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Mol. Cells , Vol. 9, No.5 , pp. 497- 503
Molecules
and
Cells
© Springer-Verlag 1999
Neuregulin Induces the Expression of Mesodermal Genes in
the Ectoderm of Xenopus iaevis
Hae Geun Chung and Hae-Moon Chung*
Department of Biology Education, Seoul National University, Seoul 151 -742, Korea.
(Received on May 24, 1999)
The primary patterning event in early vertebrate
development is the formation of mesoderm and
subsequent induction of the neural tube by the
mesoderm. Some of the transforming growth factor
(TGF)-13 famil y (Activin, Vgl) and the fibroblast
growth factor (FGF) family molecules have been
implicated for their roles in mesoderm induction. Here
we show fir st the evidence that neuregulin , an
epidermal growth factor (EGF) - like growth factor
known for its role in neural and muscle differentiation,
participates in mesoderm induction. Neuregulin could
induce the ectopic expression of mesoderm specific gene
Xbra in animal cap explants reared to the midgastrula
stage, when animal caps dissected from late blastula
were cultured with Neuregulin at a low concentration
(10 ng/ml). In situ hybridization study showed that
a-cardiac actin was expressed in animal caps that were
treated with Neuregulin overnight. Skeletal and cardiac
muscle specific genes such as MyoD family genes
(myoD, MRF4, myf5) and SLl as well as NCAM, a pan
neural marker, were also ectopically expressed by
treatment with Neuregulin. However, the expression of
N CAM is presumed to be a secondary result of the
initial mesoderm induction by Neuregulin. The
temporal expression pattern of neuregulin during the
early developmental stages was analyzed by RT-PCR in
order to determine if neuregulin is expressed at the
time of mesoderm induction. It has been found that the
neuregulin transcript was already detected from the
16-cell stage (stage 5) and continued to be expressed till
the tailbud stage (stage 25), the latest embryonic stage
analyzed in this study. Considering that the mesoderm
is induced at early blastula before the start of zygotic
* To whom correspondence should be addressed.
Tel: 82-2-880-7770; Fax: 82-2-886-2 11 7
E-mail : [email protected]
transcription, maternal neuregulin is expressed at the
right time to participate in mesoderm induction. These
data strongly suggest that neuregulin pla ys an
important role in mesoderm induction.
Keywords: Mesoderm Induction ; Muscle Specific Genes;
Neuregulin ; Xbra; Xenop us laevis.
Introduction
The primary patterning of the embryonic body relies on the
interaction between adj acent cells. During the development
of multi cellul ar eukaryotes, cell specialization and fate
determination occur in response to a series of inducti ve
signals (Kim et ai. , 1999). Mesoderm induction is the first
induction event in embryo patterning. The inducti on and
patterning of the mesoderm have been studied extensively
in Xenopus laevis. By Nieu wkoop's brilliant experiment,
so me aspects of the ori gin of the mesoderm inducti on
signal co uld be identi fied. When vegetal and ani mal
explants of bl astula are put together in a 'sandwich' and
cultured for 3 d, the region of the animal explant that is in
immediate contact with the vegetal tissue is converted into
mesoderm al tiss ue s uch as mu sc le. As th e anim al
hemi sphere originall y fo rms an epidermi s when isolated
and cul tured alone, thi s result suggests that the vegetal
hemisphere whi ch is the future endodermal tissue, released
a signal on the overlying animal explant and induced the
region abutting the vegetal explant to form the mesoderm.
Abb rev iatio ns: EGF, ep id ermal grow th factor; EGTA,
[ethylenebi s(oxyethyleneni tr i10 )]tetraace ti c ac id ; FGF,
fibro bl as t grow th fac tor; HEPES , 4-(2 -h yd roxyeth yl)- Ipiperazineethanesulfo nic acid; MMR, Marc's modi fied ringer
solution; MOPS, 3-(N-morpholino)propanesulfo nic acid; PBS,
phosp hate buffe red saline; RT-PC R, reve rse transcriptionpolymerase chain reaction; TGF- ~ , transformi ng growth fac to r-~ .
