Vertebrate heart growth is regulated by functional
antagonism between Gridlock and Gata5
Haibo Jia*, Isabelle N. King†, Sameer S. Chopra*, Haiyan Wan*, Terri T. Ni*, Charlie Jiang*, Xiaoqun Guan*, Sam Wells‡,
Deepak Srivastava†, and Tao P. Zhong*§
*Departments of Medicine and Cell and Developmental Biology, and‡Department of Physiology and Biophysics, Vanderbilt University School of Medicine,
Nashville, TN 37232; and†Gladstone Institute of Cardiovascular Disease, Department of Pediatrics, University of California, San Francisco, CA 94158
Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved July 20, 2007 (received for review March 12, 2007)
Embryonic organs attain their final dimensions through the gen-
eration of proper cell number and size, but the control mechanisms
remain obscure. Here, we establish Gridlock (Grl), a Hairy-related
basic helix–loop–helix (bHLH) transcription factor, as a negative
regulator of cardiomyocyte proliferative growth in zebrafish em-
bryos. Mutations in grl cause an increase in expression of a group
of immediate-early growth genes, myocardial genes, and devel-
opment of hyperplastic hearts. Conversely, cardiomyocytes with
augmented Grl activity have diminished cell volume and fail to
divide, resulting in a marked reduction in heart size. Both bHLH
domain and carboxyl region are required for Grl negative control
of myocardial proliferative growth. These Grl-induced cardiac ef-
fects are counterbalanced by the transcriptional activator Gata5
but not Gata4, which promotes cardiomyocyte expansion in the
embryo. Biochemical analyses show that Grl forms a complex with
Gata5 through the carboxyl region and can repress Gata5-medi-
that Grl regulates embryonic heart growth via opposing Gata5, at
least in part through their protein interactions in modulating gene
cardiomyocyte ? proliferation ? size control ? transcription
generation of myofibrillar arrays (1). Cardiac proliferation dimin-
ishes progressively after birth, and postmitotic hypertrophy in the
adult heart provides most of the adaptive responses necessary to
supply increased cardiac output (2). Despite recent progress that
has been made in the regulation of postnatal cardiac hypertrophy,
are poorly delineated. It is not known what molecular signals
The GATA zinc-finger transcription factors promote myocardial
differentiation and expansion. Among the three gata genes (gata4,
gata5, and gata6) in zebrafish, gata5 plays the most prominent role
in heart growth and development (3). Mutations in gata5 in
zebrafish cause a reduction in expression of early and late myocar-
dial genes and a decrease in cardiac progenitor cells and prolifer-
ative cardiomyocytes, resulting in small hearts. Forced gata5 ex-
pression in the zebrafish embryo increases heart size and
phenotype of zebrafish gata5 mutants closely resembles the cardiac
phenotypes in gata4 mutant mice. Myocardium-restricted deletion
reduction in cardiomyocyte proliferation and results in hypoplastic
hearts (5–7). Although it seems to be clear that certain levels of
GATA activity are required to drive myocardial proliferative
growth, opposing signals might also be necessary to constrain the
excessive cardiac growth during development.
grl encodes a hairy-related basic helix–loop–helix (bHLH) tran-
scriptional repressor and belongs to the murine hey/hrt gene family
that contains hey1, grl/hey2, and heyL (8–12). In zebrafish, grl/hey2
is the only hey gene that is expressed in the heart and aorta (8, 13).
hey1 is expressed in the presomitic mesoderm, whereas heyL shows
he embryonic heart grows through a combination of cardio-
myocyte proliferation and an increase in cell mass due to the
expression in the ventral side of the neural tube (13). These data
suggest that grl may play critical functions in both cardiac and
vascular systems. The zebrafish grlm145mutant was originally iso-
lated from a mutagenesis screen and classified as a vascular mutant
(14). The cardiovascular lesion in the grlm145mutant was identified
in the aortic bifurcation, where the lateral aortae fail to assemble,
leading to a lack of blood flow to the trunk, which resembles
coarctation of the aorta in humans (15). The mutant grlm145gene
causes a point mutation that changes a stop codon to Gly, thereby
extending the protein by 44 aa (8). Knockdown grl activity, using
antisense morpholino oligonucleotides (MO), phenocopies the
grlm145mutant and affects arterial differentiation and development
be rescued by VEGF and two structurally related small molecules
(17). Our studies demonstrate that grl promotes arterial differen-
tiation and development in part via the Notch signaling pathway
(18). The roles of grl in cardiac development and growth, however,
have not yet been examined in zebrafish. Although targeted inac-
tivation of grl/hey2 in mice results in a wide spectrum of cardiovas-
cular malformations (19–22), the mechanisms and pathways that
underlie these morphological alterations and its roles in myocardial
growth remain unclear.
