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First published online 20 September 2006
doi: 10.1242/dev.02586
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1 Brookdale Department of Molecular, Cell and Developmental Biology, Box 1020,
Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029,
USA.
2 The Burnham Institute, Center for Neurosciences and Aging, 10901 North Torrey
Pines Road, La Jolla, CA 92037, USA.
Author for correspondence (e-mail:
manfred.frasch{at}mssm.edu)
Accepted 16 August 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Dorsal vessel, Drosophila, tinman, Dorsocross, Nkx2.5, seven-up, Heart, Repressor
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Buckingham et al., 2005;
Reim and Frasch, 2005). In
most cases, the expression of each of these cardiogenic factors spans a large
developmental window of cardiogenesis and continues in the mature heart.
Consequently, the specific function of a cardiogenic factor can change during
the progression of cardiogenesis because, for example, it depends on the
presence of additional factors and signals that modulate its activity.
Although several examples of differential and stage-specific functions of
cardiogenic factors have already been described
(Pashmforoush et al., 2004;
Zeisberg et al., 2005;
Oka et al., 2006), our overall
knowledge of these dynamic changes in regulatory activities is still very
limited.
The apparent conservation of cardiogenic factors during evolution suggests
strongly that some of their specific molecular functions within the regulatory
network of cardiogenesis and heart differentiation may also be shared among
different vertebrate and invertebrate species. Prime examples of conserved
cardiogenic factors include the Drosophila NK homeodomain factor
Tinman and the homologous Nkx2.5 proteins in vertebrates, which have been
shown to play key roles during early stages of heart formation in the
respective organisms (Harvey,
1996). In Drosophila, the tinman (tin)
gene is essential for the specification of all cardiac progenitors in the
early dorsal mesoderm, and some of its target genes during this event, and the
functional Tin-binding sites in their regulatory sequences have been defined
by genetic and molecular analyses (Azpiazu
and Frasch, 1993; Bodmer,
1993; Xu et al.,
1998; Halfon et al.,
2000; Gajewski et al.,
2001; Knirr and Frasch,
2001; Han et al.,
2002). The requirement for tin during the earliest steps
of cardiogenesis is reflected in its early expression in the mesoderm, which
is controlled by two distinct enhancer elements
(Yin et al., 1997). Initially,
during the invagination and spreading of the mesoderm, tin expression
is activated by the bHLH protein Twist through an intronic enhancer in the
entire trunk mesoderm (Bodmer et al.,
1990; Yin et al.,
1997). Thereafter, tin expression becomes dependent on a
Dpp-responsive enhancer located downstream of tin, which leads to the
restriction of tin expression to dorsal mesodermal cells that receive
Dpp signals from the ectoderm (Xu et al.,
1998). This corresponds to the stage when tin is required
for the specification of myocardial and pericardial progenitors within the
dorsally located cardiogenic mesoderm. In addition, tin is essential
for the formation of other dorsal mesodermal derivatives during this stage,
which include trunk visceral mesoderm precursors and dorsal somatic muscle
progenitors. Even later, tin expression is further restricted to
cardiac progenitors and this expression persists in myocardial and pericardial
cells of the mature dorsal vessel. tin expression in cardioblasts
depends on the Tbx20-related genes midline (mid)
and H15, and is driven by another downstream enhancer
(Yin et al., 1997;
Reim et al., 2005). It is
thought that tin plays a role in the differentiation of the heart
progenitors during this late phase of expression, which is likely to include
the direct activation of Mef2 and Hand, which regulate
normal differentiation, as well as of cardiac differentiation genes such as
ß3-tubulin (Bour et al.,
1995; Lilly et al.,
1995; Gajewski et al.,
1997; Kremser et al.,
1999; Han and Olson,
2005). However, genetic analysis of the significance of late
tin expression in cardial cells has been hampered owing to the fact
that tin mutant embryos lack a heart altogether because of the early
requirement of tin for cardiogenesis. Furthermore, conditional
alleles that would circumvent this problem have not been available.
