1 Laboratório de Genética e Cardiologia Molecular InCor
HC.FMUSP 05403-900 São Paulo-SP, Brazil
2 Laboratório de Cardiologia Celular e Molecular IBCCF-UFRJ Rio de
Janeiro-RJ, Brazil
3 Departamento de Histologia e Embriologia, ICB-USP, São Paulo-SP,
Brazil
4 Pulmonary Center Boston University School of Medicine, Boston, MA,
USA
5 EMBL European Molecular Biology Laboratory Mouse Biology Programme,
Monterotondo-Scalo, Italy
Author for correspondence (e-mail:
xavier.neto{at}incor.usp.br)
Accepted 9 July 2003
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SUMMARY |
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Key words: Heart, Atrium, Ventricle, RALDH2, Retinoic acid, AMHC1, BMS493, Mouse, Chicken, Embryo
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Introduction |
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Although a few genes have been identified that play a role in chamber
formation (Bruneau, 2002), much
remains to be known about how information flows from signalling events to the
synthesis and assembly of specific contractile and electrophysiological
modules along the cardiac AP axis. Recently, much information has been
obtained showing that retinoic acid (RA) is a morphogen that communicates AP
polarity to the heart (Xavier-Neto et al.,
2001
). RA is synthesized from vitamin A through a chain of
oxidative reactions, from retinol to retinaldehyde and from retinaldehyde to
RA. The former reaction is mediated by alcohol dehydrogenases (ADHs) and the
latter by retinaldehyde dehydrogenases (RALDHs). Because ADH3 activity is
ubiquitous (Molotkov et al.,
2002
), the availability of RA is dictated by the distribution of
RALDHs. Previous studies indicate that RALDH2 is the main RALDH in early
cardiac development (Moss et al.,
1998
; Niederreither et al.,
2001
). RALDH2 is expressed in the developing heart to generate
sequential programs of RA synthesis in myocardial and epicardial layers
(Moss et al., 1998
;
Xavier-Neto et al., 2000
).
Using immunohistochemistry we previously showed that Raldh2 is
expressed in a region of the avian lateral mesoderm that contains sino-atrial
precursors in HH8 embryos. Moreover, at HH9, posterior
cardiac precursors express Raldh2
(Xavier-Neto et al., 2000
) and
Amhc1, a marker of commitment to the atrial phenotype
(Yutzey et al., 1994
). From
these stages onwards, Raldh2 expression remains associated with
sino-atrial structures until a myocardial wave takes it to ventricles and
conotruncus. This myocardial phase is then replaced by another wave of
epicardial RALDH2 that envelops the heart. Thus, these patterns provided clues
that RALDH2 plays important roles in sino-atrial morphogenesis, in the
development of the coronary circulation and in growth of the ventricular
myocardium (Xavier-Neto et al.,
2000
; Pérez-Pomares et
al., 2002
; Stuckmann et al.,
2003
).
The crucial role of RALDH2 in sino-atrial development has been established
by pharmacological, genetic and dietary manipulations
(Xavier-Neto et al., 1999;
Niederreither et al., 1999
;
Kostetskii et al., 1999
).
Although effective, these approaches were systemic and protracted, and
therefore lacked the spatial and temporal resolution required to define target
cell populations and developmental times when the endogenous RA signal
polarizes the heart. Thus, to fill these fundamental gaps in our understanding
of the developmental mechanisms that communicate and maintain sino-atrial
fates, here we describe the changing spatial relationship between cardiac
precursors and the domains of Raldh2 expression during the critical
phases of cardiac AP patterning. The different stages of the relationship
between cardiac precursors and RALDH2 were correlated to the states of
commitment of anterior and posterior cardiac precursors using treatments with
RA or with a RA pan-antagonist, respectively. We show that there are two
phases of cardiac AP patterning by RA. The first phase, the specification
phase (HH5-7), is characterized by increasing proximity between sino-atrial
precursors and the anterior margin of the RALDH2-expressing mesoderm. The
second phase, the determination phase (HH7-8), is characterized by progressive
encircling of sino-atrial precursors by a field of RALDH2 originating from a
highly dynamic caudorostral wave in the lateral mesoderm. Integrating the data
on morphology, fate mapping and states of commitment we conclude that the RA
required for cardiac AP specification is provided by the posterior mesoderm
(HH5-7). Later, the RA required for determination of AP fates is provided by
the anterior lateral mesoderm (HH7-8) in the form of a caudorostral wave of
RALDH2. Identification of the tissue sources of RA that define AP boundaries
in cardiac precursors should pave the way to a better understanding of how AP
information is relayed to the developing heart.