49S
Mesodelmal Gene Expression by Neuregulin
The mesoderm induction and its dorsoventral patterning
can be explained by the 'four signal model ' (Wolpert et al.,
1998). First, the vegetal half produces a general mesoderm
inducing signal onto the animal half, broadly specifying a
ventral-type mesoderm. Secondly, the signal specifies the
dorsal-most mesoderm, the future Spemann organizer. The
third set of signals emerges from the ventral region of the
marginal zone, while the fourth s ignal modifies the
ventralizing action of the third set of signals (Wolpert
et al., 1998). Many molecules have been reported to
participate in this process, including Activin, V g l , and
FGF. Recentl y, based on the works of Zhang et ai., ( 1998),
VegT, a T-box transcription factor and a novel model of
mesoderm induction has been proposed. It argues that the
egg contains a weak me so derm-inducin g signal of
unknown identity, as well as VegT mRNA , and at the onset
of tran scription , the maternal VegT promotes its own
endodermal fate and activates hi gh level s of TGF-~
signaling (Reviewed by Kimelman and Graffin, 1998).
There are a wide variety of tissues originating from the
mesoderm - notochord, muscle, blood, mesenchyme and
heart. Th e refore , the elucidation of the mechani s m
underlying mesoderm induction and patterning is the first
step toward under standing the formation of the se
mesodermal derivatives.
The neuregulin family of growth factors can induce a
variety of responses in cultured cells. Specifically,
neuregu lin ha s bee n observed to i nf) uenc e ce ll
proliferation , differentiation, survival or fate depending on
cell type (Caraway, 1996). There are numerous names for
these proteins, reflecting their various functions: ARIA
(Acety lcholine Receptor Inducing Activity), NDF (Neu
Differenti ation Factor), GGF2 (glial growth factor 2),
SMDF (Sensory and Motor neuron Derived Factor), and
so on.
Althou gh most attention has been focused on the
function s of these proteins in the nervous system (FIorini
et ai., 1996), they also have major effects on mu scle
differentiation. It ha s been reported th a t rhGGF2
(recombinant human Glial Growth Factor 2), is a potent
stimulator of myogenesis in L6Al myobl asts, giving a
maximal stimulation of cell fusion and creatine kinase
elevation at a concentration of 1 nglml (FIorini et al.,
1996). Di sruption of neuregulin and their receptors
resulted in aberrations in cardiac development. These
mutant embryos were fatal due to the lack of trabeculae in
the ventricular myocardium (Reviewed by Caraway, 1996).
Recently, it has been shown by the in situ hybridization
study in Xenopus that neuregulin is prominently expressed
in myotomal muscl e (Yang et ai., 1998). These former
studies implicate that neuregulin is necessary for normal
muscl e differentiation .
To elucidate the function of neuregulin, we asked if
neuregulin participates in mesoderm induction, the primary
event in musc le differentiation in the early embryonic
development of Xenopus laevis. Using the animal cap
assay, we show that neuregulin can ectopically induce the
expression of mesoderm specific genes. The analysis of the
temporal expression pattern of neuregulin showed that it is
expressed before zygotic gene expression (stage 5) , and
persists until the tailbud stage. These results suggest that
neuregulin participates in mesoderm induction .
Materials and Methods
Embryo manipulation Frogs (Xenopus laevis) were in vitro
fertilized by injec tin g 150 and 300 IU of human chori oni c
gonadotropi n (LG PhD) to the male and female, respective ly.
Embryos were dej ellied with 2.7S% L-cysteine (pH 7.S-S.0),
rin sed severa l times, and reared in 0.1 X MMR ( I X MMR ;
100 mM NaCl, 2 mM KCI, 2 mM CaCI 2 , I mM MgC I2 , 5 mM
HEPES, pH 7.4-7.6) at IS- 22°C. Staging was done according to
Nieuwkoop and Faber ( 1967).
RNA isolation RNA s from embryos and animal caps were
extracted using RNAzo lTM B. In brief, embryos were rin sed in
PBS and homogeni zed with RNAzolTM B. Chloroform was added
to it, then was centrifuged at 4°C. The upper aqueous phase was
precipitated with an equal vo lume of isopropanol. The time of
incubation at 4°C was 15 min for whole embryos, and 45 min for
animal ca ps. After centrifugation, the pellet was washed once
with 75 % ethanol. 10 units of DNase I was treated for 30 min at
37°C to eliminate the contaminating genomi c DNA. The vacuum
dried pellet was di ssolved in diethyl pyrocarbonate-treated water
and the concentrati on was determined spectrophotometrically.
