Brookdale Department of Molecular, Cell and Developmental Biology, Box 1020, Mount Sinai School of Medicine, New York, NY 10029, USA
* Author for correspondence (e-mail: manfred.frasch{at}mssm.edu)
Accepted 6 September 2005
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SUMMARY |
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Key words: Mesoderm patterning, Heart development, T-box genes, Drosophila
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Introduction |
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In Drosophila, the primordial cells generating myocardial cells
(cardioblasts) and pericardial cells are induced within mesodermal areas
receiving a combination of Dpp (i.e. BMP) and Wg (i.e. Wnt) signals
(Zaffran and Frasch, 2002).
Dpp is secreted from a dorsal domain of the ectoderm on either side of the
embryo, whereas Wg derives from transversely striped segmental domains; hence,
the cardiac progenitors are generated in bilateral, segmentally repeated areas
of the dorsal mesoderm of the early embryo. The NK homeobox gene
tinman (tin), which is crucial for the generation of all
types of cardiac progenitors and all other dorsal mesodermal derivatives, is a
direct downstream target of the Dpp signaling cascade and, as such, is induced
in the entire dorsal mesoderm (Azpiazu and
Frasch, 1993
; Bodmer,
1993
; Frasch,
1995
; Xu et al.,
1998
). In addition, Tinman protein is required to provide
mesodermal cells with the competence to respond to Dpp and other signals
during additional events in cardiac induction
(Halfon et al., 2000
;
Knirr and Frasch, 2001
;
Han et al., 2002
).
tin, potentially in combination with Dpp, is required for the
transcriptional activation of the Gata factor-encoding gene pannier
(pnr) in the cardiogenic mesoderm
(Gajewski et al., 2001
).
pnr is crucial for the specification of both cardioblasts and
pericardial cells (Gajewski et al.,
1999
; Alvarez et al.,
2003
; Klinedinst and Bodmer,
2003
). One of the earliest regulatory genes turned on specifically
in all cardioblasts downstream of pnr is the Tbx20 ortholog
midline (mid)
(Miskolczi-McCallum et al.,
2005
; Qian et al.,
2005
; Reim et al.,
2005
). Together with its paralog H15, mid serves to
activate late-stage expression of tin specifically within
cardioblasts, which in turn is required for the normal patterning of the heart
tube and the differentiation of myocardial cells
(Reim et al., 2005
).
When compared with Dpp, the exact role of Wg during cardiac induction is
less well understood. For example, the lack of spatial correlation between
pnr and wg expression suggests that pnr is not a
direct target of Wg signals. Whereas ectodermal wg is striped,
pnr expression initiates almost uniformly along the anteroposterior
axis near the dorsal margin of the mesoderm
(Gajewski et al., 1999;
Klinedinst and Bodmer, 2003
),
at a time when the segmentally distributed cardiac progenitors coalesce into a
continuous band of cells. However, one important and direct consequence of Wg
signals is the induction of sloppy paired (slp1 and
slp2) in striped domains in the early mesoderm
(Riechmann et al., 1997
;
Lee and Frasch, 2000
). The
slp genes encode forkhead domain repressor factors, which prevent the
induction of visceral mesoderm regulators by Dpp that would otherwise
interfere with cardiac induction in the presumptive cardiogenic areas
(Zaffran et al., 2001
).
In addition to excluding these inappropriate regulators from the
cardiogenic areas, wg is thought to promote the formation of both
pericardial and myocardial progenitors also in a direct fashion. Indeed, such
a mechanism operates during the specification of one specific subset of
pericardial cells, termed Eve-PCs, and the induction of the
even-skipped (eve) gene in progenitors of these cells
requires binding sites for the Wg effector Pangolin (Pan, also known as dTCF)
in combination with binding sites for Tinman and the Dpp-effectors Mad and
Medea (Halfon et al., 2000;
Knirr and Frasch, 2001
;
Han et al., 2002
). Hence, the
combinatorial inputs from Wg, Dpp and the cardiogenic competence factor Tinman
are directly integrated at the level of the enhancer of a pericardial
regulatory gene. Based upon the available genetic data on cardioblast
specification, it is likely that an analogous integration of Wg and Dpp
signals also occurs during the induction of certain early-acting myocardial
regulatory genes. However, clear candidates for common targets of Wg and Dpp
in myocardial development have not been described.
Our current study identifies the Dorsocross T-box genes as crucial new
components that mediate the combinatorial activities of Wg and Dpp during
early steps of myocardial induction. The Dorsocross (Doc) locus encodes a
cluster of three genes, Doc1, Doc2 and Doc3, that are
closely related in terms of their T-box sequences and embryonic expression
patterns (Reim et al., 2003).
