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INTRODUCTION
The vertebrate body plan develops along three geometric axes:
anterior-posterior, dorsal-ventral, and left-right. Left-right
asymmetries are not random with respect to the dorsal-ventral
and anterior-posterior axes, suggesting that the mechanisms
determining all three body axes might be linked. In sea urchin
embryos, lineage tracing and cell separation experiments
indicate that left-right asymmetry is specified with respect to
and coincident with dorsal-ventral axis specification (McCain
and McClay, 1994). In vertebrates, the left-right axis is most
evident in the heart and viscera, and the orientation of left-right
cardiac asymmetry is highly conserved (Burggren, 1988).
Examples in humans and mice indicate that left-right axis
formation may be vital to the organism since congenital heart
defects and early death often accompany problems in left-right
organization (Brueckner et al., 1991; Casey et al., 1993;
Horwich and Brueckner, 1993; Yokoyama et al., 1993). Since
the left-right axis is geometrically defined with respect to the
other two axes, it might be developmentally influenced by
mechanisms determining anterior-posterior and dorsal-ventral
axes.
Most embryonic structures are symmetric across the midline
in early development, and only display left-right asymmetries
well after dorsal-ventral and anterior-posterior differences are
visible (reviewed by Brown and Wolpert, 1990). However,
left-right axial information is set up early in development, well
before its morphological expression is discernible (reviewed
by Yost, 1994). In Xenopus laevis the heart forms from two
primordia that move to the ventral midline during gastrula and
early neurula stages and fuse to form a symmetric cardiac tube.
The cardiac tube then loops, breaking symmetry and giving
rise to an S-shaped organ in the tadpole stages (Nieuwkoop and
Faber, 1967). The left-right orientation of the heart is
dependent upon the extracellular matrix lining the blastocoel
roof of early gastrulae and appears to be transmitted to the
cardiac and visceral primordia as they move across this matrix
to the ventral midline. Experimental perturbations of the matrix
during early gastrula stages by microsurgical wounding,
treatment with RGD peptides or heparinase, result in random
orientations of the heart and gut (Yost, 1992). During early
neurula stages, when the pre-cardiac mesoderm cells move
across the matrix to the ventral midline, proteoglycan synthesis
is necessary for looping of the cardiac tube, which occurs 1.5
days later in development (Yost, 1990). Thus, early events in
the embryo appear to establish the left-right embryonic axis
well before the cardiac tube is formed.
Results presented here indicate that orientation of cardiac
left-right asymmetry is coupled with dorsal-anterior develop-
ment. The dorsal-ventral and anterior-posterior embryonic
1467
Development 121, 1467-1474 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
The left-right body axis is defined relative to the dorsal-
ventral and anterior-posterior body axes. Since left-right
asymmetries are not randomly oriented with respect to
dorsal-ventral and anterior-posterior spatial patterns, it is
possible that a common mechanism determines all three
axes in a coordinate manner. Two approaches were under-
taken to determine whether alteration in dorsal-anterior
development perturbs the left-right orientation of heart
looping. Treatments known to decrease dorsal-anterior
development in Xenopus laevis, UV irradiation during the
first cell cycle or Xwnt-8 DNA injections into dorsal blas-
tomeres, caused an increase in cardiac left-right reversals.
The frequency of left-right reversal was correlated with the
severity of dorsal-anterior perturbation and with the extent
of anterior notochord regression. Injection of Xwnt-8 DNA
into dorsal midline cells resulted in decreased dorsal-
anterior development and a correlated increase in cardiac
left-right reversals. In contrast, injection of Xwnt-8 DNA
into cardiac progenitor blastomeres did not result in left-
right reversals, and dorsal-anterior development and
notochord formation were normal. Disrupting develop-
ment of dorsal-anterior cells, including cells that give rise
to the Organizer region and the notochord, results in the
randomization of cardiac left-right asymmetry. These
results suggest dorsal-anterior development and the regu-
lation of left-right orientation are linked.
