Proc. Natl. Acad. Sci. USAVol. 93, pp. 4833-4838, May 1996Neurobiology

Neuregulins with an Ig-like domain are essential for mousemyocardial and neuronal developmentRAINER KRAMER*, NATHAN BUCAY*, DARCI J. KANE*, LAURA E. MARTINt, JOHN E. TARPLEYt,AND LARS E. THEILL*§Departments of *Molecular Biology, tLaboratory Animal Resources, and tExperimental Pathology, AMGEN Center, 1840 DeHavilland Drive, Thousand Oaks,CA 91320-1789

Communicated by Gerald D. Fischbach, Harvard Medical School, Boston, MA, January 11, 1996 (received for review November 4, 1995)

ABSTRACT Neuregulins are ligands for the erbB family ofreceptor tyrosine kinases and mediate growth and differentia-tion of neural crest, muscle, breast cancer, and Schwann cells.Neuregulins contain an epidermal growth factor-like domainlocated C-terminally to either an Ig-like domain ora cysteine-rich domain specific to the sensory and motor neuron-derived isoform. Here it is shown that elimination of the Ig-likedomain-containing neuregulins by homologous recombinationresults in embryonic lethality associated with a deficiency ofventricular myocardial trabeculation and impairment of cranialganglion development. The erbB receptors are expressed inmyocardial cells and presumably mediate the neuregulin signaloriginating from endocardial cells. The trigeminal ganglion isreduced in size and lacks projections toward the brain stem andmandible. We conclude that IgL-domain-containing neuregulinsplay a major role in cardiac and neuronal development.

The structurally related neuregulin proteins ARIA (1), glialgrowth factors (2), neu differentiation factor (NDF) (3),heregulin (4), and sensory and motor neuron-derived factor(SMDF) (5) are derived from the same gene by alternativesplicing. All described neuregulins express an a- or 3-typeepidermal growth factor-like (EGF-L) domain (6) and eitheran Ig-like (Ig-L) domain or the apolar, cysteine-rich SMDF-specific domain at the N terminus (5). The EGF-L domainbinds to the receptors (7), and the Ig-L domain binds toconstituents of the extracellular matrix such as heparin (8).Neuregulins are expressed in a variety of embryonic and adulttissues, in particularly high levels in the central and peripheralnervous systems (2, 9, 10). In vitro, neuregulins have multiplebiological activities, ranging from induction of acetylcholinereceptors and sodium channels in myotubes (1, 11), neuralcrest stem cell differentiation (12), maturation of Schwann cellprecursors (13), and Schwann cell proliferation (1) to growthand differentiation of cultured breast cancer cells (3, 4).

Neuregulins bind to the related erbB3 and erbB4 receptortyrosine kinases (14, 15) and, by heterodimerization, activateerbB2 (16). For example, in muscle cells, acetylcholine recep-tor synthesis induced by ARIA was shown to occur in thepresence of erbB2 and erbB3 (17). erbB2 is overexpressed insome aggressive human cancers and is associated withSchwann cell tumors of cranial and peripheral nerves (18).However, the developmental and physiological functions ofneuregulin ligands and their corresponding erbB receptors invivo remain to be elucidated. We report here that micedeficient in Ig-L domain-containing neuregulins have severedefects in the developing heart and nervous system.

MATERIALS AND METHODSTargeting Vector Construction. A 13-kb genomic clone

encoding the neuregulin Ig-L exon 3 was isolated from a mouse

129 SV genomic DNA library (Stratagene) by use of a ratNDF-cDNA probe (762-bp BamHI-HindIII fragment) codingfor exons 3-11 (3) by standard procedures. The 7-kb PstIfragment coding for the upstream intron and the N-terminalportion of exon 3 was fused to a vector containing PGK-Neo(19) and pMC1-tk (20). A PCR fragment coding for the 700-bpdownstream intronic sequence was inserted into a HindlIl sitebetween PGK-Neo and pMC1-tk.

