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First published online 13 February 2008
doi: 10.1242/dev.010694
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Department of Biochemistry and Biophysics, Programs in Developmental Biology, Genetics and Human Genetics, Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94158, USA.
* Author for correspondence (e-mail: dstainier{at}biochem.ucsf.edu)
Accepted 8 January 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cardiovascular development, Cox2 (Ptgs2), Microscopy, Prostaglandins, SPIM, Zebrafish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Olson, 2006). A defect at any
of these transitions leads to swift embryonic death in amniotes, while minor
congenital heart defects can lead to severe problems later in life. Thus, the
cardiac developmental program is crucial for heart function and embryonic
survival. Recent studies in zebrafish have revealed the flip side of this
relationship: heart function is also required for cardiac morphogenesis
(Hove et al., 2003;
Bartman et al., 2004;
Auman et al., 2007). For
example, altering myocardial function can change myocardial cell shape
(Auman et al., 2007).
The complex morphogenesis of the atrioventricular valve (AVV) is at the
center of this relationship between form and function. During the linear heart
tube phase, the heart may function as a suction pump
(Forouhar et al., 2006) and
does not require valves. As the heart loops, it becomes less efficient at
preventing retrograde flow, causing the need for valves
(Liebling et al., 2006). In
mice and chicks, the extracellular matrix, or cardiac jelly, of the
atrioventricular canal (AVC) swells, and its composition changes as the heart
develops (reviewed by Armstrong and
Bischoff, 2004). The atrioventricular endocardial cells undergo an
epithelial-to-mesenchymal transition (EMT), migrate into the cardiac jelly and
proliferate to form the endocardial cushions. These cushions are remodeled
into mature valve leaflets. This process is controlled by many signaling
pathways, including Notch (Timmerman et
al., 2004), ErbB (Camenisch et
al., 2002; Iwamoto et al.,
2003), Bmp (Ma et al.,
2005; Rivera-Feliciano and
Tabin, 2006), TGFβ (Potts
and Runyan, 1989), Wnt
(Hurlstone et al., 2003) and
NFAT (Chang et al., 2004).
Mutations in genes encoding components of these pathways cause valve defects
and retrograde blood flow from ventricle to atrium, which disrupts the
unidirectional flow of blood, reduces cardiac output and leads to death. The
specific roles these different pathways play in valve morphogenesis are
largely unknown.
The zebrafish AVV has been reported to form in a similar fashion to that of
higher vertebrates. By 48 hours postfertilization (hpf), the AVC is defined
molecularly by expression of versican, bmp4 and notch1b
(Walsh and Stainier, 2001),
and morphologically by the transformation of AVC endocardial cells from a
squamous to a cuboidal morphology (Beis et
al., 2005). Endocardial cells from the ventricular side of the AVC
extend processes, migrate into the space between the endocardium and
myocardium at 60 hpf, and form what appears to be an endocardial cushion in
both the superior and inferior AVC by 96 hpf
(Beis et al., 2005). These
endocardial cushions are remodeled into the adult valve leaflets. This process
requires Notch (Beis et al.,
2005; Timmerman et al.,
2004), NFAT (Chang et al.,
2004), Wnt (Hurlstone et al.,
2003) and ErbB (Goishi et al.,
2003) signaling, as in the mouse. Zebrafish embryos are suitable
for investigating this process because they are transparent, amenable to
genetic manipulation, can survive with severe heart defects until late stages
of development, getting oxygen by diffusion, and can be easily treated with
chemical inhibitors of signaling pathways
(Glickman and Yelon,
2002).