Our previous work has identified important roles of these genes during
amnioserosa development and epidermal patterning of the embryo
(Reim et al., 2003
). We now
show that, within the mesoderm, the Doc genes have an essential role for the
formation of cardioblasts and a subset of pericardial cells. The induction of
Doc gene expression occurs in dorsal mesodermal quadrants of cells at the
intersections of the Dpp and Wg domains, and requires the activities of both
Dpp and Wg, but not tin. We demonstrate that, in the absence of all
three Doc genes, only very few cardioblasts become specified and no dorsal
vessel is formed. In addition, the subpopulations of odd-skipped
(odd)-expressing pericardial cells (Odd-PCs), odd-positive
lymph gland cells and tin-only-expressing pericardial cells (Tin-PCs)
require Doc gene activity. However, eve + tin-positive
pericardial cells (Eve-PCs) and dorsal somatic muscle founders develop
independently of Doc. We observe genetic interactions among Doc, tin
and pnr, which suggests that these three cardiogenic regulators
synergize during the specification of myocardial and certain pericardial
cells. Accordingly, simultaneous ectopic expression of Doc2 with
tin, pnr or both generates large numbers of ectopic cardioblasts in
the mesoderm. We demonstrate that one of the key functions of the Doc genes
during cardiogenesis, which they exert in combination with tin, is
the activation of pnr expression in the cardiogenic mesoderm.
Altogether, the incorporation of the Doc genes into the cardiogenic network
has allowed us to close important gaps in our understanding of the regulatory
circuits operating during the induction of myocardial and pericardial
cells.
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Materials and methods |
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Staining of embryos
Immunostaining of embryos using DAB, fluorescent immunostaining and in situ
hybridization were carried out essentially as described by Knirr et al.
(Knirr et al., 1999). Primary
antibodies were detected with FITC-, Cy3- or Cy5-conjugated AffiniPure goat
anti-rabbit IgG (H+L) (1:200; Jackson ImmunoResearch Laboratories). If
necessary, Tyramide Signal Amplification (TSA) was performed using
biotinylated secondary antibodies (1:500) in combination with the Vectastain
ABC Kit (Vector Laboratories) and fluorescent Tyramide Reagent (PerkinElmer).
Primary antibodies included rabbit anti-Bap (1:500 with TSA)
(Zaffran et al., 2001
), rabbit
anti-Doc2 (1:2000), guinea pig anti-Doc2+3 (1:400), guinea pig anti-Doc3+2
(1:600) (Reim et al., 2003
),
rabbit anti-Eve (Frasch et al.,
1987
), guinea pig anti-Eve (1:400), rat anti-Odd (1:500), guinea
pig anti-Runt (1:300) (Kosman et al.,
1998
), rabbit anti-ß-Galactosidase (1:1500; Promega), mouse
polyclonal anti-ß-Galactosidase (1:200; Sigma), rabbit anti-Mef2 (1:750;
a gift from Hanh Nguyen, Albert Einstein College, Bronx, NY)
(Bour et al., 1995
), rabbit
anti-Phospho-Smad1/PMad (1:2000 with TSA; gift from C.-H. Heldin, Ludwig
Inst., Uppsala), rabbit anti-Pnr (1:3000 with TSA)
(Herranz and Morata, 2001
),
rabbit anti-Srp (1:800) (gift from D. Hoshizaki), rabbit anti-Tin (1:750)
(Yin et al., 1997
) and rabbit
anti-Zfh-1 (1:2000) (Broihier et al.,
1998
). Monoclonal anti-Wg 4D4 (1:40 with TSA) and
anti-ß-Galactosidase 40-1a (1:60 with TSA) were obtained from the
Developmental Studies Hybridoma Bank at the University of Iowa.
Digoxigenin-labeled RNA in situ probes for Doc
(Reim et al., 2003
), bkh,
hand (S. Zaffran and M.F., unpublished) and H15 were prepared
from cloned genomic fragments, and the mid in situ probe from the
BDGP EST RE27439 (Reim et al.,
2005
). Images of DAB-stained embryos were taken using Nomarski
optics and images from fluorescent staining using confocal laser scanning
microscopy with Leica TCS-SP and Zeiss LSM 510 META microscope systems.