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Materials and methods |
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In situ hybridization
In situ hybridization was performed according to Wilkinson
(Wilkinson, 1992) using probes
against chicken and mouse mRNAs such as chick GATA4
(Jiang et al., 1998
), chick
RALDH2 (Swindell et al.,
1999
), AMHC1 (Yutzey et al.,
1994
), mouse Tbx-5 (Bruneau et
al., 1999
) and mouse RALDH2
(Zhao et al., 1996
). For
double in situ hybridization, embryos were treated as described by Stern
(Stern, 1998
). GATA4 and Tbx-5
were revealed with BMPurple. RALDH2 probes were revealed using BMPurple or
INT/BCIP. RALDH2 immunohistochemistry was performed as described
(Xavier-Neto et al., 1999
).
Double mouse Tbx-5 in situ hybridization/ß-galactosidase stains in
RAREhsplacZ embryos (Rossant et
al., 1991
) were performed according to Houzelstein and Tajbakhsh
(Houzelstein and Tajbakhsh,
1999
). Paraffin sections were generated according to Sassoon and
Rosenthal (Sassoon and Rosenthal,
1993
). Isotopic (35S-labeled riboprobes) in situ
hybridization was performed on paraffin sections (6-12 µm) as described by
Cardoso et al. (Cardoso et al.,
1996
) and the labelling displayed in pseudocolor.
Image analyses
Embryos and paraffin sections were photographed on stereozoom and
fluorescence microscopes. Bright field and fluorescent pictures were taken
with a digital camera and acquired with MediaCybernetics software. Images in
slides were acquired with a slide scanner and processed with Adobe
Photoshop.
Morphometry
Expression patterns from 200 chicken embryos were quantified with the Scion
Image program (ported from NIH Image for the Macintosh by Scion Corporation
and available on the Internet at
http://www.Scioncorp.com).
Unprocessed images in TIFF format were fed into Scion to obtain grayscale
images that were calibrated to give distances in µm. Grayscale images were
submitted to density slicing which segments images on the basis of gray level.
By manipulating upper and lower threshold levels in the look up tables, pixels
representing low levels of staining were displayed in red, whereas pixels
either above (high level of staining) or below threshold (background staining)
were unchanged (Fig. 1C-E).
Changes in expression patterns were measured as distances from embryonic
structures or staining landmarks. We measured four parameters in the lateral
mesoderm: (1) Distance traveled by the RALDH2 wave (anterior expansion); (2)
Front of the RALDH2 wave (anterior limit of Raldh2 expression); (3)
Anterior limit of the cardiac field (anterior limit of chick Gata4
expression); (4) Posterior limit of the cardiac field (posterior limit of
chick Gata4 expression).
|
Anterior and posterior limits of chick Gata4 and chick Raldh2 expression were measured relative to the anterior tip of Hensen's node. Points above or below it were attributed positive or negative values, respectively. Careful examination of all parameters did not reveal differences between right and left sides. Thus, final averages include both sets of data.
Fate mapping
The fluorescent tracer DiI was diluted and loaded into glass pipettes
according to Garcia-Martinez and Schoenwolf
(Garcia-Martinez and Schoenwolf,
1993). DiI was pressure-injected as a small bolus in the left
lateral mesoderm with a Narishige micromanipulator and a Harvard Apparatus
picoinjector. After injection embryos were washed in PBS, photographed under
fluorescent and bright fields and cultured to HH11+, when they were
fixed, examined and photographed again. Injection sites were recorded using
grid system and coordinates by Redkar et al.
