, our Tbx5 over-
expressing embryos showed particularly severe eye defects such as
asymmetrically positioned eyes, fusion of eyes, and even unilateral
anophthalmia (Fig. 5). Comparable eye defects are observed in
Brg1 over-expressing zebrafish embryos . Recently the
importance of the balance between Brg1, a member of the
SWI/SNF chromatin-remodeling complex, and several cardiac
transcription factors including Tbx5, has been demonstrated .
Since Brg1 is maternally expressed , the over-expression of
Tbx5 might generate a strong imbalance between these two
factors during the first hours of development, and this imbalance
might be at the root of eye defects. At the moment we do not know
how miR-218 might also partially rescue eye defects. It is
interesting to know that Pax2, that is negatively controlled by
Tbx5 , is a predicted target of miR-218. Overall, these
observations suggest that Tbx5 over-expression affects heart and
eye development and that this might be at least partially mediated
by miR-218. Our observation that MO-218 co-injection is able to
ameliorate both heart and eye defects caused by Tbx5 injection is
consistent with this model.
Our data suggest that the haplo-insufficiency of the Tbx5 gene,
at the moment the most significant cause of HOS, does not impact
heart and upper limb formation through miR-218 misregulation.
The simplest explanation for this might be that other key RNAs
controlled by Tbx5 than miR-218 might be necessary for heart
morphogenesis by regulating mechanisms other than myocardial
cell migration. This would still be consistent with the requirement
of a tight tbx5 gene dosage regulation in space and time for proper
heart morphogenesis. More rarely, HOS has been associated with
increased Tbx5 expression by partial chromosome duplication
[6,28] or mutation resulting in Tbx5 gain of function .
However advances in DNA sequencing technology also highlight-
ed the potential role of non-coding variants in congenital
malformations. Recent studies in mice uncovered three enhancers
that collectively recapitulate the endogenous expression pattern of
tbx5 but that singularly have specific and compartmentalized
expression in the heart and forelimbs . Interestingly,
MiR-218 is a Tbx5 Effector in Heart Development
PLOS ONE | www.plosone.org8November 2012 | Volume 7 | Issue 11 | e50536
MiR-218 is a Tbx5 Effector in Heart Development
PLOS ONE | www.plosone.org9November 2012 | Volume 7 | Issue 11 | e50536
a mutation in one of these enhancer sequences was identified in
a cohort of non-syndromic patients and it has been shown to affect
the enhancer activity in mice and zebrafish transgenic models. The
population-wide frequency of this variant suggests that a significant
number of congenital heart defects (CHD) associated with Tbx5
dysregulation might arise from non-coding mutations in Tbx5
heart enhancers effectively decoupling the heart and hand
phenotypes of HOS syndrome. Therefore it is likely that
modulation of Tbx5 in general, and over-expression of miR-218
as a consequence of Tbx5 up-regulation in particular, might have
a higher impact on CHD population than previously hypothe-
Finally, our results highlight the potential advantage of using
miRNAs as target molecules for heart disease therapies. Their
potential to restore the expression of hundreds of dysregulated
mRNAs to their pre-pathological level in one shot and, in so doing
possibly reverse the disease, places miRNAs among the most
exciting molecules for the development of new therapeutic
Ensembl (http://www.ensembl.org/index.html), was used to
obtain information about chromosome location, position and
segment of the selected genes. Ensembl is a joint scientific project
between the European Bioinformatics Institute (EBI; http://www.
ebi.ac.uk/clustalw/) and the Wellcome Trust Sanger Institute
(http://www.sanger.ac.uk/). EBI provides a centralized resource
with annotations on genomes of sequenced species and the
Ensembl Perl API (Application Programming Interface) models for
access to biological objects, such as genes and proteins. Moreover
EBI allows the execution of Perl programs for retrieving data from
a public database MySQL (http://www.mysql.it/). We generated
two local databases, one for genes, and one for microRNAs. By
applying the Perl program that uses Ensembl API, we compared
the gene databases with the microRNA database using a standard
database interface module for Perl.
