Molecular Psychiatry (1998) 3, 112–115  1998 Stockton Press A l rights reserved 1359–4184/98 $12.00

NEWS & VIEWS New branches on the family tree

The are a diverse family of that play an important role in controlling the growth and differentiation of glial, epithelial and muscle cells. This review describes recent findings of novel neuregulins (NRG2 and NRG3) and summarizes the tissue distribution and receptor interaction of these ligands.

Several years ago, factors that could regulate Schwann organization, spacing and pattern of innervation of cell proliferation, induce synthesis of acetylcholine these ganglia to and from the central receptors or promote phosphorylation of the receptor is disrupted. One explanation for the striking differ- ErbB2 were isolated in different labora- ence in hindbrain phenotypes of NRG1 and ErbB4 tories and designated glial (GGF), acetyl- knockout mice was that an additional ligand distinct choline receptor inducing activity (ARIA) and from NRG1 might be recognized by ErbB4 in the CNS. heregulin/Neu differentiation factor (NDF), respect- These prescient observations were supported by the ively (for review).1,2 These factors turned out to be pro- recent discovery of two new that are structurally ducts of alternative splicing of a single that and functionally related to NRG1. Like NRG1, these yielded a variety of membrane-bound or secreted pro- genes are also ligands for members of the ErbB family teins. Collectively this group of proteins is referred to of receptor tyrosine kinases and their expression pat- as neuregulin (NRG, now called NRG1). Since its dis- tern suggests they may have important functions in the covery, NRG1 has been shown to be a mesenchymal- nervous system. derived factor that plays a critical role in the develop- Four different laboratories have recently published ment of the heart and nervous system. The diverse articles characterizing a gene called NRG2,6,7 Don-18 or activities of NRG1 are mediated by activation of the NTAK.9 NRG2/Don-1/NTAK (referred to here as NRG2) four members of the (EGF) is not an alternatively spliced variant of NRG1. NRG2 family of receptor tyrosine kinases, designated EGFR, is located on human 5q8 while NRG1 is ErbB2, ErbB3 and ErbB4. NRG1 binds directly to ErbB3 located on chromosome 8p11-22.10,11 Like NRG1, sev- and ErbB4, which can then act to induce ErbB2 and eral alternatively spliced forms of NRG2 have been EGFR activity through heterodimerization of the recep- reported. The relative distribution and significance of tors. these splice variants remain to be determined. NRG2 Analysis of mice containing targeted mutations in and NRG1 exhibit a similar domain structure: an N- either ErbB2, ErbB4 or NRG1 provided the first hints terminal domain which resembles GGF, a C2-like Ig that NRG1 might have siblings.3–5 Mice lacking ErbB2, domain, an epidermal growth factor (EGF) domain ErbB4 or NRG1 exhibit similar defects in the develop- which determines the receptor-binding specificity, a ment of myocardial trabeculae in the ventricle and die transmembrane domain and a large intracellular during mid-embryogenisis (embryonic day (E) 10.5). domain. The greatest region of homology between These results suggest that NRG1, ErbB2 and ErbB4 are NRG2 and NRG1 is seen near the transmembrane components of the same signaling pathway essential domain (91%); significant homology is also observed for the development of the heart. However, these stud- in the Ig domain (36%), and the EGF domain (50%). ies also suggested that a novel ligand for ErbB4 may While the intracellular domain (ICD) is only 24% hom- play a role in the development of the hind brain. NRG1 ologous over the entire length, there are some small is expressed in the neuroepithelium and cells arising highly conserved regions (up to 89%) within that from rhombomeres 2, 4 and 6, while ErbB4 is domain.6 expressed in rhombomeres 3 and 5. NRG1 and ErbB2 We recently identified a novel gene more distantly knockout mice exhibit a loss of cells and axons of the related to NRG1 and NRG2 named NRG3, which maps cranial sensory ganglia. In contrast, ErbB4 deficient to human chromosome 10q22.12 NRG3 has an EGF mice do not exhibit this loss of cells. Rather, the domain with 39% homology to that of NRG2 and 31% to NRG1. Like NRG2, the most highly conserved region of NRG3 with NRG1 lies in the extracellular domain Correspondence: Dr PJ Godowski, Department of Molecular very near the transmembrane domain (62%). This con- Biology, Mailstop 37, Genentech Inc, 460 Pt San Bruno Blvd, servation suggests that this region may play an South San Francisco, CA 94080, USA. E-mail:skiȰgene.com important role in molecular interactions. Unlike NRG1 News & Views 113 and NRG2, NRG3 does not contain an Ig-like or kringle does not seem to translate to similar responses of cells domain in its extracellular region. Instead, NRG3 has treated with these factors. NRG1 appears to be more a Ser/Thr rich domain with a number of potential sites potent than NRG2 in activating ErbB2 while the reverse for O-linked glycosylation. Like its siblings, NRG3 also is observed for activation of the EGFR. One explanation contains a large ICD but it is not well conserved with is that this may reflect a difference in the ability of NRGs 1 and 2. these two ligands to promote heterodimerization of Initial studies of the properties of the NRG family ErbB family members.6 members suggest they exhibit distinct biological roles. A second clue that the functions of the new neuregu- One indication stems from the analyses of the receptor- lins are different is that the expression of these ligands binding specificities and intracellular signaling path- differs both spatially and temporally.6–9,11 Both NRG1 ways that occur following receptor binding. While and NRG2 mRNA are detected in the endothelial lining NRG3 was shown to bind specifically to ErbB4, NRG1 of the heart. NRG1 is more abundant in the ventricle and NRG2 bind to both ErbB3 and ErbB4. Interestingly, than the atrium, while the converse is true for NRG2. the overlapping receptor specificity of NRG1 and NRG2 In contrast, we did not detect NRG3 in the heart endo- a c