regulator that restricts embryonic heart growth by opposing Gata5
activity in zebrafish. We reveal that the grl mutant heart increases
expression of immediate-early growth genes and myocardial genes
and contains more cardiomyocytes with increased cell size. We
show that forced grl expression in WT embryos causes a marked
reduction in heart size, due to a decrease in both cardiomyocyte
number and cell volume. Significantly, the hypoplastic heart phe-
notype induced by grl can be rescued by increasing mRNA of gata5,
but not gata4, implying that these two gata factors are not func-
tionally equivalent during heart growth. Our biochemical studies
demonstrate a physical association of Grl with Gata5 via the
carboxyl region. This association is required for inhibiting Gata5-
sive effects on myocardial proliferative growth.
grl Mutants Display Increased Embryonic Heart Growth. In zebrafish
data; and T.P.Z. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations: bHLH, basic helix–loop–helix; hpf, hours postfertilization; MO, morpholino
Data deposition: The sequence reported in this paper has been deposited in the GenBank
database (accession no. DQ886664).
§To whom correspondence should be addressed at: Vanderbilt University School of Med-
icine, 358 Preston Research Building, 2220 Pierce Avenue, Nashville, TN 37232. E-mail:
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
August 28, 2007 ?
vol. 104 ?
(ALPM) at the 3-somite stage [supporting information (SI) Ap-
pendix, section I]. Thereafter, grl expression becomes predomi-
nantly ventricular myocardial with some transcripts also detectable
in the atrioventricular boundary and the atrium (SI Appendix,
section I). To study grl function during heart development, we
examined myocardial gene expression in the cardiac primordia.
Before the heart tube assembly, nkx2.5 and gata4 are expressed at
comparable levels at the ALPM in grlm145mutant embryos, com-
in the ALPM. In grlm145embryos, these progenitor cells differen-
tiate into cardiomyocytes expressing the normal levels of cardiac
myosin light chain (cmlc2) (Fig. 1 E and F) and migrate to the
midline to properly form the heart cone (Fig. 1 G and H).
Quantitative RT-PCR analyses indicated that the levels of cmlc2
transcripts were not altered in grlm145mutants at the onset of
myocardial differentiation [17 hours postfertilization (hpf)] com-
pared with WT embryos (Fig. 1U). Together, these data suggest
that mutations in grl do not affect myocardial cell commitment and
differentiation. It is not until 48 hpf, when the myocardium under-
goes concentric growth, that the expression of the sarcomere
components cmlc2, atrial myosin heavy chain (amhc), ventricular
myosin heavy chain (vmhc), and atrial natriuretic factor (anf) are
increased in grlm145mutant hearts, when compared with WT
counterparts (Fig. 1 I–P).
We hypothesized that changes in the expression of anf and
sarcomeric components would be accompanied by changes in the
expression of growth regulatory genes. We compared transcrip-
tional profiles of grlm145hearts with WT hearts at 48 hpf, using
microarray analyses. Remarkably, expression of junb, v-fos, early
growth response-1 (egr-1), and other myocardial genes are signifi-
cantly increased in mutant hearts (SI Appendix, section II). egr-1, a
zinc-finger transcriptional factor, belonging to a group of immedi-
ate-early growth genes such as fos and jun, plays an important role
in cell growth and proliferation (1, 23). Quantitative RT-PCR
validated the up-regulation of junb, v-fos, and egr-1 in grlm145hearts
(Fig. 1U). We next examined expression of these genes in grlvu59
mutants, a recently identified nonsense mutation (SI Appendix,
in grlvu59mutant hearts (Fig. 1 Q–T). Thus, grl negatively regulates
expression of immediate-early growth genes and myocardial genes,
which may in turn control heart growth during development.
We next determined cardiomyocyte number and cell size in grl
mutants, using whole-mount confocal microscopy imaging. Confo-
cal imaging of both WT and mutant hearts that express EGFP
revealed that the grlm145heart had an apparent looping defect (Fig.
2 A and B). In addition, the size of mutant atrium is enlarged,
whereas the mutant ventricle is relatively compact and small in
comparison with WT cardiac chambers (Fig. 2 A and B). Histo-
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genes. Expression patterns of nkx2.5 (A and B), gata4 (C and D), cmlc2 (E–H),
amhc (I and J), vmhc (K and L), anf (M–P), junb (Q and R), and egr-1 (S and T)
in WT, grlm145, and grlvu59embryos were revealed by in situ hybridization at
and H), 48 hpf (I–N), and 72 hpf (O–T). wt, wild type. (U) Real-time RT-PCR
analyses showing the increased expression of cmlc2, vmhc, amhc, anf, junB,
v-fos, and egr-1 in grlm145mutants at 48 hpf. Standard deviations were
obtained and presented as standard error bars, using Excel. Asterisks indicate
statistically significant difference between grl and WT data (P ? 0.001). (A–H)
Dorsal views with anterior to the top. (I–P) Ventral views with anterior to the
top. (Q–T) Lateral views with anterior to the left. Arrow, atrium; arrowhead,
ventricle; blue arrow, heart; wt, wild type.