In this study, we used selective plane illumination microscopy (SPIM) to
investigate the changing morphology and function of the developing valve in
zebrafish embryos and larvae. SPIM allows high-speed fluorescence imaging and
optical sectioning with deep penetration at cellular resolution
(Huisken et al., 2004). This
technology allowed us to visualize changes in valve morphology over
developmental time and in various experimental conditions at cellular
resolution. We first showed that the zebrafish AVV does not form through an
intermediate stage of mesenchymal endocardial cushions as previously reported,
but directly forms leaflets by a process of invagination. We next analyzed the
changing function of the valve through development. The efficiency of the
valve, as measured by the number of blood cells flowing backwards through the
AVC during each valve closure, increases over developmental time. We also
disrupted individual aspects of valve function with inhibitors of ErbB
receptors, TGFβ receptors or Cyclooxygenase 2 [Cox2; also known as
Prostaglandin-endoperoxide synthase 2 (Ptgs2)]. These experiments revealed a
novel role for Cox2 in determining the geometry of the valve. Specifically,
Cox2 plays a role in regulating myocardial cell shape upstream of
prostaglandin F2
(PGF2) and thromboxane A2 (TXA2).
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We used
wild-type AB and the transgenic lines
Tg(flk1:EGFP)s843
(Beis et al., 2005),
Tg(gata1:dsRed)sd2
(Traver et al., 2003) and
Tg(myl7:HRAS-EGFP)s883
(D'Amico et al., 2007).
Pharmacological treatment
Embryos were dechorionated and treated with a 4 mM stock of AG1478
(Calbiochem) diluted to 4 µM, a 3 mM stock of SB431542 (Sigma) diluted to 3
µM, a 5 mM stock of AL8810 (Cayman) diluted to 5 µM, and 10 mM stocks
diluted to final concentrations of 25 µM for CAY10404, 40 µM for NS398,
100 µM for furegrelate, 10 µM for SQ29548, 10 µM for PGF2, 15
µM for indomethacin and 10 µM for U46619 (Cayman), or 1% DMSO. Embryos
were raised at 28°C until imaging or harvesting.
Microinjection
Embryos were injected at the 1-2-cell stage with 2-4 ng thromboxane
synthase MO (5'-AGCTGCATGATGGGATCTGTCAATC-3'), then raised at
28°C until harvesting.
Selective plane illumination microscopy
Animals were embedded in 1% low melting agarose in plastic syringes and
imaged in SPIM as described previously
(Huisken et al., 2004) using a
20x/0.5 NA water dipping objective. Fluorescence was excited in the
focal plane with 488 nm and 561 nm laser beams. Images were recorded on two
synchronously triggered cameras (Andor). The frame rate ranged from 70 to 160
frames per second (fps) depending on the size of the region of interest. The
two channels were merged using Matlab and saved as avi movies with a frame
rate of 15 fps.
Confocal microscopy
Animals were embedded in 4% low melt agarose and cut into 200 µm
sections with a Leica VT1000S vibratome. Where indicated, sections were
incubated overnight with 1:75 rhodamine-phalloidin (Molecular Probes). Images
were acquired with a Zeiss LSM5 Pascal confocal microscope.
Immunohistochemistry
We used mouse monoclonal antibodies zn8 (Zebrafish Stock Center and
Hybridoma Bank) at 1:10, as previously described
(Beis et al., 2005).
RT-PCR
At 48 hpf hearts were purified as previously described
(Burns and MacRae, 2006) and
total RNA was isolated with Trizol (Invitrogen). The following primers were
used to amplify each gene: 5'-ATCTGAAACCCTACACATCCTTCGC-3' and
5'-AGACGTTTTGCTAAAGTTCGCCGTG-3' for cox1,
5'-TACTCATCCTTTGAGGAGATGACAG-3' and
5'-GACCTTTTACAGCTCTGAACTCCGC-3' for cox2a,
5'-TTTCACAACAGCCCTGAACC-3' and
5'-GTTGAAGGACTCAACCAAGC-3' for cox2b
(Ishikawa et al., 2007) and
5'-GCATTTTGATGTGGTCAACG-3' and
5'-ACTTGGTGGGTTCAGTCCAG-3' for tbxas. Thirty cycles of
PCR were performed.
