Department of Biology, University of New Mexico, Albuquerque, NM 87131-1091, USA
* Author for correspondence (e-mail: rcripps{at}unm.edu)
Accepted 7 August 2002
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SUMMARY |
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Key words: Heart, Hox genes, abdominal-A, Bithorax Complex, Drosophila, Aorta, Dorsal vessel, Ostia, Ostium
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INTRODUCTION |
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Consistent with the embryological similarities, the molecular mechanisms
and transcriptional regulatory pathways that govern heart cell specification
and development are also strongly conserved between different animal species
(reviewed by Cripps and Olson,
2002). Heart cell fate is induced by signaling molecules of the
transforming growth factor ß family
(Frasch, 1995
;
Schultheiss et al., 1997
).
These signals serve to activate genes encoding the Tinman/NKX family of
homeodomain transcription factors, which are required for specification or
morphogenesis of the functional heart (reviewed by
Harvey, 1996
).
Despite a strong understanding of the signaling and transcriptional events
that specify the linear heart tube, there is still much to learn concerning
how unique cell types are specified within this organ. Although numerous genes
are known to be expressed in the heart tube at specific anteroposterior (AP)
locations, the mechanisms governing this process remain largely unknown
(Srivastava and Olson, 2000).
To elucidate the mechanisms governing AP identity in the heart, we studied
diversification in the Drosophila dorsal vessel. The dorsal vessel is
composed of a linear tube spanning segments thoracic 2 (T2) to abdominal 8
(A8). From T2 to A5 the tube is narrow and is termed the aorta, whereas the
posterior portion has a larger bore and is termed the heart. Additionally the
heart is perforated by three pairs of valve-like ostia, which serve as inflow
tracts for hemolymph (Rizki,
1978
; Bodmer and Frasch,
1999
).
We demonstrate that the location of the heart correlates precisely with the expression of the Hox segmentation gene abdominal-A (abd-A), and that heart cell identity and the formation of heart-specific structures depends upon abd-A function. These findings for the first time demonstrate a mechanism for AP patterning the developing animal heart tube.
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MATERIALS AND METHODS |
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Immunohistochemistry and in situ hybridization
Antibody staining was performed as described
(Patel, 1994). Primary
antibodies and the concentrations used were: rabbit anti-MEF2, 1:1000
(Lilly et al., 1995
); rabbit
anti-myosin heavy-chain (MHC), 1:500
(Kiehart and Feghali, 1986
);
mouse anti-UBX, 1:50 (White and Wilcox,
1984
); mouse anti-ABDA, 1:1000
(Macias et al., 1990
); mouse
anti-ABDB, 1:1000 (Celniker et al.,
1989
); and rabbit anti-Tin, 1:1000
(Yin et al., 1997
).
Fluorescent secondary antibodies were either Alexa 488 anti-mouse or Alexa 568
anti-rabbit (Molecular Probes, Seattle, WA) all used at a 1:2000 dilution.
Non-fluorescent antibody detection was using the Vectastain Elite kit (Vector
Laboratories, Burlingame, CA).
Confocal microscopy was performed using a BioRad MRC-600 confocal laser-scanning microscope using 488/568 nm excitation in the dual-channel mode (T1/T2A filter cubes, BioRad, Hercules, CA).
In situ hybridization was performed as described
(O'Neill and Bier, 1994),
using digoxigenin-labeled riboprobes (Roche Molecular Biologicals,
Indianapolis, IN). To generate a probe for the Hand and
Tina-1 genes, primers were designed to amplify sequences from
embryonic cDNA, based upon the exon structures for each gene described
(Adams et al., 2000
). Primer
sequences for Hand were: 5'-CATGTTCGACATGAAACG-3' and
5'-AAATATTATTTTTGCAAAATATG-3'. Primer sequences for
Tina-1 were: 5'-CAAGAAAATGGGCCAAGC-3' and
5'-CCACGTTATTAGTTCTGG-3'. PCR products were cloned into the pGEM-T
Easy vector (Promega, Madison, WI). Recombinant clones were sequenced for
orientation and then sense and antisense riboprobes generated following
linearization of the plasmid.