Key words: axis formation, left-right asymmetry, cardiac
development, situs inversus, Xenopus
SUMMARY
Linkage of cardiac left-right asymmetry and dorsal-anterior development in
Xenopus
Maria C. Danos and H. Joseph Yost*
Department of Cell Biology and Neuroanatomy, 4-135 Jackson Hall, 321 Church Street SE, University of Minnesota, Minneapolis,
MN 55455, USA
*Author for correspondence (e-mail: yostx001@staff.tc.umn.edu)
1468
axes are established during gastrulation by signals from cells
at the dorsal midline (for reviews, Spemann, 1938; Slack et al.,
1992). During late gastrulation, the mesoderm cells on the
dorsal midline form the notochord. During neurulation, signals
from the notochord specify cell fates along the anterior-
posterior axis of the neural tube (Hemmati Brivanlou et al.,
1990) and the dorsal-ventral axis of both the neural tube (Hatta
et al., 1991; Krauss et al., 1993; Yamada et al., 1993; Roelink
et al., 1994) and the somites (Dietrich et al., 1993; Halpern et
al., 1993; Pourquié et al., 1993).
The results presented here suggest a novel role for dorsal-
anterior midline cells: determining the orientation of left-right
cardiac asymmetries during development. Perturbation of
dorsal-anterior development, leading to diminished anterior-
dorsal structures and loss of anterior notochord, causes
reversals in the orientation of cardiac left-right asymmetry.
MATERIALS AND METHODS
Embryos
Xenopus laevis pigmented females (Xenopus I, Ann Arbor, MI) were
induced to ovulate by injection into the dorsal lymph sac of 50 units
of pregnant mare’s serum gonadotropin (Sigma), followed by 800
units of human chorionic gonadotropin (Sigma) 24 hours later. Eggs
were stripped from the ovulating females and fertilized with a minced
testis suspension in one third strength modified Ringers containing 50
µg/ml gentimycin sulfate, subsequently denoted as R/3. The fertilized
eggs were dejellied in 2% cysteine, pH 8.0. All embryo incubations
and operations were done in R/3 unless otherwise noted. Embryos
were cultured at either 21°C or 15°C and were staged according to
Nieuwkoop and Faber (1967). Heart orientation was scored in anes-
thetized embryos. Embryos and tadpoles were fixed in MEMFA (0.1
M Mops, pH 7.4; 2 mM EGTA; 1 mM MgSO4; 3.7% formaldehyde)
for 2 hours and stored in methanol at −20°C for in situ hybridization
or immunohistochemistry. To photograph hearts, embryos were
stained with MF20 (gift from David Bader) (González-Sánchez and
Bader, 1984) according to the whole mount immunohistochemistry
protocol of Hemmati-Brivanlou and Harland (1989), and epidermis
was removed from the ventral trunk of the embryos.
UV irradiation
Fertilized eggs were dejellied and placed vegetal hemispheres down
on quartz slides in R/3 within 25 minutes of fertilization and irradi-
ated with a ultraviolet (UV) mineralite lamp at 254 nm for 15-30
seconds (Scharf and Gerhart, 1980). The embryos were not disturbed
until the first cleavage had occurred and were cultured until they were
ready for scoring of DAI and heart looping. Asymmetry of the gut
was not scored in these or any of the subsequent experiments since it
does not complete development in treated embryos. A description of
the DAI scale follows: DAI 5, normal in all external respects; DAI 4,
reduced forehead, eyes smaller and sometimes joined; DAI 3, eyes
fused or cyclopic, at least some retinal pigment visible; DAI 2, no
visible retinal pigment, at least one otic vesicle present (Scharf and
Gerhart, 1983; Kao and Elinson, 1988).
Embryo injections
Four-cell stage embryos were transferred into a solution of 5% ficoll
(Sigma) in R/3. Prospective dorsal blastomeres are smaller and
pigmented more lightly than their ventral counterparts at this stage
(Nieuwkoop and Faber, 1967), and can be readily distinguished. The
two dorsal blastomeres were each injected with 100 pg of CSKA-
X8myc (Xwnt-8) DNA plasmid, or as a control the CSKA-pTCAT
DNA construct, or water (Christian and Moon, 1993). For injections
into prospective heart blastomeres, 100 pg of DNA was injected into
each of the two lateral blastomeres on the third tier adjacent to the
dorsal midline (C2 blastomeres) in 32-cell stage embryos with regular
cleavage patterns. For midline cell injections, each of the two dorsal
midline blastomeres on the third tier (C1 blastomeres) in 32-cell stage
embryos were injected. For lineage labelling, 100 pg of CSKA-X8myc
DNA and 15 ng of rhodamine dextran (polyanionic, 70×103Mrfrom
Molecular Probes in Eugene, OR) were injected per blastomere. After
injections, the embryos were transferred to R/3 and incubated until
control embryos reached stages 42 to 45 for scoring heart orientation.
