1 Department of Biochemistry and Biophysics and Cardiovascular Research
Institute, University of California, San Francisco, San Francisco, CA 94143,
USA
2 Department of Physiology, University of California, San Francisco, San
Francisco, CA 94143, USA
3 Zebrafish Neurogenetics Group, IDG, GSF-National Research Center for
Environment and Health, Ingolstaedter Landstrasse 1, 85764 Neuherberg,
Germany
4 Department of Molecular, Cellular, and Developmental Biology, University of
California Los Angeles, CA 90095, USA
¶ Author for correspondence (didier_stainier{at}biochem.ucsf.edu)
Accepted 7 July 2005
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SUMMARY |
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Key words: Heart, AV canal, Endocardium, Notch, Calcineurin, Zebrafish
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Introduction |
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The mammalian heart has four chambers: two atria, separated by an
interatrial septum; and two ventricles separated by an interventricular
septum. At the AV junction lie two valves: the tricuspid valve, with three
leaflets separating the right atrium and ventricle; and a bicuspid mitral
valve separating the left-sided chambers. These cardiac valves derive from
endocardial cushions (ECs), transient structures that form from the
cellularization and expansion of the extracellular matrix (ECM) between the
endocardium and myocardium at the AV canal. In an initial step of cushion
formation, endocardial cells at the AV canal undergo an epithelial to
mesenchymal transformation (EMT), delaminate and invade the ECM. Studies on
chick and mouse cushion development, which were aimed at identifying molecules
regulating EMT, have predominantly used an in vitro system of explanted
cushion tissue on a type I collagen gel
(Bernanke and Markwald, 1982).
Using this system, Krug et al. (Krug et
al., 1987
) showed that myocardial cells from the AV boundary, but
not those from the ventricle, induce endocardial cells to undergo EMT.
Likewise, only endocardial cells from the AV canal, but not those from the
ventricle, can undergo EMT in response to a myocardial signal
(Runyan and Markwald, 1983
).
Therefore, the endocardial and myocardial cells at the AV canal appear to have
unique properties in comparison to other myocardial cells (reviewed by
Eisenberg and Markwald,
1995
).
One key pathway in the interactions between myocardial and endocardial
cells at the AV canal appears to be Calcineurin/NFAT signaling
(Chang et al., 2004;
de la Pompa et al., 1998
;
Ranger et al., 1998
).
vegf expression is suppressed in the AV canal myocardial cells as a
direct target of the NFATc 2/3/4 transcription factors, thereby permitting the
adjacent endocardial cells to undergo EMT. At a later stage, endocardial
calcineurin function through NFATc1 is required for valve morphogenesis
(Chang et al., 2004
).
In addition to NFATc/calcineurin and Vegf, a number of
other genes have been implicated in the formation of the valves and septa. For
example, mutations in the transcription factor genes NKX25, GATA4 and
TBX5 have been found in individuals with atrial septal defects
(Basson et al., 1997;
Garg et al., 2003
;
Schott et al., 1998
). It has
also recently been described that E9.5 mouse embryos carrying mutations in
either Notch1 or the Notch transcriptional effector gene
RBPJk exhibit a collapse of the endocardium and lack mesenchymal
cushion cells, indicative of a failure in EMT
(Timmerman et al., 2004
).
These phenotypes suggest that Notch signaling is required to regulate the
morphological changes associated with the progression of differentiation of
the endocardium. However, it remains unclear at what stage the differentiation
of the endocardium fails in these mutants and how endocardial cell
disorganization leads to chamber collapse.
Studies carried out in zebrafish have identified additional signaling
pathways involved in valve morphogenesis. Zebrafish mutants in the tumor
suppressor adenomatous polyposis coli (apc) gene exhibit a profuse
endocardial layer at the AV boundary and form excessive ECs at 72 hours
post-fertilization (hpf) as a result of a constitutively active
Wnt/ß-catenin signaling pathway
(Hurlstone et al., 2003).
Other studies have demonstrated that pathways implicated in valve development
in mammals also regulate this process in zebrafish. For example, blocking
Calcineurin signaling by cyclosporine A (CsA) treatment causes EC phenotypes
in zebrafish embryos (Chang et al.,
2004
), although the cellular effects of CsA treatment on cushion
and valve formation remain to be analyzed.
The zebrafish heart consists of two chambers: an atrium and a ventricle.