b d

Figure 1 Expression of NRG3 and ErbB4 in developing and adult mouse brain. (a and b) Darkfield views of NRG3 (a) or ErbB4 (b) hybridization in E16 mouse forebrain. (c and d) Darkfield views of NRG3 (c) or ErbB4 (d) in adult mouse brain. (a) NRG3 hybridization is absent from the ventricular and subventricular zones, but is observed in the cortical subplate and differentiating fields of the neocortex (cortical plate), hippocampus, amygdala and basal ganglia. (b) ErbB4 hybridization is apparent in the subventricular and marginal zones and differentiating fields of the amygdala and basal ganglia, as well as in isolated cells in the intermediate zone and in the cortical plate of the neocortex. (c) NRG3 hybridization is uniformly present throughout layers 2–6 of the cerebral cortex, and strongly apparent in the pyramidal and granule cell layers of the hippocampus and in the thalamus. (d) ErbB4 mRNA is expressed by subsets of cells within the cerebral cortex, hippocampus and hypothalamus. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone; Ctx, cerebral cortex; HC, hippocampus; Th, thalamus. News & Views 114

Figure 2 Schematic diagram showing the interactions between neuron-derived NRGs and glial ErbB receptors. This diagram does not imply that all forms of NRGs and ErbBs are expressed in the same type of cells. For example, Schwann cells only express ErbB2 and ErbB3. Cell type-specific expression of NRG2 and NRG3 is not known, and the neuronal expression in this figure is a hypothesis. Although interactions between NRG2 and ErbB2/ErbB3, between NRG3 and ErbB4 have been demon- strated, the cellular responses of these interactions are not known (thus dashed lines are used to indicate interactions only). NRGs can be proteolyzed and the secreted forms may act as ligands.