Generation and Genotype Analysis of Neuregulin MutantAnimals. The linearized targeting vector was transfected into CJ7embryonic stem (ES) cells (21) by use of a Bio-Rad Gene Pulser(500 ,tF, 240 V). The electroporated ES cells were cultured andselected as described (21). Genomic- DNA of resistant ES cellclones was prepared from colonies in replica plates and screenedby Southern blot analysis with the intron probe. Frequency ofhomologous recombination was 3 of400 clones. Chimeric animalswere obtained by microinjecting ES cell clones CJ7/264 andCJ7/355 into C57BL/6 mouse blastocysts as described (21).Chimeric male offspring were mated to C57BL/6 females to testfor germ-line transmission of the dominant agouti coat colormarker. F1 animals heterozygous for the neuregulin Ig-L allelewere intercrossed. Embryos of heterozygous matings were dis-sected between 9.5 and 13.5 days postc.oitum (morning of detec-tion of copulation plaque is 0.5 days postcoitum), and theembryonic yolk sac (or tail tips of neonates) was digested at 55°Cfor 12 h in lysis buffer [0.5% SDS/1 mg/ml of proteinase K in 20mM (50mM for tail tips) Tris, pH 8/10mM (100mM for tail tips)EDTA, pH 8]. Debriswas pelleted by centrifugation, and genomicDNA was isolated by isopropanol precipitation. Genomic DNA(50-100 ng) was analyzed using an Expand kit (BoehringerMannheim) in a Perkin-Elmer 9600 Thermal Cycler under thefollowing conditions: 94°C/2 min, followed by 30 cycles of94°C/10 s, 65°C/1 min, and 68°C/1 min (plus 20-s increments percycle), followed by 68°G/10 min; primer a, 5'-GTT GGC ATTTCA ATT GCA TTA GAC ATC AAC ACTT-3', primer b,5'-CTT TTT CCA GCA TTG CCT CCC AGA TTG GAAGAG ATG-3'. Oligonucleotides a and b produced a 0.5-kb PCRreaction product from the wild-type allele and a 2-kb productfrom the mutant allele (see Fig. 1A), which were visualized byelectrophoresis in a 1% agarose gel.

In Situ Hybridization. In situ hybridization was performedwith mouse Ig-L (143 bp) and EGF-L (152 bp) domain-specific[35S]UTP-labeled riboprobes on deparaffinized sagittally sec-tioned E10.5 embryos (22). Templates for in vitro transcriptionwere generated by PCR with oligonucleotides specific for theIg-L domain coding exon 3 (5'-GAT GTT AAG CCA GGAGTC AGC TGC AG-3' and 5'-GTA TCT TGA TGT TTGTGG TT-3') and the EGF-L domain coding exon 6 (5'-TTCCTA TCC AGC CAC ATC TAC-3' and 5'-TTC ATT TCTTAC TTG CAC AAG-3') and were subcloned into BluescriptKS vectors (Stratagene).

Abbreviations: NDF, neu differentiation factor; SMDF, sensory andmotor neuron-derived factor; EGF-L, epidermal growth factor-like;Ig-L, Ig-like; ES, embryonic stem; DRG, dorsal root ganglia.§To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. Ig-L domain disruption by gene targeting. (A) Schematic diagram of the neuregulin gene modified from ref. 2 to include the newlyidentified SMDF-specific N-terminal domain (5), whose relative position is unknown. The mouse genomic clone 4 encoding the Ig-L domain exon3 with flanking introns was used to generate the targeting vector (19). Homologous recombination results in acquisition of an additional NcoI sitein the PGK-Neo-coding segment; sizes ofNcoI fragments are indicated (P, PstI, and N, NcoI). (B) Southern blot analysis of NcoI-digested genomicDNA of transfected, double-resistant ES cell clones. Hybridization with the intron probe shown inA demonstrated that two clones contained themutated 10-kb NcoI fragment in addition to the 15-kb NcoI wild-type fragment, indicating homologous recombination. (C) Genotype analysis ofan E10.5 litter from a heterozygous intercrossing by PCR using oligonucleotides a and b (A) and yolk sac genomic DNA. A 0.5-kb fragment wasgenerated from the wild-type allele, and a 2-kb fragment was generated from the mutant allele (20).