Animal cap assay The vitelline membrane of the late blastula
(stage 9) embryo was removed manuall y using forceps. Animal
pole ex plants were di ssec ted using tun gs ten needles in 2 %
agarose coated dishes contai nin g 0.5 X MMR, 0.1 % bovine serum
albumin and 1 X antibiotics mi x (S igma). They were cultured in
the sa m e me di a with or without gro wth fact o rs. Th e
developmental stage of the anim al caps was determined by ag ing
of the control whole embryos reared under the same conditions.
Animal caps were cultured until midgastrula (stage I I) or late
neurul a (stage IS-20) . They were then subj ected to RT-PCR or
in situ hybridization for th e analysis of gene ex pression .
IX-cardiac actin probe synthesis The digoxygenin-Iabeled
se nse a nd a nti se nse probe for in situ hy bridi za ti o n was
made by asy mmetric PCR. The template was a-cardiac actin
PCR product amplified from stage 16 cDNA . For the antisense
probe, 1 J..l1 of 500 ng/ml reverse primer and 1 J..lI of 10 ng/ml
forward primer was used in 25 J..l1 PCR reaction. The ratio of
digox igenin- I I -dUTP to dTTP was I :2. The quality of the probe
was determined by loading a fraction of the PCR product onto
agarose gel and subseq uent stai nin g with ethidium bromide.
Due to the incorporation o f di goxygenin , the probe mi grated
s lowe r th an the template a nd seemed as if it were of a
bigger size. The co ncen tratio n of the probe was determined
spectrophotometricall y.
In situ hybridization
In situ hybridi zati on was performed as
desc ribed by Hemmati -Brivanlou et al. ( 1990) and Harl and
( 1991 ) with a few mod ifications. The embryos and animal caps
Hae Geun Chung & Hae-Moon Chung
were fix ed in MEMFA (100 mM MOPS , 2 mM EGTA, 1 mM
MgS0 4 , 3.7% formaldebyde) for 2 h at room temperature. The
specimens were stored in cold methanol until use. For in situ
hybrid izatio n, th e specimens were rehydrated gradually, and
tre a ted wit h proteinase K ( 10 ]..lg/ ml ) for 10-15 min.
Afte r re fix a ti o n of s pecim e ns in 4 % parafo rmaldehyde ,
prehybridi zation was done for 6 h at 60°C with gentle shaking
followed by overni ght hybridi zation at the same temperature. BM
purple (Boehrin ger-Mannheim) was used as a substrate for
alkaline phos phatase. After the color reaction was completed, the
e mbryos were washed with PBS twi ce fo r 5 min at room
temperature. The specimens were then dehydrated for 3 min in
methanol and stored in absolute ethanol. The wild type embryos
and animal caps were bleached in 15% H 20 2/ 10% PBS .
RT-PCR 0.2- I]..lg of an imal cap and I ]..lg of whole embryo
RNA were reverse transcribed with AMY reverse transcriptase
(Promega) using oligo dT(15 ) primer. The primers used to detect
the neureguiin gene were as foll ows: 5' -GAC CTG TCA AAC
CCG TCA A-3 '(sense); 5'-GCA GTA GGC CAC CAC ACA3'(antisense). The mesoderm and neural specific genes used as
markers for the RT-PCR assay are listed in Table I. PCR was
carried out for 30 cycles as foll ows: 95 °C for I min, 50-60.9°C
for I min , 72°C for I min . The products were analyzed by
electrophoresis using 2% agarose ge l. To confirm the absence of
genomjc DNA , reverse transcription without reverse transcriptase
was performed and the product was subj ected to PCR.
499
Xbra, the pan mesodermal marker is ectopically
expressed in Neuregulin-treated animal caps The
purpose of this study was to investigate if Neuregulin
participates in mesoderm induction and patterning, the
primary event in muscle differentiation. To achieve this
goa l, an an imal cap assay was done to find out if
neuregulin could induce the ectopic expression of pan
mesodermal marker gene Xbra in animal cap explants
which is origi nally fated to form the ectoderm.