grl mutations cause an increased expression of myocardial growth
e l c i r tnevmu i r t a
h 27fph 8 4
a grl mutant heart (grlm145/grlm145, Tg(cmlc2:EGFP)/?) with an apparent loop-
ing defect, an enlarged atrium, and a compact ventricle (B). (C and D) Histo-
logical section showing the multiple cell layers in grlm145ventricles. (E and F)
Immunostaining analyses revealing more PH3-positive cells in grlm145hearts
than WT hearts. (G and H) Double exposures of fluorescent images showing
PH3-positive cells in cmcl2-EGFP transgenic hearts in WT and grlm145embryos.
(I and J) Transmission electron microscopy showing intact myofibrillar arrays
in both WT and grl mutant hearts. (K) Cell number in the atrium and ventricle
hpf. (L) Myocyte size in both grlm145and grlvu59hearts are increased at 72 hpf
but not 48 hpf compared with WT myocytes. Error bars indicate standard
deviation, and asterisks indicate statistical significance between grl and WT
data (P ? 0.01). Red arrow, ventricle; blue arrow, atrium; yellow arrow,
atrioventricular boundary; wt, wild type.
grl mutant embryos display cardiac hyperplasia and looping defects.
Jia et al.
August 28, 2007 ?
vol. 104 ?
no. 35 ?
ventricular wall resulted in multiple cell layers in a compact
ventricle, whereas the mutant atrium contains more cells in a single
quantified total cardiomyocyte number in grlm145hearts versus WT
counterparts in a series of confocal sections. When the heart tube
forms at 24 hpf, the cardiomyocyte number in grlm145embryos
(142 ? 4) is comparable with WT siblings (148 ? 5). At 48 hpf, a
large increase in the number of cardiomyocytes in grlm145mutants
is evident (328 ? 7) compared with WT embryos (281 ? 7). The
numbers of mutant ventricular myocytes and atrial myocytes in-
crease proportionally, in comparison with WT counterparts (Fig.
2K). Increased mitotic cells were observed by using antibodies
recognizing a mitotic marker phosphohistone 3 (PH3) in grlm145
mutant hearts (4.1 ? 0.3), compared with WT hearts (2.9 ? 0.4)
WT cells from 48 hpf to 72 hpf (Fig. 2L). Electron microscopy
revealed a normal and characteristic sarcomere structure in both
mutant and WT cardiomyocytes (Fig. 2 I and J). We also examined
cell number and size in grlvu59hearts and revealed a greater
proportional increase in both parameters compared with WT
hearts (Fig. 2 K and L). During early development stages, few
myocytes undergo apoptosis in WT and mutant hearts (SI Appen-
dix, section IV). Together, these data suggest that increased cell
size and number in grl mutant hearts are due to defects in cell
growth and division, a tightly coupled cellular event during heart
Forced Expression of grl in WT Embryos Causes a Marked Reduction in
Heart Size. We next investigated whether the increased expression
of grl in zebrafish embryos was sufficient to suppress myocardial
growth. Notably, we found that injection of embryos with synthetic
grl mRNA at low doses (?45 pg) caused formation of small hearts
(Fig. 3 A–J). TUNEL and acridine orange assays indicated that the
(SI Appendix, section IV). Double immunofluoresent staining,
using antibodies that recognize differentially expressed ventricular
and atrial epitopes, indicated that both ventricular and atrial
differentiation occurred in the affected hearts (Fig. 3 A–F). These
hearts correctly form myocardial and endocardial layers (Fig. 3 G
and H). It appears that the mutant ventricular growth is more
impaired than the atrial growth (Fig. 3 I and J). We next measured
the number and area of cardiomyocytes in embryos expressing
increased levels of grl mRNA. Using confocal microscopic imaging
of these EGFP-expressing hearts, we found substantially lower
cardiomyocyte number (Fig. 3K) and a marked diminished cell size
compared with controls (Fig. 3L). Elevated grl expression appears
to be cell type-specific because endothelium and overall embryonic
growth were not affected (data not shown). Interestingly, at the
onset of myocardial differentiation, the number of cardiomyocytes
expressing cmlc2 was not noticeably different in grl-misexpressing
embryos when compared with controls (data not shown). Thus, it
appears that the reduction in cardiomyocyte number is not due to
a reduced production in myocardial precursor cells but rather to a
decreased proliferative capacity of differentiating cardiomyocytes.