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Results |
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In the trunk mesoderm, Doc expression emerges first at stage 10 within
segmentally repeated patches of cells located in dorsal areas
(Reim et al., 2003). As shown
in Fig. 1A for Doc3
mRNA, the ventral borders of Doc expression coincide with the ventral border
of nuclear phosphorylated Mad (PMad) signals in the dorsal mesoderm. This
indicates that Doc expression in the dorsal mesoderm is induced by
Dpp signals from the dorsal ectoderm. The fully developed clusters of Doc
expression at mid stage 10 are reciprocal to the clusters of cells expressing
the homeobox gene bagpipe (bap), which mark the precursors
of the trunk visceral mesoderm (Fig.
1B). Ectopic activation of the Dpp signaling pathway throughout
the mesoderm via a constitutively active Dpp receptor, Thickveins,
TkvQ253D (Nellen et al.,
1994
), leads to ventral expansion of bap expression
(Yin and Frasch, 1998
), and
Doc expression expands in a similar fashion upon pan-mesodermal
TkvQ253D or Dpp expression (Fig.
1C, compare with 1B, and data not shown). TkvQ253D- or
Dpp-induced ectopic Doc and bap occur only within their appropriate
segmental domains that are defined by positive and negative Wg inputs,
respectively (Azpiazu et al.,
1996
; Lee and Frasch,
2000
; Reim et al.,
2003
). In the case of Doc, wg is essential for the
segmental induction in both mesoderm and ectoderm
(Reim et al., 2003
).
Accordingly, triple immunostaining with antibodies recognizing the Doc
proteins Doc2/Doc3, the dorsal mesoderm marker Tin and Wingless (Wg) protein
demonstrates that Doc co-localizes with Tin in the dorsal mesoderm, but unlike
Tin is restricted to the areas that are close to the Wg-secreting cells of the
ectoderm (Fig. 1D). In
addition, forced pan-mesodermal wg expression causes the induction of Doc in a
largely continuous fashion along the anteroposterior axis of the dorsal
mesoderm, in a pattern that closely resembles that of tin
(Fig. 1E). Altogether, these
data confirm that early mesodermal Doc is induced by intersecting Dpp and Wg
signaling pathways. As a consequence, Doc becomes expressed in the entire
anterior dorsal quadrant of each mesodermal parasegment (i.e. the dorsal
region of each mesodermal A domain)
(Azpiazu et al., 1996
). These
areas will eventually generate the dorsal vessel as well as dorsal somatic
muscles, as opposed to the bap-positive areas of the P domains that
contain most of the primordial cells of the visceral musculature.
|
For additional clarification of the developmental events in the early
cardiogenic mesoderm, we examined Doc expression relative to other cardiogenic
regulators, including the Gata factor-encoding gene pannier
(pnr). In addition to its expression in the cardiogenic mesoderm,
pnr is expressed in a broad band of cells along the adjacent dorsal
ectoderm (Winick et al., 1993;
Herranz and Morata, 2001
). We
first detect Pnr protein in the mesoderm at early stage 11, which is notably
later than the earliest Doc expression but coincides with the time when Doc
expression starts to refine along the dorsal margin. Doc and Pnr protein
colocalize within the most dorsal areas of the tin-positive portion
of the mesoderm (Fig. 1H;
tin expression is monitored with tinD-lacZ, a reporter
driven by the Dpp-responsive enhancer of tin)
(Yin et al., 1997
). Like Doc,
Pnr does not overlap with Eve (data not shown). Hence, the cardiac mesoderm in
the Drosophila embryo (in a more stringent definition excluding the
Eve-positive cells, which also produce dorsal muscles) is characterized by the
co-expression of tin, pnr and Doc.
During stage 12, Doc maintains its co-expression with pnr and
tin, although the expression of all three genes becomes further
restricted to the dorsal margin (Fig.
1I) (Bodmer and Frasch,
1999). For a brief period, Doc is expressed in all cardioblasts
that form during this time and are marked by expression of the Tbx20
ortholog midline (mid) or the COUP-TFII-related
gene seven-up (svp) (Fig.
1J) (Reim et al.,
2005
). Doc and Pnr gradually disappear during stage 12-13, except
for two out of six cardioblasts per hemisegment that maintain Doc expression
until the end of embryogenesis (Lo and
Frasch, 2001
; Reim et al.,
2003
). tin expression is maintained in a complementary
set of cardioblasts, as well as in certain pericardial cells
(Yin and Frasch, 1998
;
Ward and Skeath, 2000
;
Lo and Frasch, 2001
).
Next we asked whether tin and pnr regulate Doc expression
in the dorsal and cardiogenic mesoderm. Despite the importance of tin
for the specification of all dorsal mesodermal derivatives, Doc expression
initiates normally in the dorsal mesoderm of tin null mutants
(Fig. 2B, compare with 2A).