Whole-mount in situ hybridizations
RNA probes for collagen II, labelled with digoxigenin-rUTP
(Boehrigner Mannheim Biochemical), were synthesized from the
p7XK500 plasmid (Amaya et al., 1993). This plasmid was linearized
with XhoI and transcribed with T7 polymerase for the antisense probe
or linearized with KpnI and transcribed with Sp6 polymerase for the
sense control. RNA probe synthesis and whole-mount in situ hybrid-
ization were performed as described by Harland (1991), with the
exception of omitting the proteinase K step. Embryos were made
transparent in benzyl benzoate/benzyl alcohol (2:1) and pho-
tographed.
RESULTS
UV irradiated embryos lack dorsal-anterior
structures and have disturbed left-right
development
To obtain embryos defective in dorsal-anterior structures, fer-
tilized eggs were briefly treated with UV radiation, and grown
to stages 42-45. Treatment of the vegetal pole of Xenopus
embryos with UV radiation during the first cell cycle blocks
formation of microtubule arrays in the embryo. This prevents
cortical rotation and embryos fail to develop dorsal-anterior
structures such as head, central nervous system, notochord, and
somites (reviewed by Gerhart et al., 1989). External defi-
ciencies in dorsal-anterior structure can be classified into a
dorsal-anterior index (DAI) (Kao and Elinson, 1988). A short
exposure time (15-30 seconds) to UV light resulted in a het-
erogeneous population of embryos ranging from DAI 5
(normal embryos) to DAI 0 (ventralized, radial embryos).
Embryos at DAI 2 or lower had either a severely diminished
heart or no discernible heart (319 of 322 embryos), so left-right
cardiac asymmetry was scored for embryos at DAI 3 or above.
Fig. 1 depicts the hearts and the external morphologies of
normal (DAI 5) and slightly dorsal-anterior reduced (DAI 4)
embryos obtained from UV treatment. In normal heart orien-
tation (Fig. 1A; DAI 5 embryo), the ventricle is on the
embryo’s left and the outflow tract on the embryo’s right; the
atrium is positioned dorsally and leads into the ventricle from
the embryo’s left side. In contrast, some embryos derived from
UV irradiation displayed reduced dorsal-anterior development
(compare Fig. 1D with 1C) and reversal of the cardiac left-right
orientation (Fig. 1B); the ventricle is on the embryo’s right, the
outflow tract is on the embryo’s left, and the atrium is located
dorsally and leads into the ventricle from the embryo’s right
side.
Left-right reversals were correlated with UV-induced loss of
dorsal-anterior structures. Embryos that had lower DAIs
showed the highest frequencies of reversed hearts (Fig. 2).
Embryos with a DAI of 4 showed a 22% reversal frequency,
while a decrease in dorsal-anterior structures to DAI 3 resulted
M. C. Danos and H. J. Yost
1469Cardiac left-right development
in a 45% reversal frequency. The DAI 5 class served as an
internal control for this experiment; UV treated DAI 5 embryos
and untreated embryos (also DAI 5) both had basal frequen-
cies of cardiac reversals (3% and 1%, respectively). In an addi-
tional control group, embryos treated with UV irradiation late
in the first cell cycle, after the cortical rotation had occurred,
developed normal dorsal anterior structures (DAI 5) and
normally oriented cardiac left-right asymmetries (29 of 29).
Thus, there was a striking correlation between the frequency
of cardiac left-right reversals and the extent of dorsal-anterior
deficiencies due to perturbation of cortical rotation during the
first cell cycle.
