1 Department of Genetics, Washington University School of Medicine, 4566 Scott
Avenue, St Louis, MO 63110, USA
2 Program in Developmental Biology, Washington University School of Medicine,
4566 Scott Avenue, St Louis, MO 63110, USA
* Author for correspondence (email: jskeath{at}genetics.wustl.edu)
Accepted 17 March 2003
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SUMMARY |
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Key words: Drosophila, Heart development, GATA factor, ETS-domain proteins
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INTRODUCTION |
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The Drosophila heart is composed of two cell types (reviewed by
Bodmer and Frasch, 1999).
Cardioblasts express muscle specific proteins, coalesce to form the linear
heart tube and are the contractile cells of the heart. Pericardial cells are
loosely associated with and flank the cardioblasts; these cells do not express
muscle specific proteins and are thought to filter and detoxify the blood or
hemolymph of the fly. Cardioblasts and pericardial cells develop from closely
interspersed precursor cells that develop in the dorsalmost region of the
mesoderm, termed the cardiac mesoderm. After precursor divisions, heart cells
on both sides of the embryo align themselves into two rows of cells, with
pericardial cells being displaced slightly ventral and interior to the tightly
aligned row of cardioblasts at the dorsalmost extent of the mesoderm. As
dorsal closure occurs, the bilaterally symmetric rows of heart cells move
toward each other and the two rows of cardioblasts meet at the dorsal midline,
align perfectly with one another and coalesce to form a lumen between them.
Subsequently, the heart tube becomes divided into two domains: the aorta more
anteriorly and the heart proper in the posterior three segments. The heart
proper is distinguished from the aorta by a wider bore and the presence at
segmental intervals of ostia, the inflow valves of the heart. Here, we
collectively refer to the aorta and heart proper as the heart.
Gene expression, cell lineage and morphological studies indicate that
distinct subtypes of cardioblasts and pericardial cells populate the heart
(see Fig. 7)
(Gajewski et al., 2000;
Jagla et al., 1997
;
Lo and Frasch, 2001
;
Lo et al., 2002
;
Ward and Skeath, 2000
). These
studies also distinguish the development and gene expression profiles of heart
cells located in the posterior seven segments relative to those found more
anteriorly. Each hemisegment of the posterior region contains six cardioblasts
and ten pericardial cells. Cardioblasts can be roughly divided into two
classes: those that express Svp but not Tin (these are the first two
cardioblasts of a hemisegment); and those that express Tin but not Svp, the
four remaining cardioblasts of a hemisegment. The lineage of these
cardioblasts is known and shown in Fig.
7. Pericardial cells can be divided into three classes: Eve-, Tin-
and Odd-positive pericardial cells. The relative position of these cells is
shown in Fig. 7. The cell
lineage of the Odd-positive pericardial cells is known; however, the lineage
of Eve- and Tin-positive pericardial cells remains unclear.
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Genetic studies have identified the Nk2 type homeodomain protein Tin as a
key regulator of heart development (reviewed by
Bodmer and Frasch, 1999;
Cripps and Olson, 2002
).
tin expression is the earliest known marker of the cardiac mesoderm
and loss-of-function mutations in tin result in the complete absence
of all heart cells, as well as all other dorsal mesodermal derivatives. In
addition to tin, heart cell development absolutely requires the
function of the AP patterning genes wingless and
sloppy-paired. Despite the identification of a number of genes
required to promote the development of all heart cells, very few genes have
been identified that regulate the decision of cells to choose between the
cardioblast and pericardial cell fate. One such gene appears to be the GATA
zinc-finger transcription factor pnr
(Ramain et al., 1993
). Loss of
pnr function results in a significant loss of cardioblasts and an
apparent increase in at least one class of pericardial cells, the Eve-positive
pericardial cells (Gajewski et al.,
1999
). Other genes are likely to act with or in opposition to
pnr to regulate the decision of cells to acquire the cardioblast or
pericardial cell fate.
The pointed (pnt) locus encodes two protein isoforms,
both of which act as transcriptional effectors of the Ras/MAP-kinase pathway
(Brunner et al., 1994;
Klaes et al., 1994
;
Klambt, 1993
;
O'Neill et al., 1994
). The two
Pnt isoforms, PntP1 and PntP2, are members of the ETS family of transcription
factors and arise due to alternative use of two promoters separated by roughly
50 kb. PntP1 and PntP2 contain unique domains at their N terminus but share
the identical stretch of 394 amino acids at their C terminus within which
resides the ETS DNA-binding domain. The DNA-binding properties of these
proteins appear identical; however, PntP1 and PntP2 exhibit crucial
differences in their functional properties and transcriptional regulation.
PntP1 is a constitutive transcriptional activator and Ras/MAP-kinase pathway
activity induces PntP1 transcription
(Gabay et al., 1996
). By
contrast, PntP2 is not a constitutive transcriptional activator and
pntP2 transcription appears to be independent of Ras/MAP-kinase
activity. Nonetheless, PntP2 activity depends on Ras/MAP kinase activity as
Ras/MAP kinase-mediated phosphorylation of PntP2 at Thr151 turns it into a
potent transcriptional activator (Brunner
et al., 1994
; O'Neill et al.,
1994
). Although PntP1 and activated PntP2 regulate the development
of many different cell types and tissues in Drosophila, a role for
pnt function in cardioblast development has not been identified.