cardium.12 NRG1 is expressed in mesenchymal cells of ErbB4 is expressed in the subventricular and inter- various parenchymal organs, such as lung, intestine, mediate zones underlying the cortical plate (Figure 1). stomach, kidney and the genital ridge. NRG2 and Cortical neuronal precursor cells are generated in the NRG3 are expressed at highest levels in the nervous ventricular zones adjacent to the ventricle and migrate system, although low levels of NRG2 are observed in from this location to their final positions in the the lung.8 Within the CNS, the three ligands have dif- developing cortex. Thus, NRG3 seems to be expressed ferent expression patterns. For example, expression of in more differentiated neurons. In the adult mouse, NRG1 in the developing cortex of E14.5 mice or post- NRG3 is widely expressed in multiple layers of the cer- natal day 0 rats corresponded mostly to the deeper lay- ebral cortex with escalated expression in the hippo- ers (ventricular and subventricular zones) of the neur- campus (Figure 1c). In contrast, ErbB4 is expressed in oepithelium which lines the lateral ventricles. In the a punctate distribution in adult brain (Figure 1d). The E16 mouse (Figure 1, panel a), NRG3 mRNA is complementary pattern of NRG3 and ErbB4 is remi- enriched in the cortical plate. In the hippocampus of niscent of recent findings by C Rio et al, that cerebellar adult rats, no NRG1 expression can be detected, granule cells express NRG1 whereas radial glial cells whereas NRG2 expression is limited to the dentate express ErbB4, and that NRG1–ErbB interaction plays gyrus. In contrast, NRG3 is highly expressed in the hip- a crucial role in the migration of cerebellar granule pocamal regions of CA1–CA3 pyramidal cells, as well cells along radial glial fibers.13 Studies of the NRG3 as in granule cells in the dentate gyrus of adult mice expression in earlier stages are underway to determine (Figure 1, panel c). The distribution of the three NRGs if NRG3 is the ‘missing ligand’ for ErbB4 during cranial during neural development, combined with their nerve formation. NRG2 may not be such a ligand due to unique receptor-binding properties, suggests that these its absence of expression in E9.5–E10 mouse embryos.7 gene products play distinct roles in vivo. Identification of additional members of the NRG fam- A comparison of the patterns of expression of NRG3 ily affords new insight into the regulatory interactions and ErbB4 in the telencephalon of E16 mice by in situ of neuronal cells. Studies comparing NRG ligand and hybridization revealed that the ligand and the receptor receptor expression patterns during various develop- are expressed in a complementary fashion. NRG3 mental stages, as well as studies of mice containing tar- expression is confined to the cortical plate (highly geted mutations in these genes should help to elucidate expressed in the subplate) of the telencephalon while the contributions of NRGs 1, 2 and 3. A diagram show- News & Views 115 ing some of the known features of neuron-derived 5 Lee KF, Simon H, Chen H, Bates B, Hung MC, Hauser C. Require- NRG1 is shown in Figure 2. Stimulation of ErbB recep- ment for neuregulin receptor erbB2 in neural and cardiac develop- ment. Nature 1995; 378: 394–398. tors may promote the production of other surface or 6 Carraway KLR, Weber JL, Unger MJ Ledesma J, Yu N, Gassmann M, 14 secreted molecules from the recipient cells and form Lai C. Neuregulin-2, a new ligand of ErbB3/ErbB4-receptor tyrosine a regulatory loop from glia to neurons. In addition, the kinases. Nature 1997; 387: 512–516. large intracellular domains of most NRGs raise the 7 Chang H, Riese DJN, Gilbert W, Stern DF, McMahan UJ. Ligands for ErbB-family receptors encoded by a neuregulin-like gene. Nat- interesting possibility that NRGs are themselves sig- ure 1997; 387: 509–512. naling molecules. These studies, along with the 8 Busfield SJ, Michnick DA, Chickering TW, Revett TL, Ma J, Woolf detailed characterization of the consequences of recep- EA et al. Characterization of a neuregulin-related gene, Don-1, that tor activation by the ‘new neuregulins’ will keep neuro- is highly expressed in restricted regions of the cerebellum and hip- biologists busy for some time to come. pocampus. Mol Cell Biol 1997; 17: 4007–4014. 9 Higashiyama S, Horikawa M, Yamada K, Ichino N, Nakano N, Nak- agawa T et al. 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Neuregulins in development. Mol Cell Neurosci 1996; 7: 9562–9567. 247–262. 13 Rio C, Rieff HI, Qi P, Corfas G. Neuregulin and erbB receptors play 3 Meyer D, Birchmeier C. Multiple essential functions of neuregulin a critical role in neuronal migration. Neuron 1997; 19: 39–50. in development. Nature 1995; 378: 386–390. 14 Verdi JM, Groves AK, Farinas I, Jones K, Marchionni MA, Reichardt 4 Gassmann M, Casagranda F, Orioli D, Simon H, Lai C, Klein R et LF et al. A reciprocal cell–cell interaction mediated by NT-3 and al. Aberrant neural and cardiac development in mice lacking the neuregulins controls the early survival and development of sym- ErbB4 neuregulin receptor. Nature 1995; 378: 390–394. pathetic neuroblasts. Neuron 1996; 16: 515–527.