The Drosophila heart is a relatively simple linear tube that, in
spite of its overt simplicity, is highly structured and consists of a variety
of myocardial and pericardial cell types
(Rizki, 1978;
Ward and Skeath, 2000). This
organization is illustrated by the presence of different chamber-like regions
with distinct functions (the heart in the posterior and the aorta in the
anterior), and by the controlled posterior-to-anterior flow of the larval
hemolymph, which enters through valvular openings (ostia) into the heart
region. In addition to its broad anteroposterior (AP) organization, the dorsal
vessel maintains a segmental organization, with units that, in most of the
tube (i.e. from segments A2 to A7), consist of six pairs of cardioblasts in
each segment. The segmental organization and AP polarity within each segment
is revealed by the restricted expression of several transcription
factor-encoding genes (reviewed by Lo and
Frasch, 2003). In fact, the myocardial expression of tin
within the dorsal vessel is restricted to the four posterior pairs of
cardioblasts within each of these segments. Conversely, the anterior two pairs
of cardioblasts in each segment are marked by the expression of
seven-up (svp), which encodes an orphan nuclear receptor,
and of the Dorsocross T-box genes (Doc1, Doc2 and
Doc3, henceforth referred to as Doc) that are downstream of
svp in these cells (Gajewski et
al., 2000; Lo and Frasch,
2001; Reim et al.,
2003; Reim and Frasch,
2005). In the heart region, this differential organization
correlates with the formation of distinct subtypes of myocardial cells within
each segment. Specifically, the two Svp/Doc-positive cardioblasts in each
segment differentiate into ostial cells to form inflow valves, whereas the
four Tin-positive cells form `working' cardiomyocytes of the heart
(Molina and Cripps, 2001).
Currently, it is not known whether the Tin-versus Svp/Doc-positive
cells in the major part of the aorta are also distinct with regard to their
function or physiology. Likewise, it is still unclear whether the two central
pairs of cardioblasts, which express the ladybird (lb)
homeobox genes in addition to tin
(Jagla et al., 1997), are
functionally distinct from the two posterior pairs of
Lb-/Tin+ cells within each segment.
In the present study, we test genetically whether tin possesses a
later cardiogenic function specifically within the four Tin+ pairs
of cardioblasts in each segment of the dorsal vessel. Based upon the known
arrangement of the different enhancer elements, we generated genomic
tin constructs that support normal patterns of tin
expression in the early, or early plus dorsal, mesoderm, without supplying
tin expression in any cardioblasts of the dorsal vessel at later
stages. By analyzing the phenotype of embryos that carry these constructs in a
tin-null mutant background, we show that tin expression
during the early stages is sufficient to specify the dorsal mesoderm
derivatives, including cardiac progenitors, to generate a dorsal vessel, and
to allow the development of adult flies. However, the specific ablation of
functional tin in cardioblasts reveals that its function is normally
required for the normal diversification of cardial cells and the proper
differentiation of the four Tin+ cardioblast pairs within each
segment. In the heart region, this function of tin is required for
preventing `working' myocardial cells from acquiring ostia-like features,
which include specific morphological properties and the expression of
wingless (wg) (Lo et
al., 2002; Ponzielli et al.,
2002). We demonstrate that a key role of tin is the
repression of Doc and, hence, the restriction of Doc to two segmental
pairs that include the future ostial cells. This activity can also be
fulfilled by Nkx2.5, thus indicating that the repressive potential of
NK2-class homeodomain proteins has been conserved during evolution. In
addition, tin promotes the expression of particular differentiation
genes specifically in the Tin+ cardioblasts, which are
likely to contribute to the differential properties of the `working'
myocardium. We present a model in which svp in cardiac progenitors
and their descendents represses tin in two cardioblast pairs within
each segment from A2 to A7; this allows the expression of Doc by
default, which in turn contributes to the continued repression of tin
in these cells. Together, these interactions lead to the establishment and
maintenance of two mutually exclusive differentiation states of cardioblasts,
which are modulated further by differential homeotic gene activities in the
aorta versus heart regions of the dorsal vessel. In addition, we show that the
function of tin is required for normal remodeling and growth of the
dorsal vessel during metamorphosis in pupal stages and, thus, is needed for
generating the enlarged myocardium observed in adult flies.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), tinEC40
(Bodmer, 1993) and
mid1 (Buescher et al.,
2004), and the deficiency lines Df(3R)GC14
(Azpiazu and Frasch, 1993;
Bodmer, 1993),
Df(3L)DocA and Df(3L)29A6
(Reim et al., 2003) and
svpAE127 (svp-lacZ; from Y. Hiromi, National
Institute of Genetics, Mishima, Japan) were balanced with CyO,
wg-lacZ or TM3, eve-lacZ. Ectopic expression experiments with
UAS-tin (Yin and Frasch,
1998), UAS-Doc1, UAS-Doc2
(Reim et al., 2003),
UAS-svp1 (from M. Hoch, Bonn University, Germany) or
UAS-Nkx2.5 (lines 1, 2b, and 3a) were carried out at 28°C.