Likewise, early Doc expression is also unaffected in pnr-null mutants
(Fig. 2F; compare with 2E), which is consistent with the observed onset of pnr expression after
that of Doc. Therefore, Doc induction at stage 10 seems to be a direct result
of Dpp and Wg signaling and, unlike bap
(Lee and Frasch, 2005), does
not a require Tin as a mesodermal competence factor. We presume that Dpp and
Wg induce Doc expression in a similar fashion in both the dorsal mesoderm and
the dorsal ectoderm.
In contrast to stage 10, Doc expression is affected in both
tin and pnr mutants at later stages, and fades prematurely
from the dorsal mesoderm. In stage 11 tin mutants, we see only few
irregularly arranged cells that still express Doc in the dorsal
mesoderm (Fig. 2D; compare with
2C), and by mid stage 12 Doc expression is absent in this area. We
also find that pnr is never activated in the mesoderm of tin
mutants (Fig. 2D'; compare with
2C'), which is consistent with previous findings that a
pnr-lacZ reporter construct requires tin and essential
Tin-binding sites in order to be activated in the mesoderm
(Gajewski et al., 2001).
Indeed, loss of Doc expression in tin mutants might be
caused indirectly by the absence of pnr products, as high-level Doc
expression also fails to emerge in the dorsal mesoderm of stage 11
pnr mutants and is virtually absent from stage 12 onwards
(Fig. 2H; compare with 2G). The
loss of Doc expression in stage 11-12 pnr mutants is accompanied by
decreased tin expression (Fig.
2H) (Alvarez et al.,
2003
; Klinedinst and Bodmer,
2003
), with the strongest reduction in the dorsal-most cells that
would normally show high levels of Pnr and Doc.
As pnr is also required for dpp expression in the dorsal
ectoderm at this stage (Herranz and
Morata, 2001), it is conceivable that the loss of Doc
expression is caused indirectly by the lack of the ectodermal Dpp inputs. To
address this possibility, we restored either pnr expression or Dpp
signaling exclusively in the mesoderm of pnr mutant embryos. We found
that forced mesodermal pnr expression can significantly rescue
high-level Doc expression in stage 11 pnr embryos
(Fig. 2I, arrowheads; compare
with 2H). There is also a significant rescue of cardioblast specification, as
indicated by the activation of cardioblast-specific midline in this
background (Reim et al., 2005
)
and by late stage Doc and Tin expression in a complementary pattern (as in
wild-type cardioblasts, albeit in fewer cells; data not shown). By contrast,
activating Dpp signaling in the mesoderm of pnr mutants with
UAS-tkvQ253D or UAS-dpp does not result in a
significant rescue of Doc expression in the dorsal mesoderm
(Fig. 2J and data not shown;
compare with 2H,I). These data suggest that pnr is required, perhaps
directly, to regulate Doc expression in the dorsal mesoderm during
stages 11-12, although it is not involved in the initial activation of Doc
during stage 10.
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Dorsocross is required for the specification of cardioblasts
We used a recently generated small deficiency, Df(3L)DocA, that
deletes all three Doc genes, Doc1, Doc2 and Doc3
(Reim et al., 2003), to
investigate the role of the Doc genes in dorsal vessel development. As
described previously, DocA mutants (i.e. embryos homozygous for
Df(3L)DocA) do not undergo germ band retraction owing to the
requirement of Doc in the amnioserosa. To avoid this complication and rule out
indirect influences of morphogenetic defects on cardiogenesis, we performed
our analysis predominantly with DocA mutants in which germ band
retraction has been rescued by amnioserosa-specific expression of
Doc2 via c381-GAL4 (Reim
et al., 2003
). Dorsal vessel development in these embryos was
analyzed through expression of genes that normally mark all or subsets of
cardioblasts.
|
Two distinct types of cardioblasts can be discriminated by their mutually exclusive expression of either tin or svp. We performed anti-ß-Galactosidase/anti-Mef2 double staining of embryos carrying one copy of the svp-lacZ enhancer trap insertion AE127 to confirm that not only the Tin-cardioblasts (Tin-CBs) but also the Svp-cardioblasts (Svp-CBs) are affected by the loss of Doc genes. As shown in Fig. 3H, Svp-CBs are absent in DocA mutants, as is the Mef2-stained row of cardioblasts above the somatic mesoderm (compare with Fig. 3G). Heterozygous DocA embryos usually have normal numbers of cardioblasts (Fig. 3I), although a small fraction of embryos do have reduced numbers, which indicates less robust development of cardioblasts with decreased Doc activity. Indeed if Doc dose is further reduced by combining Df(3L)DocA with Df(3L)29A6, a deletion that removes Doc1, there is a significant reduction from normally 104 cardioblasts in wild-type embryos to 80±6 cells (n=10) (Fig. 3J). Although all types of cardioblasts are affected by the reduction of Doc levels, Svp-CBs appear to be more sensitive than Tin-CBs. This suggests that Doc has an early function required for all cardioblasts as well as a more specific function for Svp-CBs, in which it is expressed at later stages.