We present evidence that pnt plays a region specific role in regulating cardioblast and pericardial cell development. Loss of pnt function results in an approximate twofold increase in cardioblasts and an almost commensurate decrease in pericardial cells. This increase in cardioblasts arises largely from a specific increase in Svp-positive cardioblasts and is restricted to the posterior seven heart segments where Svp-positive cardioblasts normally develop. We demonstrate that this effect of pnt is carried out primarily if not exclusively by the PntP2 isoform, and that in this context PntP2 may act independently of Ras/MAP kinase pathway activity. Contrary to a prior study, we find that pnr acts early in mesoderm development to promote the development of the cardiac mesoderm and thus the development of all heart cells. Phenotypic analyses of pnr pnt double mutant embryos suggest a model whereby pnr acts before pnt to promote the formation of the cardiac mesoderm and that pnt acts subsequently within this domain to distinguish between cardioblast and pericardial cell fates. In addition, we present the completion of the cell lineage of all heart cells. These pedigree analyses identify a clear distinction between the lineage of anterior cardioblast and those that develop in the posterior seven segments. The transition point between these heart cell lineages correlates perfectly with the region specific effect of pnt on heart development. These results suggest independent genetic control of heart cell development in the anteriormost region of the heart relative to the posterior seven segments.
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MATERIALS AND METHODS |
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Lineage-tracing experiments
Random lacZ-expressing clones were created using the FLP/FRT
lineage tracing system as described previously
(Ward and Skeath, 2000) with
the following modifications. Three to four-hour-old embryos of the appropriate
genotype were heat-shocked for 20 minutes at 33°C to induce flp
recombinase, placed at 18°C and aged until stage 15-16, at which point
they were fixed and stained.
Antibody generation and immunohistochemistry and
immunofluorescence
Amino acids 1-230 of PntP1 were cloned into pET (Novagen) for protein
expression and purification. This protein domain is unique to PntP1. This
antigen was used to immunize rabbits at Pocono Rabbit Farm. The PntP1 antibody
is specific for PntP1 because it detects a protein expressed in a pattern
identical to pntP1 RNA and because the antibody does not detect
antigen in embryos that delete the pntP1-specific exons and
downstream exons of the pnt locus.
Single- and double-label immunohistochemistry analyses were performed as
described previously (Skeath,
1998). We used the following antibodies at the indicated
dilutions: mouse anti-Zfh1 (1:1000) (Lai
et al., 1991
); rabbit anti-Mef2 (1:1000)
(Lilly et al., 1995
); rabbit
anti-Eve (1:2000) (Frasch et al.,
1987
); mouse anti-ß-gal (1:2000; Promega); rabbit
anti-ß-gal (1:2000; Jackson); rabbit anti-Tin (1:500)
(Azpiazu and Frasch, 1993
);
rabbit anti-Pnr (1:400) (Herranz and
Morata, 2001
); rabbit anti-Odd (1:500)
(Ward and Skeath, 2000
);
rabbit anti-PntP1 (1:500).
Double stranded RNA interference (dsRNAi) and allele sequencing
RNAi was prepared as described previously
(Kennerdell and Carthew,
1998). We used dsRNA probes specific for pntP1 or
pntP2 to target each isoform independently. For pntP1 we
made dsRNA corresponding to nucleotides 1-690 of the pntP1-coding
region. This region encodes for the entire pntP1-specific domain. For
pntP2, we made dsRNA for corresponding to nucleotides 1-906 of the
pntP2 coding region. This region encodes
90% of the
pntP2-specific domain. dsRNA was injected into the posterior region
of pre-cellular blastoderm embryos at a concentration of 2 µg/µl, and
the embryos were allowed to develop until stage 15 to 16 at which point
embryos were collected and fixed for immunohistochemistry.
We identified the molecular lesions in pnt2,
pntRR112 and pnr1 by PCR-based
sequencing of the entire coding region and intron/exon boundaries of the
appropriate locus from genomic DNA obtained from each mutant background.
pnt2 contains G to A conversion at base 2653 of the
pntP1 cDNA (base 2667 of the pntP2 cDNA)
(Klambt, 1993). This mutation
converts the tryptophan (W) at amino acid 536 of PntP1 (amino acid 631 of
PntP2) to a premature stop codon, truncating both PntP1 and PntP2 roughly one
third of the way through the shared ETS-DNA-binding domain.
pntRR112 contains a G to A conversion in the splice donor
site of exon IV in pntP2. This lesion converts the GT donor site to
AT, and is expected to disrupt splicing of pntP2 but not
pntP1 because this exon is specific for pntP2.
pnr1 contains a G to A conversion at nucleotide 1034. This
mutation converts a W to a premature stop codon, truncating the pnr
midway through the first zinc finger.
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RESULTS |
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We identified eighteen clones that contained at least one Eve-positive pericardial cell. Eleven of these clones (61.1%) consisted solely of two Eve-positive pericardial cells (Fig. 1A), six clones (33.3%) consisted of two Eve-positive pericardial cells and one or two nearby heart or other mesodermal cells, and one clone (5.6%) consisted of a single Eve-positive pericardial cell. Thus, when we observe one Eve-positive pericardial cell within a clone of two or more cells a second Eve-positive pericardial cell always exists within this clone. These data demonstrate that the two Eve-positive pericardial cells within a hemisegment are siblings and arise from an Eve-positive pericardial cell precursor.