All samples were mounted in 80% (v/v) glycerol for photography. Samples
were either prepared as whole mounts, or were filleted along the ventral
midline with the viscera removed to allow detailed visualization of the dorsal
vessel and the ostia. The size of the svp-expressing cells which
formed the ostia were measured as follows. Embryos stained for expression of
tin (in black) and Myosin heavy-chain (in brown) were
filleted and photographed such that the muscular dorsal vessel and the
tin-expressing cells were visible at 600x magnification. A 10
µm graduation slide was also photographed at the same magnification. The
sizes of each non-Tin expressing cell (those cells of the dorsal vessel
lacking black nuclei) were determined on projected images by measuring the
distance from the luminal to the apical sides of the cell, and the
measurements converted to actual µm values after measuring a projection of
the calibration slide. Average cell sizes were then calculated for each
cluster of svp-expressing cells along the AP axis in y w
control embryos and in embryos ectopically expressing abd-A. Cell
sizes from at least five animals were measured, and because there are four Svp
cells at each AP location, sample sizes were relatively large
(15n
24).
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RESULTS |
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To identify genes expressed in subpopulations of the dorsal vessel, we
analyzed by in situ hybridization the expression of several muscle structural
gene isoforms. We identified a novel member of the troponin-C superfamily
termed Tina-1 (for Troponin Cakin-1; formerly
CG2803) (Adams et al.,
2000), whose expression in the dorsal vessel was detected at high
levels only in the heart (Fig.
1G,H). Tina-1 was also expressed in a subset of other
cells, including the hindgut visceral mesoderm. The full expression pattern of
this gene will be presented elsewhere (T. P. N., T. L. L. and R. M. C,
unpublished). The identification of Tina-1 as a heart-specific marker
in the dorsal vessel permitted us to follow changes in heart versus aorta fate
at both the morphological and molecular levels.
Expression of Bithorax-Complex genes in the dorsal
vessel
To identify candidate genes involved in the segmentation of the dorsal
vessel, we studied the expression of members of the Bithorax Complex
(BX-C) of homeotic segmentation genes. The functions of these genes
in controlling segmentation in the ectoderm and skeletal muscle precursors has
been well documented (Lawrence and Morata,
1994; Grieg and Akam,
1993
; Michelson,
1994
). BX-C gene expression in the dorsal vessel was
studied by double-staining embryos for the Hox gene product and the mesoderm
marker MEF2, and examining the embryos by confocal microscopy
(Fig. 2). As the dorsal vessel
curves slightly along the dorsal midline of the embryo, it was necessary to
combine several z-series images to illustrate in a single micrograph
the expression pattern of each gene along the entire length of the dorsal
vessel (Fig. 2A-C,E-G,I-K). As
a result, some ectodermal BX-C gene expression is observed in
addition to expression in the mesoderm. To demonstrate that the BX-C
gene products were co-expressed with MEF2 in the muscular cardial cells of the
dorsal vessel, high power images are also shown that represent only one or two
optical sections combined, differing by 2µm
(Fig. 2D,H,L).
|
All three BX-C gene products were detected in the dorsal vessel, albeit in strikingly different patterns. UBX was detected at low levels in dorsal vessel cells from A2 to the posterior tip of the heart, although expression was slightly lower in A5 to A8 (Fig. 2A-D). By contrast, ABDA protein was detected in cardial and pericardial cells in the heart region from A5 to A8 (Fig. 2E-H), and closer examination indicated that abd-A expression corresponded exactly to the cells forming the heart. ABDB was detected in the dorsal vessel in the most posterior four nuclei in A8, in which ABDA accumulation was reduced (Fig. 2I-L). Therefore ABDA seemed most likely to play a role in heart cell specification, and was chosen for further study. The expression of other BX-C genes in the dorsal vessel suggests that further structural and functional diversity also exists in this organ.