Whole-Mount Immunohistochemistry. Embryos were fixedin methanol/dimethyl sulfoxide (DMSO) (4:1) overnight at4°C, treated for 4 h at room temperature with methanol/DMSO/30% H202 (4:1:1), and rehydrated for 30 min at roomtemperature in PBT (PBS plus 0.5% Tween 20). Rehydratedembryos were incubated overnight at 4°C with the monoclonalanti-NF160 antibody (Sigma) diluted 1:600 in calf serum/DMSO (4:1). After five washes in PBT for 60 min at roomtemperature, embryos were incubated with horseradish per-oxidase-coupled goat anti-mouse IgG overnight at 4°C, thenwashed in PBT and developed with diaminobenzidine.

Histopathology and Image Analysis. Paraffin serial sagittalsections (3 ,um) from homozygous and wild-type E10.5 em-bryos were prepared and stained with hematoxylin and eosin.For image analysis, ganglia were manually outlined on every10th section, and images were captured from a Nikon FXAmicroscope by use of a color camera (Optronics Engineering,Santa Barbara, CA). Analysis was performed with the Meta-Morph Imaging System (Universal Imaging, Media, PA), andstatistical significance tested by Student's t test.Immunohistochemistry. Formalin-fixed, paraffin-embed-

ded embryos were serially sectioned at 3 ,m, deparaffinized,and stained on a TechMate 500 immunostainer (BioTekSolutions, Santa Barbara, CA) using an ABC (BioTek)method. Sections were blocked in normal goat serum, incu-bated with primary antibody, and subsequently treated witheither biotinylated goat anti-rabbit IgG or biotinylated goatanti-mouse ,-chain-specific secondary antibody and ABCreagent. Counterstaining was performed with hematoxylin. Anavidin-biotin block (Vector Laboratories) was used to reduce

nonspecific staining of anti-erbB antibodies. Anti-erbB anti-bodies (Santa Cruz Biotechnology) were used at followingconcentrations: polyclonal anti-erbB2 (0.5 Ag/ml), monoclo-nal IgM anti-erbB3 (4 ,ug/ml), polyclonal anti-erbB4 (1 ,ug/ml), and affinity-purified polyclonal anti-human NDFa2 an-tibody named 1915 (2 ,ug/ml) (23).

RESULTSTargeted Disruption of the Neuregulin Ig-L Domain. The Ig-L

domain of the neuregulin gene was disrupted by homologousrecombination by use of a targeting vector that replaced part ofthe Ig-like domain encoding exon 3 and the downstream intronwith a PGK-Neo cassette and introduced several stop codons inthe sense strand, eliminating the capacities for correct RNAsplicing and generation of functional protein (Fig. 1A). ES cell

Table 1. Results of Ig-L domain heterozygote mating

-No. of embryos GenotypeAge or pups +/+ +/- -I-

E9.5 44 15 19 10E10.5 123 33 48 42E11.5 10 3 3 4*E12.5 15 3 10 2*E13.5 25 7 15 3*Newborn 299 98 201 0

Genotypes of progeny of neuregulin heterozygous intercrosses wereidentified by PCR analysis as described in Materials and Methods.*Resorption sites and resorbing embryos.

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FIG. 2. Abnormal heart and cranial ganglion development in homozygous mutants. Sagittal sections of hematoxylin- and eosin-stained heart(A, B, F, and G) and trigeminal ganglion (D, E, I, and J) from wild-type (A-E) and homozygous mutant (F-J) E10.5 embryos. (C and H)Whole-mount immunohistochemistry with an anti-neurofilament NF 160 antibody. Arrowheads in B and G indicate the endocardial cell layer.Arrows in J indicate cellular debris in mutant trigeminal ganglion. At, atrium; EC, endocardial cushion; Ve, ventricle; MC, myocardium; TG,trigeminal ganglion; V, trigeminal ganglion; VII/VIII, facio-acoustic ganglion complex; IX, glossopharyngeal nerve; X, vagus nerve. (A and F,x12.5; B and G, x50; C and H, x10; D and I, x20; E and J, x100.)