Rhythmic contractions start at 22 hpf and looping occurs near 36 hpf. Although
the first morphological differences between the two cardiac chambers can be
observed after the formation of the linear heart tube, molecular differences
between atrium and ventricle are apparent much earlier (reviewed by
Yelon and Stainier, 1999). The
AV canal forms at the border between the atrium and ventricle and the first
molecular indication of AV canal specification in zebrafish occurs at
37
hpf with the restriction of bmp4 and versican expression to
the AV myocardium (Walsh and Stainier,
2001
). At
45 hpf, the expression of notch1b becomes
restricted to the AV endocardium (Westin
and Lardelli, 1997
). The differentiated AV canal suffices to
prevent retrograde blood flow in the 48 hpf zebrafish heart as mutants with
defects in AV canal differentiation display blood regurgitation. One such
mutant is jekyll, which was shown to carry a mutation in
ugdh (Walsh and Stainier,
2001
), a homologue of Drosophila Sugarless
(Hacker et al., 1997
). The
restriction of bmp4, versican and notch1b expression, as
well as the upregulation of Tg(Tie2:EGFP)s849
(Stainier et al., 2002
), all
of which mark the specification of the AV canal, fail to occur in jek
mutant embryos (Walsh and Stainier,
2001
), indicating that jek/ugdh is required upstream of
AV canal specification. A similar failure in the upregulation of
Tg(Tie2:EGFP)s849 at the AV canal is observed in
silent heart (sih) mutant embryos
(Bartman et al., 2004
).
sih corresponds to the cardiac troponin T gene and
homozygous mutant hearts do not contract
(Sehnert et al., 2002
). These
and other results (Bartman et al.,
2004
) suggest that mechanical stimuli caused by the beating heart
are essential for AV canal differentiation. Similarly, hemodynamic shear
stress has been implicated in zebrafish valve development
(Hove et al., 2003
).
The zebrafish system offers a unique combination of advantages for studying
cell biology during vertebrate organogenesis, as zebrafish embryos develop
externally and are practically transparent throughout development, thereby
allowing non-invasive observation. Especially advantageous to the study of
cardiovascular development is the fact that the embryos, because of their
small size, receive sufficient oxygen by passive diffusion alone to allow
heart morphogenesis to proceed to a late stage, even in the total absence of
circulation (Stainier, 2001).
Furthermore, the amenability to forward and reverse genetics enables the
identification of novel signaling pathways that regulate a developmental
process, as well as the further analysis of previously identified genes. In
the first forward genetic screen for heart mutations in Boston, a series of
mutants displaying blood regurgitation between the atrium and ventricle were
identified (Stainier et al.,
1996
), but unfortunately few were successfully propagated or
preserved.
In this paper, we present a detailed description of the cellular events underlying AV canal differentiation, EC formation and morphogenesis of cushions into valve leaflets. We find that AV canal endocardial cells differentiate by adopting a cuboidal shape before the onset of EC formation. We use several novel transgenic lines and immunohistochemical markers to analyze further the role of Notch and calcineurin function, as well as mechanical stimuli in AV canal differentiation. We also introduce a large set of mutants that exhibit defects in discrete stages of AV cushion development.
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Materials and methods |
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Immunohistochemistry and confocal microscopy
We used the following antibodies at the indicated dilutions: mouse
monoclonal antibodies zn5 and zn8 (Zebrafish stock center and Hybridoma Bank)
at 1:10, mouse anti ß-catenin (BD Biosciences) at 1:200
(Hurlstone et al., 2003),
rabbit anti-Myc (Sigma) at 1:200, rabbit anti-fibronectin (Sigma) at 1:200
(Trinh and Stainier, 2004
) and
mouse IgG anti ZO-1 (Zymed) at 1:200
(Trinh et al., 2005
).
Embryos were fixed for 2 hours at room temperature with 4% (zn5, anti ß-catenin, anti-Myc, anti-fibronectin) or 2% PFA (anti ZO-1). Whole-mount antibody staining was carried out in PBT (4% BSA, 0.3% Triton and 0.02% NaN3 in PBS pH 7.3). Stained embryos were embedded in NuSieve GTG low melting agarose and cut into 200 µm sections with a Leica VT1000S vibratome. Sections were incubated ON with rhodamine phalloidin (Molecular Probes) 1:50 in PBDT (PBS, 0.1% Tween, 1% DMSO) for filamentous actin staining and with topro3 (Molecular Probes; 1:5000 in PBDT) for nuclear staining. Images were acquired using a Zeiss LSM5 Pascal confocal microscope.