(Redkar et al., 2001
). Grids
were superimposed on pictures of living HH7 and HH8 embryos. Fluorescence and
bright field images were superimposed using Adobe Photoshop. Specific
information on each DiI injection point is provided as supplemental data (see
Tables S1, S2, S3 at
http://dev.biologists.org/supplemental).
We superimposed HH7 and HH8 cardiac fate maps on RALDH2 in situ
hybridization pictures from 2 embryos that represented the average patterns
determined by morphometric analysis. We adopted the procedure described by
Streit to correct for uneven shrinking induced by dehydration and in situ
hybridization (Streit, 2002).
Corrections were applied by enlarging in situ hybridization pictures of HH7
embryos by 1.0% in the left-right axis and 3.7% in the AP axis. Correction
factors for HH8 were 5% and 9%, respectively.
Treatments
Cultured embryos were treated with all-trans RA or the RA pan-antagonist
BMS493. Stock solutions of all-trans RA and BMS493 103 M in
DMSO or ethanol, respectively, were diluted in PBS to 106,
105 and 104 M. Twenty microliters of test
solution were applied over embryos beginning at HH4-9. All embryos were
harvested at HH10. Controls received vehicle for all-trans RA (DMSO 1% in PBS)
or BMS493 (ethanol 10% in PBS). For unilateral treatments of the anterior
lateral mesoderm we placed 3 cylinders of agar 1.5% (1.0 mm height, 0.5 mm
diameter) (Rugh, 1952) made in
PBS containing BMS493 104 M, on endoderm overlying the left
lateral mesoderm between Hensen's node and the headfold. Controls received
cylinders containing PBS.
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Results |
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To establish the dynamics of the RALDH2 caudorostral wave in a quantitative fashion we measured the anterior expansion of RALDH2 in the lateral mesoderm (Fig. 1B-E). As shown in Fig. 1F, anterior expansion begins slowly between HH6-7. Between HH7-8, however, it is accelerated to its maximal rate. Thereafter, it proceeds at a much slower pace until bilateral arches of RALDH2 fuse at the midline at HH9-10.
In the mouse embryo, Raldh2 expression changes are less pronounced than in chicken, but overall, display similar progression. Anterior expansion of RALDH2 in mice begins at the late allantoic bud stage. The maximal rate of anterior expansion occurs between the stages of late headfold and 1 somite (not shown). Thereafter, RALDH2 expansion is slowed between the stages of 1 somite and 2 somites, and, similar to the chicken embryo, its bilateral arches eventually join in the midline over the AIP (Fig. 2 and see RALDH2 whole-mount immunohistochemistry).
|
We performed in situ hybridization with a chick RALDH2 probe and a chick
GATA4 probe as a marker for cardiac precursors. Chick Gata4 was
chosen as a marker because its pattern of expression coincided with the
cardiac field as revealed by our fate maps (see Raldh2 expression and
the cardiac fate map). This contrasted with those of chick nkx-2.5,
which excluded most posterior cardiac precursors (data not shown)
(Redkar et al., 2001).
Fig. 3 shows the two phases of the changing relationship between chick GATA4 and chick RALDH2 patterns. The parameters utilized in this morphometric analysis are illustrated in Fig. 3A,B. At HH5 there was a gap of approximately 700 µm separating cardiac precursors from tissue synthesizing RALDH2 (Fig. 3C). This gap narrowed between HH5-7 (Phase 1) and, eventually, RALDH2 entered the cardiac field between HH7-8 (Phase 2). At HH8 RALDH2 penetrated deeper into cardiac tissue, overlapping slightly more than the lower half of the cardiac field. At HH9 chick Raldh2 expression extended over three-quarters of the cardiac field.
