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The region with the potential to form the heart has traditionally been called the heart field. This region can be approximated by, but is not identical to, the expression domain of the early cardiac gene Nkx2.5. The region expressing Nkx2.5 does not change in size, although there are major shape changes and a subdivision of the region into non-myogenic and myogenic lineages. Using a variety of embryo manipulations, we have sought to determine whether cellular interactions could change the size of the initial Nkx2.5-expressing region and thus change the size of the heart. We have shown that if the heart is isolated from the dorsal half of the embryo, the volume of tissue expressing myocardial differentiation markers increases, indicating that signals restricting the size of the heart come from the dorsal side. Despite the change in myocardial volume, the non-myogenic heart lineages are still present. The ability of dorsal tissues to restrict the size of the heart is further demonstrated by fusing two Xenopus embryos shortly after gastrulation, generating twinned embryos where the heart of one embryo would develop adjacent to different tissues of the second embryo. The final size of the differentiated heart was markedly reduced if it developed in close proximity to the dorso-anterior surface of the head but not if it developed adjacent to the flank or belly. In all cases, the manipulations that restricted the size of the myocardium also restricted the expression of Nkx2.5 and GATA-4, both key regulatory genes in the cardiogenic pathway. These results provide evidence for a model in which signals from dorso-anterior tissues restrict the size of the heart after gastrulation but before neural fold closure.
Fig. 1 A comparison of tissuespecific
markers in ventral
explants, dorsal explants, and
whole embryos. (A) Explants were
generated by cutting at the lower
boundary of the cement gland (1)
and presumptive heart (red) and
cutting at the closed blastopore
(2). This left a dorsal explant
(blue) and ventral explant (yellow
and red). Normal embryos (B, E,
H), dorsal explants (C, F, I), and
ventral explants (D, G, J) are all
shown at the same age (stage 30)
and magnification, with anterior
to the left and dorsal side up.
Neural-specific b tubulin staining
(n) is prominent in this embryo (B)
and dorsal explant (C), but only
traces are found in the ventral
explant (D). Myosin-binding
protein C marks both the somites
(s) and heart (h) in embryos. No
heart staining was found in dorsal
explants (F); in ventral explants
(G), both heart and somite
staining were seen but somite
staining was restricted to the
posterior lateral edges of the
explant. cTnI transcripts are only
detected in the heart of normal
embryos (H) and in all cases the
dorsal explants had no heart (I),
whereas all ventral explants did
(J). c, cement gland; e, eye.
Fig. 2 The heart is expanded in ventral explants. Cross-sections
through the hearts from embryos (A, C, E) and ventral explants (B,
D, F) show that the heart is expanded in ventral explants. Dorsal is
up in all cases. Sections stained for cardiac troponin I transcripts
show that the myocardium (m) is thinner and has better looping
morphology in this embryo (A) than in explants (B). Nkx2.5
transcripts can be detected in the myocardium and dorsal
pericardium (p) of embryos as well as some pharyngeal endoderm
staining above the heart. Although it is difficult to discern distinct
structures in the expanded Nkx2.5 expression domain of ventral
explants (D), the dorsal pericardial region appears thickened. An
enlarged view of embryos (E) and explants (F) stained for cTnI
expression shows that the pericardial layer (p) adjacent to the
stained myocardium (m) appears thickened. e, endocardium.
Fig. 3 The myocardial thickening is
along the full anterior-posterior
axis of the heart and there is no
loss of pericardial tissue. Sections
through the anterior-posterior axis
of the heart roughly corresponding
to the anterior heart tube (outflow
tract, oft), central heart tube
(ventricle, v), and posterior heart
tube (atrial, a) show that the
myocardial (m) thickening, as
detected by cTnI expression (AâF),
is thicker in explants (DâF) than in
normal embryos (AâC). Hand1 is
expressed in the pericardium (p),
and asymmetrically in the
myocardium (m). The pericardial
expression demonstrates that there
is no loss of pericardial tissue in
explants (JâL) as compared to
embryos (GâI). e, endocardium.
Fig. 4 The expression domains of GATA-4, Nkx2.5, and Hand1 are
expanded in ventral explants prior to differentiation. Stage 25
embryos (A, C) and ventral explants (B, D) are all positioned with
dorsal up and anterior to the left. The dorsal extent of staining for
GATA-4 transcripts is roughly equivalent in the embryo (A) and
ventral explant (B). However, the staining in ventral explants
extends much further posterior. In ventral explants stained for
Nkx2.5 transcripts (D), there is no anterior-posterior expansion of
the expression domain but there is a dorsal expansion of Nkx2.5
transcripts compared to normal embryos (C). Staining for Hand1
transcripts shows expression in the lateral mesoderm of a stage 34
embryo (E) and ventral explant (F). The posterior expansion of
Hand1 staining extends to the posterior end of the explant, and
there does not appear to be a graded reduction in staining as seen in
embryos.
Fig. 5 The heart is reduced in size when it develops in close
proximity to dorsal tissues. Removing surface ectoderm from two
stage 13 embryos and fusing them together at the wound site
generated twinned embryos. (A) An embryo with the surface
ectoderm removed but not fused to another embryo develops
normally and has a normal heart (arrow) as judged by wholemount
in situ hybridization for cTnI transcripts. (B) When embryos
were fused such that the heart developed close to the flank of the
other embryo, the heart developing near ventral tissues (b; arrows)
was apparently normal. (C, D) When embryos were fused so that
the heart developed in close proximity to the head of another
embryo (b), the heart near the head was much reduced in size
compared to the heart of the twin. e, eye; h, heart; p, endogenous
pigment.
Fig. 6 Proximity to dorsal tissues
caused a reduction in the
expression of both GATA-4 and
Nkx2.5 near the fusion site. Crosssections
through stage 25/26
embryos that have been stained by
whole-mount in situ hybridization
in order to localize GATA-4 (A, D,
G, J) and Nkx2.5 (B, E, H, K)
transcripts. A cartoon is provided
for each experiment to help with
orientation of the sections (C, F, I,
J). In sections of control embryos
(A, B), with the plane of the
section illustrated by the black line
(C), both of these transcripts are
localized to a crescent on the
ventral side with equal expression
of Nkx2.5 and GATA-4 on either
side of the midline (red line).
Twinned embryos were generated
where the heart-forming region of
the top embryo was fused to the
anterior dorsal surface (F) or the
mid-dorsal surface (I) of the
bottom embryo. GATA-4
expression is reduced in regions
fused to anterior dorsal structures
(D) and mid-dorsal structures (G).
The levels of Nkx2.5 transcripts
are reduced in regions fused to
anterior dorsal structures (E) but
not in regions fused to mid-dorsal
structures (spinal cord region; H).
The cartoon illustrating the fusion
to the mid-dorsal region (I)
illustrates the result obtained for
Nkx2.5 but not GATA-4, as GATA-
4 was inhibited by the mid-dorsal
structures. Both GATA-4 (J) and
Nkx2.5 (K) expression is normal in
twinned embryos where the heartforming
region of the top embryo
was fused to the ventral belly region
of the bottom embryo (L).