Cell shape analysis
Image stacks of ca. 70 planes of hearts were recorded on a Zeiss LSM 5
Pascal. Images and meta-information were imported from lsm-files into Matlab
(Mathworks) using tiffread2c.m (F. Nedelec) and lsminfo.m (P. Li). Each stack
was rotated in steps of 15 degrees about the y- and the
z-axis. For each angle the sum projection along z was
calculated for the front half of the stack and saved into a tif file. A
dedicated Matlab program was used to display the projections, outline cells,
annotate them and save all information. In each projection all cells with a
basal surface that appeared orthogonal to the direction of projection were
analyzed. An adaptive contour algorithm was used to precisely follow the
labeled membrane. All relevant information, including cell number, region,
area A, perimeter P and pixel size was saved to a text file. The circularity C
was calculated C=P^2/(4
A). The information in this file was processed and
the statistical analysis performed in Microsoft Excel.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To our surprise, SPIM imaging showed that by 76 hpf,
what looked by confocal imaging of fixed tissue to be the superior endocardial
cushion (Fig. 1A) was actually
a valve leaflet (Fig. 1B and
see Movie 1 in the supplementary material). This leaflet was clearly visible
at 85 hpf (Fig. 1C,D and see
Movie 2 in the supplementary material), and was composed of two layers: an
outer layer of cuboidal cells and an inner layer of larger and rounder cells.
The collapse of the heart upon fixation made it possible to misconstrue the
leaflet as an endocardial cushion in previous studies. Only high-speed imaging
of live samples allows one to see this dynamic structure. These data suggest
that the zebrafish endocardium does not form mesenchymal cushions, but instead
remains a single sheet of cells as it invaginates to directly form the AVV
leaflet. To ensure that we were not missing a stage of mesenchymal cells that
quickly resolved into a leaflet, we looked at stages between 56 and 72 hpf. At
no stage did we observe mesenchymal cells in the AVC. For example, both
confocal imaging at 62 hpf (Fig.
1E) and SPIM imaging at 63 hpf showed a sheet of connected cells
(Fig. 1F and see Movie 3 in the
supplementary material). Thus, zebrafish seem to have evolved a novel
mechanism to form the AVV.
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). The
latter transgene marks blood cells, allowing visualization and quantification
of blood flow through the AVC. At 55 hpf, before any cells had invaginated,
the valve was not very efficient in preventing retrograde blood flow. The
superior and inferior AVC were brought together mechanically by the action of
myocardial contractions (Fig.
2A and see Movie 4 in the supplementary material). They then
rolled together in an atrial-to-ventricular direction, almost touching one
another (Fig. 2B). This roll
prevented most retrograde blood flow, but the endocardium was not thick enough
to fully occlude the AVC lumen, so some blood cells slipped back into the
atrium. After the roll was complete, the AVC relaxed and opened, allowing many
blood cells to flow backwards (Fig.
2C). Altogether, approximately ten cells flowed retrogradely from
the time the superior and inferior AVC were brought together to the time the
atrium contracted again (Fig.
2L, based on three embryos and 61 heartbeats). By 72 hpf, an average of three cells had invaginated at the deepest location. At this time point, there were not enough invaginated cells to hang down as a leaflet. Instead, they acted as a compressible mass of cells to occlude the AVC, perhaps similarly to the endocardial cushion of higher vertebrates. The AVC was still closed by the myocardial contractions: they brought together the two sides of the AVC (Fig. 2D and see Movie 5 in the supplementary material), which then rolled atrially to ventricularly (Fig. 2E). The valve structure was thick enough at this time to fully occlude the lumen through the roll. The invaginated cells were compressed as they were forced against the inferior wall of the AVC. Whereas at 55 hpf the AVC relaxed and opened at the end of the roll, at 72 hpf the valve structure expanded to occlude the AVC lumen (Fig. 2F). This compression and expansion can be quantified by measuring the width of the superior valve structure at its maximum compression during the roll and again at its maximum expansion during the relaxation (Fig. 2K, for examples of the width measured see white bars in B,C,E,F). At 55 hpf, there was no significant difference between the widths of the endocardium at these two time points (based on three embryos and 29 beats). By contrast, at 72 hpf, there was a significant difference between the width of the compressed and expanded valve structure (P<1.62E-25, based on three larvae and 30 beats). The thick, compressed tissue that blocked the lumen during the roll and the expanded tissue that occluded it during the relaxation phase combined to make the valve significantly more efficient at this stage than at 55 hpf, as measured by the number of blood cells undergoing retrograde flow: approximately 0.3 cells per beat at 72 hpf (Fig. 2L, P<2.03E-15, based on six larvae and 142 beats).