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|
The changing relationship between RALDH2 and cardiac precursors was directly established in the mouse embryo by RALDH2/Tbx-5 double in situ hybridization. At early stages, mouse Gata4 and mouse Tbx5 displayed similar patterns of expression and included more posterior cardiac precursors than mouse nkx-2.5. The mouse Tbx5 probe gave stronger signals than mouse Gata4 and was chosen as the marker of cardiac cells (not shown). Fig. 5 depicts embryos at stages ranging from the late allantoic bud to the 6 somite stage. At late bud stage RALDH2 was expressed exclusively in the mesoderm caudal to the node, whereas cardiac precursors were concentrated at the anterior tip of the mesoderm (Fig. 5A). In more developed late bud embryos and in embryos at the early headfold stage (Fig. 5B,C), mouse Raldh2 expression in the lateral mesoderm expanded anteriorly towards the posterior margin of the cardiac field. Mouse Tbx5 expression was also increased, forming a stripe oriented in a posterior-lateral direction towards the tip of the advancing wave of RALDH2 (Fig. 5B-D). At late headfold stage, Raldh2 and Tbx5 expression domains converged to overlap in the most posterior cardiac precursors (Fig. 5E). The presence of posterior cardiac precursors in a field actively synthesizing and responding to RA was clearly shown in a double mouse Tbx-5 in situ hybridization/lacZ staining of a late headfold RAREhsplacZ RA-indicator embryo. At this stage only the posterior third of the mouse Tbx-5 stripe overlapped with the lacZ stain (Fig. 5F). As indicated by Fig. 5F-I, the encirclement of cardiac precursors by RALDH2 progressed steadily at 3-4 somite stages (arrows) and, eventually, the bilateral arches of RALDH2 joined at the midline over the AIP. There, they overlapped most posterior precursors as shown by RALDH2 whole-mount immunohistochemistry (Fig. 5J,K).
|
Probing commitment to AP fates in the cardiac field
To correlate commitment to cardiac AP fates with the events of chick
Raldh2 expression in the lateral mesoderm, we assessed the stage at
which cardiac precursors become determined to a specific AP fate. Commitment
to posterior fates was tested with BMS493, a RA pan-antagonist, whereas
commitment to anterior fates was tested with all-trans RA. Production of
hearts with a reduced inflow compartment and an oversized ventricle after
BMS493 was interpreted as evidence against determination of the posterior
fate. Likewise, production of hearts with inflow dominance after RA was
interpreted as evidence against determination of anterior fates. Production of
normal hearts after treatments with BMS493 or RA indicated that posterior and
anterior fates were already determined.
Fig. 6A,B shows cardiac phenotypes obtained after BMS493 104 M or all-trans RA 105-104 M, respectively. As seen in Fig. 6A, BMS493 at HH4-7 inhibited development of cardiac inflow and turned the heart into an oversized ventricle. Conversely, BMS493 at HH8-9 failed to affect cardiac morphology. Likewise, treatment with RA at HH4-7, but not at HH8-9, produced hearts with clear inflow dominance displaying reduced or absent ventricular tissue. Fig. 6C indicates that reciprocal changes in inflow architecture induced by BMS493 or RA were consistent with the patterns of Amhc1 expression, a marker for posterior cardiac cells.
|
Chick Raldh2 expression and the cardiac fate map
To determine the relationship between cardiac AP fates and chick
Raldh2 expression we generated fate maps from embryos at HH7-8, the
critical phases of commitment to AP fates. In agreement with previous reports
(Redkar et al., 2001;
Rosenquist and deHaan, 1966
),
labelling of anterior or posterior cardiac precursors with DiI was followed by
appearance of the dye in ventricles or sino-atrial region
(Fig. 7D and 7B, respectively).
This was further confirmed when we superimposed grids containing information
from all injection points obtained at HH7 and HH8 on appropriate embryos
(Fig. 7E-H, Fig. 8B).
|
|
At HH7 there was a clear separation, centered at the mid of row E, between anterior and posterior cardiac precursors. Importantly, all but three injection sites representing posterior cardiac precursors fell within the domains of chick Raldh2 expression. In contrast, all injection sites representing anterior cardiac precursors fell outside the domain of chick Raldh2 expression and were separated from it by at least 100 µm (Fig. 7G).