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FIG. 3. mRNA-encoding neuregulins with an Ig-L domain is absent in mutant tissues. In situ hybridization of wild-type (A-D) and homozygousmutant (E-H) embryos with the Ig-L (A and E) or EGF-L (B, C, D, F, G, and H) domain-specific probes. In heart sections (A, B, E, and F),arrowheads indicate endocardial cell layer. (C and G) Dorsal root ganglia. (D and H) First branchial arch. MC, myocardium; BA, first branchialarch. (A-C and E-G, x50; D and H, x 15.)

clones in which homologous targeting had occurred were iden-tified by genomic Southern blotting (Fig. 1B) and used to createtwo lines of mice heterozygous for the Ig-L domain.Embryonic Lethality of Mice Deficient in Neuregulin Ig-L

Domain. F1 mice heterozygous for the Ig-L allele appearedphenotypically normal and were intercrossed. Analysis ofnewborn F2 mice revealed a 1:2 ratio of wild-type (+/+) andheterozygous mutant (+/-) progeny (Table 1), indicating alethal homozygotic phenotype. To determine when homozy-gous mutants were dying, embryos were isolated between 9.5and 13.5 days postcoitum (E9.5 and E13.5) and genotyped bya PCR-based method (Fig. 1C). When isolated at E11.5,homozygous mutant embryos (- / -) were growth arrested andunder resorption (Table 1).Lack of Myocardial Trabeculation in Mutant Embryonic

Heart Homozygous mutant embryos isolated at E10.5 haddeveloped the same number of somites (29-36) as their wild-typelittermates but occasionally displayed a reduced heart rate.Depressed contractility, dilation of the common ventricle anddecreased emptying of the ventricle could be observed by ste-reomicroscopic analysis. It is possible that dilation of the ventricleoccurs to compensate for the reduced contractility. Although the

atrium and the outflow tract appeared normal in E10.5 homozy-gotes (Fig. 2F), ventricular trabeculation was severely impaired(Fig. 2F and G). Development of the myocardium, including thefirst stages of trabeculation, appeared normal in E9.5 embryos(data not shown). At day 10.5, however, trabeculation had notproceeded further, as evidenced by a lack of intertrabecularsinosoids (Fig. 2B and G), although endocardial and myocardialcells appeared normal. The endocardial cushion itself developedquite normally in the mutant embryo. However, the cushionappeared less contracted in the mutant (Fig. 2F) than in the wildtype (Fig. 2A).Because neuregulin expression in the heart and, in partic-

ular, in the endocardial cell layer is described (10), we per-formed in situ hybridization experiments to verify the lack ofthe Ig-L domain in the mutant heart. Using domain-specificprobes, both the EGF-L and Ig-L domains were detected in theendocardial cell layer of the E10.5 wild-type heart (Fig. 3A andB). As expected, the Ig-L domain could not be detected in thehomozygous mutant embryo (Fig. 3E). Furthermore, EGF-Ldomain expression was found in the dorsal root ganglia (DRG)of both wild-type and homozygotes (Fig. 3 C and G) and in thefirst branchial arch of the wild-type only (Fig. 3 D and H).

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Collectively, these data demonstrate that mRNAs encodingneuregulins with an Ig-L domain, and therefore their corre-sponding proteins, were eliminated in homozygous mutantembryos, whereas the mRNAs encoding other neuregulinisoforms were unaffected. The coexpression of neuregulinswith and without Ig-L domains in the endocardium is stronglysupported by our finding of EGF-L domain expression in themutant endocardium (Fig. 3F). Furthermore, the neuregulinreceptors erbB2, erbB3, and erbB4 were expressed in myocar-dial cells of wild-type (Fig. 4 A-C) and homozygous embryos(not shown). These data suggest that neuregulins with an Ig-Ldomain mediate important growth and differentiation signalsbetween endocardial and myocardial cells.