Pharmacological treatment
A 10 mM stock of DAPT (Calbiochem) in DMSO was diluted in embryo water.
Embryos were dechorionated and incubated in 10 or 100 µM DAPT in embryo
water. Control embryos were incubated in 1% DMSO in embryo water. Embryos were
fixed, immunostained and imaged. A 50 mg/ml stock of CsA in ethanol was
diluted in embryo water to a final concentration of 10 µg/ml. Embryos were
incubated within their chorions and then were fixed, immunostained and imaged.
Control embryos were incubated in 0.02% ethanol. Similar results were obtained
for concentrations of CsA ranging from 5 to 50 µg/ml.
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Results |
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AV canal differentiation
To visualize endocardial cells, we used the
Tg(Tie2:EGFP)s849
(Motoike et al., 2000) and
Tg(flk1:EGFP)s843 lines, both of which express GFP in all
endothelial cells. We counterstained all samples with rhodamine phalloidin,
which outlines all cardiac cells and strongly stains the sarcomeric actin of
the myocardial cells. In addition, we used the zn5 monoclonal antibody to
visualize cell-cell borders in the embryonic myocardium. This antibody
recognizes Dm-grasp, a cell-surface adhesion molecule of the immunoglobulin
superfamily (Fashena and Westerfield,
1999
), that we find localized to the lateral side of myocardial
cells and differentiated AV canal endocardial cells.
At 36 hpf, the zebrafish embryonic heart tube has looped, placing the ventricle to the right and the atrium to the left of the midline (Fig. 1A). Endocardial cells are squamous throughout the heart, except for a single cell at the border between the atrium and ventricle, which has a cuboidal shape and has initiated Dm-grasp expression (Fig. 1A,B arrow). This shape change and initiation of Dm-grasp expression are the earliest manifestations of endocardial differentiation in the AV canal.
Over the next 12 hours, endocardial and myocardial cells located in the AV canal further differentiate morphologically. At 55 hpf, myocardial cells at the AV boundary show stronger staining for Dm-grasp than neighboring atrial or ventricular cells (Fig. 1C,D). Endocardial cells lining the AV canal form a single layer of cuboidal cells that express Dm-grasp laterally in contrast to the squamous Dm-grasp negative endocardial cells lining the heart chambers (Fig. 1C,D). These data establish Dm-grasp as a reliable marker for differentiated AV canal endocardial cells. In addition, this cuboidal shape of AV endocardial cells has not been described before during chick or mouse EC formation, where the earliest reported cellular event is an epithelial to mesenchymal transformation. In order to confirm that cuboidal endocardial cells retain their epithelial organization, we stained 55 hpf embryos for ZO1, a molecule associated with tight junctions in epithelial cells. Fig. 1E shows that both squamous and cuboidal endocardial cells express ZO-1, while rhodamine phalloidin staining is upregulated around the basolateral extent of the cuboidal AV canal endocardial cells. A similar distribution of ß-catenin in these cells (data not shown) indicates the presence of adherens junctions and further supports the claim that cuboidal AV endocardial cells retain an epithelial organization.
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In a transverse section through the AV canal at 55 hpf, a single sheet of cuboidal endocardial cells can be seen lining the superior and another sheet lining the inferior ECFR of the AV canal (Fig. 1F). Laterally, these two sheets are interconnected by squamous cells, which we refer to as hinge cells. There are approximately 40 cuboidal endocardial cells (one sheet of 20 cells in each ECFR) in the AV canal at this stage.
Endocardial cushion formation and valve morphogenesis
In order to understand how ECs form, we analyzed subsequent stages of heart
development. By 60 hpf in the superior region of the AV canal, endocardial
cells located at the border with the ventricle have formed cellular extensions
that project into the ECM between the endocardium and myocardium, reaching
towards the base of cells located at the border between the AV canal and
atrium (Fig. 2A,
n=11). These data indicate that endocardial cells on the two
different boundaries of the AV canal have distinct developmental properties
and behavior.