ReagentsMature miRNA mimics (mmu-miR-218a, mmu-miR-
492, mmu-miR-214 and miR-Ct) were synthesized by Gene-
Pharma (Shanghai, China), morpholinos (Gene Tools, LLC
USA.), Lipofectamine 2000 TRIzol reagent, DNaseI amplification
grade, SuperScript II reverse transcriptase, RNAse out, a-minimal
Essential Medium (Invitrogen), HyPerFect Transfection Reagent,
miRNeasy mini kit, miScript Reverse Transcription kit Quantitec
Reverse Transcription kit and Quantifast SYBR Green PCR kit
(QIAGEN, Milan, Italy), LightCycler 480 SYBR Green I Master,
anti-DIG antibody-alkaline phosphatase Fab fragment, Blocking
reagent, BM Purple and DIG-RNA labeling kit (Roche Di-
agnostic, Mannheim, Germany), DIG-labeled miRCURY LNA
microRNA detectin probe (Exiqon), SP6 RNA polymerase,
RNAse free DNAse I (Fermentas International Inc), Herculase
DNA polymerase, (Stratagene), pGEM Teasy vector, (PRO-
MEGA), Fetal Bovine Serum (Lonza), zebrafish diet (SDS, Dietex,
France), Tetramisole (Sigma), mMESSAGE mMACHINEH Kit
Current italian national rules: no approval needs to be given for
research on zebrafish embryos. Wild-type AB and transgenic
Tg(flk1:eGFP), Tg(cmlc2:eGFP), Tg(cmlc2:eRFP) and Tg(tie-2:GFP)s849
transgenic lines were used in these studies. Zebrafish were raised
and maintained under standard laboratory conditions (Westerfileld
M zebrafish book) in a ZEBTEC Zebrafish Housing System
(Tecniplast, Varese, Italy).
Cell Culture and Transfection
P19CL6 cells were obtained from Dr.Antonio Baldini (Telethon
Institute of Genetics and Medicine, Napoli, Italy). Cells were
cultured in growth conditions as previously described [20,21]. For
differentiation conditions, growth medium was supplemented with
1% DMSO and the medium was replaceded every two days. Cells
were grown at 37uC in a humidified atmosphere containing 6%
CO2. The pCMV-Tbx5 plasmid expressing mouse Tbx5 (Kindly
provided by Prof. Mona Nemer, Universite ` d’Ottawa) or the
pCMV empty vector as control were transfected using Lipofecta-
mine 2000. To downregulate mouse Tbx5 the following siRNAs
were designed according to previously identified criteria :
siRNA-Tbx5-1 (59-CUGG ACCCGUUUGGACACAUU-39/59-
UGUGUCCAAACGGGUCCAGUU-39) and siRNA-Tbx5-2 (59-
GUAAUCGGGGCUU-39). To downregulate mouse slit2 the
following siRNAs were designed: siRNA-Slit2-1 (59-GCUCACU-
GAGCUU-39) and siRNA-Slit2-2 (59-GGCUCAGAAGGAAAA-
the silencing experiments in P19CL6, cells were transfected with
control siRNA or a siRNA-Tbx5-1 and siRNA-Tbx5-2 mix in
GM. 6 hrs after transfection the GM was substituted with the DM.
Four days after the first round of transfection, the cells were
transfected with the respective siRNA again. Cells were pelletted
6 h after the first transfection, 48 h and 96 h after the second
round of transfection. SiRNAs were transfected using HiPerFect
Quantitative Real Time RT-PCR
Total RNA was extracted from ,30 embryos or from P19CL6
cells cultured in 6-well plates to 80-90% confluence at using the
RNeasy mini kit. After DNase treatment, 1 mg of total RNA was
retro-transcribed using miScript Reverse Transcription kit (for
miRNA analysis) and Quantitec Reverse Transcription kit (for
gene analysis) following the manufacturer’s instruction. Real-time
PCR (qRT-PCR) was carried out using either QuantiFast SYBR
Green kit with Rotor gene (Quiagen) or LightCycler 480 SYBR
Green I Master with LightCycler 480 (Roche) following the
manufacturer’s instructions. Primers used for mRNA analysis were
as follows: for mmu-Tbx5 F 59-CCACTGGATGCGACAACTT-
Figure 6. Down-regulation of miR-218 can rescue the defects generated by tbx5 over-expression. A, quantification of the phenotypes
induced by the injection of 100 pg of tbx5a mRNA (n=199), 8 ng of MOD-218 (n=182) or by the co-injection of 100 pg of tbx5a mRNA and 8 ng of
MOD-218 (n=241). As control, non injected embryos were quantified. Each experimental point in the graph represents the mean 6 SE of at least
three independent experiments. Comparisons between groups were performed by one-way analysis of variance, followed by Bonferroni’s post-hoc
for multiple comparisons. B, phase-contrast and confocal images of representative transgenic Tg(cmlc2:eGFP) embryos at 72 hpf comparing the
phenotype of a control embryo (upper panels) to the rescued phenotype generated by the co-injection of 100 pg of tbx5a mRNA and 8 ng of MOD-
218 (lower panels). C, quantification of tie-2 mis-expression in 72 hpf Tg(tie-2:GFP) embryos after the co-injection of tbx5a mRNA (100 pg) and MO-Ct
(8 ng, n=60) or of tbx5a mRNA (100 pg) and MOD-218 (8 ng, n=62). D, Confocal images of representative 72 hpf Tg(tie-2:GFP) embryos co-injected
with tbx5a mRNA and MO-Ct (a-a9) or with tbx5a mRNA and MOD-218 (b-b9). Labels: A, atrium, V, ventricle. Scale bars: black 100 mm, red 25 mm.