The grl gene is predicted to encode a protein containing a DNA
motif near its carboxyl terminus (8). To determine which of these
domains are critical for grl to repress heart growth, we constructed
a series of grl mutant forms harboring deletions in the basic motif
(grl-basic), the HLH domain (grl-hlh), the Orange domain (grl-
orange), the YRPW motif (grl-yrpw), and the carboxyl region
(grl-trunc) (Fig. 3M). We then microinjected synthetic mRNA
encoding each of these mutants into WT embryos and examined
their effects on heart size and morphology. Although grl-orange or
grl-yrpw misexpression caused the cardiac hypoplasia, embryos
misexpressing grl-basic, grl-hlh, or grl-trunc failed to develop hypo-
domain and the carboxyl region in Grl play critical roles for
negatively regulating embryonic heart growth.
grl Regulates Embryonic Heart Growth by Action Within Cardiomyo-
cytes. To determine whether grl controls heart growth by acting
within cardiomyocytes, we directed the expression of Grl-EGFP
fusion protein in cardiomyocytes under control of the zebrafish
cmlc2 promoter. The cmlc2 promoter has been shown to confer
mosaic expression of reporter genes in zebrafish hearts (24). We
examined the proliferative status of atrial myocytes with grl ectopic
expression, as well as ventricular myocytes with increased grl
EGFP under the control of cmlc2 promoter formed clusters of cells
in the ventricle and atrium at 48 hpf (Fig. 4A; n ? 35). These
clustered cells are considered to be clones of progeny derived from
a single parental cell (24). Some of these cells were observed to be
undergoing cytokinesis, and the dividing nuclei were easily detect-
able by DsRed2-Nuc labeling (Fig. 4B; n ? 18). In contrast,
ventricular and atrial myocytes expressing grl-EGFP failed to form
such cell clusters (Fig. 4C; n ? 46). These scattered cardiomyocytes
maintaining single nuclei failed to divide (Fig. 4D; n ? 46).
Moreover, the size of cardiomyocytes expressing grl-DsRed or
grl-EGFP was substantially less than those cells expressing EGFP
alone (Fig. 4D; Fig. 4C versus Fig. 4A), which is consistent with the
observation in grl misexpression experiments (Fig. 3L).
gata5 Antagonizes the grl-Mediated Cardiac Growth Effects. Because
GATA factors promote myocardial proliferative growth in the
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(A) and an affected heart in grl-misexpressing embryos (B). (C and D) Immuno-
staining with F46 antibodies (FITC for green) showing a control atrium (C) and a
small atrium in grl-misexpressing embryos (D). (E and F) Double exposures of
ventricle and atrium in grl-misexpressing embryos (F). (G) Histological analyses
showing the normal myocardium (arrowhead) and endocardium (arrow) with
blood cells present in cardiac chambers. (H) grl-misexpressed heart develops
myocardium (arrowhead) and endocardium (arrow). (I and J) Confocal micros-
copy showing a control heart (I) and a hypoplastic heart (J). (K and L) grl misex-
significant difference between grl-misexpressing and WT data (P ? 0.001). Con,
control embryos. Red arrow and v, ventricle. Blue arrow and a, atrium. (M)
Schematic representation of the grl deletion mutants and their effects on em-
bryonic heart growth. Percentage (%) indicates the number of embryos with
embryos. n, microinjection times.
grl misexpression in WT embryos reduces the heart size. (A and B)
www.pnas.org?cgi?doi?10.1073?pnas.0702240104Jia et al.
heart, we tested whether certain GATA family members can
reverse the grl-induced cardiac hypoplasia. We coexpressed gata5
and grl at different molar ratios at one- to two-cell stages in
embryos. Remarkably, at a molar ratio of 1:1, forced expression of
gata5 completely rescued cardiac hypoplasia in embryos misex-
pressing grl (Fig. 5 C, D, G, and H and SI Appendix, section V),
whereas gata5 misexpression alone expanded the cmlc2 domain
cardiomyocyte numbers in rescued hearts were restored by coex-
pression of gata5 to near normal levels, compared with controls at
specific interaction between grl and gata5, we tested whether gata4
the small hearts induced by grl were not rescued after the coex-
pression of gata4 at any tested molar ratios. gata4 misexpression
alone caused a marginal expansion of the cmlc2-expression domain
that delineates the heart tube (SI Appendix, section V). Conversely,
we examined whether a reduced gata5 activity suppressed the
excessive cardiac growth in grl mutants. We microinjected gata5
antisense morpholinos (gata5-MO) into both mutant and WT
embryos. Although knockdown gata5 at low doses in WT embryos
a marked reduction in heart size and cardiomyocyte number (Fig.
5 I and J and SI Appendix, section VI), reduced gata5 activity in grl
mutant embryos resulted in only modest effects on heart volume
and cell number (Fig. 5 K and L and SI Appendix, section VI).
Hence, gata5 knockdown suppresses the excessive heart growth in
grl mutants. Together, our results support the notion that a func-
growth during embryogenesis.