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Tin-positive pericardial cell clones fall into two classes: those that contained two Tin-positive pericardial cells (n=23), and those that contained one Tin-positive pericardial cell and one cardioblast (n=18). These two classes of clones arise in mutually exclusive regions of the heart. Clones that contain two Tin-positive pericardial cells arise in the posterior seven segments of the heart (we refer to this region as the posterior heart domain), whereas clones that contain one Tin-positive pericardial cell and one cardioblast arise anterior to this domain (we refer to this region as the anterior heart domain). The point of demarcation between these clonal types coincides precisely with the location of the first pair of Svp-positive cardioblasts (see below). These data demonstrate that heart cells exhibit distinct cell lineages as a function of position along the anteroposterior axis.
We identified a total of 24 Tin-positive pericardial cell clones in the posterior heart domain. Fifteen of these clones (62.5%) consisted solely of two Tin-positive pericardial cells (Fig. 1B), eight clones (33.3%) consisted of two Tin-positive pericardial cells and two nearby mesodermal cells, and one clone (4.2%) consisted of a single Tin-positive pericardial cell. Thus, when we observe one Tin-positive pericardial cell within a clone of two or more cells, a second Tin-positive pericardial cell always exists within this clone. These data indicate that the four Tin-positive pericardial cells found in each hemisegment of the posterior domain arise from two Tin-positive pericardial cell precursors. Our inability to identify any clones that contain four Tin-positive pericardial cells indicates that adjacent Tin-positive pericardial cell precursors are unlikely to share a common lineage.
We identified 18 Tin-positive pericardial cell clones in the anterior heart domain. All 18 clones consisted of one Tin-positive pericardial cell and one cardioblast (Fig. 1C). These data indicate that within this region Tin-positive pericardial cells arise from bi-potent heart precursors, each of which produces one Tin-positive pericardial cell and one cardioblast. These data also demonstrate that cardioblasts and Tin-positive pericardial cells in the anterior heart domain develop via a different cell lineage than cardioblasts and Tin-positive pericardial cells that develop in the posterior domain.
The analysis of ten additional cardioblast clones in the anterior heart domain support a distinct cell lineage for anterior versus posterior cardioblasts. Nine clones consisted of one cardioblast and one non-Tin-expressing pericardial cell (Fig. 1D), whereas a single clone consisted of two cardioblasts. Thus, most, if not all, anterior domain cardioblasts share a sibling relationship with a pericardial cell. In addition, all anterior domain cardioblasts exhibit cell lineages distinct from posterior domain cardioblasts. Together with the lineage data on Tin-positive pericardial cells, these results support the idea that cardioblasts and Tin-positive pericardial cells in the anterior heart domain carry out distinct functions from those found in the posterior heart domain.
Interestingly, the lineage of the twelve cardioblasts in the anterior heart domain appears fixed with respect to whether they share a sibling relationship with a Tin-positive or Tin-negative pericardial cell. We numbered these cardioblasts 1-12 from anterior to posterior with cardioblast 12 being immediately anterior to the first Svp-positive cardioblast. We identified four clones that contained cardioblast 12 and in each clone this cardioblast shared a sibling relationship with a Tin-negative pericardial cell. By contrast, cardioblasts 10 and 11 each share a sibling relationship with a Tin-positive pericardial cell (n=3/3 and 5/5 clones, respectively). We have not obtained multiple clones for all twelve cardioblasts; nonetheless, these data suggest a fixed relationship between the position of a cardioblast and whether its sibling pericardial cell expresses Tin. We speculate that the differences in gene expression between different pairs of sibling cardioblasts and pericardial cells in the anterior domain may reflect functional differences between such pairs of heart cells.
Loss of pointed function results in excess cardioblasts
We identified pnt as an inhibitor of cardioblast development in a
screen for mutations that affect cardioblast and/or pericardial cell
development. To identify genes that regulate heart development we screened
2000 third chromosomal lethal P element lines obtained from the Hungarian
P element Stock Collection for defects in the expression of Mef2 a protein
expressed in all cardioblasts and Eve. We uncovered two P element mutations
that cause an approximate twofold increase in cardioblasts
(Fig. 2; X. Tian and J.B.S.,
unpublished). One of these P elements [l(3)S012309] maps to
cytological position 94F1-3 and was known to be an allele of pnt
(FlyBase, 2003
). To verify that
lesions in pnt result in the formation of ectopic cardioblasts, we
assayed the phenotype of five additional pnt alleles. Although the
severity of the phenotype varies for each pnt allele, all alleles
display a significant increase in cardioblast number relative to wild-type
embryos (Fig. 2). With respect
to the excess cardioblast phenotype, we can group these alleles into the
following allelic series: pntS012309,
pnt2 > pntRR112,
pntRM254 >
pnt
88,
pnt07825. The presence of excess cardioblasts in embryos
homozygous mutant for each pnt allele indicates that pnt
normally functions in heart development to repress cardioblast
development.
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Our analysis of heart development in pnt embryos indicated that the effect of pnt on cardioblast development is region specific. In wild-type embryos 12 cardioblasts develop anterior to the first pair of Svp-positive cardioblasts on each side of the embryo. As detailed in our lineage studies, these cardioblasts define the anterior heart domain. Interestingly, cardioblast development in the anterior domain is essentially normal in pnt mutant embryos (Fig. 3). In pntS012309 and in pntS012309/pnt2 embryos, an average of 10.8 (n=10) and 12.4 (n=5) cardioblasts develop anterior to the first pair of Svp-lacZ-positive cardioblasts. Thus, in pnt embryos the ectopic cardioblast phenotype is restricted to the region of the heart, the posterior domain, that normally contains endogenous Svp-lacZ-positive cardioblasts.