Misexpression of
Xwnt-8
alters cardiac left-right
orientation
UV irradiation inhibits dorsal-anterior development by
blocking the cortical rotation that occurs during the first cell
cycle. We wanted to diminish dorsal-anterior development
using another method; one that occurs later in development
than UV treatment, that can be targeted to specific cell
lineages, and that probably has an effect through a different
mechanism. Xwnt-8 ectopic expression was utilized in these
experiments to assess the effects of diminished dorsal-anterior
structures on heart orientation. Xwnt-8 is a growth factor
inducible agent that is thought to be involved in signaling the
ventral-lateral mesoderm pattern in Xenopus (Christian et al.,
1991; Christian and Moon, 1993). Ectopic expression of Xwnt-
8in dorsal cells transcribed from an injected DNA plasmid
expression construct after the mid-blastula transition (post-
MBT), results in anterior defects and a deleted or aberrant
notochord (Christian and Moon, 1993). This data suggests that
misexpression of Xwnt-8 in cells of the ‘Organizer’ field,
during the time when endogenous Xwnt-8 is expressed in
ventral-lateral cells, blocks the normal differentiation of head
and notochord (Christian and Moon, 1993).
Embryos between the four-cell and 32-cell stages were
injected in the two bilateral dorsal blastomeres near the dorsal
midline at the pigmentation boundary with 100 pg of Xwnt-8
plasmid DNA (Fig. 3). This region is fated to contribute to
dorsal structures: notochord, central neural tissue and archen-
teron (Keller, 1975, 1976; Bauer et al., 1994). Control injec-
tions were performed using CSKA-pTCAT plasmid or water.
Embryos were grown to tadpole stages and scored for DAI and
Fig. 1. Embryos derived from UV treatment, shown at stage 45. (A) Ventral view of a normal heart in a DAI 5 embryo. (B) Ventral view of a
left-right reversed heart in a DAI 4 embryo. Ventricle (v), atrium (a) and conus or outflow tract (c) are indicated; hearts were stained with
MF20 antibody and epidermis was removed. (C,D) Lateral view of a DAI 5 embryo and a DAI 4 embryo for comparison of dorsal-anterior
development. Scale bars, 0.1 mm (A and B); 1.0 mm (C and D).
Fig. 2. The frequency of cardiac left-right reversal is correlated to the
extent of dorsal-anterior deficiencies. The percentage of cardiac
reversals was scored for each DAI from UV-treated embryos (white
bars) and embryos in which Xwnt-8 was injected into dorsal cells
(grey bars). The number of embryos scored in each category are
shown beneath its abscissa.
1470
heart looping. As was seen with the UV irradiation, the extent
of diminished dorsal-anterior structures correlated with higher
frequencies of reversals. DAI 5 embryos had a basal frequency
of heart reversals (Fig. 2). In embryos with diminished dorsal-
anterior structures, scored as DAI 4 or 3, the heart reversal fre-
quencies were 24% and 45% respectively (Fig. 2).
Two different treatments, one during the first cell-cycle and
the other post-MBT, resulted in an inverse correlation between
the frequency of cardiac reversals and the extent of dorsal-
anterior development. In embryos for which the dorsal-ventral
and anterior-posterior axes were severely disrupted (DAI 3
embryos), left-right orientation was stochastically determined.
The 45% heart reversal frequency for DAI 3 embryos, from
either UV treatments or Xwnt-8 injections, was statistically
identical to the predicted frequency of randomized left-right
asymmetries (50%, P>0.25 by χ-square analysis). In these
embryos, left-right asymmetry was generated but the
mechanism that orients the left-right asymmetry with respect
to remnants of the other axes was lost.
Xwnt-8 ectopic expression in dorsal midline cells,
but not heart precursor cells, causes cardiac
reversals
Xwnt-8, as a member of the Wnt family, is thought to be a
secreted protein (Christian et al., 1991). One possible expla-
nation of heart reversals was that the Xwnt-8 expressed from
the injected plasmids was present within or secreted to the
heart precursor cells and directly altered their development. To
assess this possibility, the blastomeres in the thirty-two-cell
stage embryo that are fated to contribute to heart (Keller, 1975,
1976; Bauer et al., 1994) were injected with Xwnt-8 expression
plasmid or with water or CSKA-pTCAT plasmid as controls
(Fig. 3, blastomeres C2). To confirm that these injections
targeted the heart precursor cells, rhodamine dextran lineage
label was mixed with the DNA and injected into C2 blas-
tomeres. In accordance with the fate maps, injection of C2
blastomeres resulted in lineage labelled hearts (10 of 14
embryos), somites, lateral mesoderm, and endoderm. These
results indicated that C2 injections were into cells that con-
tributed to heart or surrounding tissue. Xwnt-8 ectopic
expression in or near heart precursor blastomeres (C2, Fig. 3)
did not result in heart reversals or in dorsal-anterior defects
(Table 1). In contrast, injection of Xwnt-8 expression plasmid
into C1 blastomeres (Fig. 3) at the dorsal midline in 32-cell
stage embryos resulted in lower DAIs and a corresponding
increase in heart reversals (Table 1). These results indicate that
ectopic expression of Xwnt-8 specifically in dorsal midline
cells, which normally give rise to part of the Organizer region
and notochord, results in altered dorsal-anterior development
and a corresponding loss of left-right orientation in the heart.