UAS-Nkx2.5 was generated from a full-length cDNA of
Flag-Nkx2.5 (Kasahara et al.,
2001), initially subcloned as a HindIII/NotI
blunted fragment into EcoRV of pBS-SK and then into EcoRI of
pUAST vector (Brand and Perrimon,
1993). S59-Mef2-Ht
D-Gal4 (line
10-2a) contains a minimal cardioblast enhancer of Mef2 (Hanh
Nguyen, AECOM, Bronx, NY, unpublished) and a minimal skeletal muscle enhancer
of S59/slouch (M.F., unpublished) in pGAL4-221 (from Christian
Klämbt, Münster University, Germany), and is active in cardioblasts,
in S59-positive somatic muscles and, weakly, in some pericardial cells.
Embryo staining
We used rabbit anti-ß3-Tubulin (1:1500, TSA; from Renate
Renkawitz-Pohl, Philipps University Marburg, Germany), rat anti-Bin (1:500)
(Zaffran et al., 2001),
anti-
-actinin (Saide et al.,
1989), rabbit anti-Homothorax (1:500) (from Richard Mann, Columbia
University, New York, NY), monoclonal rat anti-Tropomyosin (1:500), mouse
anti-ß-galactosidase 40-1a (1:60, TSA), rabbit anti-Toll (1:500, TSA;
from Steve Wasserman, UC San Diego, CA) and mouse anti--Spectrin 3A9
(1:10, TSA) (from the Developmental Studies Hybridoma Bank, University of
Iowa, developed under the auspices of NICHD). Other antibodies and the in situ
probes for bkh, mid, H15, svp1 and Sur are described by Reim
et al. (Reim et al., 2005) and
by Lo and Frasch (Lo and Frasch,
2001). A Zeiss Axiophot and the confocal Leica TCS-SP and Zeiss
LSM 510 META systems were used for analysis.
Generation of tin rescue constructs
Restriction fragments from the genomic region of tin in
pCaSpeR-Re28 (Azpiazu and Frasch,
1993) were subcloned into pBluescript KS (Stratagene). For,
tin-AB, the large genomic EcoRI fragment was cloned into pCaSpeR-3.
The tin-D enhancer element (Yin et al.,
1997) was cloned downstream of the tin-AB fragment to generate
tin-ABD. For tin-D, the EcoRV/EcoRI fragment from the
tin cDNA was used to replace the corresponding genomic fragment in
tin-ABD, leaving intact the minimal promoter. All pCaSpeR constructs were
sequenced and several transformant lines from each were analyzed. For rescue
with tin-ABD, the insertions T003-1B2 (2nd chromosome) and/or
T003-1C1 (3rd chromosome) were used.
Cardiac pacing and survival assays
Females and males were separated and aged to 2-3 days post-eclosion. Flies
were aligned between two electrodes on a glass microscope slide and then paced
to 6 Hz for 30 seconds using a square wave stimulator
(Wessells and Bodmer, 2004;
Wessells et al., 2004). Heart
failure rate is defined as the percentage of flies that either arrest or
fibrillate during or immediately after pacing. Flies that have undergone heart
failure were observed for 2 minutes to calculate the percentage of flies that
recover to a normal resting heartbeat [recovery rate; see Wessells et al.
(Wessells et al., 2004) for
the survival assay].
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Bodmer,
1993), each regulated by a separable enhancer module that is
subject to distinct regulatory inputs (Yin
et al., 1997; Venkatesh et
al., 2000). The earliest tin expression, which is
observed in the entire trunk mesoderm during gastrulation, is recapitulated by
constructs containing the enhancer element tinB
(Fig. 1A,D). The Dpp-dependent
expression in the dorsal mesoderm, which includes the primordia of the dorsal
vessel, visceral muscles and somatic dorsal muscles, during stage 10-11 is
driven by tinD (Fig.