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Owing to the specific increase in Svp-lacZ cardioblasts in
pnt embryos, we also performed a detailed analysis of the development
of Svp-lacZ cardioblasts and pericardial cells. In wild-type embryos,
two Svp-lacZ heart precursors arise in each hemisegment during stage
11 (Ward and Skeath, 2000).
Each precursor divides during stage 12 to yield one Svp-lacZ
cardioblast and one Svp-lacZ pericardial cell. In pnt
embryos the formation and division of each endogenous Svp-lacZ heart
precursor is normal and wild-type numbers of Svp-lacZ pericardial
cells persist throughout embryogenesis (data not shown). Thus,
Svp-lacZ pericardial cells appear to develop normally in pnt
embryos. However, we observe ectopic Svp-lacZ cardioblasts during
late stage 12/early stage 13 in pnt embryos and these cells are found
in locations normally occupied by pericardial cells
(Fig. 4; not shown). These
results, together with those detailed above, suggest that pnt
normally functions in presumptive non-Svp-lacZ-expressing pericardial
cells to repress the development of the Svp-lacZ cardioblast
fate.
Our quantification of cardioblasts and pericardial cells in pnt
embryos suggests the excess cardioblast phenotype does not arise solely from a
conversion of pericardial cells into cardioblasts, as we observe a net loss of
30 pericardial cells and a net gain of
50 cardioblasts per embryo
side. To investigate whether loss of pnt affects the proliferative
potential of heart cells, we created and analyzed cardioblast clones in
pnt embryos. We identified 124 clones in pnt embryos that
contained at least one cardioblast. 97 clones (78%) consisted of either two
cardioblasts or one cardioblast and one pericardial cell; 21 (17%) clones
consisted of two cardioblasts and one or two pericardial cells whereas two
(1.6%) consisted of one cardioblast. These clone types as well as their
frequencies are similar to that observed for cardioblast clones in wild-type
embryos (Ward and Skeath,
2000
) (data not shown). However, in addition to these clones, we
identified four (3.2%) that consisted of between six and 12 cardioblasts. We
have never observed clones of more than four cardioblasts in wild-type embryos
(n>200 clones). These data suggest that loss of pnt leads
to a slight but perceptible increase in the proliferative capability of
cardioblast precursors. However, the weak increase in cardioblast
proliferation and the apparent conversion of pericardial cells into
cardioblasts still appear insufficient to account for the approximate twofold
increase in cardioblasts in pnt embryos. We hypothesize that loss of
pnt also causes other dorsal mesodermal cells to acquire the
cardioblast fate inappropriately. Consistent with this, we observe loss of
specific dorsal muscles in pnt embryos.
pointed may regulate cardioblast development independently
of the Ras pathway
Through the use of alternative promoters, the pnt locus encodes
two distinct protein isoforms: PntP1 and PntP2. Both isoforms act as effectors
of the Ras/MAP kinase pathway in multiple developmental contexts
(Brunner et al., 1994;
Klambt, 1993
;
O'Neill et al., 1994
). This
raises the possibility that the role of pnt during heart development
is mediated through Ras/MAP kinase activity. Thus, we examined whether loss or
reduction in the function of different members of the Ras/MAP kinase pathway
also increased cardioblast number. We assayed cardioblast development in
homozygous embryos singly mutant for spitz, Star, rhomboid, heartless
and heartbroken. We also assayed cardioblast number in embryos in
which we expressed dominant-negative forms of ras as well as the
EGF- and FGF-receptors specifically in the mesoderm to
reduce the activity of these genes in this tissue (see Materials and Methods).
In all genetic backgrounds tested, we never observed an increase in
cardioblast number. For the experiments involving dominant-negative
constructs, we verified dominant-negative activity of the expressed protein by
assaying ras-dependent developmental events that occur in the
mesoderm prior to the role of pnt in cardioblast development. In all
cases, the ras-dependent developmental events were perturbed (data
not shown). Thus, we are confident that our failure to observe an effect on
cardioblast number is not simply due to an inability of the dominant-negative
proteins to inhibit the function of the targeted proteins in a timely manner.
We interpret these results to suggest that pnt may regulate
cardioblast development in a Ras-MAP kinase-independent manner.
PointedP2 regulates cardioblast number
The presence of two Pnt isoforms raises the question as to whether PntP1
and/or PntP2 carry out the function of the pnt locus during heart
development. To address this issue, we used isoform-specific RNAi and
isoform-specific rescue of the pnt cardioblast phenotype. We first
generated double-stranded RNA probes to the unique 5' regions of the
PntP1 and PntP2 transcripts, and injected these separately into presynctial
stage Drosophila embryos. We then labeled all such embryos either for
Mef2 (to follow cardioblast development) or Mef2 and PntP1 protein (to follow
cardioblast development and PntP1 protein levels). Embryos treated for
pntP1 RNAi exhibit severe morphological defects and a complete loss
of PntP1 protein expression. In many embryos the extent of the morphological
defects preclude a clean analysis of cardioblast development; however, it is
possible to score cardioblast number in a subset of these embryos. We only
scored cardioblast number in embryos devoid of detectable PntP1 protein. In
these embryos, we observe an average of 53.2 cardioblasts per embryo side
(n=11), nearly identical to the 52 cardioblasts that develop on each
side of wild-type embryos. These results suggest that pntP1 does not
play a significant role in the regulation of cardioblast number by
pnt.