Together, abd-A and Tina-1 represent the first two genes known whose expression patterns differentiate between the heart and the aorta cells of the dorsal vessel.
abd-A function controls dorsal vessel cell identity
To determine if abd-A functions as a selector gene in the
Drosophila dorsal vessel to distinguish between heart and aorta cells
we used the GAL4-UAS system (Brand and
Perrimon, 1993) to express abd-A ectopically in different
germ layers, and monitored the formation of the dorsal vessel by studying
expression of Mef2, Hand, Mhc and the heart-specific marker
Tina-1 (Fig. 3).
Initially, we induced abd-A using the ectodermal driver
69B-gal4 (Brand and Perrimon,
1993
), to determine if expression of abd-A in an adjacent
germ layer might affect dorsal vessel identity. These animals did not undergo
dorsal closure to form a linear heart tube
(Fig. 3A-D); however, we were
still able to distinguish between heart and aorta cells. This was apparent
when studying Hand expression, where the posterior cells of the
dorsal vessel had a larger volume than the more rounded cells of the aorta
(Fig. 3B), a characteristic of
heart cells. In addition, MHC accumulated at higher levels in the presumptive
heart region (Fig. 3C).
Furthermore, Tina-1 expression was restricted to the presumptive
heart cells and was not detectable in the aorta
(Fig. 3D). Clearly no
alteration in dorsal vessel cell identity occurred upon ectopic ectodermal
expression of abd-A.
|
By contrast, expression of abd-A in the mesoderm alone using
either the 24B-gal4 driver or a twist-gal4 driver
(Brand and Perrimon, 1993;
Baylies and Bate, 1996
)
resulted in a strong a transformation into heart cell fate for all dorsal
vessel cells (Fig. 3E-L). There
was a greater distance between MEF2-postive cells in the dorsal vessel,
suggesting a large lumen running the length of the dorsal vessel
(Fig. 3E,I). In addition,
visualizing Hand expression and MHC accumulation indicated that most
of the dorsal vessel cells assumed a larger volume characteristic of cells of
the heart (Fig. 3F,G,J,K). Most
striking was the appearance of Tina-1 transcripts throughout the
dorsal vessel, indicating that all the dorsal vessel cells had assumed a heart
fate (Fig. 3H,L).
These results strongly suggested that ABDA plays an important instructive role in the dorsal vessel, directing cells to take on a heart fate. To confirm this, we studied heart formation in mutants lacking abd-A function, as this would be predicted to result in an heart-to-aorta transformation in the posterior region of the dorsal vessel. Many homozygous combinations of abd-A mutants do not complete development sufficiently to answer all of these questions; however, in the absence of abd-A function we saw phenotypes consistent with a loss of heart cell identity (Fig. 4). Despite the lack of dorsal closure, Hand expression persisted in the presumptive dorsal vessel cells; however, we never saw any size dimorphism in these cells as was observed upon ectodermal expression of abd-A (in which the mutant individuals also failed to complete dorsal closure; Fig. 3B). Furthermore, there was no enrichment of MHC in the posterior group of dorsal vessel cells (Fig. 4C,D). Tina-1 expression in the dorsal vessel was undetectable in the absence of abd-A function (Fig. 4E).
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Taken together, the gain- and loss-of function experiments described here identify the homeotic selector gene abd-A as specifying heart cell identity in the Drosophila dorsal vessel.