1B,D) (Xu et al.,
1998). At later stages, the enhancer tinC mediates
expression in a segmental subset of cardioblasts of the developing and mature
dorsal vessel (Fig. 1C,D)
(Lo and Frasch, 2001). The maintained tin expression in the dorsal vessel indicates a yet undefined role for tin in the proper differentiation and normal physiology of myocardial cells. To test this possibility, we aimed to restore early tin activity in an otherwise tin-null mutant background in order to permit proper specification of cardiac progenitors without allowing tin expression in cardial cells themselves. To this end, we produced a series of genomic tin rescue constructs containing specific subsets of these enhancer elements without the cardioblast-specific enhancer `C' (Fig. 1D), and brought them into tin mutant backgrounds (Fig. 1E,F; note that the known inactivity of the tinD and tinC enhancers in embryos lacking early tin activity prevented us from performing analogous rescue experiments with tin driven solely by tinD or tinC).
First, we tested whether the early phases of tin expression in the
entire trunk mesoderm and/or the dorsal trunk mesoderm would be sufficient for
cardiac specification and dorsal vessel formation. Surprisingly, tin
mutant embryos that express Tin protein from a transgenic rescue construct
driven by the twist-dependent enhancer (tin-AB) only in the
early mesoderm are able to specify progenitors of all derivatives of the
dorsal mesoderm. For example, staining for Biniou, a FoxF-related factor
expressed in the visceral mesoderm
(Zaffran et al., 2001), shows
that the visceral muscle precursors are present in tin-AB;
tin mutant embryos (Fig.
2A,B). The progenitors of cardioblasts and pericardial cells, as
marked by their expression of brokenheart (bkh)
(Fremion et al., 1999) and
even-skipped (eve)
(Frasch et al., 1987), are
also present in these mutants, although their number is variably reduced when
compared with wild-type embryos (Fig.
2C-F). Also at later stages, cardioblasts and all types of
pericardial cells are present in tin-AB; tin mutant embryos,
although the dorsal vessel appears discontinuous, the arrangement of
cardioblasts and pericardial cells is irregular, and lymph gland formation is
impaired (Fig. 2E-H).
Altogether, these and other data suggest that the early phase of tin expression is necessary and sufficient to specify the progenitors of all cell types generated from the dorsal mesoderm, but specification occurs with reduced efficiency. This activity of tin in tin-AB; tin mutant embryos in the absence of the second, dorsal-specific phase of tin expression is probably due to Tin protein perduring in the dorsal and cardiac mesoderm from its earlier phase of twist-driven mRNA expression. However, the combination of the first two phases of tin expression, in both early in the entire early mesoderm and subsequently in the dorsal mesoderm, leads to an improved rescue of cardiac specification and allows the formation of lymph glands (shown below). This demonstrates that Dpp-induced tin does contribute to the full biological activity of tin. We conclude that the absolute requirement for dpp during the specification of dorsal mesodermal derivatives is mainly due to the requirement of synergistic Tin and activated Smads during the induction of Tin/Dpp targets, and to a lesser degree to the induction of tin expression itself by Dpp.
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;
Lilly et al., 1995) is
detectable along the dorsal midline of tin-ABD;
tin346 mutant embryos (compare
Fig. 3B with 3A). The slight
and variable decrease in cell numbers when compared with the wild type could
be due to variable and still suboptimal tin expression from the
transgenic tin-ABD rescue construct. In addition, cardioblasts of
tin-ABD; tin346 mutant embryos show nearly normal
expression of general cardioblast markers, including the
Tbx20-homologs H15 and midline (mid)
(Miskolczi-McCallum et al.,
2005; Qian et al.,
2005; Reim et al.,
2005), the bHLH gene Hand
(Kölsch and Paululat,
2002), the sarcomeric protein Tropomyosin
(Wolf et al., 2006) and the
transmembrane receptor encoding gene Toll
(Wang et al., 2005)
(Fig. 3A-H). These data lead to
the conclusion that cardiac precursors differentiate into myocardial cells in
the absence of cardioblast-specific tin expression. However, staining
for Toll, which marks the basolateral membrane of the cells, and for the
cytoskeletal protein -Spectrin demonstrate that in absence of
tin, the morphology of the posterior `heart' region in particular is
abnormal (Fig. 3H, and data not
shown). The cardioblast rows are uneven, some cardioblasts appear
intercalated, and the shape of the cardioblasts is less regular, indicating
that tin is needed for some aspects of proper specification or
differentiation of myocardial cells.
