Katsev S et al. (2000), BMP signaling is required for heart formation i...

XB-ART-10565

Dev Biol 2000 Aug 15;2242:226-37. doi: 10.1006/dbio.2000.9802.

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BMP signaling is required for heart formation in vertebrates.

Shi Y, Katsev S, Cai C, Evans S.

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In these studies, we have taken advantage of a transient transgenic strategy in Xenopus embryos to demonstrate that BMP signaling is required in vivo for heart formation in vertebrates. Ectopic expression of dominant negative Type I (tALK3) or Type II (tBMPRII) BMP receptors in developing Xenopus embryos results in reduction or absence of heart formation. Additionally, blocking BMP signaling in this manner downregulates expression of XNkx2-5, a homeobox gene required for cardiac specification, prior to differentiation. Notably, however, initial expression of XNkx2-5 is not affected. Mutant phenotypes can be rescued by co-injection of mutant with wild-type receptors or co-injection of mutant receptors with XSmad1, a downstream mediator of BMP signaling. Whole-mount in situ analyses indicate that ALK3 and XSmad1 are coexpressed in cardiogenic regions. Together, our results demonstrate that BMP signaling is required for maintenance of XNkx2-5 expression and heart formation and suggest that ALK3, BMPRII, and XSmad1 may mediate this signaling.

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Species referenced: Xenopus laevis
Genes referenced: bmp4 bmpr1a bmpr2 myl2 nkx2-5 smad1 tbx2 tnni3

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	FIG. 1. Whole-mount RNA in situ analysis of heart differentiation markers following injection of dominant negative BMP receptor constructs. Embryos were injected into one cell at the two-cell stage with CS2n-bgal, tBMPRII, or tALK3, harvested at various stages postdifferentiation (St. 27/28), and stained for expression of mRNAs encoding XMLC2 or XcTnI. Embryos shown are anterior ventral views. In each series, the leftmost embryo is a control injected with CS2n-bgal, illustrating normal heart development. In embryos injected with DNAs encoding tBMPRs, note abnormal fusion and reduced or absent staining on one side of the embryo. Later stage embryos (C and D) exhibit distinctly abnormal heart morphology. Embryos shown were stained for XMLC2 mRNA, with the exception of the embryos shown in A, 5 and B, 5, which were stained for XcTnI mRNA. Comparable results were obtained with either marker. C, cement gland. White arrows indicate asymmetric staining. (A) St. 29/30: (1) CS2n-bgal injected, (2) tBMPRII injected, (3) tALK3 injected, (4) tALK3 injected, (5) tALK3 injected. (B) St. 31/32: (1) CS2n-bgal injected, (2) tBMPRII injected, (3) tALK3 injected, (4) tALK3 injected, (5) tALK3 injected. (C) St. 33/35: (1) CS2n-bgal injected, (2) tBMPRII injected, (3) tALK3 injected. (D) St. 38: (1) CS2n-bgal injected, (2) tBMPRII injected, (3) tBMPRII injected, (4) tALK3 injected.
	FIG. 2. Section analyses of embryos stained by whole-mount in situ for expression of XMLC2 mRNA. Sections shown are from the stage 35 embryos shown in Fig. 1C. (A) Control-injected embryo, showing normal heart formation. (B) tBMPRII-injected embryo, exhibiting abnormal cardiac development, with rather than a single uniform heart tube, a bipartite structure, in which one side exhibits both reduced heart tissue and absence of a lumen (green arrowhead). (C) tALK3-injected embryo, in which staining for expression of XMLC2 mRNA reveals partially fused heart tubes. E, endocardium; Mi, midgut; My, myocardium; N, notochord; NT, neural tube; O, otic vesicle.
	FIG. 3. Whole-mount RNA in situ analysis of XNkx2-5 expression following injection of dominant negative BMP receptor constructs. Embryos were injected into one cell at the two-cell stage with CS2n-bgal, tBMPRII, or tALK3 harvested from stages 14 through 30, and stained for expression of XNkx2-5 mRNA. Embryos in A are anterior views. The remaining images show anterior ventral views. In each, the leftmost embryo is a control injected with CS2n-bgal, illustrating normal XNkx2-5 expression. Following injection with either tBMPR, embryos harvested from stages 14 through 20 exhibited normal XNkx2-5 expression, as represented in A and B. Embryos from later stages, however, exhibited asymmetric XNkx2-5 staining, at both pre- and postdifferentiation stages, as represented in C and D. Embryos shown were injected with DNA encoding tBMPRII. Comparable results were obtained with tALK3. (A) St. 15, (B) St. 19, (C) St. 25, (D) St. 28.
	FIG. 4. Section analyses of embryos stained by whole-mount in situ for expression of XNkx2-5 mRNA. Green arrowheads indicate regions where XNkx2-5 staining is reduced in response to ectopic expression of tBMPRs. (A) Control St. 25 (predifferentiation) embryo, demonstrating XNkx2-5-positive staining in both cardiogenic mesoderm and pharyngeal endoderm. (B) St. 25 embryo which was injected with tALK3, exhibiting downregulation of XNkx2-5 in both pharyngeal endoderm and cardiogenic mesoderm. (C) St. 29/30 embryo (postdifferentiation) which was injected with tALK3, exhibiting asymmetric downregulation of XNkx2-5 in cardiac mesoderm. CM, cardiogenic mesoderm; NT, neural tube; Mi, midgut; N, notochord; PE, pharyngeal endoderm.
	FIG. 5. Whole-mount in situ analysis of ALK3 and Smad 1 mRNA expression during Xenopus development. To examine expression of ALK3 and Smad1 in cardiogenic regions during development, we performed whole-mount RNA in situ analyses. Overall, expression of both mRNAs was relatively weak, yet specific staining was observed. Cardiogenic regions are indicated by black arrowheads (for reference, compare to staining for XNkx2-5 as seen in Fig. 3). (A–D) Embryos stained for expression of ALK3 mRNA. (E–H) Embryos stained for expression of Smad1 mRNA. The staining patterns of the two genes are overlapping, but distinct. In stage 21/22 and early tailbud embryos, staining is observed in both anterior and posterior regions. This pattern overlaps with that previously observed for BMP4 and BMPRII (Fainsod et al., 1994; Frisch and Wright, 1998). ALK3 is strongly expressed in eye and is also expressed in neural tube, head, and pharyngeal structures. Smad1, as previously published (Thomsen, 1996), is relatively strongly expressed in migrating cephalic neural crest and its derivatives, neural tube, head, and eye. For A, B, E, and F, upper embryos are shown in anterior view, oriented with dorsal at the top; lower embryos are ventral views, oriented with anterior at the top. For C, D, G, and H, upper embryos are lateral views, oriented with anterior at the top; lower embryos are ventral views, oriented with anterior at the top. (A and E) St. 17/18, (B and F) St. 21/22, (C and G) St. 25/26, (D and H) St. 28/29.