Development 99, 145-154 (1987)
Printed in Great Britain © The Company of Biologists Limited 1987
145
Induction of myofibrillogenesis in cardiac lethal mutant axoloti hearts
rescued by RNA derived from normal endoderm
LYNN A. DAVIS and LARRY F. LEMANSKI
Department of Anatomy and Cell Biology, State University of New York, Health Science Center at Syracuse, 766 Irving Avenue, Syracuse,
New York, 13210, USA
Summary
A strain of axoloti, Ambystoma mexicanum, that
carries the cardiac lethal or c gene presents an
excellent model system in which to study inductive
interactions during heart development. Embryos
homozygous for gene c contain hearts that fail to beat
and do not form sarcomeric myofibrils even though
muscle proteins are present. Although they can survive for approximately three weeks, mutant embryos
inevitably die due to lack of circulation. Embryonic
axoloti hearts can be maintained easily in organ
culture using only Holtfreter's solution as a culture
medium. Mutant hearts can be induced to differentiate in vitro into functional cardiac muscle containing
sarcomeric myofibrils by coculturing the mutant heart
tube with anterior endoderm from a normal embryo.
The induction of muscle differentiation can also be
mediated through organ culture of mutant heart tubes
in medium 'conditioned' by normal anterior endoderm. Ribonuclease was shown to abolish the ability
of endoderm-conditioned medium to induce cardiac
muscle differentiation. The addition of RNA extracted
from normal early embryonic anterior endoderm to
organ cultures of mutant hearts stimulated the differentiation of these tissues into contractile cardiac
muscle containing well-organized sarcomeric myofibrils, while RNA extracted from early embryonic
liver or neural tube did not induce either muscular
contraction or myofibrillogenesis. Thus, RNA from
anterior endoderm of normal embryos induces myofibrillogenesis and the development of contractile
activity in mutant hearts, thereby correcting the
genetic defect.
Introduction
of mutant embryos including small gills, microcephaly and ascites, can be prevented by providing
the mutant embryo with a source of circulation. Thus,
the only tissue that appears to be adversely affected
by gene c is the heart. Lemanski and workers
(Lemanski, Mooseker, Peachy & Iyengar, 1976;
Lemanski, Fuldner & Paulson, 1980) and Starr, Diaz
& Lemanski (1985) have demonstrated that the
mutant hearts contain the contractile proteins actin,
o--actinin and myosin. However, the mutant heart is
distinctive in its lack of organized sarcomeric myofibrils.
Two sets of experiments provide evidence that
there is a lack of inductive stimulus for heart differentiation in c/c embryos. First, in transplantations
performed by Humphrey (1972) mutant heart tissue
placed in a normal host differentiates into functional
contractile cardiac muscle. On the other hand, normal presumptive heart tissue placed into a mutant
It has been established in chicks (Orts-Llorca & Gil,
1965) and salamanders (Jacobson & Duncan, 1968)
that anterior endoderm is the most potent inducer of
heart differentiation. However, as in most inductive
interactions that have been studied, the mechanism
by which the inductive stimulus is passed from one
tissue to another remains unknown.
The strain of Ambystoma mexicanum that carries
the cardiac lethal or c gene provides an excellent
model system in which to study inductive interactions
that occur during development of the heart. Embryos
that are homozygous for the c gene do not develop
beating hearts and inevitably die from the resulting
lack of circulation. Experiments using parabiotically
joined normal and mutant embryos (Humphrey,
1972) have shown that the characteristic morphology
Key words: myofibrillogenesis, RNA, anterior endoderm,
cardiac mutant, axoloti, Ambystoma mexicanum.
146
L. A. Davis and L. F. Lemanski
embryo fails to contract. The second piece of evidence comes from experiments in which hearts were
removed from mutant embryos and maintained in
organ culture. Mutant hearts cultured in this manner
do not beat and do not develop sarcomeric myofibrils
(Hill & Lemanski, 1979). However, if anterior endoderm is cocultured with mutant heart tubes, the
mutant heart tissue differentiates into functional
contractile cardiac muscle containing normally organized myofibrils (Lemanski, Paulson & Hill, 1979).
This paper describes our efforts to elucidate the
mechanisms by which anterior endoderm is able to
influence the differentiation of mutant heart tissue.
First, by using medium conditioned with normal
anterior endoderm to culture mutant hearts we were
able to show that a diffusible factor(s) must be
involved in this interaction. Second, experiments
were performed to identify and characterize the
active factor(s) present in conditioned medium.