By 76 hpf, a valve leaflet was apparent in the superior AV endocardium (Fig. 1B), but its role in valve function could be most clearly seen at 96 hpf. In these samples, the sides of the AVC were still brought together by the myocardial contractions (Fig. 2G and see Movie 6 in the supplementary material). Even though a leaflet was present, the valve was not closed by hemodynamic forces. There was still the atrial-to-ventricular roll, with the thickness of the leaflet occluding the canal (Fig. 2H). The difference of this stage from 72 hpf was at the relaxation phase: instead of the tissue expanding to block the lumen of the AVC, the leaflet hung down to prevent retrograde blood flow (Fig. 2I). At this stage, the valve appeared to be 100% efficient, as we never observed any retrograde flow of blood cells (based on four samples and 113 heartbeats).
By 102 hpf, the valve was similar to the adult valve, in that both a superior and an inferior valve leaflet were present (Fig. 2J and see Movie 7 in the supplementary material). However, the AVC was still closed by the myocardial contractions instead of hemodynamic forces, and the rolling persisted. Both leaflets hung down across the AVC lumen to prevent retrograde flow.
Disrupting valve function
In this analysis, four main aspects of valve function stand out. First, the
AVC closed by myocardial action. Next, the AVC rolled through the beat. Third,
the valve tissue compressed and expanded to occlude the lumen during the
relaxation phase. And finally, the valve structure increased in thickness to
occlude the lumen during the roll. We tested the importance of these aspects
for valve function and efficiency. Unfortunately, the first aspect could not
be disrupted because it would lead to a complete loss of heart function.
However, we identified chemical inhibitors that affected AVC rolling, valve
compression and the increasing thickness of the endocardial tissue at the
AVC.
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;
Iwamoto et al., 2003), as well
as in valve function (Goishi et al.,
2003) and AVC specification
(Milan et al., 2006) in
zebrafish. Because of the modified morphogenesis in zebrafish valve
development, we wanted to investigate the role this signaling pathway might
play in the process of invagination, and we used the ErbB receptor inhibitor
AG-1478 (Goishi et al., 2003).
We began treating embryos at 56 hpf, just before the invagination began, and
imaged them with SPIM at 72 hpf. In the treated animals, the sides of the AVC
were still brought together by the myocardial contractions, but because of a
changed geometry of the invaginating tissue the AVC rolled in the reverse
direction, i.e. ventricularly to atrially
(Fig. 3A,B and see Movie 8 in
the supplementary material). This altered roll resulted in the AVC lumen
opening during the relaxation phase, allowing severe retrograde blood flow
(Fig. 3C). The valve was
significantly less efficient than wild type at the same stage, allowing the
retrograde flow of approximately 8.6 cells per beat
(Fig. 3F,
P<2.52E-11, based on three larvae and 42 heartbeats). The subtly
altered morphology of the valve was also apparent in confocal images of fixed
tissues (Fig. 3E). The AVC
endocardial cells did not fully invaginate and were separated from the cells
on the outer surface of the leaflet by a space. These data suggest that the
atrial-to-ventricular rolling is required for efficient valve function and
that this mechanism plays a role in ensuring that the invaginated cells are in
place for the relaxation phase.
