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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). References Carraway, K. (1996) Involvement of the neuregu]jns and their receptors in cardiac and neural development. BioEssays 18, 263-266. Chambers, A . E., Kotecha, S. , Towers, N ., and Mohun, T. J. ( 1992) Muscle-specific expression of SRF-related genes in the early embryo of Xenopus laevis. EMBO J. 11, 4981-4991. Chambers, A. E. , Logan, M. , Kotecha, S. , Towers, N. , Sparrow, D. , and Mohun, T. J. (1994) The RSRF-MEF2 protein SLl regulates cardiac muscle-specific transcription of a myosin light-ch a in gene in Xenopus embryos. Genes Dev. 8 , 1324-1334. Fiorini , J. R. , Samuel , D. S., Ewton, D. Z. , Kirk, c., and Sklar, R. M. ( 1996) Stimulation of myogenic differentiation by a neureg ulin , glial growth factor 2. J. BioI. Chem. 271 , 12699-12702. Harland, R. M. (1991) In situ hybridi zation: an improved wholemount method for Xenopus embryos. Methods Cell BioI. 36 , 685-695. Hemmati-Brivanlou, A., Frank, D. , Boice, M. E. , Brown , B. D. , Sive. H. L. , and Harland, R. M. (1990) Localization of specific mRNA s in Xenopus embryos by whole-mount in situ hybridization. Development 110, 325-330. Hopwood, N. D., Pluck, A. , and Gurdon, J. B. (1991) Xenopus Myf-5 marks early muscle cells and can activate muscle genes ectopically in early embryos. Development 111, 551-560. Jennings, C. G. B. (1992) Expression of the myogeI1lc gene MRF4 during Xenopus development. Dev. BioI. 150, 121-132. Kessler, D. S. and Melton, D. A. (1995) Induction of dorsal mesoderm by soluble, mature V g I protein. Development 121, 2155-2164. Kim , S. 1. , Sh in , 1. H. , Kim, J., Kim, S. H., Chae, J. H., Park, E. J., Seong, R. H., Hong, S. H., Park, S. D. , Jeong, S., and Kim, 503 C. G. (1999) Isolation of developmentally regulated novel genes based on sequence identity and gene expression pattern . Mol. Cells 9, 207-218. Kimelman, D. and Griffin, K. J. P. (1998) Mesoderm induction: A postmodern view. Cell 94, 419-42l. Krieg, P. A. , Varnum , S. M., Wormington, W. M. , and Melton, D. A. (1989) The mRNA encoding Elongation Factor I-a (EFI -a) is a major transcript at the midblastula transition in Xenopus. Dev. BioI. 133, 93-100. Lamb, T. M ., Knecht, A . K. , Smith, W. C. , Stachel , S. E., Economides, A. N. , Stahl, N ., Yancopolous, G. D. , and Harland, R. M. (1993) Neural induction by secreted polypeptide, noggin. Science 262, 713-:-718. . . Meyer, D. and Birchmeier, C. (1995) Multlple essentlal functIOns of neuregulin in development. Nature 378, 334-335. Mohun, T. J. , Brennan , S. , Dathan, N. , Fairman, S. , and Gurdon. J. B. (1984) Cell type-specific activation of actin genes in the early amphibian embryos. Nature 311 , 716-721. Nieuwkoop, P. D. and Faber, J. (1967) Normal Table of Xenopus laevis (Daud in). Amsterdam, North Holland. Smith, J. c., Price, B. M. J. , Green, J. B. A. , Weigel , D. , and Herrmann, B. G. (1991) Expression of Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction. Cell 67, 79-87. Wolpert, L. , Beddington, R. , Brockes, J. , Jessell, T. , Lawrence, P. , and Meyerowitz, E. (1998) Principles of development. Current Biology, Oxford. Yang, 1. F. , Zhou, H. , Pun, S. , Jp, N. Y. , Peng, H. B. , and Tsim, K. W. K. (1998) Cloning of cDNAs encodi ng Xenopus neuregulin: expression in myotomal muscle during embryo development. Mol. Brain Res. 58, 59-73. Zhang, J. , Houston, D. w., King, M. L., Payne, c., Wylie, c., and Heaseman, J. (1998) The role of maternal vegT in establishing the primary germ layers in Xenopus embryos . Cell 94 , 515-524. Zhao, J. J. and Lemke, G. (1998) Selective disruption of neuregulin-l function in vertebrate embryos using ribozymetRNA transgenes. Development 125, 1899- 1907. Zhao, Y. Y., Sawyer, D. R. , Baliga, R. R. , Opel, D. J. , Han, X. , Marchionni , M. A. , and Kelly, R. A. (1998) Neuregulins promote survival and growth of cardiac myocytes. Persistence of ErbB2 and ErbB4 expression in neonatal and adult ventricular myocytes. J. BioI. Chem. 273, 10261-10269.