Enzyme-inactivation experiments showed that the
activity of conditioned medium was destroyed by
ribonuclease. Mutant hearts were then organ cultured in Holtfreter's solution containing RNA
extracted from normal embryonic anterior endoderm. The addition of this RNA to the culture
medium stimulated differentiation of mutant heart
tissue into functional cardiac muscle composed of
cells containing well-organized sarcomeric myofibrils.
RNA derived from other embryonic axolotl tissues
was not able to induce differentiation of mutant
myocardium. Thus, RNA from normal anterior endoderm is able to induce myofibrillogenesis in the
mutant heart.
Organ culture of embryonic hearts
Stage-34 embryos were removed from their jelly coats
in sterile modified Holtfreter's solution containing 1 %
antibiotic/antimycotic. The embryos were classified as
normal or mutant by whether or not they possessed a
beating heart. As expected, there were approximately 25 %
mutant embryos in +/c x +/c spawnings. The normal and
mutant hearts were removed using glass needles to minimize damage during dissection and placed in lO^tl of
Holtfreter's solution, conditioned medium or Holtfreter's
solution containing RNA on a small piece of sterile dental
wax in a Falcon tight-sealing culture dish. The cultures were
maintained at 18 °C for 24-48 h and their activities were
monitored periodically under a dissecting microscope during the culture period.
Materials and methods
Enzyme inactivation of conditioned medium
Insoluble enzymes attached to agarose (Sigma) were used
to treat the conditioned medium. The enzyme/agarose
complexes were washed three times with Holtfreter's solution prior to use. 200 u\ of bovine pancreatic ribonuclease
A (0-65 units), 200/il of bovine pancreatic trypsin (2units)
or 200/d of type VI-A neuraminidase from Clostridium
perfringens (2-44 units) were added to 200 /A of conditioned
medium. The mixture was agitated at 18°C for l h . Enzymes attached to agarose were removed by centrifugation
and the enzyme-treated conditioned medium was then used
for organ cultures of normal and mutant hearts. As a
control in these experiments, 200/jl conditioned medium
was diluted with 200 $ of Holtfreter's solution and treated
in the same manner as conditioned medium containing
enzymes. Conditioned medium was placed in a boiling
water bath for 3min to denature proteins. The boiled
conditioned medium was then used to culture mutant
hearts. The activity of ribonuclease attached to agarose was
tested under the conditions used to inactivate conditioned
medium. Polycytidylic acid was incubated with 200/J of
insoluble ribonuclease for time periods of up to 1 h at 18 °C.
Degradation of polycytidylic acid was measured spectrophotometrically at 280/on (Zimmerman & Sandeen, 1965).
Preparation of conditioned medium
Ambystoma mexicanum embryos were removed from their
jelly coats at various times between stages 29 and 33
(Schreckenberg & Jacobson, 1975). Embryos from both
+ / + x + / + matings and + / + x +/c matings were dissected; however, the spawnings were used separately. The
embryos were placed in sterile modified Holtfreter's solution (3 • 5 g NaCl, 100 mg CaCl2, 50 mg KC1, 204 mg MgSO4
and 200 mg NaHCO 3 /litre) containing 1 % antibiotic/
antimycotic (Gibco). Anterior endoderm was dissected
from ten embryos and placed in 150 /JI of sterile Holtfreter's
solution in a single well of a Falcon 24-well tissue culture
plate. The plate was covered and placed in an 18CC
incubator in air for 48 h. Following this culture period the
supernatant was drawn off and centrifuged at 15 000 g for
lOmin to remove any cellular debris. At this point, the
conditioned medium was divided into equal samples and
used for organ cultures, biochemical analysis or frozen in
liquid N2 for later use.
RNA extraction
Anterior endoderm, presumptive liver and neural tube
tissues were dissected from 50 stage-29 + / + normal embryos. The tissues were homogenized in 6 M-guanidinium
isothiocyanate, 5mM-sodium citrate (pH7-0), 0-1M-/3mercaptoethanol and 0-5 % N-lauroylsarcosine. Following
homogenization, lg of caesium chloride was added to the
mixtures. The homogenates were then layered onto a 1 -2 ml
cushion of 5-7M-CSC1 in 0-1M-EDTA (pH7-5) in a polyallomer tube and centrifuged at 31000 revs min"1 for 12 h at
20°C in a Beckman SW60 Ti rotor. Following centrifugation, the supernatants were discarded and the pellets of
RNA were dissolved in 10 mM-Tris-HCl (pH7-4), 5mMEDTA and 1 % SDS. The RNAs were then extracted with a
4:1 mixture of chloroform and 1-butanol, centrifuged at
2000g for lOmin at 4°C and the aqueous layer recovered.