MiR-218 is a Tbx5 Effector in Heart Development
PLOS ONE | www.plosone.org 10November 2012 | Volume 7 | Issue 11 | e50536
39, R59-GCATGGAGTTCAGGATA ATGTG-39; for mmu-Slit2
GACTGTCCGGAATGACA-39, R 59-GGCTTCTTGTTGGA-
CACGACATCT-39; for mmu-Oct4 F 59-TCAG CTTGGGCTA-
GAGAAGG-39, R 59-GGCAGAGGAAAGGATACAGC-39and
for mmu-PAX6 F 59-CCT CCTTCACATCAGGTTCC-39, R
59-CATAACTCCGCCCATTCACT-39. For dre-Tbx5a F 59-
TCCTTTCAAGGTCTCCG TTC-39, R 59-AGGCCTTTGG-
GAAGTTCAGT-39. Transcript values were normalized with
those obtained from the amplification of mmu-a-actin with the
following primers: F (59-CGAGCTGTCTTCCCATCCA-39), R
(59-TCACCAACGTAGCTGTCTTTCTG-39) for P19CL6 anal-
ysis and from the amplification of dre-a-actin or dre-EF1
respectively with the following primers F 59-CGAGCTGTCTT
CCCATCCA-39, R 59-TCA CCAACGTAGCTGTCTTTCTG-
39 and F 59-CTGGAGGCCAGCTCAAACAT-39, R 59-ATCAA-
GAAGAG TAGTACCGCTAGCATTAC-39 for zebrafish em-
bryos analysis. The primers for mature miR-218a, mmu- or dre-U6
TTGTGCTTGATCTAACCATGT-39, 59-CGCAAG GATGA-
39. To amplify both pre-miRNAs of 218 in mouse, the following
primers were used: for pre-miR-218-1 F 59-GATAATGGAGC-
GAGATTTTCT G-39, R 59-TAGAAAGCTGCGTGACGTTC-
39 and for pre-miR-218-2 F 59-AGTTGCCGCGGGGCTTTC-
39, R 59-AGGAGAGAGCGATGCTTTC-39.
All reactions were performed in triplicate. Relative quantifica-
tion of gene expression was calculated as described .
59- for dre-Slit2F
Cardiac tissue-enriched total RNA extracted from 5 day old
embryos was used to clone zebrafish tbx5a. dre-tbx5a cDNA was
directely amplified by RT-PCR (forward primer 59-AGATCTA-
GAC ATCGTACAGGC-39, reverse primer 59-CTGCATGT-
TAGCTGGCTTCGT-39) The tbx5a PCR fragment was cloned
into the pGEM-T Easy Vector (Promega) and sequenced by
Genechron C.R.ENEA (Roma, Italy). For zebrafish tbx5a mRNA
production the entire open reading frame was subcloned in pCS2-
GFP vector using BamHI and XbaI restriction sites. The construct
was linearized with NotI and the capped mRNA was synthetized
with mMESSAGE mMACHINEH Kit (Applied Biosystems). As
control, capped mRNA of GFP obtained from pCS2-GFP
linearized template was used.