By contrast, PntP2-RNAi indicates that pntP2 function is necessary to regulate cardioblast number. Embryos treated for PntP2 RNAi exhibit wild-type morphology, a clear excess of cardioblasts and an essentially normal pattern of PntP1 expression (Fig. 5). In these embryos, we observe an average of 80.1 cardioblasts per embryo side (n=22; ranging from 62 to 108 cardioblasts). The most severe pntP2 RNAi phenotypes are as severe as those observed for pnt2 or pntS012309 embryos. We attribute the variable expressivity of the pntP2 RNAi phenotype to the technique of RNAi as we observe a large variance in expressivity of the RNAi phenotype for all genes we have assayed in this manner.
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We also addressed the relative roles of pntP1 and pntP2 by assaying the effect generalized mesodermal expression of each pnt isoform has on cardioblast development in wild-type and pnt embryos. In these experiments, we used the Twist-GAL4 driver line to drive either pntP1 or pntP2 under UAS control throughout the mesoderm of homozygous wild-type or pntS012309 embryos. We find that mesodermal expression of pntP2 in pnt embryos is sufficient to rescue to wild-type the pnt cardioblast phenotype (Fig. 5). In addition, we find that mesodermal expression of pntP2 in otherwise wild-type embryos has no effect on cardioblast development (not shown). By contrast, we find that mesodermal expression of pntP1 in wild-type or pnt embryos leads to a near complete loss of all cardioblasts and pericardial cells (Fig. 5). This drastic effect of pntP1 on heart development may arise because of an effect of pntP1 overexpression on early steps of mesodermal development prior to heart cell development. This possibility makes interpretation of whether pntP1 can rescue the pnt cardioblast phenotype difficult. Nonetheless, these experiments clearly show that pntP2 is sufficient to rescue the pnt heart phenotype. Together with the RNAi experiments and the phenotypic analysis of a pntP2-specific allele, these results demonstrate that pntP2 is necessary and sufficient for the cardioblast and pericardial cell development, and suggest that pntP1 is irrelevant in this developmental context.
pannier acts as a general promoter of dorsal mesoderm
development
The published heart phenotype of the GATA transcription factor pnr
is opposite to that of pnt. In pnr mutant embryos, too many
pericardial cells and too few cardioblasts are thought to develop
(Gajewski et al., 1999). As a
first step towards examining the potential regulatory interactions between
pnr and pnt, we carried out a detailed analysis of heart
development in pnr mutant embryos. We used
pnrVX6, a null allele that contains a small deletion that
removes all but the N-terminal nine amino acids of pnr
(Ramain et al., 1993
), as well
as pnr1, a molecularly uncharacterized allele. In contrast
to a prior study, we find a loss of both cardioblasts and pericardial cells in
pnr embryos (Fig. 6).
We quantified the dorsal mesodermal phenotypes for Eve-positive pericardial
cells as well as for all pericardial cells using the pan-pericardial marker
Zfh1. In wild-type embryos we observe an average of 22.7 Eve-positive
pericardial cells (n=32) and 61.1 Zfh1-positive pericardial cells
(n=17) per embryo side. pnrVX6 embryos exhibit
the most severe effect with an average of 9.4 (n=11) and 16.9
(n=21) Eve- and Zfh1-positive pericardial cells, respectively.
pnrVX6/pnr1 embryos exhibit an intermediate
phenotype with an average of 16.4 Eve-positive pericardial cells
(n=25) and 27.4 Zfh1-positivepericardial cells (n=18), while
pnr1 embryos exhibit the mildest phenotype with an average
of 21.2 and 37.4 Eve- (n=10) and Zfh1- (n=11) positive
pericardial cells, respectively. We also observed a severe loss of
cardioblasts and Odd-positive pericardial cells in these backgrounds although
we did not quantify these phenotypes. The loss of cardioblasts and
Odd-positive pericardial cells is most severe in pnrVX6
embryos and least severe in pnr1 embryos where short
stretches of cardioblasts are still visible
(Fig. 6). These results
indicate that pnr normally functions to promote the development of
all heart cells.
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The above results indicate that pnr1 is a hypomorphic
allele. Sequence analysis identified a single mutation in the
pnr1-coding region that converts a tryptophan residue at
amino acid 180 to a premature stop codon roughly halfway through the first
zinc finger. Using an antibody specific to epitopes N-terminal to this
premature stop codon (Herranz and Morata,
2001), we find that the pattern and level of the mutant
Pnr1 protein in homozygous pnr1 embryos are
identical to those of the wild-type Pnr protein. However, while wild-type Pnr
protein localizes predominantly to the nucleus, we find that Pnr1
protein localizes predominantly to the cytoplasm (not shown). These data
together with the mild pnr1 phenotype relative to the
pnrVX6 null allele suggest that the truncated
Pnr1 protein retains residual activity.
pannier acts upstream of pointed in a developmental
pathway
Our studies on pnr and pnt suggest these genes act in a
developmental pathway in which the prior function of pnr to promote
cardiac mesoderm formation is required for the subsequent action of
pnt to specify between pericardial cell and cardioblast fates. If
this model is correct, pnr pnt double mutants should display the
pnr phenotype, as neither pericardial cells nor cardioblasts will
arise in the absence of cardiac mesoderm. Consistent with this,
pnrVX6 pntS012309 embryos lack cardioblasts and
pericardial cells, and appear phenotypically indistinguishable with respect to
heart development from pnrVX6 embryos (not shown). To test
our model more stringently, we assayed heart development in
pnr1 pntS012309 embryos. We used
pnr1 embryos because small regions of cardiac mesoderm
develop in pnr1 embryos and these regions produce short
strings of cardioblasts that maintain the wild-type 1:2 ratio of
Svp-positive:Svp-negative cardioblasts
(Fig. 6). We reasoned that if
pnr and pnt act in a developmental pathway, then we should
observe the pnt mutant phenotype in those regions of
pnr1 pntS012309 embryos in which cardiac
mesoderm develops. In agreement with this, we observe local overproduction of
cardioblasts in pnr1 pntS012309 embryos and the
vast majority of these cardioblasts are Svp-lacZ positive. These
double mutant studies support the model that pnr acts upstream of
pnt in a developmental pathway.