We tested whether AV endocardial cells undergo EMT after sending cellular protrusions into the ECM, by immunostaining 60 hpf embryos for ZO1. Fig. 2B shows that endocardial cells in the ECM (arrow) have downregulated and delocalized ZO1, indicative of EMT. In the superior region of the AV canal, an EC forms on the basal side of a single layer of cuboidal Dm-grasp positive endocardial cells (Fig. 2C,D, n=6). The endocardial cells at the ventricular border of the inferior region of the AV canal begin to form cellular extensions into the ECM at 80 hpf in a pattern similar to that observed in the superior cushion, indicating a delayed initiation of the formation of the inferior EC relative to the superior one (Fig. 2D). By 96 hpf, both cushions have formed (Fig. 2E, arrows). In transverse sections, the mesenchymal cushions are located between the AV myocardium and a layer of cuboidal endocardium (Fig. 2F). AV ECs are transient structures; by 105 hpf, both cushions have started morphogenetic rearrangements that ultimately lead to the formation of the valve leaflets (Fig. 2G,H). Both AV ECs extend into the ventricular lumen (Fig. 2G, arrows). These extensions consist of an outgrowth formed by two layers of cells separated by a layer of fibronectin-containing ECM (Fig. 2G). At 96 hpf, we also observe Tg(0.7her5:EGFP)ne2067 expression in a subset of cells in the extending ECs (Fig. 2H). Tg(0.7her5:EGFP)ne2067 expression is observed in a subpopulation of AV canal endocardial cells from 48 until 120 hpf (data not shown), although the biological significance of this expression is unclear at this time.
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Notch signaling restricts the differentiation of cuboidal endocardium to the AV canal
In zebrafish embryonic hearts, notch1b expression is distributed
throughout the ventricular endocardium at 24 hpf and then becomes restricted
to the AV canal endocardium around 48 hpf
(Walsh and Stainier, 2001;
Westin and Lardelli, 1997
). In
jek (ugdh) mutant embryos, which fail to form ECs, this
restriction of notch1b expression fails to occur
(Walsh and Stainier, 2001
). In
a recent study, Notch1 signaling has been shown to be required for the
progression of differentiation of the endocardium in mouse
(Timmerman et al., 2004
). In
order to test whether Notch signaling is required for the regulation of AV
canal endocardial cell differentiation, we manipulated Notch signaling in the
Tg(0.7her5:EGFP)ne2067 background. We incubated
Tg(0.7her5:EGFP)ne2067 embryos with different
concentrations of the
-secretase inhibitor DAPT.
-Secretase is
required for the activation of the Notch signaling pathway (reviewed by
Mumm and Kopan, 2000
) and
DAPT-treated zebrafish embryos have somitogenesis and neurogenesis phenotypes
identical to those caused by a loss of Notch signaling
(Geling et al., 2002
). We
found that zebrafish embryos treated with 10 and 100 µM DAPT from 24-60 hpf
exhibited ectopic expression of Dm-grasp in endocardial cells throughout the
ventricular chamber at 60 hpf (10 µM DAPT n=5; 100 µM DAPT
n=8). These cells had characteristics of AV canal endocardial cells
such as a cuboidal appearance and upregulated actin staining
(Fig. 5B, arrow). In addition,
some of the ectopic Dm-grasp-positive cells also expressed
Tg(0.7her5:EGFP)ne2067
(Fig. 5B, arrowheads). These
results indicate that Notch signaling is required in the ventricular
endocardial cells to maintain their squamous morphology and inhibit an AV
canal fate. When embryos were treated with 100 µM DAPT from 24 to 80 hpf,
embryonic hearts formed hypercellular ECs in the superior region of the AV
canal (Fig. 5D, arrow,
n=8), suggesting that inhibiting Notch signaling does not block EC
formation. These hypercellular cushions could result from the aggregation of
supernumerary cuboidal endocardial cells or a hyperactivation of EMT. In order
to test whether DAPT treatments affect EMT, as has been suggested previously
(Timmerman et al., 2004
), we
treated embryos between 36-80 hpf (n=6) and 50-80 hpf
(n=12). We found that in both cases, ECs were comparable in size to
the DMSO controls (n=11), although they appeared disorganized and
Dm-grasp expression was downregulated in cuboidal endocardial cells
(Fig. 5E,F). These data
indicate that Notch signaling regulates cell differentiation and patterning
during AV canal EC formation.
In order to test whether constitutive Notch signaling is sufficient to
inhibit AV canal endocardial cell differentiation, the intracellular domain of
Notch was expressed in all endothelial cells of the zebrafish embryo using the
GAL4/UAS binary expression system.
