XB-ART-9384Dev Biol April 1, 2001; 232 (1): 191-203.
A role for BMP signalling in heart looping morphogenesis in Xenopus.
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.
PubMed ID: 11254357
Article link: Dev Biol
Genes referenced: actc1 actl6a bmp4 myl2 nodal nodal1 nodal2 nog pitx2 tbx5
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|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 40 embryo 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 32 embryo 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 42 embryo 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 45 tadpole, 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.|