Formation of ostia in ectopically generated heart
A unique characteristic of the Drosophila heart is the presence of
inflow tracts termed ostia. There are three pairs of ostia located at the
segmental boundaries of A5/A6, A6/A7 and A7/A8, and each ostium is visible in
larvae as a broadening of the width of the heart, at the peak of which are
small openings (Rizki, 1978;
Bodmer and Frasch, 1999
;
Molina and Cripps, 2001
). No
ostia form in the aorta during embryonic or larval development. Recently, we
demonstrated that the ostia which form at each segmental boundary develop from
two pairs of cells expressing the orphan nuclear receptor gene sevenup
(svp). The remaining four pairs of cardial cells in each segment express
the homeobox-containing gene tinman (tin) and form the heart wall
(Molina and Cripps, 2001
).
Close examination of MHC-stained wild-type hearts from embryos indicated that the wall of the heart curved sharply outwards close to the segment borders (bracketed regions in Fig. 5A), whereas no such broadening occurred in the aorta (Fig. 5B). At these locations, two cardial cells were morphologically distinct in that they had oval-shaped nuclei, rather than the round nuclei of the remaining cells. Given the locations of these morphologically distinct cells close to the segmental boundary and the similarity of this structure to the organization of the larval heart, we reasoned that the sharp curves in the outer heart wall corresponded to the locations of the ostia. In support of this, we occasionally saw indentations at the tip of these cell pairs, suggesting that the heart wall was perforated at these locations. To confirm our identification of these cells as ostia, we double-stained wild-type embryos with an antibody to Tin (to identify the heart wall cell nuclei) and with an antibody to muscle MHC (to visualize the shape of the heart). The sharp curves in the heart wall corresponded to the locations of ostia, as they were formed by the non-Tin expressing population of cardial cells (Fig. 5C). In the aorta of wild-type embryos, svp-expressing cells were still detected; however, the wall of the aorta was uniform (Fig. 5D).
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As ectopic mesodermal expression of abd-A resulted in ectopic heart formation, we studied these ectopic heart structures for the presence of cells forming ostia. In many cases, sharp curves in the wall of the heart tube in locations more anterior to those found in wild type indicated the presence of ectopic ostia, formed by cells more elongated than their neighbors (asterisks in Fig. 5E). Furthermore, by staining these embryos with anti-Tin and anti-MHC, as we did for wild type, we found that these elongated cells precisely corresponded to those expressing svp (Fig. 5F). Although it is difficult to visualize the openings of the ostia, the most likely conclusion from these observations is that ectopic ostia were formed in the presence of ectopic ABD-A. Furthermore, these ostia were positioned appropriately within the segment, only at the coincidence of abd-A expression and svp expression.
To quantify more precisely the alteration in Svp cell morphology upon the induction of ectopic heart structures, we determined the size of each svp-expressing cell by measuring the distance from the luminal surface of the Svp cells to the outer wall of the dorsal vessel. In wild-type embryos there are seven segmentally repeating groups of Svp cardial cells in the dorsal vessel, four cells in each group. To distinguish between groups located at unique positions along the AP axis, we refer the groups as S1 to S7, from anterior to posterior in the embryo. Thus, the Svp cells of clusters S1 to S4 do not form ostia in wild type, whereas S5 to S7 form the ostia of the heart.
In control embryos, clusters S1 to S4 contained cells measuring approximately 5 µm, whereas the Svp cells of the heart were significantly larger (7-8 µm; Fig. 6). Upon overexpression of abd-A in the mesoderm there was a large increase in the sizes of cells in groups S1 to S4, many of which were indistinguishable from those in the wild-type heart (Fig. 6). These results clearly show the effects of abd-A expression upon aorta cell fate, transforming Svp cells of the aorta into ostia.
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Ubx function is not required for heart formation
The data above suggest strongly that autonomous action of abd-A in
the dorsal vessel promotes heart cell fate at the expense of aorta cell fate.
A formal alternative possibility is that a reduction in UBX levels could
specify heart fate, as we have also shown that UBX levels are slightly lower
in the heart versus the aorta (Fig.
2). Indeed, ectopic expression of abd-A in the mesoderm
reduced UBX accumulation (Fig.