The morpholino antisense oligonucleotide MO-Tbx5a (59-
GAAAGGTGTCTTCACTGTCCGCCAT-39) and MO-Tbx5b
(59-GGATTCGCCATATTCCCGTCTGAGT-39) were designed
against the translational start sites of tbx5a and tbx5b respectively.
To test the specificity of the two MOs two plasmids pCS2-Tbx5a-
GFP and pCS2-Tbx5b-GFP were generated by fusing a 180-bp
fragment of tbx5a and a 219-bp fragment of tbx5b, both including
the morpholino target sides, into GFP producing vector. The
primers used for fragment amplifications were: for Tbx5a F 59-
AAGCTTCAACCGCTAGTGCTGGAAG-39, R 59-GGATCC
TTCGCTGTCACTGGGAGAG-39 for Tbx5b F 59-AAGCTT-
GATTCGCCATATTC-39 where the underlined sequences rep-
resent anchored Hind III (F primers) and Bam HI (R primers)
restriction sites used for subcloning in pCS2 vector. tbx5a and
b PCR fragments were cloned in pGEM-T Easy Vector,
sequenced and subcloned in pCS2-GFP vector.
The morpholino antisense oligonucleotide MOM-218 (59-
(59-TGCATGGTTAGATCAAGCACAAGGG-39) were designed
against the mature form of miR-218a and the Drosha cleavage site
of pre-miR-218a respectively. The sequence of the control MO was
The one- and two-cell stage embryos were injected with
a constant injection volume (,1 nl, confirmed by volume analysis)
using a microinjector (Tritech Research, Los Angeles CA USA).
Optical Microscopy and Confocal Analysis
Optical microscopy was performed with Olympus SZH
microscope, images were acquired with Nikon DS-Fi1 camera
and NIS-Elements F 3.0 software. Images were processed with
Gimp-2.6 software. For confocal analysis embryos were fixed in
4% PFA for 1 h at room temperature under slow agitation and
embedded in 2% low-melt agarose. Images were acquired with
a Leica DM IRE 2 confocal microscope. Image stacks were
processed with FIJI-WIN32 by projection. For migration analysis,
embryos were injected with the indicated miRNA mimics or
morpholinos and allowed to develop at 26uC until the 14-somite
stage. Then they were embedded in 1% low-melting soft agarose
and imaged at 26uC with Leica DM IRE 2 confocal microscope to
obtain stacks. To calculate cell velocity stacks were processed with
Zebrafish Tg(tie-2:GFP-cmlc2:eRFP) embryos were injected and
raised in standard conditions until 72 hpf. Next embryos
(n=15225 embryos for each analysis) were collected and treated
with trypsin 0,125 mg/l at 37uC. After a 30 min incubation,
trypsin was inactivated, samples were filtered with 50 mm cell-
strainer and processed with FACScalibur BD.
Whole Mount in situ Hybridization
slit2 and slit3 clones for in situ hybridization were kindly
provided by Dr. Hitoshi Okamoto (Laboratory for Development
of Gene Regulation, RIKEN, Brain Science Institute Japan).
Whole mount in situ hybridization was performed as previously
described  with some modification: pre-hybridization temper-
ature was 62uC; hybridization temperature was 62uC for gene
probes and 52uC for miR-218 probe. 500 ng of antisense DIG
labelled RNA probe was added to the hybridization mix. The anti-
DIG antibody-alkaline phosphatase Fab fragment was diluted
1:4.000 in MABlock buffer (2% Blocking reagent in 100 mM
Maleic acid and 150 mM NaCl) and incubation was performed at
+4uC for gene probes and at room temperature for miR-218 probe.
After incubation with the anti-DIG antiserum, embryos were
washed in PBT and then incubated in the alkaline Tris buffer
solution containing 2 mM Tetramisole (Sigma). The final staining
step was performed in BM Purple AP-Substrate, precipitating
buffer according to the manufacturer’s recommendations. Labeled
MiR-218 is a Tbx5 Effector in Heart Development
PLOS ONE | www.plosone.org 11November 2012 | Volume 7 | Issue 11 | e50536
embryos older than 24 h were bleached as follows: embryos were
washed at room temperature under slow agitation in PBS changed
2x at 5 min. intervals. PBS was removed and embryos were
incubated for 10 minutes in pre-bleaching buffer (SSC 0,5X and
6% Formamide). After discarding the pre-bleaching buffer,
bleaching buffer (pre-bleaching buffer containing 33% H2O2)
was added to the embryos which were subsequently exposed to
a bright source of light for 15 minutes. Afterwards embryos were
transferred back to 87% glycerol in PBS incubated at room
temperature under slow agitation for 4 h and then stored at –
Data were analyzed using GraphPad Prism (GraphPad Soft-
ware, San Diego, CA USA). Statistical differences were de-
termined by unpaired t-test, with values of P,0.05 considered
statistically significant. Each experimental point in the graph
represents the mean 6 SE of at least three independent
experiments. In the graph of Fig. 6A comparisons between groups
were performed by one-way analysis of variance, followed by
Bonferroni’s post-hoc for multiple comparisons.