tinman expression in developing cardioblasts is required for the diversification of myocardial cell types
Based on the expression patterns of various genes, cardioblasts are thought
to acquire at least three different identities within each segment of the
dorsal vessel, the Tin-positive cells, the Tin/Lb-positive cells and the
Svp/Doc-positive cells (Lo and Frasch,
2003). The Svp/Doc-positive cells in the posterior three segments
of the dorsal vessel become different from the remaining cardioblasts
morphologically and functionally, as these are the cells that form the ostia
(inflow valves) of the larval heart
(Molina and Cripps, 2001). In
addition, based on the restricted expression of certain genes specifically in
the Tin-positive cells such as Sulfonylurea receptor (Sur),
which encodes a K+ channel subunit
(Nasonkin et al., 1999), and
the structural protein ß3-tubulin
(Kremser et al., 1999),
Tin-positive and Tin-negative cells are thought to possess different
physiological properties. To test for possible alterations in the identities
of myocardial cells in mutants lacking tin expression in the
developing dorsal vessel, we examined the expression of the available identity
markers in tin-ABD; tin346 mutant embryos
(Fig. 4). Strikingly, Doc,
which is normally expressed in the two Tin- pairs of cells within
each segment, is expressed ectopically and detected in all cardioblasts in
these embryos (compare Fig. 4B with
4A). Conversely, the expression of Lbe, which is normally
co-expressed with Tin in subsets of cardioblasts, is lost
(Fig. 4C,D)
(Jagla et al., 1997). The
expanded expression of Doc is highly reminiscent of what has recently been
described for mid mutants (Reim
et al., 2005). These mutants also lose Tin from developing
cardioblasts, which explains the similarities of the phenotypes with regard to
Doc expression and of other phenotypes such as disrupted cardioblast
diversification in the `heart' region (Fig.
3H) (Reim et al.,
2005). Furthermore, in both cases high-level expression of the
Sur gene, a likely target of Tin in the Tin-positive cells, is lost
(Fig. 4E,F)
(Reim et al., 2005; Akasaka et
al., 2006). Surprisingly, ß-Tubulin at 60D
(ßTub60D, also known as ß3-tubulin), which has
also been reported to be a target gene activated by Tin in the dorsal vessel
(Kremser et al., 1999), is
still expressed in the tin-ABD; tin346 mutant
background and maintains its intrasegmental modulation, albeit with a slightly
disturbed pattern (Fig. 4G,H).
This observation suggests that the ß3-tubulin gene is driven by
both tin-dependent and tin-independent enhancers in the
Tin+ cardioblasts. In addition, the segmental pattern of
ß3-tubulin in tin-ABD; tin346
mutants shows that there must be factors other than Tin and Doc that can still
generate a patterned expression of certain differentiation genes in the dorsal
vessel. svp, which is activated during early stage 12 possibly in
response to Hh signaling (Ponzielli et
al., 2002), also maintains its segmental expression in this
genetic background (Fig. 4I,J),
and hence may act as a repressor that antagonizes the activation of
ß3-tubulin by a uniformly expressed activator in myocardial
cells. We predict that other markers like ß3-tubulin exist that
are regulated differentially by svp but do not depend on
tin.
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), also
shows ectopic Doc expression in the absence of myocardial Tin
(Fig. 4B,D), whereas the
expression of the anterior-specific marker Homothorax (Hth;
Perrin et al., 2004) is
unaffected (see Fig. S1 in the supplementary material). In addition, the near
absence of Tin in pericardial cells causes a loss of pericardial
ladybird and a reduction of pericardial zfh-1 expression,
but the only known marker specific for Tin- pericardial cells, Odd,
does not exhibit any concomitant ectopic expression (see Fig. S2 in the
supplementary material).
Mutual repression of Tin and Doc maintains differential cardial cell identities
The observed expansion of Doc expression, as well the
morphological abnormalities in the heart region, suggest a switch in cardial
cell identities in dorsal vessels lacking Tin. In order to test this notion
further and to establish the underlying regulatory mechanisms, we examined the
expression of regulatory factors associated with cell diversifications into
different cardioblast subtypes under various genetic conditions.