Xbra is first expressed in the marginal zone of the
gastru la embryo, the future mesoderm, and later, in the
notochord and posterior tissue (Smith et ai. , 1991). Since
the expression of Xbra is considered as the immediate
response to mesoderm induction (Smith et al., 1991), it
was of interest if neuregulin could activate the expression
of this gene ectopically.
neuregulin
Results
RT+
Temporal expression pattern of the neuregulin
gene To address the issue of stage-specific expression of
the neuregulin gene, RT-PCR was performed. One ~g of
RNA from different embryonic stages was reverse
transcribed and set to PCR using neuregulin gene specific
primers. The neuregulin transcripts were already detected
at stage 5, the 16 cell ~ tage, reflecting that it is a maternal
message. The transcripts were also present at the gastrula
and neurula. It continued to be expressed until the tailbud
stage (stage 25), the latest embryonic stage analyzed in this
study (Fig. I).
EFl-a
RTFig. 1. The temporal expression pattern of neuregulin during
early developmental stages. RNA from various stages of the
embryo was reverse transcribed and amp lified with neureguiin
specific primers. Neureguiin is already expressed at stage 5, the
advanced 16-ceJl stage. EFI -a , reverse transcription and loalling
control. Reverse transcriptions were done with (RT +) or without
(RT- ) reverse transcriptase.
Table 1. Marker genes used for RT-PCR assay.
Gene
Expression domain
Reference
Xbra
a -cardiac actin
myoD
Presumptive mesoderm and later, in notochord and posterior ti ssue
Predomjnantly in heart tissue
Mainly in skeletal muscle, low level in heart
myf5
MRF4
Somites
Skeletal muscle
Skeletal muscle and heart
Nervous system
Anterior domain at neurulation, later in brain and eyes
Ubiquitous
Smith et ai., 1991
Mohun et ai. , 1984
Jennings, 1992
Chambers et ai. , 1994
Hopwood et ai. , 1991
Jennings, 1992
Chambers et ai., 1992
Lamb et ai., 1993
Lamb et ai., 1993
Krieg et ai., 1989
SLl
NCAM
otxA
EFJ-a
Mesodermal Gene Expression by Neuregulin
500
Animal caps, which were treated with Neuregulin and
cultured until the mid gastrula stage, were subjected to
RT-PCR. Neuregulin treatment was done at various
co nce ntration s - from 1 ng/m l to 100 ng/ml - to
determine the least amount of neuregulin required to
induce the expression of mesodermal genes. Activin , a
potent mesoderm inducer, was used at 50 ng/ml for a
positive control. The results showed that Neuregulin could
induce the expression of Xbra at the concentration of
10 ng/ml but not at 1 ng/ml (Fig. 2) .
Muscle specific genes are expressed from animal caps
treated with Neuregulin To investigate if mu scle
spec ifi c genes are expressed in ectoderm treated with
neuregulin, the animal caps of late blastula were treated
with Neuregulin and cultured until the late neurul a stage
(stage 18-20). The control animal caps and Neuregulin
treated animal caps were fix ed in MEMFA and hybridized
animal cap
WE
Neuregulin
(nglml)
C~
(1
10
ART+RT-
100)
Xbra
EFl-a.
Fig. 2. Induction of the pan mesodermal marker, Xbra by
Neuregulin. Animal pole explants fro m late blastula were treated
with increasing doses of Neuregulin (1, 10, and 100 ng/ml) and
cul tured until the midgastrula stage. The gene expression was
ana lyzed by RT-PCR. With 10 ng/m l of Neuregulin , the
express ion of Xbra was induced. Xbra express ion was not
detected in untreated animal caps (C) . Activin effect was tested at
50 ng/ml in the same condition as a positive control (A). The
staging of the animal caps were done by comparing the stage of
the same batch of control embryos reared under the same
conditions. The gene express ion of the whole embryos (WE)
reared with animal cap till the midgastrula stage is shown (RT +) .