The organic phases were re-extracted with the Tris buffer
and the two aqueous phases combined. RNAs were precipitated by adding 0 1 (v/v) of 3M-sodium acetate (pH5-2)
and 2-2 (v/v) of 95% ethanol (Glisin, Crkvenjakov &
Induction of myofibrillogenesis by endoderm RNA
Byus, 1974; Foster, Rich, KarT & Przybyla, 1982). Prior to
use in organ cultures, the RNAs were reprecipitated from
RNase-free distilled water and dissolved in modified Holtfreter's solution. The concentration and purity of RNA in
each sample was determined by spectroscopy at 260 nm and
280 nm.
Electron microscopy
At the end of the culture period, hearts were fixed in a
glutaraldehyde-paraformaldehyde-picric acid mixture (Ito
& Karnovsky, 1968), postfixed in OsO4, dehydrated and
embedded in Epon. Thin sections were stained with lead
citrate and uranyl acetate.
Results
Hearts removed from normal embryos and maintained in organ culture beat in a strong and rhythmic
manner. Ultrastructurally they showed the same
myofibrillar organization as normal hearts in vivo
(Figs 1, 2). In contrast to the normal hearts, mutant
hearts both in vivo and in organ culture in modified
Holtfreter's solution did not beat or contain organized myofibrils, although amorphous areas and scattered filaments were present (Figs 3,4).
Experiments using medium conditioned by normal
anterior endoderm to organ culture mutant hearts
showed that the endoderm tissue itself was not
necessary for differentiation of the presumptive heart
tissue to occur (Table 1). The active factor(s) produced by anterior endoderm was released into the
culture medium and retained its ability to stimulate
myofibrillogenesis in mutant hearts as indicated
macroscopically by the development of propagated
rhythmic contractions in the heart tubes and ultrastructurally by the presence of organized sarcomeric
myofibrils in the myocardium (Fig. 5).
Conditioned media from different sources were not
uniformly effective in inducing myofibrillogenesis in
mutant hearts. We found that conditioned medium
produced by anterior endoderm removed from embryos of a homozygous ( + / + x + / + ) spawning was
significantly more effective than that produced by
anterior endoderm from heterozygous ( + / + x +/c)
embryos (Table 1). Another set of experiments
suggested that anterior endoderm from younger embryos was more effective in promoting differentiation
of mutant heart tissue than from older embryos
(Table 1). In fact by stage 33 it appears that the
anterior endoderm is no longer able to provide the
active factor(s) for differentiation of mutant hearts in
culture.
Enzyme-inactivation studies were done in an effort
to discover the identity of the active factor(s) in the
conditioned medium. The treatment of conditioned
medium with neuraminidase had no effect on the
147
ability of conditioned medium to correct mutant
hearts. Boiling of conditioned medium did not affect
its activity. Treatment with trypsin resulted in a
reduction of the activity of conditioned medium,
although it retained the ability to induce rhythmic
contractions in 38 % of the cultured mutant hearts.
The only enzyme that completely abolished the
activity of conditioned medium was ribonuclease
(Table 2). An increase in the acid-soluble absorbance
of polycytidylic acid incubated with the ribonuclease
at times up to 1 h indicated that the enzyme was able
to degrade RNA under the experimental conditions
used. In further studies, RNA was extracted from
normal embryonic anterior endoderm and added to
organ cultures of mutant hearts. Mutant hearts organ
cultured in modified Holtfreter's solution plus RNA
were indistinguishable from those cultured in conditioned medium. The hearts beat rhythmically
(Table 3) and ultrastructural examination revealed
the presence of sarcomeric myofibrils (Fig. 6). In
contrast to the effects of endoderm RNA on mutant
heart tissue, RNAs extracted from embryonic liver
and neural tube were not able to induce differentiation of functional cardiac muscle from mutant
heart tubes. Mutant hearts cultured in modified
Holtfreter's solution containing either liver RNA or
neural tube RNA did not beat (Table 3) and did not
contain sarcomeric myofibrils. Ultrastructurally,
these hearts looked like mutant hearts in vivo (Fig. 3)
or mutant hearts cultured only in Holtfreter's solution
(Fig. 4).