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DISCUSSION |
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The effect of pnt on heart development is restricted to the
posterior seven heart segments where Svp cardioblasts normally develop.
Interestingly, our lineage studies identify a clear difference in the cell
lineage of cardioblasts that develop in the posterior seven heart segments
versus those that develop more anteriorly
(Fig. 7)
(Ward and Skeath, 2000). These
results identify a genetic and developmental distinction between these two
regions of the heart. In addition, they suggest that cells in different
regions of the heart carry out different functions and that these functions
are probably under homeotic gene control. Future work that addresses the
physiological role of these cells in heart function and the control of their
development by homeotic genes should provide a more comprehensive
understanding of heart development.
Does PntP2 act independently of the Ras/MAPK pathway?
Our data suggest that PntP2 may regulate cardioblast and pericardial cell
development independently of Ras/MAP kinase activity. Given that every other
developmental function of pnt has been traced back to receptor
tyrosine kinase/Ras signaling activity, the apparent Ras independent activity
of PntP2 is puzzling. As PntP2 is expressed broadly throughout the mesoderm
(data not shown) (Klambt,
1993), a number of models can explain the apparent Ras-independent
activity of PntP2 in the heart. For example, PntP2 may not require
MAP-kinase-mediated phosphorylation to carry out a subset of its function.
Consistent with this, phosphorylation of PntP2 does not appear to affect its
DNA-binding ability (O'Neill et al.,
1994
). Thus, in the absence of MAP-kinase stimulation, PntP2 is
still probably able to bind target promoters alone or in complexes with other
proteins. Such an activity of PntP2 could on its own regulate target gene
expression by blocking the ability of other transcriptional effectors to bind
to and activate target gene transcription, or through an obligate association
with other proteins required to activate (or to repress) target genes.
Significant precedent exists for such activity. For example, the Su(H)/CSL and
pangolin/TCF proteins are the transcriptional effectors of the Notch
and wingless pathways, respectively, and in the absence of
Notch or wingless activity these proteins can repress target
gene transcription (Cavallo et al.,
1998
; Li et al.,
1997
; Mumm and Kopan,
2000
; van de Wetering et al.,
1997
).
A second model is that PntP2 requires MAP kinase activation but that this
activity is carried out by one of the other MAP kinase pathways in
Drosophila: the JNK pathway or the p38 pathway. Preliminary
phenotypic analyses indicate that heart development is normal in embryos
mutant for basket, the Drosophila JNK-kinase (J.B.S.,
unpublished). Analysis of p38 kinase activity is presently limited because of
the absence of suitable genetic backgrounds. A third possibility is that a
novel Ras-dependent pathway does in fact activate PntP2 during heart
development. This model is consistent with the recent identification of a
novel receptor tyrosine kinase expressed in the developing visceral mesoderm
(Loren et al., 2001). Our
experiments that failed to identify a pnt-like excess cardioblast
phenotype upon mesodermal overexpression of a dominant-negative form of Ras
argue against this model. However, Ras is maternally loaded and it is
extremely difficult to eliminate all Ras activity in this manner. Thus, even
though we observed Ras-like mesodermal phenotypes in these experiments, we
still may have missed a role for Ras in regulating cardioblast number because
of differential sensitivity of different developmental pathways to partial Ras
inactivation. Future work that (1) addresses the ability of MAP-kinase
insensitive forms of PntP2 to regulate heart development, and (2) identifies
PntP2 target genes in the heart and elucidates how PntP2 regulates such genes
should help clarify the molecular basis through which PntP2 governs heart
development.
Can Pannier function independent of its DNA binding ability?
Our phenotypic analysis of pnr conflicts with a prior study that
showed an increase in pericardial cells in pnr mutants
(Gajewski et al., 1999). This
study used Eve to identify a subset of pericardial cells in
pnr1 embryos. We attribute the difference in our results
to our use of the pnrVX6 null allele, our ability to
distinguish unambiguously Eve-positive pericardial cells from Eve-positive
somatic muscle progenitors, and to specific defects in dorsal closure
exhibited by pnr embryos that result in the local aggregation of
cells in the dorsal region of the embryo. Our genetic results identify
pnr1 as a hypomorphic allele and we find that Eve-positive
pericardial cell formation is almost wild type in this background. In these
experiments, we unambiguously identified Eve-positive pericardial cells via
their co-expression of Zfh1 and were thus able to quantify precisely
Eve-positive pericardial cell number in pnr1 embryos. This
is important as one can observe local increases in Eve-positive mesodermal
cells in pnr embryos. However, such apparent increases arise from the
local aggregation of dorsal mesodermal cells in pnr1
embryos caused by defects in dorsal closure and not by an overall increase in
Eve-positive mesodermal cells.