At HH8, a significant region of overlap, centered at grid square F3, developed between anterior and posterior cardiac precursors. Nonetheless, most anterior and posterior cardiac precursors remained at their respective rostrocaudal sections in the lateral mesoderm. At this stage all injection sites representing posterior precursors were contained within the domains of chick Raldh2 expression. Most injection sites representing anterior precursors also fell within the domains of RALDH2 such that only the rostral-most anterior precursors located at square B2 were outside the RALDH2 domain (Fig. 7H).
Thus, we demonstrated that at stage HH7, RALDH2 is present in the lateral mesoderm at a position consistent with the location of posterior, but not of anterior cardiac precursors. In contrast, at stage HH8, RALDH2 in the lateral mesoderm reaches most cardiac cells and no longer discriminates between anterior or posterior precursors.
RA signalling controls cardiac fates and is a local requirement for
atrial differentiation
To establish whether RA inhibition affects specification of AP identities
in the cardiac field we generated cardiac fate maps in the presence of BMS493.
As shown in Fig. 8A, RA
inhibition at HH7 changed the cardiac fate map. In the presence of BMS493,
ventricular precursors were found in the posterior cardiac field between rows
G and H, which, in the absence of treatment, contained only sino-atrial
precursors (Fig. 8B). This is
consistent with RA determining sino-atrial fates in posterior cardiac
precursors and suggests that conversion of sino-atrial precursors to a
ventricular fate is important as a mechanism of ventricular dominance after RA
inhibition (Fig. 6A,
Fig. 8D).
To determine the role played by local RA in the anterior lateral mesoderm we performed unilateral treatments with BMS493. Three agar cylinders containing BMS493 104 M were placed on the endoderm overlying the left lateral mesoderm between Hensen's node and headfold (Fig. 8E). As shown in Fig. 8F, RA inhibition in the left lateral mesoderm repressed expression of the atrial marker AMHC1 exclusively on the left side. This indicates that local RA signalling in the lateral mesoderm is necessary to induce atrial differentiation.
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Discussion |
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Using expression of RALDH2 to understand cardiac RA signalling
Recent advances in retinoid biology made clear that the dynamic and
elaborated patterns of RA signalling during embryogenesis require more
sophisticated regulatory options than those provided by RA receptors and their
patterns of expression. In short time, work on RALDHs and RA-degrading enzymes
have confirmed that retinoid signalling cannot be understood without knowing
how these enzymes are regulated (Duester et
al., 2003; Swindell et al.,
1999
).
Amongst the RALDHs, RALDH2 is the first expressed and its appearance
coincides with initiation of RA synthesis in the mouse embryo
(Ulven et al., 2000).
Furthermore, Raldh2 expression coincides with the response to
endogenous RA in hearts from immediately after fusion of cardiac primordia, up
to looping and wedging stages (Moss et
al., 1998
). In this study we extend these findings to show that
Raldh2 expression faithfully represents RA signalling in cardiac
precursors even before their fusion (Fig.
5E,F). In addition, ablation of the Raldh2 gene abrogates
atrial development, promotes premature differentiation of ventricular cells
and leads to embryonic death
(Niederreither et al., 2001
).
Thus, although there is evidence for novel, as yet uncharacterized, RALDH
activities in the developing heart (Mic et
al., 2002
; Niederrheither et al., 2002), RALDH2 is a major RALDH
activity in cardiac development, suggesting that Raldh2 expression is
an accurate readout of RA signalling in cardiac precursors.
Recently, Halilagic et al. indicated that RA signalling in chicken embryos
starts much earlier than previously thought
(Halilagic et al., 2003).
However, the putative contribution of this early RA signalling to cardiac AP
patterning needs to be evaluated in the light of previous work indicating that
cardiac cells commit irreversibly to their AP phenotypes between stages HH7
and 8, but not earlier (Orts-Llorca and
Collado, 1967
; Satin et al.,
1988
; Inagaki et al.,
1993
; Yutzey et al.,
1995
; Patwardhan et al.,
2000
) (for a review, see
Xavier-Neto et al., 2001
).