We next studied inhibitors that block the compression and expansion of the
valve. TGFβ signaling plays a role in endocardial EMT in amniotes
(Potts and Runyan, 1989). As
zebrafish valves form by a different mechanism, we wanted to investigate the
potential role of TGFβ signaling in this process. Treating embryos with
SB431542, an inhibitor of Alk4 (Acvr1b), 5 (Tgfbr1) and 7, from 56-72 hpf did
not block endocardial invagination. However, in treated animals the
invaginating cells retained a cuboidal appearance at 72 and 96 hpf in contrast
to the large round cells seen in control larvae
(Fig. 4A-D). The invaginating
cells also failed to downregulate Alcam, an adhesion molecule expressed in the
cuboidal cells on the outer surface of the leaflets
(Fig. 4E,F)
(Beis et al., 2005). Because
these cells remained cuboidal, they appeared unable to compress and expand
(see Movie 9 in the supplementary material). The thickness of the valve
structure in treated larvae remained constant during all phases of the beat
(Fig. 4G, based on three
samples and 42 heartbeats). The valve in treated larvae had significantly
decreased efficiency compared with control larvae at this stage, allowing the
retrograde flow of approximately 1.9 cells per beat
(Fig. 3F,
P<2.1E-27, based on three samples and 124 heartbeats). Thus, it
appears that compression and expansion of the valve tissue is important for
valve function.
Next, we looked at the role of the increased thickness of the valve tissue during the rolling of the AVC. During a screen for chemical compounds that cause retrograde blood flow, we identified a number of Cox2 inhibitors. Treatment with two Cox2 inhibitors, NS-398 and CAY10404, from 56-72 hpf caused the invaginating structure to be shifted ventricularly (Fig. 5A-C and see Movie 10 in the supplementary material; data not shown). When the AVC closed and rolled, it was actually closing and rolling at a point where there was only a monolayer of endocardial cells (Fig. 5A,B). The valve structure only became involved at the end of the roll, the relaxation phase (Fig. 5C). Thus, for the initial part of the valve closure, the valve resembled the 55 hpf AVC, with no thick superior AV endocardium. As with the 55 hpf AVC, the endocardium in the treated larvae was unable to fully occlude the lumen, so many cells moved retrogradely, approximately three per beat, and the valve was significantly less efficient than control (Fig. 3F, P<1.62E-14, based on three larvae and 30 heartbeats). These data suggest that the thickening of the endocardium is important for valve function during the rolling phase.
Cox2 and myocardial cell shape
Why do Cox2 inhibitors cause invaginating cells to be translated towards
the ventricle? When we looked at the myocardium using a myocardially expressed
membrane-bound GFP in the
Tg(myl7:HRAS-EGFP)s883 line
(D'Amico et al., 2007), we
found that the myocardium above the superior AVC bent inwards towards the
ventricle in larvae treated with either of two Cox2 inhibitors, NS-398 or
CAY10404 (Fig. 5E and data not
shown). As the invaginating cells in these larvae were at the tip of this
myocardium, they also moved towards the ventricle.
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|
) recently
showed that myocardial cells are larger and more elongated in the outer
curvature of the ventricle than in the inner one. If cell shapes changed, then
the shape of the whole ventricle might change. Analysis of 3D projections of
hearts treated with Cox2 inhibitors suggested that cells in the posterior
outer curvature were smaller and rounder than in control hearts
(Fig. 5F,G). We measured
myocardial cell shape using dedicated software that creates a 3D
reconstruction of the heart based on confocal images and allows segmentation
of myocardial cells and quantification of area and circularity (B. Jungblut,
C. Munson, J.H., L. A. Trinh and D.Y.R.S., unpublished). Because there is an
intrinsic difference between the outer and inner curvature of the ventricle,
we analyzed these two areas separately. As we observed changes in the anterior
inner curvature (the ventricle-wards bend) and the posterior outer curvature,
we analyzed the anterior and posterior heart separately as well, giving four
separate zones for analysis (Fig.
5H). Significant differences were observed only in the posterior
outer curvature, hence we focus our discussion on this area
(Fig. 5H, asterisk). In animals
treated with Cox2 inhibitors, there was a significant decrease in the cell
surface area (P<0.0014) as well as a significant increase in the
cell circularity (P<4.75E-5)
(Fig. 5I,J, based on four
samples and 48 cells for wild-type measurements and five samples and 81 cells
for CAY10404-treated measurements). Thus, inhibition of Cox2 caused the cells
of the posterior outer curvature of the ventricle to become smaller and
rounder.