To distinguish whether Doc genes are required for the specification versus
survival of cardioblasts, we examined the expression of a number of markers
for newly specified cardioblasts at early stages of cardiogenesis. Cardioblast
expression of mid (Fig. 3L;
compare with 3K), svp-lacZ
(Lo and Frasch, 2001), the
G0
-subunit gene brokenhearted (bkh)
(Fremion et al., 1999
) and
hand (Kölsch and Paululat,
2002
) (data not shown) never initiates during early stage 12 in
DocA mutants, although bkh and hand are still
expressed in Eve-PC progenitors as in wild-type embryos. Cardiac expression of
several markers, including mid, H15, Mef2-Ht
D-lacZ,
svp-lacZ, hand and bkh, is partially rescued by forced
expression of either Doc1, Doc2 or Doc3 via
tinD-Gal4 in the dorsal mesoderm in the background of
Df(3L)DocA (Fig. 3M;
see also Fig. 7; other data not
shown; Doc1 and Doc3 rescuing activity was only tested with
mid and Mef2-Ht
D-lacZ). As observed
previously for the amnioserosa (Reim et
al., 2003
), the best rescue was consistently obtained with
Doc2. These data demonstrate that mesodermal expression of Doc is
sufficient to rescue cardioblast specification and that it is the Doc genes
and not any of the other genes uncovered by Df(3L)DocA that are
responsible for the observed cardiac phenotypes. We conclude that Doc genes
have an essential role and act in a dose-dependent manner in specifying
cardioblasts.
Doc is required for Odd-positive pericardial and lymph gland cells but not for Eve-positive pericardial cells and dorsal somatic muscles
The dorsal quadrants of Doc-expressing mesodermal cells from stage 10
embryos not only give rise to cardioblasts, but also to pericardial cells and
dorsal somatic muscle founders. The observed reduction of Tin-positive cells
already indicates that the pericardial cells expressing tin but not
eve, called Tin-PCs, are also drastically reduced in number in Doc
loss-of-function mutants (Fig.
3D,F). To complete the analysis of dorsal mesodermal cell types,
we analyzed DocA mutants for the presence of Tin
pericardial cells that express odd-skipped (odd). Two out of
the four Odd-positive pericardial cells (Odd-PCs) in each hemisegment are
Svp-CB siblings, while the other cells derive from a different lineage
(Ward and Skeath, 2000). All
Odd-PCs are missing in the mutants, as are Odd-positive cells that originate
from the thoracic dorsal mesoderm and normally form the lymph glands
(Fig. 4B; compare with 4A). This result is consistent with an early defect in the specification of a
common pool of precursors of pericardial/lymph gland cells and cardioblasts
(see Mandal et al., 2004
). By
contrast, and as described above, progenitors that produce Eve-pericardial
cells and Eve-expressing founders of muscle 1 (DA1) from a distinct pool of
cells are specified normally in the absence of Doc
(Fig. 4B; compare with 4A). Runt marks another somatic muscle founder and the corresponding muscle #10
(DO2) that are derived from the early Doc-expressing domains. As for Eve, the
Runt pattern in DocA mutants with rescued germ band retraction is
very similar to wild type, even at later stages
(Fig. 4D, compare with 4C).
Likewise, staining for 1010 B2-lacZ, a reporter gene fortuitously
expressed in all dorsal muscles (Yin and
Frasch, 1998
), shows that the dorsal muscles are present in
DocA mutants, although they are distorted because of the defects in
embryo morphology without the amnioserosa
(Fig. 4F, compare with 4E).
Therefore Doc genes, although present in the entire dorsal quadrants of the
early mesodermal A domains, are specifically required for pure cardiac cell
lineages, but not for those that generate in addition or solely somatic
muscles. This function correlates with the restricted Doc expression at stage
11-12, where Doc expression is excluded from the Eve-positive clusters.