Tg(flk1:Gal4-UAS:EGFP)s848 fish were crossed with the
Tg(UAS:myc-Notch1a-intra)kca3 line
(Scheer, 1999). Immunostaining
for the myc-tag was used to visualize embryos positive for myc-Notch-intra
(Fig. 5H, blue; there is
overlap with EGFP, which is also under the control of flk1:GAL4).
These experiments showed an absence of cuboidal cells and ECs in the AV canal
at 96 hpf (Fig. 5H;
n=12). By contrast, wild-type embryos have fully developed ECs at 96
hpf (Fig. 5G, arrowheads).
These data show that constitutive activation of Notch signaling in endocardial
cells is able to suppress their transition from squamous to cuboidal, an
essential step for EC development.
Calcineurin signaling is required for AV endocardium EMT and subsequent valve morphogenesis
Chang et al. (Chang et al.,
2004) recently reported that myocardial NFATc through suppression
of VEGF signaling allows EMT to occur and that subsequent NFATc signaling is
required for proper valve morphogenesis. Using the tools reported in this
paper, we wanted to further analyze the cellular basis for these requirements.
We observed that raising zebrafish embryos from the one cell stage in medium
with 10 µg/ml CsA, which blocks NFATc signaling, resulted in a very
specific heart defect with no other obvious morphological phenotypes
(Fig. 6A; n>100).
Embryos developed outflow tract stenosis and blood regurgitation between the
atrium and ventricle by 60 hpf (Fig.
6A; see Movie 1 in the supplementary material). The myocardium
appeared thinner at 96 hpf, as previously described
(Molkentin et al., 1998
) and
had little to no trabeculae (compare Fig.
6C with 6B). However, despite these myocardial defects,
contractility appeared unaffected up to 96 hpf (see Movie 1 in the
supplementary material). Interestingly, although AV endocardial cells
initiated cell shape changes and upregulated
Tg(Tie2:EGFP)s849 (Fig.
6C, arrows; n>10), they failed to undergo EMT and form
ECs (Fig. 6C, arrows). Applying
CsA at 48 hpf did not interfere with the initiation of EMT but resulted in
disorganized ECs (Fig. 6D;
n>10), supporting the report of an additional, later role of NFATc
signaling in mouse valve morphogenesis
(Chang et al., 2004
).
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In contrast to the s204 phenotype, the s22 mutation
causes the ventricular lumen to be filled with cuboidal
Tg(Tie2:EGFP)s849-expressing cells
(Fig. 7C). This observation
suggests that endocardial cells in the ventricle undergo an ectopic
differentiation reminiscent of AV canal endocardial cells. The cellular
structure of the atrium in s22 mutant embryos appears normal with
squamous endocardial and myocardial cells. We observed a somewhat similar
phenotype in the apc mutant
(Hurlstone et al., 2003) where
the atrium appears normal, while the ventricular endocardial cells appear
mesenchymal and fill the ventricular lumen
(Fig. 7D).
In s624 mutant embryos at 72 hpf, AV endocardial cells form extensions into the ECM in a disorganized fashion and the ECs are absent (Fig. 7F). The s624 phenotype suggests that a tight spatial regulation of the behavior of these cells is important for AV cushion morphogenesis.
The s266 mutation results in a single layer of cuboidal endocardial cells in the AV canal as late as 96 hpf with no cellular extensions observed (Fig. 7G). These data indicate that the s266 gene is required for the physical process of EMT during EC formation.
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Discussion |
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AV canal differentiation
In mouse and chick embryos, the differentiation of the AV canal comprises
not only a thickening of the ECM in the AV canal but also the acquisition of
specific developmental properties by both endocardial and myocardial cells in
this region (reviewed by Eisenberg and
Markwald, 1995). Similarly, we find that in zebrafish embryos,
endocardial and myocardial cells differentiate morphologically before the
onset of EMT. The AV endocardium undergoes a transition from squamous to
cuboidal cell shape and starts expressing Dm-grasp, a cell-adhesion molecule
that becomes localized laterally. Cuboidal AV endocardial cells show a
characteristic pattern of actin and ß-catenin staining indicative of
adherens junctions. This process starts around 36 hpf and is completed by 55
hpf, when the superior and inferior regions of the AV canal are each lined by
a sheet of cuboidal endocardial cells interconnected by squamous cells. As the
AV canal must function to ensure unidirectional blood flow prior to the
formation of the cushions or valves proper, we suggest that this spatial
organization of squamous and cuboidal cells is required to prevent blood
regurgitation. Others have shown that at 4.5 dpf the diameter of the AV canal
changes 2.8-fold between systole and diastole
(Hove et al., 2003
). It is
likely that the specific arrangement of cuboidal and squamous cells allows for
this change in diameter and that the two opposing sheets of cuboidal cells
ensure closure of the AV canal during ventricular systole.