Embryos with lower DAIs lack anterior notochord
The above results suggest that dorsal midline cells, which are
fated to contribute to part of the Organizer and to notochord,
are involved in the developmental regulation of cardiac left-
right orientation. To assess the extent of notochord develop-
ment in axial-deficient embryos, in situ hybridization was
performed using a collagen II mRNA probe that stains the
notochord (Su et al., 1991; Amaya et al., 1993). Embryos
(stages 34-38), from the UV treatment and Xwnt-8 injections
described above, were fixed and in situ hybridizations were
performed (Harland, 1991). Notochords were present in DAI
4 embryos derived from both treatments (Fig. 4C,D). This is
in contrast to a report that notochords were not detected by
scanning electron microscopy of younger embryos (stages 22-
24) that had received mild UV treatment, including embryos
with very little loss of dorsal-anterior structure (DAI 4; Youn
and Malacinski, 1981). In the present study, both UV treatment
and Xwnt-8 injections during early development gave rise to
embryos that had specific deficiencies in notochord develop-
ment. Notochords in DAI 4 embryos did not extend as far ante-
riorly as in DAI 5 embryos (Fig. 4C,D and A,B). Notochords
in DAI 3 embryos from either treatment were more severely
regressed, ventrally displaced and lacked the vacuoles seen in
normal embryos (Fig. 4E,F and A,B).
The deficiency in notochord development was correlated
with a decrease in DAI as assessed by a ratio of notochord
length to body length. Embryos from both UV treatment and
M. C. Danos and H. J. Yost
B1
C2 C1
ANIMAL
VENTRAL DORSAL
VEGETAL
Fig. 3. Drawing of 32-cell stage embryo to show injection locations,
labelled using the nomenclature of Nakamura and Kishiyama (1971).
The C2 blastomeres are fate mapped to give rise to precursor heart
cells. The stippling shows the region that will give rise to the
notochord (Keller, 1975, 1976; Bauer et al., 1994). Injections done at
this stage were either into C1 blastomeres to target the notochord and
other axial structures (see text), or into C2 blastomeres to target the
heart.
Table 1. Xwnt-8 injections into the dorsal midline but not
the heart progenitor cells at the 32-cell stage cause higher
incidence of heart reversals and lower DAI scores
Injection site n% Reversal Average DAI
Dorsal midline (C1) - Xwnt-8 110 16 3.8
Dorsal midline (C1) - Control 130 2 4.9
Heart progenitor (C2) - Xwnt-8 122 5 4.8
Heart progenitor (C2) - Control 108 7 5
Uninjected 151 2.6 5
Fig. 4. Notochord deficiencies in dorsal-anterior deficient embryos
(stages 33-38) were detected by in situ hybridizations with the
collagen II probe. Embryos treated with UV during the first cell
cycle, scored as DAI 5 (A), DAI 4 (C), and DAI 3 (E), stained with
the antisense probe. Embryos dorsally injected with Xwnt-8, scored
as DAI 5 (B), DAI 4 (D), or DAI 3 (F), stained with the antisense
probe. Arrows mark the anterior extent of the notochords. As
hybridization controls, DAI 5 (G) and DAI 3 (H) embryos (obtained
from Xwnt-8 injections) were stained with the sense probe. Scale bar,
1.0 mm.