Genetic interactions between Doc, pnr and tin
The observed co-expression of Doc, tin and pnr during
stages 11 and 12, and defective cardioblast specification upon loss of
function of these genes suggest that these three genes act in a common pathway
during early cardiogenesis. The occurrence of genetic interactions among these
genes would be further indicative of this possibility. Because we observe mild
defects in cardioblast specification in embryos with a reduced Doc dose
(Df(3L)29A6/Df(3L)DocA,
Fig. 3J), we asked whether
reduction of tin and pnr gene dose would have any further
impact on cardiogenesis. In order to test this possibility, the null alleles
pnrVX6 and tin346 were individually
recombined onto the Df(3L)DocA chromosome. In addition, triple
mutants carrying Df(3L)DocA, pnrVX6 and
tin346, as well as pnrVX6
tin346 double mutants, were generated. Heterozygous
individuals of all combinations are viable, although heterozygous triple
mutants have a slightly reduced viability (63%) when compared with
tin346 heterozygotes. If Df(3L)DocA/TM3
heterozygous flies are crossed to Df(3L)29A6/TM3 flies, all
non-balanced transheterozygotes die at pupal stages
(Reim et al., 2003). We were
also able to obtain pupae of the genotypes Df(3L)29A6/Df(3L)DocA
pnrVX6 and Df(3L)29A6/Df(3L)DocA
tin346, but not with Df(3L)29A6/Df(3L)DocA
pnrVX6 tin346 among about 200 progeny, indicating
more severe defects with the latter combination.
Fig. 5 shows representative phenotypes of transheterozygous embryos. As described above, Df(3L)29A6/Df(3L)DocA embryos, with one copy each of Doc2 and Doc3 and no Doc1, have a reduced number of cardioblasts (Fig. 5B; compare with 5A). Although all types of cardioblasts are affected, the number of Doc-positive cardioblasts is most strongly reduced, and not all of the residual svp-lacZ-positive cells retain Doc expression (Fig. 5B and data not shown, compare also with Fig. 3J). If one copy of tin or pnr is removed in the Df(3L)29A6/Df(3L)DocA background, even fewer cardioblasts are specified, which frequently causes gaps in the dorsal vessel (Fig. 5C,D). This synergistic effect becomes very strong in Df(3L)29A6/Df(3L)DocA pnrVX6 tin346 embryos, in which less than half the normal number of cardioblasts are formed (Fig. 5E). However, if only one Doc gene copy (Doc1) is removed, the reduction of one copy of pnr and tin has only mild effects (Df(3L)29A6/pnrVX6 tin346; Fig. 5F). Odd-pericardial cells and the lymph glands are also strongly reduced in Df(3L)29A6/Df(3L)DocA pnrVX6 tin346 embryos, suggesting that Doc interacts with pnr and tin already early during the specification of the cardiac mesoderm (Fig. 5H; compare with 5G). The impact on Eve-pericardial cells is much weaker (data not shown), which is consistent with the observation that these cells are not affected upon complete loss of the Doc genes in DocA mutants. These observations indicate that Doc genes, tin and pnr cooperate or depend on each other during the specification of cardiac progenitors.
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|
As the observed genetic interactions suggest a synergistic action of
Doc with tin and pnr, we asked whether combined
expression of Doc2 with tin and pnr would increase
the efficiency of generating ectopic cardioblasts. Ectopic expression of
pnr alone in the entire mesoderm has been reported to increase
cardioblast numbers (Gajewski et al.,
1999), but with the more restricted and slightly weaker driver
tinD-GAL4, we find only few extra cardioblasts (data not shown). No
increase in cardioblast number was seen in embryos in which only tin
is overexpressed (data not shown), but co-expression of tin and
pnr produces more cardioblasts than pnr alone
(Fig. 8C). Although this
increase in cardioblasts is similar to that of Doc2 expression, the
numbers of pericardial cells are only slightly reduced
(Fig. 8K). If Doc2 is
expressed together with tin or pnr, or with both, large
numbers of extra cardioblasts are formed
(Fig. 8D,E,F). Combined
expression of Doc2 and pnr consistently produces more
cardioblasts than expression of Doc2 and tin, and nearly as
many as the triple co-expression of Doc2, pnr and tin, under
which conditions cardioblast numbers are roughly doubled.
Broader ectopic expression of Doc2, via the early pan-mesodermal
driver 2xPE-twi-GAL4, causes even stronger expansion of early
cardioblast markers such as mid, H15, Toll305-clacZ and
tinC7-lacZ in the mesoderm of stage 12-13 embryos
(data not shown) (Wang et al.,
2005
). However, even under these conditions, the effects tend to
be stronger in dorsal areas of the mesoderm, suggesting that Doc cooperates
with other dorsally localized activities to promote cardioblast fates. Likely
candidates include tin and pnr, because the combined ectopic
expression of Doc2, tin and pnr causes widespread ectopic
cardioblast formation (Fig. 8H; compare
with 8G) to a much greater extent than compared with Doc2
alone or the combination of tin plus pnr (data not shown)
(Klinedinst and Bodmer, 2003
).
