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Dev Biol
2000 Feb 15;2182:326-40. doi: 10.1006/dbio.1999.9579.
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Subdivision of the cardiac Nkx2.5 expression domain into myogenic and nonmyogenic compartments.
Raffin M
,
Leong LM
,
Rones MS
,
Sparrow D
,
Mohun T
,
Mercola M
.
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Nkx2.5 is expressed in the cardiogenic mesoderm of avian, mouse, and amphibian embryos. To understand how various cardiac fates within this domain are apportioned, we fate mapped the mesodermal XNkx2.5 domain of neural tube stage Xenopus embryos. The lateral portions of the XNkx2.5 expression domain in the neural tube stage embryo (stage 22) form the dorsal mesocardium and roof of the pericardial cavity while the intervening ventral region closes to form the myocardial tube. XNkx2.5 expression is maintained throughout the period of heart tube morphogenesis and differentiation of myocardial, mesocardial, and pericardial tissues. A series of microsurgical experiments showed that myocardial differentiation in the lateral portion of the field is suppressed during normal development by signals from the prospective myocardium and by tissues located more dorsally in the embryo, in particular the neural tube. These signals combine to block myogenesis downstream of XNkx2.5 and at or above the level of contractile protein gene expression. We propose that the entire XNkx2.5/heart field is transiently specified as cardiomyogenic. Suppression of this program redirects lateral cells to adopt dorsal mesocardial and dorsal pericardial fates and subdivides the field into distinct myogenic and nonmyogenic compartments.
FIG. 1. Expression domains of XNkx2.5 and myocardial differentiation
markers during heart tube formation. (A, B, and C)
Embryos were stained for expression of XNkx2-5, XMLC-2a,
XMHCa, and XTn-Ic genes at stages 28 (A) and 32 (B) by RNA
whole-mount in situ hybridization. Stained embryos were embedded
in paraffin wax and (C) shows representative transverse
sections through the anterior, middle, and posterior portions of
a stage 28 heart region.
FIG. 1. Expression domains of XNkx2.5 and myocardial differentiation
markers during heart tube formation. (A, B, and C)
Embryos were stained for expression of XNkx2-5, XMLC-2a,
XMHCa, and XTn-Ic genes at stages 28 (A) and 32 (B) by RNA
whole-mount in situ hybridization. Stained embryos were embedded
in paraffin wax and (C) shows representative transverse
sections through the anterior, middle, and posterior portions of
a stage 28 heart region.
FIG. 2. 3D models of linear heart tube formation. 3D models showing the expression domains for XNkx2.5 (gray) and myocardial
differentiation markers XMLC-2a and XTn-Ic (red) during heart tube formation. Endocardial tissue (yellow) was identified solely by
histological criteria and is most prone to distortion during the wax embedding procedure. The dorsal boundary of XNkx2.5 expression is
necessarily imprecise because of the low levels of transcript (see Fig. 1C and text), while the boundaries of myocardial differentiation are
relatively precise. (A) Stage 28 embryo showing progressive dorsal constriction of the XNkx2-5 domain along the AP axis; tissue ventral to
the constriction expresses cardiac muscle markers. (B) Stage 32 embryo showing dorsal closure of the linear heart tube. (C) A posterior
portion of the Stage 32 model, seen from a lateral oblique view to show attachment of the heart tube to the pericardial roof via the dorsal
mesocardium.
FIG. 3. Fate map of the XNkx2.5 expression domain. (A and B) Three small multicellular patches within the XNkx2.5 domain of early tail-bud
stage (stage 22) embryos were microinjected with the lipophilic dye, CM-DiI. Injections were positioned in a line extending dorsally from the
posterior margin of the cement gland with site A aligned with the bottom of the eye vesicle as visible externally, site B aligned with the dorsal
border of the cement gland, and site C just off the ventral midline. (C) XNkx2.5 mRNA in a stage 22 embryos. Site A corresponded to the dorsal
border of the stage 22Nkx2.5 domain, site B corresponded to the lateral region, and site C was near the ventral midline. (D, E, and F) The positions
of cells labeled at the three sites were examined at stage 28 in transverse Vibratome sections. (G) The data set (n 5 7 for site A, n 5 15 for site
B, and n 5 12 for site C) is summarized in the schematic (also in Table 1). Purple, yellow, and blue hatchlines represent locations of cells injected
at sites A, B, and C, respectively. Labeling of overlying ectoderm and underlying endoderm occasionally occurs and can serve as a reference point
for the injection site (white arrowheads in D and E). Note that mesodermal cells labeled by A- and B-site injections (black arrowheads in D and
E) shift ventrally between stages 22 and 28 such that cells from site A contribute to the dorsolateral edge of pericardial roof while cells from site
B contribute to dorsal mesocardium and the adjoining portion of the pericardial roof (and, infrequently, the adjoining myocardium). Site C cells,
in contrast, form myocardium and endocardium. Thus, cells within the lateral portion of the XNkx2.5 domain at stage 22 (sites A and B) shift
ventrally but do not lose XNkx2.5 expression as the sheet of cardiac cells folds to form the heart tube. Dorsal is up in all images. Abbreviations:
en, endoderm; me, mesocardium; my, myocardium; se, surface ectoderm; pc, pericardium.