To exclude the possibility of genomic DNA contamination, the
RNA of the whole embryo was reverse transcribed without
reverse transcri ptase (RT-).
in situ with th e a-cardiac actin probe. An in situ
hybridization study showed that animal caps treated with
Neuregulin expressed a-cardiac actin (Fig. 3A). Control
animal caps remained round and darkl y pi gmented and
showed no signal of a-cardiac actin transcripts (Fig. 3B).
In normal neurula embryos, a-cardiac actin was expressed
in the somati c mesoderm (Fig. 3C). Embryos and animal
cap explants hybridi zed with the sense probe gave no
significant signal (Fig. 3D).
As a-cardiac actin is expressed from Neuregulin
treated animal caps, it was very likely that expression of
other muscle specific genes could also be induced. A
RT-PCR assay showed that MyoD family genes such as
MyoD , Myf5, MRF4 were expressed with 5 ng/ml
Neuregulin (Fig. 4).
We next investigated if Neuregulin induces SL-J. SL-J
is known to li e down strea m of th e My o D family
transcription factors, and it is also known to be involved in
the process of myogenesis. Our results show that SL- J was
induced by Neuregulin treatment (Fig. 4).
Also, from these results, we were able to characterize
the mesoderm induced by Neuregulin . Since the muscle
genes analyzed in this study were skeletal and cardiac
specific, and as the skeletal and cardiac muscle mostly
comprise the dorso-lateral part of the body, it was
concluded that the mesoderm induced by Neuregulin was
probably of the dorso-lateral type.
Neural specific genes are secondarily induced It has
been reported that mesoderm-inducing fac tors such as
Activin and Vg 1 (Kessler and Melton, 1995) also induce
the expression of neural specific genes. Thi s phenomenon
has been presumed to occur through the initi ally induced
dorsal mesoderm, in turn inducing the ectoderm to take on
a neural fate. If the character of the mesoderm induced by
Neuregulin is genuinely dorso-lateral, it is quite possible
for the animal cap explants to express neural genes when
treated with neuregulin. To check the possibility of this, the
specimens, which were shown to express muscle specific
genes, were also analyzed for the expression of neural
genes . A RT-PCR assay showed that the pan neural marker
NCAM and anterior brain marker olxA were expressed,
impl yi ng that seco nd ary neural induction may have
occurred (Fig. 5).
Discussion
In the present study, we describe the temporal express ion
pattern of neuregulin in the early embryonic development
and its function in the mesoderm induction of Xenopus
laevis. In the first stage of this study, a partial clone of the
neuregulin spa nnin g th e EGF-like dom ain a nd th e
transmembrane domain was actually obtained. Then , we
intended to attain the whole neuregulin clone by using the
PCR strategy for th e Xenopus brain cDNA library.
Hae Geun Chung & Hae-Moon Chung
B
A
c
50 1
D
~.
•.
Fig. 3. Ectopic expression of a -cardiac actin in animal caps treated with Neuregulin. Animal caps from late blastula were dissected
and cultured in media containing Neuregulin until contro l embryos reached the late neurula stage. The animal pole explants and contro l
neurul a embryos were hybridized with the a-cardiac actin probe made by asymmetric PCR. A. Animal caps treated with Neuregulin.
The blue color signal corresponds to a-cardiac actin transcripts. B. Control animal caps show no signal. C. Whole em bryos of late
neurul a. The so mati c mesoderm is stained blue. The embryos in the bottom left and right panels each correspond to the cross sectional
views of the anterior and posteri or parts of the neurul a embryo. D. Whole embryos and animal caps hybridized with sense probe show
no significant signal.
Meanwhile, another group was reported to have cloned
neuregulin by screening the Xenopus cDNA library using
chi ck ARIA , a member of chick neuregulins, as a probe
(Yang et at., 1998). The cloned neuregulins had ex and P
isotypes, and the neuregulin cloned by us was a portion of
the p isoform.
Since mesoderm is actively induced in early bl astula
before the start of zygotic transcription, maternal genes
are thought to be responsible fo r mesoderm induction .
The te mporal expression patte rn of neuregulin shows
that it is ex pressed at the ri ght time to participate In
mesoderm induction.
MyoD, MyfS, Myogenin and MRF4 comprise a small
family of myogenic tra nsc ripti o nal re gulator s in
vertebrates. It is known that these factors are capable of
converting a large number of differe nt cell types into
striated muscle. The results of the RT-PCR assay and
in situ hybridi zation study led us to think that neuregulin
has altered the ectodermal fate of the animal cap ti ssue to
a mesodermal one.