Therefore, although earlier RA signalling by enzymes other than RALDH2 may
play a major role in cardiac development and be necessary or permissive for
induction of RALDH2 in the appropriate regions of the cardiogenic plate, the
data available indicate that the crucial decision between anterior or
posterior fates occurs at developmental times when RALDH2 is the only RALDH
enzyme expressed in a clear AP pattern in the cardiac mesoderm.
GATA4 as a marker for the early cardiac field
In this study we utilized GATA4 instead of Nkx-2.5 as a marker for the
cardiac field. This choice was validated by comparing our cardiac fate maps
with typical in situ hybridization patterns for GATA4. As shown in
Fig. 7I, the GATA4 expression
domain matched the distribution of the cardiac field at stage HH8. This
observation is consistent with previous studies showing that GATA4 is highly
expressed in the lateral mesoderm from the level of the AIP to somite 3
(Jiang et al., 1998;
Kostetskii et al., 1999
)
(Fig. 4F), a domain which
encompasses all cardiac precursors as determined by our fate map in
Fig. 7I or almost all cardiac
precursors according to a previous fate map study
(Redkar et al., 2001
). In
contrast, in agreement with the study of Redkar et al.
(Redkar et al., 2001
), the
Nkx-2.5 domain fell short of labelling all cardiac precursors, leaving behind
the caudal third of the cardiac field where sino-atrial precursors predominate
(data not shown). Thus, our data indicate that GATA4 is a better marker for
the early cardiac field than Nkx-2.5.
The patterns of Raldh2 expression are consistent with roles
for RA in specification and determination of cardiac AP fates
We showed that Raldh2 expression and RA signalling were confined
to mouse sino-atrial tissues from 8.25 to 9.5 dpc
(Moss et al., 1998). Using
chicken embryos we demonstrated that atrial precursors co-expressed
Amhc1 and chick Raldh2 as early as stage
9, indicating that the association between atrial precursors
and RALDH2 could be pushed further back in time
(Xavier-Neto et al., 2000
).
Although these observations were consistent with a role of RA in the
maintenance and development of the sino-atrial phenotype, they were not
sufficient to prove that an endogenous RA signal was a determinant factor at
the earlier developmental periods when the sino-atrial fate is determined.
Therefore, this study was performed to fill the gap in our knowledge of the
relationship between RALDH2 and the cardiac field at the critical stages of AP
differentiation.
The progressive adherence of cardiac precursors to their AP phenotypes has
been studied. Using different techniques, several investigators established
that cardiac AP fates are specified between HH4-7 and determined around HH7-8
(Orts Llorca and Collado,
1967; Satin et al.,
1988
; Inagaki et al.,
1993
; Yutzey et al.,
1995
; Patwardhan et al.,
2000
). We extend these findings by showing, through reciprocal
manipulations of RA signalling, that cardiac AP fates remain plastic from
HH4-7, but not after HH8.
We describe two phases of the dynamic relationship between Raldh2
expression and cardiac precursors that fit into the paradigms of specification
and determination. In the chick embryo Phase 1 spans stages HH4-7, and is
characterized by progressive closure of a spatial gap that separates RALDH2
from the cardiac field (Fig.
3C, Fig. 4). The
patterns of Raldh2 expression during Phase 1 suggest that RA
concentrations reaching the posterior cardiac field increase gradually from
low values at stage HH4-5 to higher values at HH7, as the distance between
source and target tissue decrease. Such profile of increasing RA
concentrations in posterior cardiac precursors would be consistent with the
pattern of increasing association of these cells to the sino-atrial phenotype.