Fig. 5A visualizes the
expression of the three known regulatory genes, tin, Doc and
svp (as detected by svp-lacZ expression from a
heterozygous lacZ enhancer trap insertion in svp), which are
expressed in the described `4+2' pattern in an otherwise wild-type embryo.
Whereas in the absence of Tin in developing cardioblasts Doc expression is
expanded into all cardioblasts, svp-lacZ maintains its restriction to
two pairs of cardioblasts per segment (Fig.
5B, yellow arrows). Although we have shown previously that
svp is normally required for Doc expression in the two
Svp+ cells in each hemisegment
(Lo and Frasch, 2001), our
present observation shows that Doc expression can occur in
Svp-negative cardioblasts, provided that Tin is not present. Hence, we propose
that Doc is under the repressive control of Tin rather than being
activated directly by Svp. To confirm this hypothesis, we analyzed
Doc expression in double mutants lacking Tin and Svp in cardioblasts.
Although the dorsal vessels of such embryos (tin-ABD;
svpAE127 tin346) display some morphological
defects, we still observe an expansion of Doc expression into all
cardioblasts present (Fig. 5C).
Thus, in the normal situation, the presence of Tin in four cardioblasts per
hemisegment is essential for the repression of Doc in these cells.
Accordingly, analysis of embryos in which tin is ectopically
expressed in most (although not all) cardioblasts using
S59-Mef2-HtD-Gal4 confirms the repressive activity of
Tin towards Doc (Fig.
5D,E; the S59 enhancer-driven component serves as an
internal expression control). The presence of
svp-lacZ-positive Tin+/Doc- cells
suggests that Tin did not repress svp in these experiments. In
conclusion, these data demonstrate that Tin acts as a repressor of
Doc but not svp expression in cardioblasts.
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; Lo and Frasch,
2001; Molina and Cripps,
2001) (see also Fig.
6B,C). This repressive effect of Svp towards tin is
confirmed upon ectopic expression of Svp in the cardioblasts of the dorsal
vessel (Fig. 5F). In addition,
ectopic Svp leads to an expansion of svp-lacZ expression into some,
but not all, of the Doc-positive cells, arguing for the presence of a positive
feedback loop in those cells.
We further considered the possibility that Doc can also act as a repressor
of tin. When UAS-Doc2 is ectopically expressed throughout
the dorsal vessel (Fig. 5G), we
observe a reduction or loss of tin expression in most of the
Doc-expressing cells, particularly in the posterior aorta and heart regions.
Repression of tin by Svp in the misexpression assay was consistently
more robust throughout the dorsal vessel when compared with repression of
tin by Doc. However, the repression of tin by
Doc is independent of svp, as svp is not activated
downstream of Doc in Tin-negative cardioblasts
(Fig. 5G), and tin
repression is also seen when Doc2 is misexpressed in a homozygous
svpAE127 mutant background (see Fig. S3 in the
supplementary material). In addition, analyses of embryos with genotypes in
which Doc1 is deleted and the gene dose of Doc2 and
Doc3 is reduced by half (Reim et
al., 2003; Reim and Frasch,
2005) also support the notion that Doc acts as a repressor during
cardioblast diversification. In these embryos, we detect ectopic expression of
tin in a number of Svp-positive cells, presumably because the lowered
dose of Doc provided insufficient repressive activity towards
tin (Fig. 5H). Taken
together these findings show that Doc and Tin have mutually repressive
functions in cardioblasts of the dorsal vessel.
The expression of Wingless (Wg) in the late stage embryonic heart in three
segmentally repeated double pairs of cardioblasts
(Fig. 6A) marks the
differentiation of the Svp/Doc-positive cardioblasts within the heart region
into ostia (inflow valves) (Lo et al.,
2002). Previously, it has been shown that wg expression
depends on svp and abd-A activities; it is missing in
svp mutants, but expanded if svp is expressed ectopically
(Lo et al., 2002;
Perrin et al., 2004) (see also
Fig. 6B,C). However, based on
these data, it is not possible to discriminate between a direct activation of
wg by Svp, an indirect activation by Svp via Doc or an
absence of repression of wg by Tin in the Svp+
cardioblasts. In order to distinguish between these possibilities,
Doc2 was activated ectopically in the dorsal vessel
(Fig. 6D), which produced a
moderate expansion of Wg, with some but not all Doc+ cardioblasts
in the `heart' region being positive for Wg. tin-ABD;
tin346 mutant embryos, in which svp is not
affected, display ectopic activation of wg in all cardioblasts of the
`heart' region (Fig. 6E).