Grl Forms a Protein Complex with Gata5 and Represses Gata5-
Mediated Transcription. In zebrafish, both grl and gata5 are ex-
pressed in myocardial precursor cells and the embryonic heart (SI
Appendix, section I) (4). We hypothesized that the opposing effects
between Grl and Gata5 may be mediated through protein–protein
interactions. Coimmunoprecipitations of Myc-tagged Gata5 and
EGFP-tagged Grl in COS1 cells revealed the physical association
between Gata5 and Grl (Fig. 6A). Subsequent experiments done
Grl-yrpw mutants were able to associate with Gata5, whereas
failed to interact with Gata5 (Fig. 6A). These results indicate that
the carboxyl region containing the Orange domain and its contig-
uous regions (but not YRPW domain) is necessary for the physical
association. In vivo, forced expression of grl-trunc failed to inhibit
association with Gata5, suggesting that protein interactions are
required for the inhibitory effects of Grl on heart growth. The Grl
mutants lacking a basic or a helix–loop–helix domain were able to
bind to Gata5 but did not result in cardiac hypoplasia when
misexpressed (Fig. 3M), implying that protein interactions between
Grl and Gata5 are necessary but not sufficient to cause grl-induced
We reasoned that Grl mutant proteins lacking a basic or a
helix–loop–helix domain, although not devoid of associative capac-
to inhibit embryonic heart growth. To test this hypothesis, we
performed luciferase assays, using an anf promoter that is normally
activated by GATA factors in a vitro system. HeLa cells were
cotransfected with gata5 and with grl or grl mutant expression
constructs. The native Grl protein strongly repressed gata5-
transactivation of the anf reporter (Fig. 6B). Grl-orange and
Grl-yrpw preserved the transcriptional repressive activity and also
resulted in inhibition of heart growth in certain degrees (Figs. 3M
and 6B). In contrast, Grl-basic and Grl-hlh bound to Gata5 but
demonstrated a loss of repressive activity and failed to develop
cardiac hypoplasia (Figs. 3M and 6 A and B). These data revealed
the possible existence of a transcriptional repression domain (the
bHLH domain) along with a separate protein binding domain (the
carboxyl region) within Grl. Surprisingly, deletion of the carboxyl
region, the putative Gata5 binding region, did not result in a
complete loss of repressive activity. Because Hey2/Hrt2 protein
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the embryonic heart. Microinjection of cmlc2:EGFP and cmlc2:grl-EGFP into
WT embryos (A and C), and microinjection of cmlc2:DsRed-Nuc and cmlc2:grl-
DsRed-Nuc into cmlc2-EGFP transgenic embryos (B and D) at one- to two-cell
analyses, using Zeiss optics (A and C) and double-channel confocal optics (B
and D). A, atrium; V, ventricle.
Increased grl activity inhibits cardiomyocyte proliferative growth in
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heart growth. (A and E) cmlc2 in situ hybridization revealing the normal size
of the heart tube (A) and heart (E) in egfp-misexpressing embryos. (B and F)
Forced gata5 expression (90 pg) in WT embryos causes a marginal expansion
causes hypoplasia of both the heart tube (C) and heart (G). (D and H) Coex-
in a relatively normal size for both the heart tube (D) and heart (H). (I) A
normal-sized heart labeled by cmlc2 in situ in WT embryos microinjected with
gata5-control morpholinos. (J) Microinjection of gata5-MO (?13 ng) in WT
embryos causes development of a hypoplastic heart. (K) grl mutants display a
hyperplastic heart. (L) gata5 morpholino injection (?13 ng) into grl mutants
results in a modest reduction of heart size. (A–D) Lateral views. The red line
indicates the length of the heart tube. (E–L) Ventral views.
grl and gata5 function antagonistically in controlling embryonic
Jia et al.
August 28, 2007 ?
vol. 104 ?
no. 35 ?
result may be explained by the possibility that the bHLH domain
might recruit other DNA-binding factors or Gata5-interacted pro-
teins in HeLa cells that restore Grl repressive ability, but this may
not occur in vivo and in cultured cardiomyocytes (Fig. 3M and SI
Appendix, section VII). The presence of these unidentified cellular
factors may provide an additional level to tissue specificity to the
regulation of Gata5 activity. Thus, our in vitro observations are
support the notion that the Grl cardiac growth effect is mediated
at least in part through the interaction with Gata5 resulting in
inhibition of Gata5-mediated transcription.
In this study, we describe a regulatory circuit that involves Gridlock
and a well known transcriptional activator Gata5 in controlling
myocardial proliferative growth. We establish that Hairy-related
transcriptional repressor Grl is a negative regulator of Gata5
responsive heart growth. Mutations in grl cause up-regulation of
of cardiac hyperplasia. This study is the first demonstration of the
ability of a bHLH transcriptional repressor to inhibit cardiogenesis
in the context of developing embryo. Importantly, we link the
opposing effects between Grl and Gata5 to their transcriptional
The normal role of grl in the heart appears to prevent excessive
growth of the myocardium and serve to limit embryonic heart size.
compliments our finding that reduced grl activity causes excessive
myocardial proliferative growth. The cell-autonomous inhibitory
developing heart and in cultured cardiomyocytes (SI Appendix,
section VII). These data suggest that cell growth and division are
tightly coupled in the embryonic heart.