matic tool developed to identify in silico Tbx5-controlled
miRNAs. Ensembl was used to obtain information about
chromosome location, position and segment of the selected genes.
Ensembl is a joint scientific project between the European
Bioinformatics Institute (EBI; http://www.ebi.ac.uk/clustalw/)
and the Wellcome Trust Sanger Institute (http://www.sanger.ac.
uk/). EBI provides a centralized resource with annotations on
genomes of sequenced species and the Ensembl Perl API
(Application Programming Interface) models for access to bi-
ological objects, such as genes and proteins. Moreover EBI allows
the execution of Perl programs for retrieving data from a public
database MySQL (http://www.mysql.it/). We generated two local
databases, one for genes, and one for microRNAs. By applying the
Perl program that uses Ensembl API, we compared the gene
databases with the microRNA database using a standard database
interface module for Perl.
Schematic representation of the bioinfor-
tissues. A-D, relative expression of tbx5, slit2, slit3 and miR-218 as
evaluated by q-RT-PCR in different newborn mouse tissues.
Results are standardized against GAPDH for genes, and against
U6 for miRNAs. Values represent the averages and standard
deviations of at least two independent experiments.
tbx5 and miR-218 are co-expressed in mouse
mature miR-218 during mouse differentiation and tbx5
modulation. Q-RT-PCR detection of pre-miR-218-1 and pre-
miR-218-2 relative expression in P19CL6 cells during differenti-
ation (A), or 48 h after plasmids or siRNA transfection (B). In B
fold changes of CMV-Tbx5 and siRNA-Tbx5 are relative to
CMV-empty and siRNA-Ct values, respectively. Results are
standardized against GAPDH. *, P,0.05 (Student’s t-test). C,
Q-RT-PCR detection of slit2 and mature miR-218 in P19CL6 cells
transfected with a mix of two siRNAs against slit2 or with a siRNA-
Expression of pre-miR-218-1 parallels that of
218a over-expression was accomplished by co-injecting
Rescue of cardiac defects induced by miR-
MO-218a. A, qRT-PCR analysis of miR-218a relative expression
in 24 hpf embryos microinjected with 12 ng of control morpholino
(MO-Ct) or MOD-218a and with 260 pg of miR-Ct or miR-218a
mimic. miR-218a relative expression was calculated as the ratio
between the expression of injected and the expression of non
injected embryos. B, representative transgenic Tg(cmlc2:eGFP)
embryos at 72 hpf showing heart morphological defects induced
by the injection of 260 pg of miR-218a mimic in the absence (a,b)
or in the presence (c,d) of MOD-218a (12 ng). Labels: a, atrium, v,
ventricle. Black scale bars: 100 mm, red scale bars 25 mm.
Tg(flk1:eGFP) embryos injected with 260 pg of miR-Ct (A),
260 pg of miR-218 mimic (B) or 8 ng MOD-218 (C). Black and
white scale bars: 100 mm.
miR-218 dysregulation does not affect vascu-
Confocalimagesof representative72 hpf
alter amhc and vmhc cardiac marker expression in
zebrafish embryos. Ventral views of 48 hpf embryos injected
with the indicated miRNA mimics (260 pg) or MOs (12 ng MO-
Ct and MOD-218a, 3 ng MO-Tbx5a) after mRNA in situ
hybridization. Scale bar 100 mm.