Furthermore, svpAE127 tin346 double mutant
embryos carrying the tin-ABD transgene also show expanded wg
expression (Fig. 6F). Hence,
wg may either be activated by Doc, or it may be repressed by Tin
while being activated by another factor that could be active in all
cardioblasts. In summary, these data prove that svp is not directly
required for Doc and wg gene activation, but rather as a
repressor of tin, which in turn represses Doc and perhaps
wg. These findings agree with a role of Svp as a transcriptional
repressor, a function also fulfilled by its mammalian homolog COUP-TFII
(Nr2f2) (Pereira et al.,
1999).
|
; Ranganayakulu et al.,
1998), we observed that Doc and Wg expression are strongly
repressed when Nkx2.5 is expressed in cardioblasts, similar to the
effects seen with ectopic tin (compare
Fig. 6H with 6I). These data
indicate that Nkx2.5, like Tin, can have a repressive function towards some
cardiac regulators.
Cardiac tinman function is required for proper ultrastructure, remodeling and functionality of the larval and adult heart
In both larval and adult dorsal vessels of wild-type animals, the
myofibrils are arranged spirally with essentially transverse orientations
around the heart and aortic tubes (Fig.
7A,A') (Molina and
Cripps, 2001; Monier et al.,
2005). By contrast, larval dorsal vessels from animals in which
tin was never expressed in myocardial cells (tin-ABD,
tin346/tinEC40) show a very different
ultrastructure. In these dorsal vessels, the myofibrils are arranged almost
exclusively in an AP orientation, which leads to the appearance of striations
(Fig. 7B,B'). The only
exceptions are seen in the heart, where several abnormal cross-shaped or
`knotted' patterns of myofibrils are present, particularly near the posterior
end. In addition, the aortae in these larvae appear thinner when compared with
the wild type, whereas the heart frequently has a wider diameter
(Fig. 7B,B', see Fig. S4
in the supplementary material; data not shown). Hence, myocardial activity of
tin is required for establishing the normal ultrastructure of the
contractile fibers and is, perhaps, related to this function, for generating a
morphologically normal dorsal vessel. An almost identical alteration of the
myofiber orientation has been reported for adult animals with ectopic
expression of abd-A in the aorta
(Monier et al., 2005);
however, it is presently not known whether there is any mechanistic connection
with our observation.
In adult animals lacking cardiac tin expression, we observe a much
thinner heart tube (Fig. 7D,F, compare with
7C,E). As in larvae, the myofibrils are arranged longitudinally
and transverse spirally arranged myofibrils are almost completely absent in
these mutants (Fig. 7F, compare with
7E) (see also Molina and
Cripps, 2001; Monier et al.,
2005). The adult heart is generated by remodeling of the posterior
larval aorta, which is accompanied by a significant widening of the tube and
myocardial cell growth and the histolysis of the larval heart
(Molina and Cripps, 2001;
Monier et al., 2005;
Sellin et al., 2006).
Obviously, the absence of tin activity in myocardial cells impedes
this process of heart remodeling. As a consequence, the small adult dorsal
vessel in tin-ABD, tin346/tinEC40
flies probably represents the largely unchanged larval aorta after the larval
heart has been histolyzed.
The altered ultrastructure of myocardial cells, as well as the observed
changes in the expression of cardiac differentation genes and in cardial cell
identities, would be expected to affect the functionality of the dorsal
vessel. In order to examine the role of tin in the function of the
adult heart, we measured cardiac responses to acute stress in
tin-depleted adult flies and in controls. External electrical stimuli
were applied to pace the heart of the fly to an elevated rate compared with
wild type (Wessells et al.,
2004). In response to this stress, flies either recover to a
regular heart beat or else fail (defined as cardiac arrest or fibrillation).
tin-deficient hearts showed a dramatic increase in heart failure
rate, while those that are heterozygous for tin in the heart failed
at the same rate as wild-type controls (20-30%;
Fig. 7G; see Movies 1 and 2 in
the supplementary material). In parallel, we monitored the recovery of flies
that underwent either fibrillation or arrest. Two minutes after the pacing
protocol, almost all wild-type and heterozygous flies recovered to a normal
resting heartbeat, whereas in the absence of cardiac tin, only 40% of
flies were able to recover (Fig.