The genetic identification of pnr1 as a hypomorphic
allele is intriguing given that molecular and expression analyses indicate the
pnr1 lesion results from a premature stop codon in the
middle of the first zinc finger and that the Pnr1 protein localizes
predominantly to the cytoplasm. This lesion is expected to abrogate the
DNA-binding ability of the Pnr protein. However, our genetic experiments
indicate that the Pnr1 protein retains residual activity at least
with respect to heart development. These results raise the possibility that
Pnr may be able to carry out some of its functions independently of DNA
binding. Precedence for such an activity comes from studies on a genetically
engineered form of the homeodomain transcription factor Fushi-tarazu that
lacks the homeodomain but retains significant biological activity
(Copeland et al., 1996). Future
work that focuses on a detailed structure function analysis of the Pnr protein
should clarify whether Pnr can act independently of its DNA-binding ability in
some developmental contexts.
We should also note that our pnt allelic series indicates that
pnt88 exhibits a milder
excess cardioblast phenotype than pntS012309,
pnt2, and pntRR112. This result is
surprising as pnt
88
deletes the exons pntP2 shares with pntP1 and as a result
was assumed to be an amorphic allele of the pnt locus
(Scholz et al., 1993
). Using
antisense RNA probes specific for the unique exons of pntP2, we
observe an essentially wild-type pattern of pntP2 transcription in
pnt
88 mutant embryos
(data not shown). These data raise the possibility that the N-terminal regions
of pntP2 may also retain partial activity. Studies along the lines of
those suggested for Pnr should also help elucidate whether truncated forms of
PntP2 retain residual activity.
Do vertebrate ETS transcription factors regulate heart
development?
As noted, significant similarity exists between the embryology and
molecular regulation of early heart development in Drosophila and
vertebrates. In this context, the identification of a role for pnt, a
member of the evolutionarily conserved ETS transcription factor family, in
Drosophila heart development raises the possibility that ETS family
proteins regulate vertebrate heart development. Consistent with this, ETS1 and
ETS2, the two most closely related vertebrate ETS proteins to pnt,
are expressed in the developing vertebrate heart; functional studies indicate
these genes regulate the expression of specific genes in the heart
(Majka and McGuire, 1997;
Macias et al., 1998
). However,
knockout studies have not yet revealed a clear role for ETS1 or ETS2 in the
morphological development or differentiation of the vertebrate heart. The
existence of multiple vertebrate ETS-family members highly homologous to
pnt, as well as a total of 25 ETS family members in humans suggests
the possibility of functional redundancy in ETS protein function during
vertebrate and mammalian heart development. Thus, a full understanding of ETS
protein function during heart development awaits construction and analysis of
animals multiply mutant for different ETS family members.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Azpiazu, N. and Frasch, M. (1993). tinman and bagpipe: two homeo box genes that determine cell fates in the dorsal mesoderm of Drosophila. Genes Dev. 7,1325 -1340.[Abstract]
Bodmer, R. and Frasch, M. (1999). Genetic determination of Drosophila heart development. In Heart Development (ed. N. Rosenthal), pp.65 -90. San Diego: Academic Press.
Borkowski, O. M., Brown, N. H. and Bate, M.
(1995). Anterior-posterior subdivision and the diversification of
the mesoderm in Drosophila. Development
121,4183
-4193.
Brunner, D., Ducker, K., Oellers, N., Hafen, E., Scholz, H. and Klambt, C. (1994). The ETS domain protein pointed-P2 is a target of MAP kinase in the sevenless signal transduction pathway. Nature 370,386 -389.[CrossRef][Medline]
Carmena, A., Gisselbrecht, S., Harrison, J., Jimenez, F. and
Michelson, A. M. (1998). Combinatorial signaling codes
for the progressive determination of cell fates in the Drosophila embryonic
mesoderm. Genes Dev. 12,3910
-3922.
Cavallo, R. A., Cox, R. T., Moline, M. M., Roose, J., Polevoy, G. A., Clevers, H., Peifer, M. and Bejsovec, A. (1998). Drosophila Tcf and Groucho interact to repress Wingless signalling activity. Nature 395,604 -608.[CrossRef][Medline]
Copeland, J. W., Nasiadka, A., Dietrich, B. H. and Krause, H. M. (1996). Patterning of the Drosophila embryo by a homeodomain-deleted Ftz polypeptide. Nature 379,162 -165.[CrossRef][Medline]
Cripps, R. M. and Olson, E. N. (2002). Control of cardiac development by an evolutionarily conserved transcriptional network. Dev. Biol. 246,14 -28.[CrossRef][Medline]
FlyBase (2003). The FlyBase database of the
Drosophila genome projects and community literature. Nucleic Acids
Res. 31,172
-175.
Frasch, M., Hoey, T., Rushlow, C., Doyle, H. and Levine, M. (1987). Characterization and localization of the even-skipped protein of Drosophila. EMBO J. 6, 749-759.[Abstract]
Gabay, L., Scholz, H., Golembo, M., Klaes, A., Shilo, B. Z. and
Klambt, C. (1996). EGF receptor signaling induces
pointed P1 transcription and inactivates Yan protein in the Drosophila
embryonic ventral ectoderm. Development
122,3355
-3362.
Gajewski, K., Choi, C. Y., Kim, Y. and Schulz, R. A. (2000). Genetically distinct cardial cells within the Drosophila heart. Genesis 28,36 -43.[CrossRef][Medline]
Gajewski, K., Fossett, N., Molkentin, J. D. and Schulz, R.
A. (1999). The zinc finger proteins Pannier and GATA4
function as cardiogenic factors in Drosophila.
Development 126,5679
-5688.
Herranz, H. and Morata, G. (2001). The
functions of pannier during Drosophila embryogenesis.