Discussion
How one tissue influences the developmental pathway of another is an old but still unanswered question. For example, it has been established that the
anterior endoderm induces differentiation of the
overlying mesoderm tissue to form the heart at a very
early stage in development (Orts-Llorca & Gil, 1965;
Jacobson & Duncan, 1968). However, it is not clear
how the inductive process or transfer of information
takes place. The cardiac lethal mutation in the
axolotl, Ambystoma mexicanum, provides a unique
opportunity to explore the induction phenomenon. In
homozygous c/c mutant embryos there is apparently
a lack of appropriate inductive stimulus to the presumptive heart (Hill & Lemanski, 1979). The heart
does not develop beyond stage 34 and remains a
quiescent structure (Humphrey, 1972). Although
muscle proteins are present (Lemanski et al. 1976,
1980; Starr et al. 1985) they are not organized into
myofibrillar structures. While other investigators
have provided evidence of coordinated muscular
contractions propagated throughout the lengths
of mutant heart tubes in culture (Kulikowski &
148
L. A. Davis and L. F. Lemanski
"
•
•
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;
-
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•
•
'
•
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Figs 1, 2. For legends see p. 150
-
•
•
*
Induction of myofibrillogenesis by endoderm RNA
L
Figs 3, 4. For legends see p. 150
149
150
L. A. Davis and L. F. Lemanski
Manasek, 1978), we only occasionally find contractions in the conus region of the heart. We have never
observed contractions propagated throughout the
lengths of mutant heart tubes either in vivo or when
cultured in Holtfreter's solution alone. Neither Kulikowski & Manasek (1978) nor our laboratory has
been able to demonstrate by electron microscopy the
presence of sarcomeric myofibrils in cardiac tissue of
mutant embryos (Lemanski, 1973; Hill & Lemanski,
1979). Thus, we conclude that either in vivo or in
organ culture, presumptive heart tissue from cardiac
lethal mutant embryos is unable to differentiate fully
due to the lack of a final inductive stimulus. What
makes this mutation so useful in studying heart
development is that the mutant heart can be 'rescued'
in vitro by culturing it with normal anterior endoderm
(Lemanski et al. 1979). Thus, in this experimental
system we have a tissue that failed to receive the
proper input for differentiation at the usual time in
development. However, we can still influence the fate
of this tissue by providing it with an exogenous
inductive stimulus.
Since previous work in this laboratory had shown
that mutant heart tissue could be induced to differentiate by cocultunng with normal anterior endoderm
(Lemanski et al. 1979) our first question was whether
cell-to-cell contact with the endoderm tissue was
necessary for this induction to occur or whether a
diffusible substance might be involved. We found that
the inductive stimulus produced by anterior endoderm was indeed able to be transmitted via the
conditioned medium to mutant heart tissue without
the simultaneous presence of endoderm in the culture. Clearly, the endoderm must be producing a
diffusible factor(s) which presumptive mutant heart
Fig. 1. Myocardium of a normal stage-39 axolotl embryo.
Note the presence of sarcomeric myofibrils. A, A-band; /,
I-band; L, lipid; Z, Z-line. Bar, 0-5um (X38600).
Fig. 2. Myocardial cells of normal embryonic heart
explanted into Holtfreter's solution organ culture at stage
34 and maintained for 48 h. These hearts are comparable
to stage-39 in vivo and also contain sarcomeric myofibrils.
A, A-band; /, I-band; v, yolk; Z, Z-line. Bar, 0-5/an
(X38600).
Fig. 3. Myocardium of a mutant stage-39 axolotl embryo.
No organized myofibrils are present; however, scattered
15 nm filaments (arrows) and amorphous areas {am) are
often seen. Bar, 0-5/xm (X38600).
Fig. 4. Myocardial cells of a mutant axolotl embryo heart
explanted into Holtfreter's solution organ culture at stage
34 and maintained for 48 h. These hearts are comparable
to mutant stage-39 hearts in vivo. No sarcomeric
myofibrils are present. Instead the tissue contains patches
of amorphous material {am) and randomly arranged
15 nm filaments (arrows). ,L, lipid; y, yolk. Bar, 0-5/en
(X38600).