7H). This suggests that tin function is required for a
properly functioning adult heart. Moreover, flies without cardiac tin
have a reduced lifespan (Fig.
7I), consistent with a possible link between cardiac function and
aging. Taken together, these data suggest that tin is required for
the formation of the adult heart, in addition to its early requirement for the
embryonic dorsal vessel. As a consequence, the lack of cardiac tin
causes severe disruptions in cardiac contractility and rhythmicity (see Movies
1 and 2 in the supplementary material), and leads to a much elevated risk of
heart failure in response to stress.
|
|
DISCUSSION |
|---|
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Qian et al.,
2005; Reim and Frasch,
2005; Reim et al.,
2005), and mid then feeds back to activate tin
expression specifically in myocardial cells
(Reim et al., 2005). However,
the expression of the COUP-TFII-related gene svp, which is activated
during stage 12 in two cardioblast progenitors in each hemisegment between A2
and A7, prevents mid from activating tin in these cells
(Gajewski et al., 2000;
Lo and Frasch, 2001).
tin then represses Doc, which appears to be activated by
default (via yet unknown activators) in the Tin- cardioblasts and
in turn contributes to the continued repression of tin in them. These
interactions lead to the stabilization of two mutually exclusive
differentiation states of myocardial cells that are defined presumably to a
large extent by their differential expression of tin versus
Doc and of their respective target genes. The particular features of
these two segmental types of myocardial cells are further modulated by the
activities of Hox genes such as abd-A, which leads to the
differential formation of `working' (Tin+) versus ostial myocardial
cells (Doc+, Wg+) that form the inflow valves in the
heart region (Fig. 6G).
Whereas previous data have documented that Tin functions as a direct
activator of specific target genes, our present data implicate Tin strongly in
the repression of certain myocardial genes, including Doc and
potentially wg. Indeed, interactions of Tin with the corepressor
Groucho have been demonstrated in biochemical and cell culture experiments
(Choi et al., 1999), and the
N-terminal TN domain of Tin is proposed to function as an EH1 repressor domain
(Copley, 2005). We propose that
the activity of Tin either as an activator or a repressor is context-specific
and is ultimately determined by the enhancer architecture of a particular
target gene. For example, during early stages of cardiac induction by Dpp,
combinatorial binding of Tin and Smads promotes activating functions of Tin,
whereas in the Tin+ cardioblasts during later stages, Tin can
either activate (e.g. Mef2, Sur) or repress (e.g. Doc).
These opposite activities in the same cells are probably determined by the
presence of additional binding sites for tissue-specific or ubiquitous
co-factors that can switch Tin activity.
|
;
Tanaka et al., 1999) (our
present data). Although at first it was thought that, in the mouse, closely
related Nkx genes could partially compensate for the functional loss of
Nkx2.5, it is now widely assumed that cardiogenic genes encoding
other classes of transcription factors, particularly those of the GATA and
T-box families, exert most of the compensatory effects
(Tanaka et al., 1998;
Olson and Schneider, 2003).
The latter type of compensation appears also to operate upon
cardioblast-specific ablation of tin function, where the derepressed
Doc T-box genes are presumably able to regulate myocardial
development in the cells that would normally be under the control of
tin. It will be instructive to determine the phenotype of animals in
which the cardioblast expression of both tin and Doc has
been ablated simultaneously. Likewise, the analysis of cardiogenesis with
analogous double mutants in the mouse will be highly informative.
Additional phenotypic similarities between our tin mutant animals
and mouse Nkx2.5 mutants include the failure to remodel the linear
heart tube, which in the mouse leads to defects in looping morphogenesis and
chamber formation and in Drosophila to a failure of converting the
larval aorta into an adult heart. As in adult flies, a conditional knockout of
mouse Nkx2.5 also leads to defects in ventricular cell lineage
specification and maturation, which is accompanied by the aberrant down- or
upregulation of cardiac differentiation genes
(Pashmforoush et al., 2004).
Notably, a major difference between the vertebrate and Drosophila
systems is the early and broad mesodermal expression of Tin, which is
essential for Drosophila cardiac induction and is not compensated for
by other factors.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/20/4073/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Developmental Biology Institute of Marseille-Luminy, CNRS
URM 6216, Campus de Luminy, Case 907, 13288 Marseille Cedex 9, France
|
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