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), rather than cell shape changes causing the bending. To
determine which defect arises first, we performed timecourses of Cox2
inhibitor treatment. We found that myocardial bending and cell shape changes
both arose 4 hours after initial treatment, either from 56-60 hpf
(Fig. 5M,N) or 68-72 hpf (data
not shown). We did not find one phenotype without the other. Although it is
possible that changes in flow may swiftly lead to changes in myocardial shape,
the close temporal relationship between the two defects supports a mechanism
by which cell shape changes deform the ventricle, leading to the myocardial
bending.
The balance of PGF2 and TXA2 signaling regulates myocardial cell shape
Cyclooxygenases transform the lipid arachidonic acid into prostaglandin H2
(PGH2) (see Fig. S1 in the supplementary material) (reviewed by
Hata and Breyer, 2004;
Cha et al., 2006b). PGH2 is
the precursor used by prostanoid synthases to generate the five major
prostaglandins produced in the embryonic zebrafish: prostaglandin E2 (PGE2),
prostaglandin D2 (PGD2), PGF2, prostacyclin and TXA2
(Cha et al., 2005;
Yeh and Wang, 2006). These
ligands bind to cell surface receptors and regulate a number of physiological
and developmental processes, including, in zebrafish, gastrulation movements,
vasculogenesis, renal patterning and hematopoietic stem cell homeostasis
(Cha et al., 2005;
Cha et al., 2006a;
North et al., 2007). To
identify the downstream effectors of Cox2 in the heart, we overexpressed or
inhibited each of the downstream pathways. Manipulation of PGE2, PGD2 or
prostacyclin signaling had no effect on heart function or myocardial bending
and failed to rescue the Cox2 inhibition defect (data not shown). By contrast,
inhibition of TXA2 synthase (Tbxas; also known as Tbxas1 - ZFIN) with
furegrelate, or inhibition of the TXA2 (TP) receptor with SQ29548 caused the
same bending of the AVC myocardium into the ventricle as in larvae treated
with Cox2 inhibitors (Fig. 5A
and data not shown). Similarly, the cells of the posterior outer curvature of
the ventricle in SQ29548 treated larvae were significantly smaller
(P<0.004) and rounder (P<0.000725) than in control
larvae (Fig. 5I,J,
Fig. 6B based on five samples
and 59 cells).
To further test the role of Tbxas in this process, we used translation-blocking morpholinos (MO). Injection of 2 ng of MO led to pericardial edema at 48 and 72 hpf, with generally no other body defects (see Fig. S2 in the supplementary material; 114/140 (81%) from three injections). Injection of 4 ng caused pericardial edema and curving of the tail. Confocal microscopy revealed that the injected embryos exhibited the same myocardial cell shape changes (Fig. 6C,D) as those in which Tbxas, TP receptor or Cox2 had been inhibited. RT-PCR analysis revealed the expression of tbxas, cox1 (ptgs1), cox2a and cox2b in isolated embryonic hearts (Fig. 6E).
Treatment with PGF2 beginning at 56 hpf had the same effect as
inhibition of Cox2 or TXA2 signaling. The myocardium overlying the superior
AVC bent towards the ventricle (Fig.
6F), and the cells of the posterior outer curvature of the
ventricle were smaller (P<0.0043) and rounder
(P<2.79E-5) than in control larvae
(Fig. 5I,J,
Fig. 6G, based on three samples
and 31 cells). These results suggest that PGF2 or TXA2 signaling may be
perturbed when Cox2 is inhibited. Studies in mammals have shown an involvement
of PGF2 and TXA2 in myocardial hypertrophy
(Adams et al., 1996;
Lai et al., 1996;
Ponicke et al., 2000;
Jovanovic et al., 2006); thus
they are good candidates for pathways that affect myocardial cell shape.