In chick, rat and mouse embryos, `rounded' AV endocardial cells have been
described to extend cellular protrusions into the ECM, a hallmark for the
onset of EMT (Markwald et al.,
1977; Timmerman et al.,
2004
). A recent report has identified an early pro-valve enhancer
in the first intron of NFATc1. This enhancer is active specifically
in endocardial cells at the AV boundary and the outflow tract at stages before
EMT, and not in transformed endocardial cells invading the ECM
(Zhou et al., 2005
). These
data suggest that this enhancer marks the mammalian equivalent of the earliest
Dm-grasp-positive AV endocardial cells reported here. Other cuboidal
endothelial cells have been described in the high endothelial venules (HEV)
present in the paracortex of lymph nodes, tonsils and interfollicular areas of
Peyer's patches. HEVs are lined by a high-walled endothelium that functions in
the homing of lymphocytes (Miyasaka and
Tanaka, 2004
). The function of zebrafish cuboidal endocardium is
obviously different from that of HEVs. The transition of a squamous to
cuboidal endocardium in the AV canal of zebrafish is another milieu in which
basic cell biological questions regarding the regulation of changes in cell
shape, organization and cell-cell adhesion can be addressed.
Differentiation of AV canal endocardial cells depends on cardiac contractility
We analyzed the role of mechanical function of the heart in AV canal
endocardial cell differentiation. We find that in sih mutants, which
lack heart contraction, AV canal endocardial cells fail to express Dm-grasp
and to change shape. It remains to be determined whether the required
mechanical stimulus comes from the wall forces of heart muscle contraction or
from hemodynamic shear stress (reviewed by
Bartman and Hove, 2005). In
cell culture, bovine aortic endothelial cells respond to shear stress by
changing cytoskeletal organization, cell-adhesion complexes, cell morphology
and gene expression (reviewed by McCue et
al., 2004
). Intra-cardiac shear stress can be calculated by
indirect methods in live embryonic zebrafish hearts, and in the 37 hpf
embryonic heart wall shear forces of 2.5 dyn/cm2 have been
estimated (Hove et al., 2003
).
Hemodynamic shear stress is therefore a good candidate to cause changes in
endocardial cell shape, adhesion and gene expression. Likewise, passive
cyclical mechanical stretch of skeletal myocytes in culture (i.e. in the
absence of flow) appears to be sufficient to transdifferentiate these cells
into cardiomyocytes (Iijima et al.,
2003
). It has not been tested whether similar mechanical forces in
the absence of flow can also affect the differentiation of endothelial cells.
Future experiments should address the source and role of shear stress in the
differentiation of AV canal cells, and aim to identify the molecular triggers
for this process.
Notch and calcineurin signaling are involved in the spatiotemporal control of AV canal specification and differentiation
The characteristic changes in cell shape and organization together with the
specific expression of Dm-grasp are reliable markers for the specification and
differentiation of the AV canal endocardial cells. Here, we made use of these
cellular and molecular changes to show that Notch signaling is required to
restrict this program of differentiation to the endocardial cells of the AV
canal. When Notch signaling was inhibited by DAPT treatment, cells with AV
canal endocardial morphology and protein expression were found within the
ventricle. Conversely, constitutive activation of Notch signaling inhibited
the transition of AV endocardial cells from squamous to cuboidal, as well as
the subsequent formation of ECs by EMT. These results indicate that Notch
signaling inhibits the differentiation of the ventricular endocardium into
cells with AV canal-like, cuboidal, Dm-grasp-expressing properties.
A different experimental protocol, the early and widespread expression of
NotchICD following mRNA injection at the one-cell stage, led to hypertrophic
ECs, suggesting that Notch activation induces excessive EMT
(Timmerman et al., 2004).