However, twi-driven Doc2 by itself is sufficient to repress
the visceral mesoderm markers bagpipe (bap) and
biniou (bin) (data not shown).
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![]() |
Discussion |
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Drosophila offers a simpler system to study regulatory networks in
cardiogenesis. Recently, the Tbx20-related T-box genes mid
and H15 were shown to play a role in cardiac development downstream
of the early function of the NK homeobox gene tin and the Gata gene
pannier (pnr)
(Miskolczi-McCallum et al.,
2005; Qian et al.,
2005
; Reim et al.,
2005
). Whereas the role of these genes in the morphogenesis of the
cardiac tube is minor, they are involved in processes of cardiac patterning
and differentiation during the second half of cardiogenesis, which includes
the activation of tin expression in myocardial cells
(Reim et al., 2005
). In our
present report, we have characterized the roles of the Tbx6-related
Dorsocross T-box genes (which may actually have arisen from a common ancestor
of the vertebrate Tbx4, Tbx5 and Tbx6 genes)
(Reim et al., 2003
), in
Drosophila cardiogenesis. We have shown that the Doc genes have a
fundamental early role that is required for the specification of all cardiac
progenitors that generate pure myocardial and pericardial lineages. They are
not required for generating dorsal somatic muscle progenitors and lineages
with mixed pericardial/somatic muscle, even though their early expression
domains also include cells giving rise to these lineages.
The new information on the regulation and function of Doc fills a major gap
in our understanding of early Drosophila cardiogenesis. Previous data
have shown that the combinatorial activities of Wg and Dpp are required for
the formation of both myocardial and pericardial cells
(Frasch, 1995;
Wu et al., 1995
;
Carmena et al., 1998
). In
addition, the homeobox gene even-skipped (eve) is a direct
target of the combined Wg and Dpp signaling inputs in specific pericardial
cell/dorsal somatic muscle progenitors
(Halfon et al., 2000
;
Knirr and Frasch, 2001
;
Han et al., 2002
). Our current
data identify the Doc genes as downstream mediators and potential direct
targets of combined Wg and Dpp signals during the induction of myocardial and
Eve-negative pericardial cell progenitors. The induction of Doc expression by
Wg and Dpp occurs concurrently with the induction of tin by Dpp
alone, at a time when the mesoderm still consists of a single layer of cells
(Fig. 9). As a result,
tin and Doc are co-expressed in a segmental subset of dorsal
mesodermal cells that include the presumptive cardiogenic mesoderm.
Conversely, in the intervening subset of dorsal mesodermal cells (the
presumptive visceral mesoderm precursors) tin is co-expressed with
bagpipe (bap) and biniou (bin), which are
negatively regulated by Wg via the Wg target sloppy paired
(slp) (Lee and Frasch,
2000
; Zaffran et al.,
2001
). Ultimately, these shared responses to Dpp, differential
responses to Wg and the specific genetic activities of Doc versus bap
and bin lead to the reciprocal arrangement of cardiac versus visceral
mesoderm precursors in the dorsal mesoderm
(Zaffran et al., 2002
;
Lee et al., 2004
).
|
A key gene requiring combinatorial Doc and Tin activities for its
activation in the cardiac mesoderm is the Gata factor-encoding gene
pannier (pnr) (Fig.
9). pnr expression is activated in the cardiac mesoderm
shortly after the induction of Doc and tin, at a time when Doc
expression has narrowed to the mesodermal precursors giving rise to pure
cardiac lineages. The mechanisms restricting Doc expression to the cardiac
mesoderm are currently not known, but as a consequence, pnr
expression is also limited to the cardiac mesoderm. It is conceivable that Doc
receives continued inputs during this period from the ectoderm through Dpp,
whose expression domain narrows towards the dorsal leading edge by then (as
was proposed for pnr) (Klinedinst
and Bodmer, 2003). Together with the observed feedback regulation
of pnr on tin and Doc, this situation leads to a prolonged
co-expression of Tin, Doc and Pnr in the cardiac mesoderm of stage 11 to stage
12 embryos. Based upon the onset of the expression of early markers such as
mid and svp, this is precisely the period when cardiac
progenitors become specified.
We anticipate that the activation of some downstream targets in presumptive cardiac progenitors requires the combination of two, or perhaps all three, of these cardiogenic factors. Potential target genes include mid, svp and hand. However, none of these candidates is essential for generating cardiac progenitors, although mid and svp are known to be required for the normal diversification of cardioblasts within each segment.