7A).
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To determine if a loss of Ubx function could induce heart fate in the aorta, we studied dorsal vessel formation in Ubx9.22 homozygotes (Fig. 7B-D). These mutant individuals frequently showed a range of defects in dorsal vessel development, including incomplete dorsal migration of the cardiac precursors, and more minor morphological defects in the dorsal vessel. However, staining for MHC accumulation (Fig. 7B) demonstrated that a clear distinction could still be detected between the heart and aorta in these individuals; the heart showed a larger lumen and cell size compared with more anterior cells in the dorsal vessel, and cells forming ostia could also be distinguished in the heart region only (Fig. 7B, arrow). Consistent with a failure of fate change, Tina-1 expression was also confined to the heart in Ubx mutants (Fig. 7C). Interestingly, there was a slight increase in abd-A expression in cells of the aorta in Ubx mutants (Fig. 7D), however this was clearly insufficient to alter cell fate.
We also studied the effects of high-level mesodermal expression of Ubx upon the formation of the heart, to determine if this gain-of-function assay might inhibit heart cell specification. Upon induction of Ubx expression by the mesodermal driver 24B-gal4, UBX protein was detectable at high levels in the mesoderm (Fig. 7E). There was no inhibition of heart cell specification in segments A5-A8, as visualized by MHC accumulation, as might be predicted if UBX were an inhibitor of heart fate. However, we frequently observed an increase in cell size at the A4/A5 boundary, consistent with the formation of an ectopic ostium within the aorta (Fig. 7F, arrow). Furthermore, Tina-1 expression in the dorsal vessel was also broadened upon ectopic expression of Ubx (Fig. 7G). These effects upon dorsal vessel morphology and fate were not due to alterations in the levels or expression of abd-A, which was still restricted to the heart (Fig. 7H). Taken together, these findings indicate that high levels of Ubx expression are capable of inducing some aspects of heart cell fate in more anterior locations of the dorsal vessel. Owing to a lack of markers for the aorta cell fate, we cannot determine if Ubx loss of function affects any aspects of aorta fate.
Ectopic expression of Abd-B inhibits cardiac and skeletal
myogenesis
Given the restricted expression pattern of Abd-B in the dorsal
vessel, we also studied the effects of ectopic mesodermal expression of this
gene upon cardiac development. However, we were unable to define the role that
this gene might play using these experiments, as forced expression of
Abd-B caused severe defects in muscle development. In addition to
massive derangements of the skeletal muscle pattern, no dorsal vessel cells
were detected based upon MHC accumulation
(Fig. 8A). By visualizing
cardiac precursor cells using an anti- Tin antibody, we found that these
defects in dorsal vessel formation resulted from a loss of
tin-expressing cells commencing around stage 13, such that by stage
16 very few Tin-positive cells were present
(Fig. 8B-D). These results
clearly indicated that inappropriate expression of Abd-B will inhibit
cardiac and skeletal myogenesis. However, as ABDB is detectable in the mature
heart tube at stage 16 (Fig.
2), the presence of ABDB cannot always be inhibitory to dorsal
vessel formation. Defining a more clear role for Abd-B in the dorsal
vessel must await the use of GAL4 lines with more restricted temporal or
spatial patterns of expression.
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DISCUSSION |
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Does the mechanism of AP heart patterning that we have uncovered in
Drosophila apply to higher animals? The vertebrate heart initially
forms as a linear tube in much the same manner as the Drosophila
heart, and numerous genes are known to be expressed in unique domains along
the AP axis in the developing vertebrate heart
(Srivastava and Olson, 2000).
