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The heart develops from a linear tubular precursor, which loops to the right and undergoes terminal differentiation to form the multichambered heart. Heart looping is the earliest manifestation of left-right asymmetry and determines the eventual heart situs. The signalling processes that impart laterality to the unlooped heart tube and thus allow the developing organ to interpret the left-right axis of the embryo are poorly understood. Recent experiments in zebrafish led to the suggestion that bone morphogenetic protein 4 (BMP4) may impart laterality to the developing heart tube. Here we show that in Xenopus, as in zebrafish, BMP4 is expressed predominantly on the left of the linear heart tube. Furthermore we demonstrate that ectopic expression of Xenopus nodal-related protein 1 (Xnr1) RNA affects BMP4 expression in the heart, linking asymmetric BMP4 expression to the left-right axis. We show that transgenic embryos overexpressing BMP4 bilaterally in the heart tube tend towards a randomisation of heart situs in an otherwise intact left-right axis. Additionally, inhibition of BMP signalling by expressing noggin or a truncated, dominant negative BMP receptor prevents heart looping but allows the initial events of chamber specification and anteroposterior morphogenesis to occur. Thus in Xenopus asymmetric BMP4 expression links heart development to the left-right axis, by being both controlled by Xnr1 expression and necessary for heart looping morphogenesis.
FIG. 1. BMP4 expression during early heart morphogenesis. Whole-mount in situ hybridisation to endogenous BMP4 RNA at stages 30
(A), 33 (E), and 35 (I). (B, C, and D) Transverse sections through the anterior, middle, and posterior, respectively, of the heart tube of the stage
30 embryo pictured in A. The future cardiac tissue is present as a layer under the ventral surface of the embryo. BMP4 expression is uniform
along the left–right axis at this stage. (F, G, and H) Transverse sections through the anterior, middle, and posterior, respectively, of the heart
tube of the stage 33 embryo pictured in E. Note that at this stage the cardiac tissue forms a tube at the posterior end (H). Furthermore BMP4
expression is asymmetric, being present more on the left side of the tube than the right (H). (J, K, and L) Transverse sections through the
anterior, middle, and posterior, respectively, of the heart tube of the stage 35 embryo pictured in I. Note that BMP4 expression is detected
throughout the heart precursor, predominantly in the dorsal area of the presumptive atrium (a) and ventricle (v). (N) A longitudinal section
through the heart region of a stage 32 embryo. Note the predominantly left-sided expression of BMP4 in the posterior region of the heart
(red arrow). (O) An enlargement of the region in H demarked by a dotted square. Dotted lines in A, E, I, and M correlate with the respective
plane of sections in B–D, F–H, J–L, and N.
FIG. 2. Xenopus nodal related 1 (Xnr1) but not Xnr2 affects the asymmetric expression of BMP4 in the linear heart tube. (A) Section
through the heart of a stage 34 embryo previously injected with XNR1 DNA mixed with b-galactosidase RNA into the dorsal right
blastomere at the 4-cell stage and stained for BMP4 expression (purple) and b-gal activity (light blue). The blue arrow points at up-regulation
of BMP4 expression on the right side of the linear heart. (B) Section through the heart of a stage 34 embryo previously injected with XNR2
DNA mixed with b-galactosidase RNA into the dorsal right blastomere at the 4-cell stage and stained for BMP4 expression (purple) and b-gal
activity (light blue). The red arrow points at the unaffected BMP4 expression in the heart tube, which at this stage is predominantly on the
left side.