1471Cardiac left-right development
Xwnt-8 injections were stained with the collagen II probe,
sorted by DAI, and photographed. Measurements of body
length and notochord length were made, respectively, from the
anterior extent of the head and the notochord to the blastopore,
on embryos between stages 34 and 38. The ratio of notochord
length to body length was calculated per embryo for each DAI
and treatment. The two different treatments had similar effects
on the extent of anterior notochord regression (Fig. 5); decrease
in DAI was correlated with regression of anterior notochord.
Both an increase in the frequencies of left-right reversals
1472
(Fig. 2) and progressive loss of anterior notochord (Fig. 5) were
correlated with a decrease in DAI. Therefore, loss of notochord
was correlated with loss of normal left-right orientation.
DISCUSSION
Linkage of left-right and dorsal-anterior
development
In order to co-ordinate the formation of the embryo as it
develops along three geometric axes, at some point in devel-
opment the mechanisms that establish asymmetries along one
axis must interact with the mechanisms that establish asym-
metries along the perpendicular axes. Most studies of embryo
development focus on one axis. Experimental results described
here demonstrate that there is linkage between left-right devel-
opment and dorsal-anterior development in vertebrates.
There is a striking correlation between experimentally
diminished dorsal-anterior development, as scored on the DAI
scale, and increased frequencies of cardiac left-right reversal.
Embryos with reduced dorsal-anterior development (lower
DAIs) were obtained either by perturbation of the cytoplasmic
rotation in the first cell cycle that establishes the dorsal-anterior
axes or by ectopic expression of Xwnt-8 in the dorsal regions
of the embryo after the mid-blastula transition. Although these
two treatments were applied at different stages and presumably
act through different mechanisms, the effects on both dorsal-
anterior and left-right development were the same. Dorsal-
anterior development occurs along a continuum from normal
(DAI 5) to the absence of all dorsal-anterior asymmetry (DAI
0). Development of left-right asymmetries is apparently dis-
continuous; it requires both a mechanism by which asymmetry
is generated and a mechanism that regulates the orientation of
asymmetry along the left-right geometric axis. Dorsal-anterior
development is linked with the latter: in DAI 3 embryos, partial
reduction of dorsal-anterior development results in loss of the
mechanism that regulates the orientation of left-right asymme-
tries, resulting in stochastically oriented left-right structures,
but the mechanism by which left-right asymmetries are
generated is retained.
The roles of dorsal midline cells in left-right
development
The misexpression of Xwnt-8 can be regionally specified by
injection of selected cells. Ectopic expression of Xwnt-8 in
dorsal-most blastomeres (C1 blastomeres, Fig. 3) resulted in
anterior-dorsal defects (decreased DAI), regression of anterior
notochord, and loss of cardiac left-right orientation. In contrast,
injections into heart progenitor cells (C2 blastomeres, Fig. 3)
did not alter anterior-dorsal development and did not cause
cardiac reversal. These results indicate that loss of left-right
orientation is not due to a direct influence of Xwnt-8 in devel-
oping heart cells, but is correlated with perturbed anterior-
dorsal development and defective notochord development. By
fate-mapping, the dorsal-most (C1) blastomeres have been
shown to give rise to subblastoporal endoderm, bottle cells and
dorsal blastoporal lip (Organizer region) at the gastrula stage.
In the neurula stages, the progeny of C1 blastomeres are inter-
digitated throughout the notochord, head mesoderm and
archenteron (Keller, 1975, 1976; Bauer et al., 1994). The
progeny of the dorsal midline cells could regulate cardiac left-
right development as Organizer cells during gastrulation or as
notochord during neurulation, although it should be noted that
less-well-characterized dorsal midline cells might be involved
in left-right cardiac development.