Table 1. Results of mutant heart cultures in
conditioned medium
Source of
conditioned
medium
Number of
cultured
mutant
hearts
Number of
beating
mutant
hearts
Corrected
mutant
hearts
(%)
Stage 29-31
36
26
72-2
homozygous
normal ( + / + X + / + )
anterior endoderm
47
24
51-0*
Stage 29-31
heterozygous
normal ( + / + x +/c)
anterior endoderm
51
32
63-0
Stage 29
heterozygous
normal ( + / + X +/c)
anterior endoderm
32
18
56-0
Stage 30-31
heterozygous
normal ( + / + x +/c)
anterior endoderm
6
0
00
Stage 33
heterozygous
normal (+/+ x +/c)
anterior endoderm
Holtfreter's
36
1
2-8
solution controls
* Statistically significant difference ( P S 005) when compared
to conditioned medium from homozygous stage 29-31 normal
endoderm by both the Student's f-test and the G-test.
tissue responds to. Endoderm obtained from embryos produced by a homozygous normal ( + / + )
mating was more effective in inducing cardiac muscle
differentiation than that taken from embryos of a
heterozygous spawning ( + / + x +/c). Not surprisingly, since Jacobson & Duncan (1968) have shown
that the inductive action of endoderm in vivo is
greatest at early stages of development in both the
California newt, Taricha torosa, and the salamander,
Fig. 5. Myocardium of a mutant stage-34 axolotl embryo
that has been cultured in conditioned medium prepared
using normal anterior endoderm. This 'corrected' mutant
heart was beating strongly at the end of the culture
period and ultrastructurally resembles the normal hearts
seen in Figs 1 and 2. Sarcomeric myofibrils are prominent
throughout the myocardium. A, A-band; /, I-band; L,
lipid; .y, yolk; Z, Z-line. Bar, 0-5/an (x38600).
Fig. 6. Myocardium of a mutant stage-34 axolotl embryo
organ cultured in modified Holtfreter's solution
containing RNA extracted from normal anterior
endoderm. This mutant heart is also considered to be
'corrected' as it was beating rhythmically at the end of
the culture period and contains well-organized myofibrils.
A, A-band; /, I-band; L, lipid; y, yolk; Z, Z-line. Bar,
0-5/an(x38600).
Induction of myofibrillogenesis by endoderm RNA
Ambystoma tigrinum, we found that conditioned medium produced from stage-29 endoderm was more
potent than that produced from stage-33 endoderm.
151
However, even using stage-29 homozygous embryos
to provide the endoderm with which to condition the
medium we were only able to correct approximately
152
L. A. Davis and L. F. Lemanski
Table 2. Results of mutant heart cultures using enzyme-inactivated conditioned medium
Conditioned* medium
plus enzyme
Number of cultured
mutant hearts
Number of beating
mutant hearts
Corrected mutant
hearts (%)
Conditioned medium controls
(no enzyme); diluted 1:1 with
Holtfreter's solution
Boiled conditioned medium
Neuraminidase
Trypsin
Ribonuclease
Holtfreter's solution controls
26
14
54
12
4
13
18
12
58
50
38
0
0
'The conditioned medium was derived from stage 29-31 homozygous normal ( + / + x + / + ) embryonic anterior endoderm.
Table 3. Results of mutant heart cultures using
Holtfreter's solution and RNA
Culture medium
Stage 29
anterior endoderm
RNA (0-4mgmr')
Stage 29
liver RNA
Number of
cultured
mutant
hearts
Number of
beating
mutant
hearts
Corrected
mutant
hearts
(%)
10
9
90
12
0
0
11
0
0
12
0
0
(0-5mgmr')
Stage 29
neural tube RNA
(O^mgmT 1 )
Holtfreter's
solution controls
70 % of the mutant hearts. There are several possible
reasons why the other 30 % do not develop the ability
to contract. First, even though the hearts are
removed very carefully for organ culture, it is possible
that some damage occurs to the structure and thus
inhibits its further differentiation. Another explanation might be that the heart normally receives its
inductive signals much earlier in development than
we are able to reproduce in vitro, since it is not
possible at present to distinguish a mutant embryo
from its normal siblings until stage 34, when the
normal heart begins to beat. Thus, by the time the
mutant hearts are exposed to conditioned medium in
organ culture they are very possibly near the end of
the time they are competent to respond to an inductive stimulus.
Enzyme inactivation was chosen as the method to
identify the class of compounds to which the active
factor belonged. To overcome the problem of having
to add both enzyme and enzyme inhibitor to the
conditioned medium we used enzymes attached to
agarose, which then could be removed by centrifugation following the incubation period. Of the three
enzymes added to conditioned medium, i.e. trypsin to
degrade proteins, neuraminidase to cleave sialic acid
residues from glycoproteins and ribonuclease to inactivate RNAs, only treatment with ribonuclease completely abolished the activity of the conditioned
medium. The corollary of this experiment, namely
adding RNA from normal anterior endoderm, was
then done. In two separate experiments, using mutant embryos from two different spawnings, endoderm RNA added to the Holtfreter's solution used to
organ culture the mutant hearts resulted in the
development of contractile cardiac muscle. To corroborate the macroscopic evidence of differentiation,
namely development of contractile activity, organ
cultured hearts were examined ultrastructurally.