tbx5 and miR-218a misexpression does not
zebrafish embryos. Top: schematic representation of sensors
and miRNAs used for in vivo sensor assay. Bottom: examples of
24 hpf embryos microinjected with 40 pg of RFP mRNA, 400 pg
of 39UTR robo 1 sensor and 160 pg of miR-Ct (a,c,e) or miR-218a
(b,d,f). In figures C and D the percentage of the relative
phenotypes were indicated. ,30 embryos for each thesis were
injected. Scale bars 50 mm.
miR-218 targets the 39 UTR of robo1 in
down the two zebrafish tbx5 isoforms. A-D, 35 pg of pCS2
plasmid expressing GFP fused with MO-Tbx5a or MO-Tbx5b
target sequences were injected in one-cell stage embryos in the
absence (A and C) or in the presence (B and D) of 1,5 ng of the
relative morpholino. Representative fluorescent images of 24 hpf
embryos. ,20 embryos for each thesis were analysed. E-F, Tbx5
morphants analysis. Phenotypic analysis of Tbx5a (E) and Tbx5b
(F) morphants: 2 ng of MO-Tbx5a, or 4 ng of MO-Tbx5b, were
injected in Tg(cmlc2:eGFP) embryos. Phase-contrast images show-
ing pericardial edema (arrowheads) and fin absence (brackets) or
presence (arrows); in the bottom right corner of figures E and F,
fluorescent images showing heart morphology. Quantification of
Tbx5a (G) and Tbx5b (H) morphant phenotypes. The percentage
of embryos with the indicate defects was averaged across multiple
independent experiments. ,100 embryos for each thesis were
analysed. Black scale bars: 100 mm, red scale bars 25 mm.
MO-Tbx5a and MO-Tbx5b effectively knock-
increases the severity of heartstring phenotype. A,
phenotypic analysis of Tbx5a morphants co-injected with 1 ng
of MO-Tbx5a and either 130 pg of miR-218a mimic or 130 pg of
miR-Ct. B, representative images of 48 hpf embryos showing the
edema expansion caused by the co-injection of miR-218a mimic.
C, phenotypic analysis of embryos co-injected with sub-phenotypic
doses of both MO-Tbx5a(0.5 ng) and miR-218a mimic (35 pg). For
comparison the same dose of MO-Tbx5a was co-injected with
35 pg of miR-Ct. (D-E) Representative confocal images showing
heart morphology of transgenic Tg(cmlc2:eGFP) embryos injected
Injection of miR-218a in Tbx5a morphants
MiR-218 is a Tbx5 Effector in Heart Development
PLOS ONE | www.plosone.org12 November 2012 | Volume 7 | Issue 11 | e50536
with a sub-phenotypic dose of MO-Tbx5a and 35 pg of either
miR-Ct (D) or miR-218a mimic (E). Embryo in D has normal
looping while co-injected embryos in E show absence of looping,
although with different degrees of heart defects. a, atrium, v,
ventricle, e, cardiac edema. Black and white scale bars: 100 mm,
red scale bars 25 mm.
We thank Dr. Antonio Baldini (Telethon Institute of Genetics and
Medicine, Napoli, Italy) for kindly providing the P19CL6 cells, Prof.
Mona Nemer, (Universite ` d’Ottawa) for pCMV-Tbx5 plasmid, Dr. Jeroen
Bakkers (Hubrecht Institute, Developmental Biology and Stem Cell
Research, Utrecht, The Netherlands) for the Tg(Tie-2-GFP) line, Dr.
Didier Stainier (Departments of Biochemistry and Biophysics, Cardiovas-
cular Research Institute, University of California, San Francisco, USA) for
the Tg(flk1:eGFP) and the Tg(cmlc2:eRFP) lines and Dr. Massimo Santoro
(Molecular Biotechnology Center, University of Torino, Torino, Italy) for
theTg(cmlc2:eGFP) line. Moreover, we thank Dr. Davide Moroni (Institute
of Information Science and Technologies, CNR Pisa) for cell migration
analysis, Dr. Giuseppe Rainaldi and Dr. Marcella Simili (Institute of
Clinical Physiology, CNR Pisa), Dr. Michela Ori (Biology Department,
University of Pisa) and Dr. Debora Angeloni (Scuola Superiore S.Anna,
Pisa) for critical reading of the manuscript.
Conceived and designed the experiments: LP. Performed the experiments:
EC LD LV FM AM ME. Analyzed the data: GG FC LP. Contributed
reagents/materials/analysis tools: SWW FC LP. Wrote the paper: FC LP.
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