FIG. 4. Myocardial differentiation in lateral heart field (LHF) mesoderm is suppressed by dorsal tissue. Explants were dissected from stage
22 embryos, cultured to stage 41, and scored for rhythmic beating. For reference to the dye injection sites (Fig. 3), the border between the
ventral heart field (VHF) and the LHF explants was positioned just ventral to the site B microinjections such that LHF explants contained cells
labeled by microinjection at site B (prospective dorsal pericardium and dorsal mesocardium) and VHF explants encompassed site C
(prospective myocardium). Note that stage 22 LHF explants formed beating tissue efficiently when maintained in isolation from dorsal and
ventral tissues. When explants included dorsolateral somitic tissue (LHF-DL) or somitic, notochordal, and neural tissues (LHF-N) myocardial
differentiation was significantly reduced. Importantly, tissue just dorsal to the XNkx2.5 domain never formed beat in culture (DL explants),
indicating that it resides outside the classically defined heart field. Numbers of explants are given in parentheses and error bars indicate
standard error of the mean.
FIG. 5. Suppression of LHF myogenesis by individual dorsal tissues
in recombinants. Ventral and lateral heart field/Nkx2.5 field explants
at stage 22 were recombined with dorsal tissues as indicated
and beating incidence is presented. Note that neural tubes showed
strong anticardiomyogenic activity when recombined with lateral
heart field tissues. Lack of an effect on ventral field explants may
reflect commitment by this stage. Numbers of recombinants are
given in parentheses and error bars indicate standard error of the
mean.
FIG. 6. Differential regulation of contractile gene expression by
dorsal tissue. (A–C) Explants were isolated at stage 22, cultured,
and subsequently assayed for gene expression and beating (see
Materials and Methods). Contractile protein mRNA expression is
represented by the bars in the histogram. The beating frequency is
indicated by the dashed horizontal line. XNkx2.5 is normally
expressed in the LHF region in intact embryos (Figs. 1–3) and is
likewise expressed in the corresponding tissue when explanted
either with (A) or without (B) dorsal tissues (neural tube and dorsal
mesoderm). Contractile protein genes are not normally expressed
in LHF cells (Figs. 1–3). Absence of ventral (prospective myocardial)
tissue caused expression of contractile protein genes ectopically in
the LHF region (LHF-N explants, A), suggesting that signals from the
prospective myocardium suppress myocardial genes in the lateral
portion of the heart field. Note that the fraction of explants
expressing Tn-Ic and cardiac actin transcripts exceeded beating
incidence, while the fraction expressing XMLC-2a coincided with
beating incidence. Removal of neural and dorsal mesodermal tissue
in LHF explants (B) increased the incidence of expression of genes
(XTn-Tc, XMHCa, and XMLC-2a) such that all markers were
expressed in a large proportion of the explants. Beating incidence
also increased. (C) Control explants of prospective myocardium
(VHF) generally beat and expressed all markers. Expression of all
markers, with the exception of cardiac actin, was in the LHF region
(see Fig. 7). For cardiac actin, only heart and not somitic expression
was scored. Results are statistically significant (P , 0.05).
FIG. 7. Examples of heart gene expression in isolated explants. Examples of the most frequently observed expression patterns of explants
described in Fig. 6. Note the absence of XMLC-2a and XMHCa in explants containing neural, notochordal, and somitic tissues (LHF-N
explants). The monoclonal antibody CT3 was used to recognize Tn-Tc.
FIG. 8. Nonbeating LHF and LHF-N explants form tubes with lumens lined by endothelial cells. Explants were removed at stage 22, cultured
to stage 40, and subsequently separated into beating and nonbeating classes at which time they were probed for XTn-Ic mRNA (blue stain)
by in situ hybridization. Beating LHF (A) as well as nonbeating LHF (B) and LHF-N (C) explants formed a myocardial tube lined with unstained,
endothelial cells (arrows) that resembles a normal heart tube in cross section.