Development 128,4837
-4846.
Jagla, K., Frasch, M., Jagla, T., Dretzen, G., Bellard, F. and
Bellard, M. (1997). ladybird, a new component of the
cardiogenic pathway in Drosophila required for diversification of heart
precursors. Development
124,3471
-3479.
Kennerdell, J. R. and Carthew, R. W. (1998). Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95,1017 -1026.[Medline]
Klaes, A., Menne, T., Stollewerk, A., Scholz, H. and Klambt, C. (1994). The Ets transcription factors encoded by the Drosophila gene pointed direct glial cell differentiation in the embryonic CNS. Cell 78,149 -160.[Medline]
Klambt, C. (1993). The Drosophila gene pointed
encodes two ETS-like proteins which are involved in the development of the
midline glial cells. Development
117,163
-176.
Lai, Z. C., Fortini, M. E. and Rubin, G. M. (1991). The embryonic expression patterns of zfh-1 and zfh-2, two Drosophila genes encoding novel zinc-finger homeodomain proteins. Mech. Dev. 34,123 -134.[CrossRef][Medline]
Li, P., Yang, X., Wasser, M., Cai, Y. and Chia, W. (1997). Inscuteable and Staufen mediate asymmetric localization and segregation of prospero RNA during Drosophila neuroblast cell divisions. Cell 90,437 -447.[Medline]
Lilly, B., Zhao, B., Ranganayakulu, G., Paterson, B. M., Schulz, R. A. and Olson, E. N. (1995). Requirement of MADS domain transcription factor D-MEF2 for muscle formation in Drosophila. Science 267,688 -693.[Medline]
Lo, P. C. and Frasch, M. (2001). A role for the COUP-TF-related gene seven-up in the diversification of cardioblast identities in the dorsal vessel of Drosophila. Mech. Dev. 104, 49-60.[CrossRef][Medline]
Lo, P. C., Skeath, J. B., Gajewski, K., Schulz, R. A. and Frasch, M. (2002). Homeotic genes autonomously specify the anteroposterior subdivision of the Drosophila dorsal vessel into aorta and heart. Dev. Biol. 251,307 -319.[CrossRef][Medline]
Loren, C. E., Scully, A., Grabbe, C., Edeen, P. T., Thomas, J.,
McKeown, M., Hunter, T., Palmer, R. H. (2001)
Identification and characterization of DAlk: a novel Drosophila melanogaster
RTK which drives ERK activation in vivo. Genes Cells
6, 531-544.
Macias, D., Perez-Pomares, J. M., Garcia-Garrido, L., Carmona, R. and Munoz-Chapuli, R. (1998). Immunoreactivity of the ets-1 transcription factor correlates with areas of epithelial-mesenchymal transition in the developing avian heart. Anat. Embryol. 198,307 -315.[CrossRef][Medline]
Majka, S. M. and McGuire, P. G. (1997). Regulation of urokinase expression in the developing avian heart: a role for the Ets-2 transcription factor. Mech. Dev. 68,127 -137.[CrossRef][Medline]
Mlodzik, M., Hiromi, Y., Weber, U., Goodman, C. S. and Rubin, G. M. (1990). The Drosophila seven-up gene, a member of the steroid receptor gene superfamily, controls photoreceptor cell fates. Cell 60,211 -224.[Medline]
Molina, M. R. and Cripps, R. M. (2001). Ostia, the inflow tracts of the Drosophila heart, develop from a genetically distinct subset of cardial cells. Mech. Dev. 109, 51-59.[CrossRef][Medline]
Mumm, J. S. and Kopan, R. (2000). Notch signaling: from the outside in. Dev. Biol. 228,151 -165.[CrossRef][Medline]
O'Neill, E. M., Rebay, I., Tjian, R. and Rubin, G. M. (1994). The activities of two Ets-related transcription factors required for Drosophila eye development are modulated by the Ras/MAPK pathway. Cell 78,137 -147.[Medline]
Park, M., Yaich, L. E. and Bodmer, R. (1998). Mesodermal cell fate decisions in Drosophila are under the control of the lineage genes numb, Notch, and sanpodo. Mech. Dev. 75,117 -126.[CrossRef][Medline]
Ramain, P., Heitzler, P., Haenlin, M. and Simpson, P.
(1993). pannier, a negative regulator of achaete and scute in
Drosophila, encodes a zinc finger protein with homology to the vertebrate
transcription factor GATA-1. Development
119,1277
-1291.
Scholz, H., Deatrick, J., Klaes, A., Klambt, C.
(1993). Genetic dissection of pointed, a Drosophila gene encoding
two ETS-related proteins. Genetics
135,455
-468.
Skeath, J. B. (1998). The Drosophila EGF
receptor controls the formation and specification of neuroblasts along the
dorsal-ventral axis of the Drosophila embryo.
Development 125,3301
-3312.
Struhl, G. and Basler, K. (1993). Organizing activity of wingless protein in Drosophila. Cell 72,527 -540.[Medline]
Sulston, J. E. and Horvitz, H. R. (1977). Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev. Biol. 56,110 -156.[Medline]
van de Wetering, M., Cavallo, R., Dooijes, D., van Beest, M., van Es, J., Loureiro, J., Ypma, A., Hursh, D., Jones, T., Bejsovec, A. et al. (1997). Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF. Cell 88,789 -799.[Medline]
Ward, E. J. and Skeath, J. B. (2000).
Characterization of a novel subset of cardiac cells and their progenitors in
the Drosophila embryo. Development
127,4959
-4969.
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