Expression patterns of transmembrane and released forms of neuregulin during spinal cord and neuromuscular synapse development

Jeffrey A. Loeb 0 Tejvir S. Khurana 0 Janet T. Robbins 0 Ann G. Yee 0 Gerald D. Fischbach 0 0 Department of Neurobiology, Harvard Medical School , Boston, MA 02115 , USA SUMMARY We mapped the distribution of neuregulin and its transmembrane precursor in developing, embryonic chick and mouse spinal cord. Neuregulin mRNA and protein were expressed in motor and sensory neurons shortly after their birth and levels steadily increased during development. Expression of the neuregulin precursor was highest in motor and sensory neuron cell bodies and axons, while soluble, released neuregulin accumulated along early motor and sensory axons, radial glia, spinal axonal tracts and neuroepithelial cells through associations with heparan sulfate proteoglycans. Neuregulin accumulation in the synaptic basal lamina of neuromuscular junctions occurred ARIA (acetylcholine receptor-inducing activity), a protein purified on the basis of its ability to stimulate the synthesis of acetylcholine receptors (AChRs) in embryonic myotubes, is a member of a family of growth and differentiation factors, called neuregulins (NRGs), that bind to and activate members of the EGF receptor family of tyrosine kinases erbB2, erbB3 and erbB4 (Peles and Yarden, 1993; Lemke, 1996; Burden and Yarden, 1997; Gassmann and Lemke, 1997; Fischbach and Rosen, 1997). Until recently, it was thought that all NRGs arose by alternative mRNA splicing from a single gene NRG1 (Marchionni et al., 1993) but, within the past year, two additional genes have been discovered (Chang et al., 1997; Carraway et al., 1997; Busfield et al., 1997; Zhang et al., 1997). All NRG isoforms from the NRG1 gene thus far examined have an EGF-like domain necessary for activation of their receptors. Most forms, like ARIA, also have an Ig-like domain that binds heparan sulfate proteoglycans (HSPGs) and leads to the deposition of these forms in the extracellular matrix (Loeb and Fischbach, 1995). There is a growing body of evidence suggesting that ARIA, and perhaps other members of the NRG1 family, promote the local synthesis of acetylcholine receptors at developing and mature neuromuscular synapses. NRG1 mRNAs are concentrated in motor neurons, and the protein accumulates in motor nerve terminals at developing and mature endplates (Goodearl et al., 1995; Moscoso et al., 1995; Jo et al., 1995). significantly later, coincident with a reorganization of muscle extracellular matrix resulting in a relative concentration of heparan sulfate proteoglycans at endplates. These results demonstrate an early axonal presence of neuregulin and its transmembrane precursor at developing synapses and a role for heparan sulfate proteoglycans in regulating the temporal and spatial sites of soluble neuregulin accumulation during development. NRG receptors, erbB2, erbB3 and erbB4 have been detected by immunohistochemistry in the region of the postsynaptic membrane (Moscoso et al., 1995; Zhu et al., 1995; Altiok et al., 1995). ARIA also promotes the expression of voltage-gated sodium channels in chick muscle cells (Corfas and Fischbach, 1993), and, in mammals, it enhances expression of the AChR epsilon subunit (Martinou et al., 1991). Both effects would be expected to increase the efficacy of synaptic transmission as the target muscle fiber increases in size and the neuromuscular junction (NMJ) matures. Most significantly, mice in which one Ig-like domain of the NRG1 allele is disrupted by homologous recombination, exhibit a 50% reduction in the density of AChRs in the postsynaptic membrane and, when challenged by low doses of curare, a reduced safety factor for neuromuscular transmission can be demonstrated (Sandrock et al., 1997). Unfortunately, homozygous mice with disruptions of the NRG1 gene die from cardiac defects around E10 before nerve-muscle synapses form (Meyer and Birchmeier, 1995; Kramer et al., 1996). Proteins encoded by the other NRG genes have not yet been studied in detail. For ARIA and other NRGs to act as AChR inducers at neuromuscular junctions, they must be expressed in motor neurons early in development and transported orthogradely to nerve terminals. Those isoforms that are synthesized as part of a transmembrane precursor must be cleaved and released into the synaptic cleft. NRG mRNA has been detected in embryonic chick, mouse and rat motor neurons when neuromuscular synapses are first forming (Falls et al., 1993; Orr-Urtreger et al., 1993; Marchionni et al., 1993; Corfas et al., 1995; Goodearl et al., 1995). Little is known about the axoplasmic transport, proteolytic cleavage of transmembrane NRG isoforms, or where and how released forms accumulate. Earlier studies showed that proARIA is expressed on the surface of transfected CHO cells and that the ectodomain (ARIA) can be detected in conditioned medium (Burgess et al., 1995; Loeb et al., 1998). Activation of protein kinase C greatly enhances the release of ARIA into the medium of these transfected cells as well as cultured sensory neurons expressing endogenous NRG forms (Loeb et al., 1998). Once proteolytically cleaved and released, NRG isoforms containing an Ig-like domain adhere to the cell surface in vivo and can be released from the extracellular matrix by high salt, heparin or limited proteolysis (Loeb and Fischbach, 1995). Using domain-specific antibodies, we now present evidence that the transmembrane precursor, proNRG, is expressed in developing chick embryonic motor neurons shortly after they merge from the germinal epithelium. We show that proNRG immunoreactivity is transported down motor axons as they first emerge from the neural tube. Once released from the transmembrane precursor or from other secreted isoforms, NRG immunoreactivity accumulates in the extracellular matrix within the spinal cord and at the NMJ through interactions with developmentally regulated HSPGs. These observations suggest a novel means for concentrating NRGs at specific sites within the developing central and peripheral nervous system. MATERIALS AND METHODS Antibodies and reagents Mouse monoclonal IgG supernatants against Islet-1/2 (4D5) (Ericson et al., 1992) and HSPG (33-1) (Bayne et al., 1984) were obtained from the Developmental Studies Hybridoma Bank at the University of Iowa under contract N01-HD-7-3263 from the NICHD. mAb 6D2 against agrin was from Willi Halfter (University of Pittsburgh). BODIPYBTX was from Molecular Probes (Eugene, OR). Whitehorn chick embryos were obtained from Spafas (Preston, CT), grown in an humidified, rocking incubator (Kuhl, Flemington, NJ), and staged according to Hamburger and Hamilton (1951). Timed-pregnant Swiss Webster mice were from Harland (Indianapolis, IN). NRG antibody preparations and characterizations Affinity-purified rabbit antisera 1310, directed against the peptide CNSFLRHARETPDSYRDS within the proximal COOH terminus of proNRG, was provided by Theresa Burgess (AMGEN). This peptide corresponds to the extreme COOH terminus of a 2C NDF and is identical to chick (...truncated)