Mesodermal Gene Expression by Neuregulin
502
an imal cap
WE
animal
WE
cap
Neuregulin
(ng/ml)
C~ART+RT­
(5
50)
Neuregulin
(ng/ml)
myoD
(5
myfS
50)
MRF4
NCAM
a-cardiac actin
SLI
otxA
EFl -a
Fig. 4. Ex pression of mu sc le specific genes by Ne ureg ulin .
Stage 18-20 animal caps treated with Neuregulin (5, 50 ng/ml )
were subjected to RT-PCR to analyze the expression of skeletal
and cardi ac muscle specific genes. The characteristics of the
ge nes analyzed are presented in Table 1. MyoD fa mil y genes and
SL- J were induced by 5 ng/ ml of Neureg ulin . A, Ac ti vin
treatment (50 ng/ml) as positive control; C, co ntro l untreated
animal caps; RT+, whole embryo at the neurula stage; RT - ,
reverse transcriptio n without reverse transcriptase
Although Neuregulin is capabl e of inducing mesoderm
specif ic gene ex press io n, it may not be the prim ary
mesoderm indu cer co nsiderin g its spati al ex press io n
pattern in the embryo. An in situ hybridizati on study on
neuregulin has sho wn th at neureg ulin transcripts are
locali zed at the animal hemisphere of the fo ur-ce ll stage
and the early blastul a embryos (Yang et aI. , 1998). Since
the results presented in thi s study are strictl y from in vitro
ex perime nts, the role ne uregulin plays in the natural
mesoderm inducing process is not clear. With the intenti on
of investi gating the in vivo rol e of neuregulin , we are
presently mi croinjecting anti sense RNA of neuregulin into
the earl y embryo. By depleting the neuregulin sig nal
present in the animal hemi sphere, we are expecting to
e lucidate the in vivo role of thi s molecule.
It is parti cul arl y interesting that Neuregulin induces
both the skeletal and cardiac mu scle gene express io n.
Knockout mi ce of neuregulin and their receptors have been
Fig. 5. Seco nd ary indu cti o n of ne ural s pec ifi c ge nes by
Neureg ulin . Animal ca ps treated with Ne ureg ulin exp ressed
ne ural ge nes at th e late ne urul a stage pres um abl y by th e
secondary effect of the primaril y fo rmed dorsal mesoderm . The
same spec imens analyzed fo r their ex pression of muscle specific
ge nes were used for thi s study. The concentration of Neuregulin
is 5, 50 ng/ml. Note the ex press ion of the pan neural marker
NCAM, anteri or neural marker otxA. C, untreated animal caps as
a negati ve contro l; WE; whole embryos at stage 18-20.
reported to be fatal to the embryo, due to the failure of the
heart to beat. The main cause of this heart malfunction was
the lack of trabecul ae projection (Meyer and Birchmeier,
1995). The di srupti on of the neuregulin function in chick
us ing ribozy me-tRN A tran sgenes was also a faithful
phenocopy of the neuregulin in knockout mice, leading to
fa ilure of ventricul ar trabecul ati o n (Zhao and Le mke,
1998). It has been reported th at gli al growth fac tor 2
(rhGGF2) promotes the prolife ration of cardi ac myocytes
at the concentrati on of 30 ng/ml (Zhao et aI. , 1998). These
findings together de monstrate that neuregulin signaling is
pi votal for normal heart development, and the present
results support the above noti on.
The low amount of Neuregulin required to induce the
mesodermal gene expression (as low as 5 ng/ml ) implies
that neuregulin may functi on in a simil ar way in vivo, as
the concentration is within a physiologicall y meaningful
range. In the preliminary stud y, we applied treatment with
Hae Geun Chung & Hae-Moon Chung
a range of Neuregulin dosages from 5 ng/ml to 10 Ilg/ml,
and they showed similar results . Altogether, our results
provide some evidences that neuregulin participates in the
formation of the mesoderm.
Acknowledgments
This work was supported by the Korea
Research Foundation ( 1996-1998).
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