The hey2 mutant mouse displays a wide spectrum of cardiac
morphologic malformations and occasionally develops cardiomy-
opathy (19–22). Our present study shows that alterations of cardi-
omyocyte proliferative growth develop in grl mutant hearts. Addi-
tionally, Hairy-related transcription factors are involved in
patterning the atrioventricular canal (28, 29). These data suggest
that cellular derangements in combination with patterning defects
may underlie the morphologic alterations in these malformed
hearts and potentially provide a cellular basis for some forms of
congenital heart disease. In zebrafish, reduced grl activity up-
regulates expression of both atrial and ventricular genes (Fig. 1).
However, deletion of hrt2/hey2 in murine cardiomyocytes causes
ectopic atrial gene expression in the ventricle (30). This might be
due to the difference of expression patterns of hrt2/hey2 and grl in
mice versus fish. Murine hey2 is expressed in the ventricle and not
the atrium (27), whereas grl is the only hey gene that is expressed
in both cardiac chambers (SI Appendix, section I). Thus, murine
hey1 and possible heyL may play a compensatory function in the
atrium or even the whole heart in the absence of hey2 activity.
grl/hey2 that acts as an important negative regulator of myocardial
We observed that gata5 but not gata4 can rescue the grl-induced
cardiac hypoplasia, demonstrating that these two gata factors have
distinctive functions in regulating heart growth in zebrafish. Our
results highlight the specificity and effectiveness of gata5 in coun-
teracting the effects of a bHLH transcriptional repressor on heart
growth. We further link the transcriptional interactions to func-
tional antagonism between Grl and Gata5 in embryonic heart
growth. This may provide a mechanistic explanation for Grl-
dependent cardiac repressive effects. In this context, Grl interacts
with Gata5 via the carboxyl region and results in inhibiting the
Gata5-mediated gene transcription via its bHLH domain. This
cross-transcriptional interaction prevents excessive heart growth
and thus ensures an optimal mass for the embryonic heart. The
ability of transcriptional repression and physical association of
murine Hey2/Hrt2 with Gata4 has been shown to lie in the same
basic domain (25, 26). In our study, we have found that the Grl
carboxyl region containing the Orange domain is required for
physical association with Gata5, whereas the bHLH domain is
critical for inhibiting the Gata5 transcriptional activity. These data
separate the protein-binding region from the transcriptional re-
pression domain. The different physical capacities of murine Hey2/
two homologues or species-dependent differences on two Gata
factors. Because the expression of early growth genes is up-
regulated in grl mutant hearts, further elucidation of these factors
in this important developmental pathway may lead to a better
understanding the mechanisms that control embryonic heart
(A) Interaction of Grl and Gata5 depends on the carboxyl region. COS1 cells
were cotransfected with EGFP-tagged Grl, Grl-trunc, Grl-basic, Grl-hlh, Grl-
orange, Grl-yrpw constructs, and Myc-tagged Gata5 or empty PcDNA3.4 vec-
tor. Output: Protein interactions were detected by immunoblotting, using
anti-EGFP antibody after immunoprecipitation, using anti-Myc antibody. In-
put: Gata5, Grl, and Grl mutant proteins were stably expressed and detected
in the transfected lysate, using anti-Myc and anti-EGFP antibody. (B) The
repressive activity of Grl is found in the bHLH domain. Fold activation of the
anf promoter-driven luciferase reporter in the presence of Gata5-myc with
EGFP-tagged Grl, Grl-basic, Grl-hlh, Grl-orange, Grl-yrpw, or Grl-trunc. (C)
Western blot analyses showing expression of Grl and Grl-mutant proteins.
Fold activation of anf luciferase activity was normalized by dividing the
luciferase activity with the relative amount of proteins. Grl and Grl mutants
www.pnas.org?cgi?doi?10.1073?pnas.0702240104 Jia et al.
Materials and Methods
Zebrafish Strains and Molecular Constructs. The zebrafish strains
used in this study were raised according to standard procedures
(31). grlm145mutants and cmlc2:EGFP line are described in refs. 16
and 32. grlvu59mutants were generated by TILLING method (33)
(SI Appendix, section III). grl and gata5 deletion constructs were
generated by PCR-based subcloning technique (SI Appendix, sec-
tion VIII). The zebrafish gata4 and gata5 cDNAs were identified
and cloned by using Titan One Tube RT-PCR System (Roche
Diagnostics, Indianapolis, IN). The zebrafish whole-length gata4
cDNA sequence has been deposited in GenBank (DQ886664) and
used in this study.