As shown by Yutzey et al. only 67% of explants containing posterior cardiac
cells at stage HH5-6 expressed Amhc1 after 2 days of culture
(Yutzey et al., 1995). In
contrast, 95% of posterior explants at stage HH7-8 expressed Amhc1,
indicating a stronger adherence of posterior cardiac cells to the sino-atrial
phenotype. However, cardiac AP fates are not determined even at HH7
(Fig. 6). In fact, posterior
cardiac precursors commit irreversibly to their sino-atrial fates only between
HH7-8 when Raldh2 expression is at Phase 2 and invading the cardiac
field. Therefore, the patterns of Raldh2 expression at Phase 2
suggest that RA concentrations reaching prospective sino-atrial precursors
would attain a maximal value when RALDH2 encircles these cells, eliminating
the distance between source and target tissue
(Fig. 4J,K). Thus, at Phase 2,
direct exposure of the posterior cardiac field to high concentrations of RA
produced in situ would be consistent with an irreversible attainment of
sino-atrial identity. In summary, our data indicate that Raldh2
expression is present at the right times and places to direct both
specification and determination inside each AP domain. It is probable,
however, that fate determination at the cardiac AP boundary is more complex
than inside each AP domain. At stage HH7 we detected a very limited degree of
overlap between anterior and posterior cardiac fields
(Fig. 7E). Although this may
reflect an intrinsic limitation of fate mapping techniques, which cannot offer
more than an approximate view of dynamic events, fate determination at the AP
boundary will probably involve an interplay of position, movement as well as
extent and timing of exposure to RA signalling.
Is anterior the myocardial default?
Because RA signalling is required in the posterior cardiac field to induce
the sino-atrial phenotype in cells that would otherwise differentiate into
anterior cell types (Fig. 8),
it is tempting to speculate that the default fate of the myocardium is an
undifferentiated anterior cell. Evidence from RA-insufficiency studies
supports this notion (Heine et al.,
1985; Niederreither et al.,
1999
; Chazaud et al.,
1999
; Xavier-Neto et al.,
1999
). Moreover, several morphogens induce ectopic cardiac tissue
expressing vmhc1, but not the atrial marker Amhc1
(Lopez-Sanchez et al., 2002
).
This is reminiscent of the patterns of AP patterning in caudal hindbrain,
where RA is required to specify rhombomeres (r) 5-8 acting on a tissue whose
default is r4 (Dupé and Lumsden,
2001
). In fact, cardiac AP differentiation parallels caudal
hindbrain patterning. It is even probable that the somitic mesoderm
constitutes a shared source of RA for AP specification of heart and hindbrain.
Although somites may provide all the RA required for hindbrain patterning, our
data suggest that a new strategy evolved in the form of a caudorostral wave of
RALDH2 to provide the RA concentrations that pattern cardiac precursors in the
AP axis. Experience with other systems, however, suggests that a double
assurance mechanism may operate in cardiac AP patterning, such that there may
be separate determinants for each cardiac AP fate. Whatever is the identity of
the putative anterior cardiac inducer it is clear that its actions must be
recessive to the posteriorizing RA signal.
The fate of cardiac precursors after manipulations of RA
signalling
Inhibition of RA signalling by BMS493 repressed AMHC1 expression and
produced hearts with ventricular dominance, whereas RA increased AMHC1
expression and produced hearts with inflow dominance
(Fig. 6). Inflow/outflow
dominance after manipulation of RA signalling could be caused by multiple
mechanisms such as conversion between atrial and ventricular phenotypes,
selective proliferation or apoptosis. In
Fig. 8 we showed, in a fate map
performed under BMS493, that ventricular precursors were found in regions of
the cardiac field, which, in the absence of treatment, contained only
sino-atrial precursors. This suggests that atrial precursors converted to
ventricular phenotypes in the absence of RA signalling. Conversely, previous
studies by Yutzey et al. showed that exogenous RA increased the domain of
AMHC1 without interfering with VMHC1 expression or heart size and induced
AMHC1 expression in ventricular precursors
(Yutzey et al., 1994;
Yutzey et al., 1995
). These
experiments suggested that increased RA signalling converted ventricular
precursors into atrial cells. Moreover, we showed in transgenic mice that
exogenous RA induced expression of the atrial-specific marker SMyHC3-HAP in
cells that already expressed MLC2-V, a ventricular-specific marker
(Xavier-Neto et al., 1999
).
This experiment provided direct in vivo evidence that exogenous RA can induce
an atrial program in ventricular cells.