Our observation that forced expression of Pnr in the absence of any Doc
partially rescues cardiogenesis could indicate that the early, combinatorial
functions of tin and Doc are primarily mediated by pnr.
Alternatively, or in addition, this observation and the fact that a few
cardioblasts can be generated without Doc could point to the existence of some
degree of functional redundancy among these three factors. In the context of
the latter possibility, it is tempting to speculate that the functional
redundancy among T-box, Nkx and Gata factors during early cardiogenesis has
further increased during the evolution of the vertebrate lineages. This would
explain the less dramatic effects of the functional ablation of Tbx5,
Nkx2-5 and Gata4/5/6 on vertebrate heart development (reviewed
by Harvey, 1996;
Peterkin et al., 2005
;
Plageman and Yutzey, 2005
) as
compared to the severe effects of Doc, tin or pnr mutations
on dorsal vessel formation in Drosophila. Like the related
Drosophila genes, these vertebrate genes are co-expressed in the
cardiogenic region and developing heart of vertebrate embryos, which at least
for Nkx2.5 and Gata6 also involves cross-regulatory interactions that
reinforce their mutual expression
(Molkentin et al., 2000
)
(reviewed by Bruneau,
2002
).
The observed co-expression of Doc, Tin and Pnr allows for the possibility
that, in addition to combinatorial binding to target enhancers, protein
interactions among these factors play a role in providing synergistic
activities during cardiac specification. Physical interactions of Tbx5 with
Gata4 and Nkx2-5, as well between Nkx2-5 and Gata4 in vitro as well as
synergistic activities cell culture assays have been demonstrated in mammalian
systems and may be relevant to human heart disease
(Durocher et al., 1997;
Bruneau et al., 2001
;
Hiroi et al., 2001
;
Garg et al., 2003
) (reviewed
by Bruneau, 2002
). In
Drosophila, the genetic interactions between Doc, tin and
pnr observed both in loss- and gain-of-function experiments reveal
similar synergistic activities of the encoded factors during early
cardiogenesis. Altogether, our observations make it likely that these
Drosophila factors also act through combinatorial DNA binding and
mutual protein interactions to turn on target genes required for the
specification of cardiac progenitors.
Whereas pnr is expressed only transiently during early
cardiogenesis, tin and Doc continue to be expressed in developing
myocardial cells, suggesting that they act both in specification and
differentiation events. We recently showed that the T-box gene mid is
required for re-activating tin in cardioblasts
(Fig. 9)
(Reim et al., 2005). Of note,
owing to the action of svp, Doc and tin are expressed in
complementary subsets of cardioblasts within each segment
(Lo and Frasch, 2001
). This
mutually exclusive expression of tin and Doc implies that they are
not acting combinatorially but, instead, act differentially during later
stages of myocardial development. Hence, their activities could result in the
differential activation of some differentiation genes such as Sulfonylurea
receptor (Sur), which is specifically expressed in the four
Tin-positive cardioblasts in each hemisegment
(Nasonkin et al., 1999
;
Lo and Frasch, 2001
), and
wingless (wg), which is only turned on in the two
Doc-positive cells in each hemisegment of the heart that generate the ostia
(Lo et al., 2002
).
Surprisingly, even the activation of some genes that are expressed uniformly
in all cardioblasts has turned out to result from differential regulation
within the Tin-positive versus Doc-positive cardioblasts. For example,
regulatory sequences from the Mef2 gene for the two types of
cardioblasts are separable and those active within the four Tin-positive cells
are directly targeted by Tin (Gajewski et
al., 1997
; Cripps et al.,
1998
; Nguyen,
1998
; Gajewski et al.,
2000
). Likewise, regulatory sequences from a cardioblast-specific
enhancer of Toll have been shown to receive differential inputs from
Doc and Tin, respectively, in the two types of cardioblasts
(Wang et al., 2005
). In
parallel with this differential regulation, we anticipate that yet unknown
differentiation genes are activated uniformly in all cardioblasts downstream
of mid/H15 and hand. The integration of the new
information on the roles of Doc in cardiogenesis has now provided a basic
framework of signaling and gene interactions through all stages of embryonic
heart development, which in the future can be further refined upon the
identification of new components and additional molecular interactions.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Recent data showed that twi-GAL4/UAS-Doc2 expression in wg mutant embryos partially rescues cardioblast specification by producing mid/hand/Mef2-positive cardioblasts in about one to three hemisegments per embryo on each side, further supporting the notion of Doc as a key downstream mediator of Wg signals during cardiogenesis.
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