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The rightward looping of the primary heart tube is dependent upon upstream patterning events that establish the vertebrate left-right axis. In Xenopus, a left-sided Vg1 signaling pathway has been implicated in instructing cells to adopt a 'left-sided identity'; however, it is not known whether 'right-sided identity' is acquired by a default pathway or by antagonism of Vg1 signaling. Here, we propose that an antagonistic, BMP/ALK2/Smad-mediated signaling pathway is active on the right side of the Xenopus embryo. Truncated ALK2 receptor expression on the right side of the blastula elicits heart reversals and altered nodal expression. Consistent with these findings, constitutively active ALK2 (CA-ALK2) receptor expression on the left side of the blastula also elicits heart reversals and altered nodal expression. Coexpression of CA-ALK2 with mature Vg1 ligand results in predominantly left-sided nodal expression patterns and normal heart looping, demonstrating that the ALK2 pathway can 'rescue' left-right reversals that otherwise occur following right-sided misexpression of mature Vg1 ligand alone. Results with chimeric precursor proteins indicate that the mature domain of BMP ligands can mimic the ability of the ALK2 signaling pathway to antagonize the Vg1 pathway. Consistent with the observed antagonism between BMP and Vg1 ligands, left-sided ectopic expression of Xolloid results in heart reversals. Moreover, ectopic expression of Smad1 or Smad7 identified two downstream modulators of the BMP/ALK2 signaling pathway that also can regulate cardiac orientation. Collectively, these results define a BMP/ALK2-mediated pathway on the right side of the Xenopus embryo and, moreover, suggest that left-right patterning preceding cardiac morphogenesis involves the activation of two distinct and antagonistic, left- and right-sided TGF(beta)-related signaling pathways.
Fig. 1. Perturbation of nodal
expression by CA-ALK2.
Xenopus blastulae were injected
with CA-ALK2 RNA alone or in
combination with AVg RNA, as
described in Table 3. Embryos
were collected at stages 23-25
and processed for whole-mount
in situ hybridization to detect
nodal expression in the lateral
plate mesoderm (arrows). The
orientation of embryos shown is a
dorsal view, with anterior to the
right. Control embryos showed
normal, left-sided nodal
expression (A). Approximately
half of embryos injected with
CA-ALK2 RNA showed bilateral
(B), right only, or absent nodal
expression. Coinjection of AVg
RNA with CA-ALK2 RNA
resulted in embryos showing left
only (C) or bilateral nodal expression that was enhanced on the left
side relative to the right. See Table 3 and text for details.
Fig. 2. Non-cardiac defects elicited by expression of CA-ALK2. Day
5 tadpoles resulting from injection of CA-ALK2 RNA in left (A) or
right (B) cell lineages were separated into two groups prior to
scoring heart orientation. The orientation of embryos shown is a
dorsal view, with anterior at the top. Some embryos showed normal
morphology, with the exception of reversed heart situs and
ambiguous gut coiling, as indicated in Table 2 and text. Embryos
classified as having posterior mesodermal defects are shown for each
group, and the percentage of heart reversals for these types of
embryos are reported separately from embryos not showing other
defects (Table 2). Embryos with reduced dorsoanterior development
(DAI<5) were excluded from laterality analyses. A similar range of
phenotypes was also observed following injection of tALK or Smad7
RNA, as described in the text.
Fig. 3. Cardiac orientation of
embryos injected with CA-ALK2
RNA. Embryos were injected with
CA-ALK2 RNA in right or left cell
lineages, and day 5-7 tadpoles
were stained by
immunofluorescence with
monoclonal antibody CT3 to
demonstrate heart and gut
orientation. The orientation of
embryos shown is a ventral view,
with anterior at the top. CA-ALK2
expression in right cell lineages
results in normal, rightward heart
looping and ambiguous gut coiling
(A). In contrast, CA-ALK2
expression in left cell lineages
results in reversed, leftward heart
looping and ambiguous gut coiling
(B). Coinjection of AVg RNA with
CA-ALK2 in left cell lineages
results in normal, rightward heart
looping and normal gut coiling
(C). Arrowheads point to the
conotruncus (outlet) of the heart.
Fig. 4. Schematic model of left-right axis formation in Xenopus. A
left-sided signaling pathway mediated, at least in part, by Vg1 is
activated in cells of the left-right coordinator (LRC), thereby
imparting left identity to cells in this region of the early blastula.
Right cell lineages become specified for right identity as a result of
activation of the BMP/ALK2 pathway. The Vg1 and BMP/ALK2
signaling pathways are mutually antagonistic, and a balance of
signaling between these pathways establishes the left-right axis.