If overactivation of PGF2 signaling or loss of TXA2 signaling is the
result of Cox2 inhibition on valve formation, then the Cox2 inhibition
phenotype should be rescued by treating with an inhibitor of PGF2
signaling or an activator of TXA2 signaling. Indeed, when we treated embryos
with a Cox2 inhibitor and a PGF2 (FP) receptor inhibitor, the
myocardium overlying the superior AVC did not bend and the cells of the
posterior outer curvature of the ventricle were significantly larger than in
larvae treated with a Cox2 inhibitor alone (P<6.3E-7), but not
significantly different from control (Fig.
6H,I,L, based on three samples and 41 cells). The circularity of
the myocardial cells in the posterior outer curvature of the ventricle in
these treated larvae was intermediate between that of Cox2 inhibitor-treated
and control larvae, but these differences were not significant
(Fig. 6M). Thus it appears that
inhibiting PGF2 signaling can rescue all the phenotypes due to Cox2
inhibition, except the increased circularity of myocardial cells.
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These data suggest that the balance between PGF2 and TXA2 signaling
regulates myocardial cell shape in the developing heart. In other systems of
prostanoid signaling, the balance between different pathways is crucial. For
example, prostacyclin causes vasodilation and inhibits platelet clotting
whereas TXA2 causes vasoconstriction and promotes platelet clotting
(McAdam et al., 1999;
Cheng et al., 2002;
Egan et al., 2004;
Fitzgerald, 2004). If
prostacyclin signaling is lost, then excess TXA2 signaling causes thrombotic
events. If such a balance is important in regulating myocardial cell shape,
then inhibiting the FP receptor should rescue the TP receptor inhibition
phenotype. Indeed, larvae treated with both inhibitors did not have a bent
myocardium and the cells were significantly larger than in larvae treated with
the TP receptor inhibitor alone (Fig.
7A,B,E, P<1.65E-5, based on four larvae and 38 cells).
Again, the circularity of the cells was not rescued, as the double-treated
larvae had cells that were significantly more circular than controls
(Fig. 6F,
P<0.02).
Thus it seems that the PGF2 and TXA2 pathways at least partially
antagonize each other with regard to myocardial cell size. It is clear how
Cox2 might be required for TXA2 signaling by providing the PGH2 precursor in
cells producing TXA2 (see Fig. S1 in the supplementary material). Previous
studies in adult rodent hearts show that Cox2 activity can give rise to TXA2
production (Grandel et al.,
2000; Zhang et al.,
2003). The question remains how inhibiting Cox2 might produce an
excess of PGF2 signaling. One possibility is that Cox1 alone can
provide the precursor for PGF2 production, whereas Cox2 provides the
precursor for TXA2 production (Fig.
7G). Inhibition of Cox2 would remove TXA2, but Cox1 would continue
producing PGH2 for PGF2. Therefore the relative balance of the two
would be skewed in favor of PGF2 signaling. One can test this
hypothesis by inhibiting both Cox1 and Cox2. If this hypothesis is correct,
then treated larvae should not show the Cox2 phenotype but have a phenotype
akin to that of the larvae treated with both the TP and FP receptor
inhibitors. We treated embryos with indomethacin, which inhibits both Cox1 and
Cox2. As predicted, the larvae were phenotypically similar to the TP- and
FP-receptor-inhibited larvae. The myocardium did not bend towards the
ventricle, and the cells were significantly larger than in Cox2-inhibited
larvae (P<0.00033), but not significantly different in area from
controls (Fig. 7C-E, based on
four larvae and 45 cells). These cells were significantly more circular than
controls (Fig. 6F,
P<0.04). Thus, inhibiting Cox1 partially rescues Cox2 inhibition
in a way similar to FP receptor inhibition. This finding suggests that Cox1
activity may be responsible for the continued PGF2 signaling in
Cox2-inhibited animals.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Based on
our results, TGFβ signaling seems to regulate these processes. The
development of antibodies and new transgenic lines that allow the examination
of subcellular structures during invagination will be required to determine
how closely this process resembles EMT.