Impaired Cranial Nerve Development in Mutant Embryos.Whole-mount immunohistochemistry using an anti-neurofila-ment NF160 antibody demonstrated morphologically abnormalcranial ganglia in E10.5 homozygous mutants (Fig. 2 C and H).The proximal part of the trigeminal ganglion, its mandibularbranch, and the projection toward the brain stem were missing(Fig. 2H). Furthermore, the proximal part of the facio-acoustic

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ganglion complex appeared to be compromised (Fig. 2H). Theseobservations were corroborated by determination of the averageganglion area on serial sagittal sections (Fig. 2D and I) using theMetaMorph Imaging System (Universal Imaging). The meanarea of the facio-acoustic ganglion was reduced by about 30% inthe mutant embryo (23,940 lum2 + 1,355 SEM vs. 16,630 ium2 +1,206 SEM, P = 0.0008). The average area of the trigeminalganglion was reduced by about 66% in the homozygous mutants(68,211 11m2 + 11,586 SEM vs. 22,622 ,um2 ± 2,579 SEM, P =0.0004). In the proximal portions ofcranial nerves IX and X, theirprojections to the brain stem were compromised (Fig. 2H),whereas the distally located petrosal and the nodose ganglionappeared to be normal in the homozygous mutant (Fig. 2H).Other noted defects in the mutant trigeminal ganglion were adisruption of the cellular organization, vacuolization, and kary-orrhexis (Fig. 2J). Neurons of the cranial ganglia originate fromectodermal placodes and neural crest. In avians neural-crest-derived neurons are found in the proximal part of the trigeminalganglion, in the facial root ganglion, and the proximal ganglioniccomplex of cranial nerves IX and X (24). Placode-derived

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FIG. 4. Localization of erbB receptor and neuregulin expression. Immunohistochemistry of wild-type (A-D) and homozygous mutant (E-H) E10.5embryonic tissues stained with anti-erbB2 (A and E), anti-erbB3 (B and F), anti-erbB4 (C and G), and anti-NDFa2 #1915 (D and H) antibodies. (A-C)Wild-type heart; (E-G) mutant trigeminal ganglion; (D and H) neural tube with lateral motor column (filled arrowhead) and DRG (open arrowhead).Specific immunostaining for erbB2, erbB3, and erbB4 was localized to the cytoplasm/cell membrane of myocardial cells (A, B, and C, respectively), andcells in the trigeminal ganglia (E, F, and G, respectively). Strong and specific immunoreactivity for neuregulin EGF-L domain was observed in bothwild-type (D) and mutant (H) neural tube, especially the lateral motor column, and in the DRG cells. (All photographs, X40.)

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neurons are located distal to the neural-crest-derived cells andfound in the branches of the trigeminal ganglion, in the geniculateganglion, the vestibular and acoustic ganglia, and the petrosal andnodose ganglia (24). By analogy, it is likely that the parts of theganglia that were affected in the homozygous mutant (Fig. 2)originated from cranial neural crest. Interestingly, trunk neural-crest-derived DRG appeared morphologically unaffected asshown by cross-sections (Fig. 4 D and H) and whole-mountimmunohistochemistry (not shown).

Expression of neuregulins in E1i mouse cranial ganglia iswell documented (2, 9). The corresponding receptors erbB2,erbB3, and erbB4 were also expressed in the trigeminal gangliaof homozygous mutant (Fig. 4 E-G) and the wild-type embryo(not shown). ErbB2 and erbB4 were highly expressed through-out the trigeminal ganglion (Fig. 4 E and G), whereas erbB3expression was scattered in the ganglia and its periphery andoften found associated with damaged cells (Fig. 4F). SMDF,which lacks an Ig-L domain and therefore is intact in mutantanimals, is known to be highly expressed in the lateral motorcolumn, DRG, and the trigeminal ganglion (5). Immunohis-tochemical localization of the EGF-L domain with an anti-NDFa2 antibody (23) in these areas indicates normal SMDFexpression in the homozygous mutant (Fig. 4H). Furthermore,the wild-type and the homozygote expressed equal amounts ofa 31 kDa protein presumed to be SMDF, as assessed byWestern blot of proteins extracted from E10.5 mouse embryos,probed with the anti-NDFa2 antibody (data not shown).Therefore, described defects result specifically from the loss ofneuregulins with an Ig-L domain.