Thus, although our experiments here were not designed to address
specifically the fates of cardiac precursors after manipulations of RA
signalling, data in this manuscript as well as in previous studies support a
role for conversion between atrial and ventricular phenotypes in cardiac
chamber dominance. Alternative possibilities include: cell-cycle withdrawal,
apoptosis, delayed AP differentiation or switch to a non-cardiac fate. A
quantitative assessment of the role played by these mechanisms is not yet
available. However, it is unlikely that atrial precursors exposed to BMS493
would take on mesodermal fates other than the cardiac, because at the stage
when we performed these experiments (HH6) cardiac precursors are already
determined as such (Montgomery et al.,
1994). Therefore, although multiple mechanisms can contribute to
cardiac chamber dominance after changes in RA status, the evidence strongly
indicates that conversion between atrial and ventricular does play a role in
this process.
Role of RA signalling after determination of AP fates
RALDH2-null embryos display an abnormal ventricular phenotype as early as
8.5 dpc, suggesting a role for RALDH2 in ventricles at this stage
(Niederreither et al., 2001).
However, at this time no mouse Raldh2 expression can be detected in
wild-type ventricles (Moss et al.,
1998
). Moreover, although RA diffuses to several hundred
micrometers (Eichele and Thaller,
1987
), there is no evidence, before 12.5 dpc, for an endogenous RA
response in the ventricles of RA-indicator embryos
(Moss et al., 1998
).
Our results suggest an explanation for this apparent paradox. In the chick
embryo, stages HH8-10 constitute a previously undetected window for transient
expression of the chick Raldh2 gene in ventricular precursors before
fusion of cardiac primordia (Fig.
1, Fig. 3C,
Fig. 7). Because cardiac AP
fates are already determined at HH8 (Fig.
6), exposure of anterior cardiac precursors to RA at HH8-9 must
serve a developmental program distinct from AP patterning. Expression of
Raldh2 in ventricular precursors prior to fusion of cardiac primordia
may activate RA-dependent pathways later in chicken ventricles. It remains to
be established by fate-mapping whether mouse ventricular precursors also
express Raldh2 before cardiac fusion. If this proves to be the case,
the RALDH2 caudorostral wave may constitute the long sought non-epicardial
source of RA inhibiting precocious differentiation and maintaining
proliferation in mouse 8.5 to 9.5 dpc ventricles
(Kastner et al., 1997).
RALDH2 and cardiac AP differentiation: updating the model
A few years ago we proposed a model for cardiac AP patterning based on
selective signalling by RA (Rosenthal and
Xavier-Neto, 2000; Xavier-Neto
et al., 2001
). According to the model, RA signalling in posterior
cardiac precursors determines the sino-atrial fate, whereas absence of it
determines ventricular and conotruncal fates. Our data support the model as
proposed initially and also refine it. New findings include description of
tissue sources of RA for cardiac AP patterning and evidence for active roles
of cardiac precursors in the interpretation of RA concentrations. Paraxial and
lateral mesoderm are probable sources of RA for the specification of
sino-atrial identities, whereas RA in the anterior lateral mesoderm is
critical for expression of Amhc1 and determination of the sino-atrial
fate. Our results suggest that cardiac precursors must read RA concentrations
in a stage-dependent fashion. In fact, at stage HH7, anterior cardiac
precursors at the edge of RALDH2 expression must be exposed to RA
concentrations much higher than the ones experienced by posterior precursors
at earlier stages (Fig. 7E),
and yet they do not differentiate in sino-atrial cells, indicating that there
is no single RA threshold that will, at all times, push a given cardiac
precursor towards a sino-atrial fate.
In summary, our results are consistent with a two-step model of cardiac AP patterning. First, posterior cardiac precursors are specified to a sino-atrial fate by low concentrations of RA reaching the posterior cardiac field through diffusion from lateral and paraxial mesoderm. Later, posterior cardiac precursors commit irreversibly to a sino-atrial fate in response to increased concentrations of RA produced by the caudorostral wave of RALDH2.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work
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