Why is this process so different from that in mouse and chick? One possible
explanation is that this invagination is the more basal mechanism for valve
development, with large mesenchymal cushions arising in tetrapods due to the
demands of the more complicated, septated hearts of higher vertebrates.
However, this explanation does not seem to hold true, as histological analyses
of the hearts of both the dogfish (Gallego
et al., 1997), a chondrichthyan, and the sturgeon
(Icardo et al., 2004), a more
basal fish than zebrafish, show mesenchymal cushions, suggesting that the
mechanism of forming mesenchymal cushions by EMT is more ancient than simple
invagination. An alternative explanation is that invagination is a novel
mechanism that evolved at some point in the zebrafish lineage since its
divergence from sturgeon. Perhaps because of the small size of its embryonic
heart, zebrafish bypassed a complicated and apparently unnecessary step of
valve development.
Interestingly, the invagination process still requires the signaling pathways that are necessary for the development of endocardial cushions in mice and chicks. Notch, NFAT, ErbB and TGFβ signaling are all required in zebrafish valve development. They have been harnessed for a seemingly different process. Our understanding of the role of these pathways at the level of the individual cell is relatively poor because of the lack of readouts in higher vertebrates. The phenotypes of EMT mouse mutants are basically bimodal: either EMT occurs or it does not. In vitro explants allow a greater degree of discrimination, but it is difficult to know how closely in vitro systems parallel the in vivo situation. Although the overall process of valve formation is different in zebrafish and amniotes, at the level of the single cell a signaling pathway may be functioning in similar ways. Thus, investigations into how these pathways regulate zebrafish valve development may shed light on their specific function in higher vertebrates. Importantly, though, these results suggest the need for care in making comparisons among the species and the need to look more carefully into the exact effects of experimental manipulations.
In this study, we have also analyzed how valve function changes throughout the development of the AVV leaflets. Function improves with the changing morphology, but some aspects remain the same. For example, at all stages examined, the two sides of the AVC were brought together mechanically by the action of myocardial contraction. Again, at all stages, the AVC rolled together in an atrial-to-ventricular direction, and if this movement was disrupted, retrograde blood flow resulted.
Our results suggest that the most important changes are the two ways in
which the developing leaflets function to better occlude the lumen and prevent
retrograde blood flow. First, the growing thickness of the developing leaflet
alone helped to block off the AVC during the rolling motion. Second, the
developing leaflet blocked the lumen after the roll was over and the valve
relaxed. At earlier stages it achieved this task simply by expanding. This
function of the forming leaflet may be similar to that of the mesenchymal
cushions in other vertebrates. Although structurally distinct (mesenchyme with
an overlying endothelium versus endothelium elaborated into the beginnings of
a leaflet), at a functional level they are similar, a compressible mass of
cells. At later stages, the leaflet hung down to block the AVC lumen. Again,
valve function at this stage could be similar to that in higher vertebrates
when the endocardial cushions remodel into valve leaflets. These observations
support the results of Liebling et al.
(Liebling et al., 2006), who
observed that after the initial inefficiency of the looped heart, the heart
becomes more efficient, and prevents all retrograde flow by 96-111 hpf.
In our attempts to alter valve function, we made another significant
finding: the importance of Cox2-dependent myocardial cell shape changes for
AVV function. Our results suggest that Cox2 is required to control the balance
between PGF2 and TXA2 signaling in the heart. This balance ensures the
elongation of myocardial cells in the posterior outer curvature of the
ventricle. Studies in mice have shown a function for these pathways in
myocardial hypertrophy. It will be important to study whether there is any
resemblance between the embryonic and adult mechanism of prostanoid-dependent
cell shape changes, as well as the effect of these inhibitors on adult
hearts.
Together, these disruptions of valve morphogenesis indicate the complex nature of heart development, a process that can only be fully evaluated by careful high-speed SPIM analyses. The results herein illustrate how subtle alterations in cell shape and movement caused by inhibition of distinct signaling pathways can lead to severe disruptions in heart function.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/6/1179/DC1
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ACKNOWLEDGMENTS |
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