However, this approach resulted in a high mortality rate (80% by 48 hpf). In
our experiments, the use of the GAL4/UAS system to express NotchICD only in
endocardial/endothelial cells from
24 hpf onwards avoids the indirect
effects associated with mRNA injections. Indeed,
Tg(flk1:Gal4-UAS:EGFP)s848;
Tg(UAS:myc-Notch1a-intra)kca3 embryos exhibited pericardial
edema starting at 3 dpf but appeared unaffected otherwise and survived up to 7
dpf, without developing any ECs. Our observations with DAPT treatments were
also different from those reported by Timmerman et al.
(Timmerman et al., 2004
).
Whereas they reported that treating embryos with 50-100 µM DAPT from 36 hpf
to 5 dpf resulted in the formation of atrophied cushions, in our hands, such
treatment led to 100% lethality by 96 hpf in two independent experiments.
Therefore, we reduced the exposure time and found that DAPT treatments from 36
to 80 hpf and from 50 to 80 hpf resulted in the formation of normal size
superior ECs. However, these ECs appeared disorganized and Dm-grasp was
downregulated in the cuboidal endocardial cells, suggesting that Notch
signaling is required to maintain the correct pattern of cell fates in the
forming EC. It is possible that this function of Notch signaling is required
to maintain an efficient development and/or growth of ECs from 60 to 96 hpf,
which would explain the atrophied appearance of the ECs by Timmerman et al.
(Timmerman et al., 2004
).
These data indicate, in a model consistent with the pattern of
notch1b expression in the endocardium, that Notch signaling plays
multiple roles in EC formation, starting with the repression of the AV canal
endocardial phenotype in the ventricle.
Work in mouse has shown that while the later steps of EMT require NFATc1
function in endocardial cells, at earlier stages myocardial Vegf expression is
repressed by NFATc2/3/4 signaling and this repression is required for EMT
initiation (Chang et al.,
2004). Inhibiting calcineurin function from an early stage in
zebrafish embryos blocked the differentiation of the AV canal endocardium (as
demonstrated by the absence of Dm-grasp expression in the AV canal endocardial
cells and their failure to undergo EMT). However, some aspects of AV canal
specification, as illustrated by the Tg(Tie2:EGFP)s849
upregulation, were not affected. These results suggest that calcineurin
signaling is also required for the differentiation of the AV endocardium.
Systematic analysis of the identified mutants will help elucidate mechanisms of cushion and valve morphogenesis
Our data show that in zebrafish, as in amniotes, ECs are formed by the
migration into the ECM of a subset of AV canal endocardial cells followed by
their EMT. We found that only AV endocardial cells located at the ventricular
boundary migrate in the direction of the atrial boundary. This finding
suggests that cells along the AV canal have different developmental
properties, whereby only cells at the ventricular boundary are able to respond
to a localized guidance cue coming from the direction of the atrial boundary.
Detailed analysis of EC morphogenesis mutants such as s266 and
s624 will be instrumental in understanding the underlying molecular
and cellular mechanisms of these processes.
AV ECs are transient structures in zebrafish embryos. Their formation begins around 60 hpf and their transformation into valve leaflets around 96 hpf. In comparison to the four-chambered hearts of mouse and chick, where ECs are involved in both the development of valves and interventricular and interatrial septae, in the zebrafish heart valve morphogenesis is a simpler process giving rise initially to two leaflets and the AV septum. At a later stage, the two valve leaflets in the AV canal are remodeled to give rise to four leaflets.
The power of the zebrafish system revolves around its amenability to forward and reverse genetics, as well as several methods for studying organogenesis at the cellular level, allowing a high degree of integration of genetic and cellular studies. In our screen, we identified mutations affecting distinct stages of AV cushion development. The systematic analysis of this mutant collection will be instrumental in furthering our understanding of the molecular regulation of AV canal differentiation and subsequent valve development. Furthermore, zebrafish embryos are also ideal for the pharmacological analysis of specific signaling pathways in various developmental processes. The combination of genetic and pharmacological studies, along with detailed analyses of the cell biology of cushion/valve development, should provide novel insights into the developmental biology of cardiac organogenesis and provide relevant information to the clinicians who care for individuals with valvular and septal malformations.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/18/4193/DC1
* Present address: Foundation for Biomedical Research of the Academy of
Athens, Basic Research Center, Athens, Greece
These authors contributed equally to this work
Present address: Divisions of Neonatology, Pulmonary Biology and
Developmental Biology Cincinnati Children's Hospital Medical Center, OH 45229,
USA
Present address: National Institute for Medical Research, Division of
Developmental Biology, Mill Hill, London NW7 1AA, UK
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