However, there is much to learn concerning the factors that determine this AP
pattern. Treatment of chick and zebrafish embryos with retinoic acid results
in a loss of anterior heart structures and a broadening of the domain forming
more posterior structures (Stainier and
Fishman, 1992
; Yutzey et al.,
1994
; Yutzey et al.,
1995
), suggesting that retinoic acid can influence the AP
patterning of the heart. Retinoic acid also directly activates a number of Hox
genes in the trunk of the embryo (reviewed by
Krumlauf, 1994
). Taking these
findings together, it is tempting to speculate that Hox segmentation genes in
vertebrates also function to control cell identity in the heart. In support of
this are recent demonstrations of Hox gene expression in the developing heart
(Searcy and Yutzey, 1998
;
Shin et al., 1998
) and the
finding that treatment of cardiogenic explants with retinoic acid can alter
the expression of Hox genes (Searcy and
Yutzey, 1998
).
Our results also indicate that two distinct patterns of gene expression converge to control the differentiation of the Drosophila dorsal vessel. Superimposed upon the expression of abd-A in the heart segments, is the pattern of tin-expressing versus svp-expressing cells observed in cardial cells in every segment. Formation of the ostia in the heart occurs only at the intersection of abd-A expression and svp expression, and ectopic ostia form in the presence of ectopic ABD-A, but only in svp-expressing populations of cells.
Whether svp function is required for ostium formation in
Drosophila remains to be determined. A vertebrate homolog of the Svp
protein is chick ovalbumin upstream promoter transcription factor II (COUP-TF
II) (Tsai and Tsai, 1997),
which in mice is expressed in and is required for the formation of the atria
and sinus venosus (Pereira et al.,
1999
). The atria and sinus venosus carry out functions in the
mouse analogous to the ostia in Drosophila, acting as the inflow
tracts for blood to enter the heart. It will be interesting to determine
whether the homologous expression patterns of svp and COUP-TF
II reflect an homologous function in development.
It is interesting that Ubx and Abd-B are also expressed
in unique cells in the dorsal vessel. Although our loss-of-function
experiments have not demonstrated a role for Ubx in the formation of
the heart, it is still possible that Ubx plays a role in the
specification of more anterior structures in the dorsal vessel. There are a
number of cardial cells in an anterior location that do not express
Ubx, suggesting an as-yet undetermined function for Ubx in
the dorsal vessel. Along these lines, it is interesting to note that the
domain of Ubx expression in the aorta roughly corresponds to the
region of the dorsal vessel remodeled during pupal development to form the
adult heart (Molina and Cripps,
2001). Furthermore, Ubx is required to repress lymph
gland fate in the pericardial cells adjacent to the dorsal vessel
(Mastick et al., 1995
;
Rodriguez et al., 1996
),
suggesting a broad requirement for members of the BX-C in patterning
the dorsal vessel and its associated cells.
We have also shown that expression of Ubx throughout the mesoderm is capable of inducing a partial heart fate upon the cells of the aorta. This was apparent with the observation of single pairs of ectopic ostia, as well as an expansion of Tina-1 expression throughout the dorsal vessel. Given the intensity of UBX accumulation in these embryos (compare Fig. 7E with Fig. 2A), it is likely that this function of UBX occurs at higher levels of expression than are normally found in the embryonic dorsal vessel. Furthermore, even at these high levels, the transformation to heart fate is only partial, compared with the apparently complete transformation effected by abd-A expression.
The similarity in function between UBX and ABDA illuminated in our
gain-of-function experiments is not unusual given previous studies of these
genes. Either gene can promote haltere formation
(Casares et al., 1996), and
both Ubx and abd-A expression have similar effects upon the
patterning of somatic muscle precursors
(Michelson, 1994
). There are
also examples where Ubx and abd-A influence different target
genes (e.g. Brodu et al.,
2002
). It is therefore likely that the induction of a
fully-functional heart in the dorsal vessel results from the activation of
targets both specific to abd-A and common to abd-A and
Ubx. A major challenge in the future will be to identify the various
targets of both the HOX proteins and cardiac-restricted transcription factors
which realize heart fate.
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ACKNOWLEDGMENTS |
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