DISCUSSIONOur results suggest an essential biological role for neuregulinswith an Ig-L'domain during heart and cranial nerve develop-ment. Meyer and Birchmeier (25) recently observed similarphenotypic defects in the developing heart and cranial gangliaof mice lacking all neuregulin isoforms. Taken together, thesestudies imply that neuregulins with an Ig-L domain are limitingneuronal and cardiac development.The data suggest a paracrine mechanism for neuregulin func-

tion in formation of the trabeculated ventricular myocardium.Neuregulin secreted from endocardial cells binds to one or acombination of erbB receptors present on myocardial cells andmediates a growth and differentiation signal essential for thecontinued development of the myocardial musculature. Theexistence of such signals from the endocardial lining to themyocardium has been anticipated but not demonstrated (26) invivo. Fibroblast growth factor receptor 1 expression affectschicken cardiac myocyte proliferation but at comparably laterdevelopmental time points (27). Disruption of the endothelial-specific TEK receptor tyrosine kinase in mice results in dimin-ished trabeculation (28), an effect that may be secondary todegeneration of endocardial cells. Targeting of the transcriptionfactors RXRa, TEF, and Nkx2-5 also affects trabeculation (29-32) but cell-cell signaling was not shown to be affected. Neu-regulin is continually expressed in the endocardium during laterembryonic development (10) and in adult heart (4). The extentand exact nature of neuregulins trophic support for embryonicand adult myocardium demands further investigation. It is pos-sible, for instance, that neuregulins regulate expression of sodiumchannels and acetylcholine receptors in myocardial cells as it wasshown for skeletal muscle cells (1, 11).

Cranial nerve development in mutant embryos was impaired inregions corresponding to neural-crest-derived structures in birds.Two different mechanisms could account for the diminished sizeof the cranial ganglia: impaired migration of neural crest cells orlack of trophic support by neuregulins in early gangliogenesis. Thelatter is supported by signs of cell death in the ganglia. We notedthat damaged cells tended to express erbB3, but it is so far unclearwhether these are neuronal or glial cells. It is possible that theneuregulins with an Ig-L domain expressed by neurons are

required for survival and differentiation of glial cells, which thenin turn support neuronal survival or differentiation. Such amechanism would be supported by the observation that pluripo-tent neural crest cells become committed to a glial phenotype inthe presence of neuregulins in vitro (12) and our finding that noneof the remaining neurons in the mutant trigeminal ganglion wereable to extend processes toward the brain stem.

In gene knock-out experiments, redundant factors oftencompensate for the lack of a related protein. However, neu-regulin isoforms lacking the Ig-L domain had no compensatoryeffect, suggesting a unique role for the Ig-L domain. It ispossible that the Ig-L domain functions through associationwith the extracellular matrix, or perhaps by facilitating neu-regulin dimerization through homophilic interactions thatsubsequently support erbB receptor heterodimerization.

We thank Dr. D. Wen and D. Chang for the anti-human NDFa2antibody; A. Janssen and R. Basu for sequencing; Dr. K. Stark foradvice regarding targeting vectors and ES cells; and Drs. A. Welcher,J. Verdi, and D. Ingram for revision of the manuscript. We also thankDrs. L. Souza, B. Ratzkin, S. Suggs, D. Lacey, and D. Danilenko fortheir support and Drs. G. E. Lemke and K. F. Lee for sharing theirobservation about erbB4 and erbB2 mutants.

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