RNA Purification, Microarray Analysis, and Real-Time PCR.Microarray
analysis was performed with a Zebrafish Genome Array (Af-
fymetrix, Santa Clara, CA) (SI Appendix, section II). Real-time
PCR was performed with the SYBR green Kit in the 7900HT
Systems (Applied Biosystems, Foster City, CA) (SI Appendix,
In Situ Hybridization, Immunofluorescent Staining, TUNEL, and Acri-
dine Orange Assay. RNA in situ hybridization was carried out as
using antibodies MF20, S46, and phosphohistone3 (Santa Cruz
Biotechnology, Santa Cruz, CA). TUNEL assay was performed
with the Cell Death Detection KIT TMR red (Roche Diagnostics)
(35). Live embryos were used for acridine orange staining (Sigma–
Aldrich, Piscataway, NJ) (35). For histological analysis, specimens
were fixed in 4% paraformaldehyde, dehydrated, embedded in
plastic (JB-4), and sectioned at 5 ?m. Nomarski and fluorescent
photomicroscopy were performed by using Axioplan 2 and Axio-
cam camera (Carl Zeiss, Thornwood, NY).
Microinjection of mRNA and MO. Capped mRNAs were synthesized
by using an mMESSAGE mMACHINE kit (Ambion, Austin, TX)
with gata4, gata5, grl, grl-basic, grl-hlh, grl-orange, grl-yrpw, or
grl-trunc expression plasmids and microinjected into one- to two-
cell embryos. gata5-MO and control-MO targeting ATG (36) were
synthesized from Gene Tools (Philomath, OR). For DNA micro-
injections, linearized cmlc2-egfp, cmlc2-grl-egfp, cmlc2-dsred-Nuc,
and cmlc2-grl-dsred-Nuc at 100 ng/?l were used.
Whole-Mount Confocal Microscopy and Morphometric Analyses. To
examine the embryonic hearts, the embryos were ventrally posi-
tioned toward the microwell with a 45° right-sided orientation in a
special viewing chamber. Confocal images were captured by using
a Zeiss LSM 510 Confocal Microscope System with ?20/0.75
planapochromat objective. The confocal pinhole was adjusted to 1
Airy unit for optimal z resolution with the 0.75 N.A. objective
resulting in serial sections 0.9 ?m thick. Images were acquired at
0.45-?m intervals. 3D projections were constructed by using the
LSM Browser software (Zeiss), and analysis was performed by
clearly visualizing each cell’s surface margin, embryos were com-
cardiomyocytes were chosen from each group of grl mutant,
grl-misexpressing, and control embryos, and their cell sizes were
measured. Cell numbers in the heart tube, atrium, and ventricle
were determined by counting 10 embryos in each group of grl
mutant, grl-misexpressing, and control embryos at 24 and 48 hpf.
Several (five to two) representative embryos were chosen for
counting cell number from groups of normal, medium, and small
heart phenotypes, which had been subjected to injection of
grl?gata5 or grl?gata4 mRNAs to WT embryos or to injection of
gata5-MO to grl mutant and WT embryos. Numbers of cells were
counted in each confocal section by using Pickpointer embedded in
the Image J. Pickpointer permits a single user-defined mark to
appear throughout z stacks of images, allowing the tracking of a
single cell in overlapped z sections to avoid double counting.
Cell Transfection, Immunoprecipitation, and Luciferase Assays. Co-
immunoprecipitation assays were performed by using a modified
protocol (25). COS1 cells were cotransfected with the indicated
expression vectors for Myc- or EGFP-tagged proteins. Luciferase
assays were conducted in HeLa cells cotransfected with the appro-
priate reporter and expression constructs, and cell lysates were
assayed as described in ref. 25.
We thank John Guan, Mingwei Ni, and Rebacca Coyle for invaluable
assistance and David Bader, Joey Barnart, Lilianna Solnica-Krezel,
Xiaolei Xu, and members of our laboratories for comments on the
manuscript and helpful discussions. This research was supported in part
by grants from the National Institutes of Health (I.N.K., T.T.N., D.S.,
and T.P.Z.), the March of Dimes (D.S. and T.P.Z.), and the American
Heart Association (D.S. and T.P.Z.).
1. MacLellan WR, Schneider MD (2000) Annu Rev Physiol 62:289–319.
2. Olson EN (2004) Nat Med 10:467–474.
3. Peterkin T, Gibson A, Loose M, Patient R (2005) Semin Cell Dev Biol 16:83–94.
4. Reiter JF, Alexander J, Rodaway A, Yelon D, Patient R, Holder N, Stainier DY (1999)
Genes Dev 13:2983–2995.