An effect that is common to UV treatment during the first
cell cycle and Xwnt-8 misexpression in post-MBT dorsal
midline cells is deficient anterior notochord development (Figs
3 and 4). Although the present results indicate that there is a
correlation between defective notochord development and loss
of cardiac left-right orientation, it cannot be concluded that
notochord directly regulates left-right development. The most
likely explanation for progressive loss of anterior notochord,
caused by either UV treatment or Xwnt-8 ectopic expression,
is that the amount of Organizer activity is diminished. Embryo
recombinant experiments show a correlation between the
amount of Organizer and the extent of dorsal-anterior devel-
opment, as assessed by DAI (Stewart and Gerhart, 1990). It is
possible that the Organizer directly regulates left-right devel-
opment during gastrulation. Then, diminished notochord and
loss of left-right orientation would be independent conse-
quences of diminished Organizer activity. The Organizer
interacts with dorsolateral mesoderm to allow it to develop
heart-forming potency (Sater and Jacobson, 1990); perhaps it
also interacts with pre-cardiac mesoderm to specify left-right
orientation. If decreased DAI reflects decreased Organizer
activity (Stewart and Gerhart, 1990), the threshold of
Organizer activities required to make a normal-sized heart
(DAI 3 and above) must be lower than that required to con-
sistently establish cardiac left-right orientation (DAI 5). Alter-
natively, Organizer activity could indirectly establish cardiac
left-right asymmetry by working through intermediary tissues.
For example, planar signals from the Organizer are transmit-
ted through the ectoderm to establish the anterior-posterior axis
of the neural plate (for reviews, Doniach, 1992; Ruiz i Altaba
and Jessell, 1993). The extracellular matrix of the ectoderm is
necessary for normal left-right development (Yost, 1992).
Perhaps the Organizer transmits planar signals through the
ectoderm to establish left-right asymmetries, which are then
transmitted to the cardiac mesoderm by way of the ectodermal
extracellular matrix. UV-treated embryos that have no
M. C. Danos and H. J. Yost
Fig. 5. Decrease in anterior notochord development is correlated
with dorsal-anterior deficiencies. The amount of notochord for each
DAI from UV-treated embryos (white bars) and embryos in which
Xwnt-8 was injected into dorsal cells (grey bars) was measured. The
ratio of notochord length to body length was calculated for each
embryo (10-14 embryos in each category) and normalized by
dividing this ratio by the mean ratio in untreated embryos (83±1,
from measurements of 10 untreated embryos).
1473Cardiac left-right development
Organizer activity (DAI 0) (Stewart and Gerhart, 1990) appear
to form normal ectodermal extracellular matrix, at least as
assayed by fibronectin immunohistochemistry (Yost, 1992).
However, other aspects of ectodermal matrix might be
regulated by planar signals from the Organizer to regulate left-
right development of the heart.
The notochord is derived from the Organizer, and has
inductive properties for establishing the floor plate and orga-
nizing dorsal-ventral polarity of neural structures in chick
(Placzek et al., 1990; Yamada et al., 1991, 1993). By analogy,
the notochord might inductively signal left-right asymmetry to
the cardiac cells. Alternatively, the elongation and stiffening
of the notochord in neurula stages (Keller et al., 1989; Adams
et al., 1990) mechanically stretches the embryo along the
anterior-posterior axis. Embryo shape changes may be
necessary for orientation of left-right asymmetries, perhaps by
aligning the extracellular matrix that is necessary for normal
cardiac left-right asymmetry (Yost, 1992).
In Xenopus mid-neurulae stage embryos, extirpations of
dorsal-anterior tissue, including but not exclusive to the
notochord, result in randomization of cardiac asymmetry (Danos
and Yost, unpublished data), suggesting that the orientation of
left-right cardiac asymmetry can be perturbed after the
Organizer activity is diminished. In order to distinquish between
the roles of the Organizer and subsequent roles of dorsal midline
cells in regulating left-right development, it is important to
identify the developmental periods during which left-right ori-
entation is dependent on dorsal midline cells. The activities of
notochord and other dorsal-anterior tissues in left-right orienta-
tion are currently being explored in explant and in vitro systems.
Vertebrates are not bilaterally symmetric; the left side differs
from the right, and the orientation of left-right asymmetries is
highly conserved in vertebrates. The present results indicate
that the developmental regulation of left-right orientation in
Xenopus is linked to the developmental regulation of the other
axes. The mechanism of linkage between the embryonic axes
is not yet elucidated, but the linkage is dependent upon normal
development of dorsal midline cells.
We are grateful to M. L. Condic and A. L. Teel for comments on
the manuscript, and to them and members of our laboratory for
insights and encouragement throughout the course of this study. We
thank E. Amaya and J. L. Christian for DNA clones, and D. Bader
for monoclonal supernatants. This work was supported by a grant-in-
aid from the American Heart Association.
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(Accepted 6 February 1995)
M. C. Danos and H. J. Yost
