Raffin M et al. (2000), Subdivision of the cardiac Nkx2.5 expression do...

XB-ART-11553

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.

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Species referenced: Xenopus
Genes referenced: actc1 actl6a myl1 myl2 nkx2-5 tnnt2

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	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 22 Nkx2.5 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.