5. Pu WT, Ishiwata T, Juraszek AL, Ma Q, Izumo S (2004) Dev Biol 275:235–244.
6. Zeisberg EM, Ma Q, Juraszek AL, Moses K, Schwartz RJ, Izumo S, Pu WT (2005) J Clin
7. Xin M, Davis CA, Molkentin JD, Lien CL, Duncan SA, Richardson JA, Olson EN (2006)
Proc Natl Acad Sci USA 103:11189–11194.
8. Zhong TP, Rosenberg M, Mohideen MA, Weinstein B, Fishman MC (2000) Science
9. Leimeister C, Externbrink A, Klamt B, Gessler M (1999) Mech Dev 85:173–177.
10. Chin MT, Maemura K, Fukumoto S, Jain MK, Layne MD, Watanabe M, Hsieh CM, Lee
ME (2000) J Biol Chem 275:6381–6387.
11. Kokubo H, Lun Y, Johnson RL (1999) Biochem Biophys Res Commun 260:459–465.
12. Nakagawa O, Nakagawa M, Richardson JA, Olson EN, Srivastava D (1999) Dev Biol
13. Winkler C, Elmasri H, Klamt B, Volff JN, Gessler M (2003) Dev Genes Evol 213:541–553.
14. Driever W, Solnica-Krezel L, Schier AF, Neuhauss SC, Malicki J, Stemple DL, Stainier DY,
Zwartkruis F, Abdelilah S, Rangini Z, et al. (1996) Development (Cambridge, UK) 123:37–46.
15. Weinstein BM, Stemple DL, Driever W, Fishman MC (1995) Nat Med 1:1143–1147.
16. Zhong TP, Childs S, Leu JP, Fishman MC (2001) Nature 414:216–220.
17. Peterson RT, Shaw SY, Peterson TA, Milan DJ, Zhong TP, Schreiber SL, MacRae CA,
Fishman MC (2004) Nat Biotechnol 22:595–599.
18. Zhong TP (2005) Curr Top Dev Biol 71:53–81.
19. Donovan J, Kordylewska A, Jan YN, Utset MF (2002) Curr Biol 12:1605–1610.
20. Gessler M, Knobeloch KP, Helisch A, Amann K, Schumacher N, Rohde E, Fischer A,
Leimeister C (2002) Curr Biol 12:1601–1604.
21. Sakata Y, Kamei CN, Nakagami H, Bronson R, Liao JK, Chin MT (2002) Proc Natl Acad
Sci USA 99:16197–16202.
22. Kokubo H, Miyagawa-Tomita S, Tomimatsu H, Nakashima Y, Nakazawa M, Saga Y,
Johnson RL (2004) Circ Res 95:540–547.
23. Madden SL, Cook DM, Morris JF, Gashler A, Sukhatme VP, Rauscher FJ (1991) Science
24. Rottbauer W, Saurin AJ, Lickert H, Shen X, Burns CG, Wo ZG, Kemler R, Kingston R,
Wu C, Fishman M (2002) Cell 111:661–672.
25. Kathiriya IS, King IN, Murakami M, Nakagawa M, Astle JM, Gardner KA, Gerard RD,
Olson EN, Srivastava D, Nakagawa O (2004) J Biol Chem 279:54937–54943.
26. Fischer A, Klattig J, Kneitz B, Diez H, Maier M, Holtmann B, Englert C, Gessler M (2005)
Mol Cell Biol 25:8960–8970.
27. Nakagawa O, McFadden DG, Nakagawa M, Yanagisawa H, Hu T, Srivastava D, Olson EN
(2000) Proc Natl Acad Sci USA 97:13655–13660.
28. Rutenberg JB, Fischer A, Jia HB, Gessler M, Zhong TP, Mercola M (2006) Development
(Cambridge, UK) 133:4381–4390.
29. Kokubo H, Tomita-Miyagawa S, Hamada Y, Saga Y (2007) Development (Cambridge, UK)
30. Xin M, Small EM, van Rooij E, Qi X, Richardson JA, Srivastava D, Nakagawa O, Olson
EN (2007) Proc Natl Acad Sci USA 104:7975–7980.
31. Westerfield M (2000) The Zebrafish Book (Univ of Oregon Press, Eugene, OR).
32. Burns CG, Milan DJ, Grande EJ, Rottbauer W, MacRae CA, Fishman MC (2005) Nat Chem
33. Sood R, English MA, Jones M, Mullikin J, Wang DM, Anderson M, Wu D, Chan-
drasekharapp SC, Yu J, Zhang J, Liu PP (2006) Methods 39:220–227.
34. Xu X, Meiler SE, Zhong TP, Mohideen M, Crossley DA, Burggren WW, Fishman MC
(2002) Nat Genet 30:205–209.
35. Parng C, Anderson N, Ton C, McGrath P (2004) Methods Cell Biol 76:75–85.
36. Trinh le A, Yelon D, Stainier DY (2005) Curr Biol 15:441–446.
Jia et al.
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