Mutant hearts cultured in the presence of endoderm
RNA resembled hearts from normal embryos, containing organized sarcomeric myofibrils (Figs 1,6).
Mutant hearts cultured with liver or neural tube RNA
did not contain these structures. Thus, RNA derived
from normal anterior endoderm is able to substitute
for the endoderm tissue itself in inducing myofibrillogenesis in mutant heart tissue.
The idea that RNA can be used to direct specific
differentiation pathways is not new. Early work, by
Niu (1958) using embryonic ectoderm from Ambystoma tigrinum, and by Sanyal & Niu (1966), Butros
(1965) and Niu & Deshpande (1973) using postnodal
pieces of chick blastoderm, described a variety of
differentiation patterns when these tissues were cultured with the addition of RNA from several different
sources. When axolotl ectoderm treated with thymus
RNA is implanted into host flank tissue, it is capable
of differentiating into small bodies resembling thymus (Niu, 1958). When postnodal chick blastoderm is
exposed to brain RNA, neural tissue results (Sanyal
& Niu, 1966). The application of liver RNA leads to
more variable results with some neural tissue developing (Sanyal & Niu, 1966) and in other cases
myoblasts, mesonephric tubules and endoderm tissue
being present (Butros, 1965). Kidney RNA, on the
other hand, does not seem to induce neural differentiation but instead tubular structures are formed
Induction of myofibrillogenesis by endoderm RNA
(Sanyal & Niu, 1966). The RNA most extensively
studied using chick postnodal blastoderm is that from
embryonic heart. Postnodal chick blastoderm cultured with RNA derived from embryonic chick heart
is able to differentiate into pulsating structures containing actin and myosin. These proteins are arranged
into organized sarcomeric myofibrils similar to those
seen in the heart in vivo (Deshpande & Siddiqui,
1977). The inducer RNA has been characterized as a
7S poly(A)RNA which hybridizes to repetitive chick
DNA and shares homology with noncoding segments
of myosin genes (Khandekar, Saidapet, Krauskopf,
Zarraga, Lin, Mendola & Siddiqui, 1984). There is
evidence in chick blastoderm (Siddiqui, 1983) and in
primary cultures of chick myoblasts (Mroczkowski,
Dym, Siegel & Heywood, 1980), that muscle cells can
take up and use exogenously supplied RNA. The
addition of RNA extracted from adult chicken heart
to cultures of embryonic chick myocardial cells results
in the maturation of cell membranes (McLean,
Renaud, Niu & Sperelakis, 1977). Following an
incubation period of 6-14 days, myocardial cells
cultured with this RNA developed adult-type electrophysiological properties such as fast sodium channels
which were sensitive to tetrodotoxin. This effect is
abolished by the administration of cyclohexamide.
In the present studies, we have demonstrated that
RNA isolated from anterior endoderm induces myofibrillogenesis in mutant axolotl heart tissue. Nevertheless, we have no firm evidence yet that its effect
on mutant heart tissue in vitro is the same as during
in vivo embryonic development. The precise mechanism^) by which anterior endoderm RNA acts on
mutant hearts remains unknown. At the present time
we do not know whether the active RNA is functioning as messenger RNA or if it is acting in some other
capacity, such as regulating transcription or translation (Mroczkowski, McCarthy, Zezza, Bragg &
Heywood, 1984; Siddiqui, Khandekar, Krauskopf,
Mendola, Zarraga & Saidapet, 1984), or affecting
the cell membranes of the developing myocardium
(McLean etal. 1977). Studies are currently in progress
to characterize the RNA that is able to overcome the
lack of myofibrillogenesis in cardiac lethal mutant
embryos.
We would like to thank Linda Riles and Jos6 Diaz for
their excellent technical assistance. Special thanks are due
also to Chris Starr for technical support and advice in RNA
extraction procedures. The axolotl colony at Indiana
University kindly supplied many of the embryos for this
study. This work was supported by a Muscular Dystrophy
Postdoctoral fellowship to L.A.D. and NIH Grants
HL37702, HL32184 and a Grant from the American Heart
Association to L.F.L.
153
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