FIG. 3. Embryos expressing green fluorescent protein from the Xenopus light chain 2 (XMLC2) and cardiac actin promoters. (A–C)
Transgenic embryos expressing GFP from the XMLC2 promoter. (A) Stage 32 embryos stained for GFP RNA expression by whole-mount
in situ hybridisation. (B) Section through the heart of the embryo shown in A. (C) Live stage 40embryo expressing GFP from the XMLC2
promoter. GFP fluorescence in the beating heart is clearly visible at this stage. (D–G) Transgenic embryos expressing GFP from the cardiac
actin promoter. (D) Stage 32embryo stained for GFP RNA expression by whole-mount in situ hybridisation. Note expression in the heart
(h) and somites (s). (E) Section through the heart of the embryo shown in D. (F) Live stage 42embryo expressing GFP from the cardiac actin
promoter. GFP fluorescence is clearly visible in the somites (s), heart (h), and muscles of the mouth (m). (G) View of the heart region in a
live stage 45tadpole, expressing GFP from the cardiac actin promoter. At this stage, GFP fluorescence allows the fine structures of the
ventricle (v) and outflow tract (oft) to be visible. The gallbladder (gb) exhibits autofluorescence under GFP illumination.
FIG. 4. Expression of BMP4 throughout the heart tube randomises cardiac situs. (A and B) Ventral views of GFP fluorescence in stage 40
transgenic embryos expressing BMP4 from the XMLC2 promoter and GFP from the cardiac actin promoter. (A) Dextrocardia, with a
right-sided ventricle (v), and (B) levocardia, with a left-sided ventricle (v). (C) Transverse section though the middle of an embryo expressing
BMP4 from the XMLC2 promoter, showing an intact notochord (n). (D) RT-PCR analysis of embryos transgenic for BMP4 shows that
transgene expression is seen in embryos exhibiting levocardia (lanes under the red line) and dextrocardia (lanes under the purple line). (1)
Plasmid control, (2) blank control, (g) GFP1 embryo not transgenic for BMP4. (E and F) Right and left views, respectively, of a wild-type
stage 32 embryo stained for Pitx2 expression by whole-mount in situ hybridisation, showing left-sided expression. (G and H) Right- and
left-sided views of a transgenic stage 32 embryo expressing BMP4 from XMLC2 promoter (blue stain), showing unaltered left-sided
expression of Pitx2 (purple stain). (I and J) Right- and left-sided views of a transgenic stage 32 embryo expressing BMP4 from the cardiac
actin promoter (blue stain) in the heart and somites (s). The embryo appears generally abnormal, with Pitx2 (purple stain) expressed
bilaterally, but predominantly on the right, suggesting randomisation of the left–right axis.
FIG. 5. Expression of noggin throughout the heart tube prevents
dextral cardiac looping, but does not affect chamber specification.
(A) Stage 34 embryo expressing noggin from the cardiac actin
promoter and stained for noggin (blue) and cardiac actin (purple) by
double whole-mount in situ hybridisation. Note normal morphology
of embryo. (B) Stage 40 double-transgenic embryo expressing
both GFP and noggin in the heart and somites from the cardiac
actin promoter. Note the developing morphology of chambers in
the unlooped heart. For comparative wild-type stage views see Figs.
3C and 3F. Atrium (a), ventricle (v), outflow tract (oft). (C) Nontransgenic
stage 36 embryo stained for endogenous cardiac actin
RNA by whole-mount in situ hybridisation. Note left-sided ventricle
(v), relative to the position of the outflow tract (oft). (D) Stage
36 transgenic embryo expressing noggin from the cardiac actin
promoter and stained for noggin expression by whole-mount in situ
hybridisation (dark blue stain). Note the persistence of an unlooped
heart with the atrial precursor area (a) caudal to ventricular
precursor (v). Also note the presence of an atrioventricular constriction.
(E) Ventral view of nontransgenic stage 35 whole-mount in
situ hybridisation to XTbx5 antisense RNA probe. XTbx5 is a
transcription factor expressed predominantly in the atrial precursors
and venous pole of the heart. Note sinus venosus (sv) staining.
(F) Stage 35 transgenic embryo expressing noggin from the cardiac
actin promoter and stained for noggin (light blue stain) and Tbx5
(purple stain) by whole-mount double in situ hybridisation.
