Neuregulin Signaling Research Articles

Corrigendum: Maf links Neuregulin1 signaling to cholesterol synthesis in myelinating Schwann cells.

Corrigendum: Maf links Neuregulin1 signaling to cholesterol synthesis in myelinating Schwann cells.

Neuregulin 1 regulates amyloid precursor protein cell surface expression and non-amyloidogenic processing

The amyloid precursor protein (APP) is a key molecule in Alzheimer's disease. The prevailing view is that APP is initially transported to the plasma membrane as a full-length protein. Its localization at the cell surface can trigger downstream signaling and APP cleavage. Our previous work has shown that Neuregulin 1 (NRG1) has neuroprotective effects in an Alzheimer's disease model. In the present study, we examine whether NRG1 signaling is involved in APP expression and non-amyloidogenic processing in neuronal cells. Here we show that NRG1 increased the cell surface expression of APP without changing the total amount of APP mRNA or protein expression in SH-SY5Y cells and in rat primary cortical neurons. In addition, NRG1 significantly increased the levels of the secreted form of APP, sAPPα, in the conditioned media but did not change the expression of ADAM10 on the cell surface or in the cell lysates. Furthermore, we found that the protein level of NRG1 was reduced in the hippocampus of Alzheimer's disease (AD) patients. Our results demonstrate that NRG1 increased APP expression on the cell surface and sAPPα secretion into the media of neuronal cell cultures. Taken together, these results suggest a role for NRG1 in non-amyloidogenic processing.

Antibody-mediated stabilization of NRG1 induces behavioral and electrophysiological alterations in adult mice

Neuregulin 1 (NRG1) is required for development of the central and peripheral nervous system and regulates neurotransmission in the adult. NRG1 and the gene encoding its receptor, ERBB4, are risk genes for schizophrenia, although how alterations in these genes disrupt their function has not been fully established. Studies of knockout and transgenic mice have yielded conflicting results, with both gain and loss of function resulting in similar behavioral and electrophysiological phenotypes. Here, we used high affinity antibodies to NRG1 and ErbB4 to perturb the function of the endogenous proteins in adult mice. Treatment with NRG1 antibodies that block receptor binding caused behavioral alterations associated with schizophrenia, including, hyper-locomotion and impaired pre-pulse inhibition of startle (PPI). Electrophysiological analysis of brain slices from anti-NRG1 treated mice revealed reduced synaptic transmission and enhanced paired-pulse facilitation. In contrast, mice treated with more potent ErbB4 function blocking antibodies did not display behavioral alterations, suggesting a receptor independent mechanism of the anti-NRG1-induced phenotypes. We demonstrate that anti-NRG1 causes accumulation of the full-length transmembrane protein and increases phospho-cofilin levels, which has previously been linked to impaired synaptic transmission, indicating enhancement of non-canonical NRG1 signaling could mediate the CNS effects.

Proteomic Analysis of Early Diabetic Retinopathy Reveals Mediators of Neurodegenerative Brain Diseases.

PurposeCurrent evidence suggests that retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy. Our main goal was to examine whether, in the diabetic human retina, common proteins and pathways are shared with brain neurodegenerative diseases.MethodsA proteomic analysis was performed on three groups of postmortem retinas matched by age: nondiabetic control retinas (n = 5), diabetic retinas without glial activation (n = 5), and diabetic retinas with glial activation (n = 5). Retinal lysates from each group were pooled and run on an SDS-PAGE gel. Bands were analyzed sequentially by liquid chromatography-mass spectrometry (LC/MS) using an Orbitrap Mass Spectrometer.ResultsA total of 2190 proteins were identified across all groups. To evaluate the association of the identified proteins with neurological signaling, significant signaling pathways belonging to the category “Neurotransmitters and Other Nervous System Signaling” were selected for analysis. Pathway analysis revealed that “Neuroprotective Role of THOP1 in Alzheimer's Disease” and “Unfolded Protein Response” pathways were uniquely enriched in control retinas. By contrast, “Dopamine Degradation” and “Parkinson's Signaling” were enriched only in diabetic retinas with glial activation. The “Neuregulin Signaling,” “Synaptic Long Term Potentiation,” and “Amyloid Processing” pathways were enriched in diabetic retinas with no glial activation.ConclusionsDiabetes-induced retinal neurodegeneration and brain neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, share common pathogenic pathways. These findings suggest that the study of neurodegeneration in the diabetic retina could be useful to further understand the neurodegenerative processes that occur in the brain of persons with diabetes.

Maf links Neuregulin1 signaling to cholesterol synthesis in myelinating Schwann cells.

Cholesterol is a major constituent of myelin membranes, which insulate axons and allow saltatory conduction. Therefore, Schwann cells, the myelinating glia of the peripheral nervous system, need to produce large amounts of cholesterol. Here, we define a crucial role of the transcription factor Maf in myelination and cholesterol biosynthesis and show that Maf acts downstream from Neuregulin1 (Nrg1). Maf expression is induced when Schwann cells begin myelination. Genetic ablation of Maf resulted in hypomyelination that resembled mice with defective Nrg1 signaling. Importantly, loss of Maf or Nrg1 signaling resulted in a down-regulation of the cholesterol synthesis program, and Maf directly binds to enhancers of cholesterol synthesis genes. Furthermore, we identified the molecular mechanisms by which Nrg1 signaling regulates Maf levels. Transcription of Maf depends on calmodulin-dependent kinases downstream from Nrg1, whereas Nrg1-MAPK signaling stabilizes Maf protein. Our results delineate a novel signaling cascade regulating cholesterol synthesis in myelinating Schwann cells.

Modulation of the Neuregulin 1/ErbB system after skeletal muscle denervation and reinnervation

Neuregulin 1 (NRG1) is a growth factor produced by both peripheral nerves and skeletal muscle. In muscle, it regulates neuromuscular junction gene expression, acetylcholine receptor number, muscle homeostasis and satellite cell survival. NRG1 signalling is mediated by the tyrosine kinase receptors ErbB3 and ErbB4 and their co-receptors ErbB1 and ErbB2. The NRG1/ErbB system is well studied in nerve tissue after injury, but little is known about this system in skeletal muscle after denervation/reinnervation processes. Here, we performed a detailed time-course expression analysis of several NRG1 isoforms and ErbB receptors in the rat superficial digitorum flexor muscle after three types of median nerve injuries of different severities. We found that ErbB receptor expression was correlated with the innervated state of the muscle, with upregulation of ErbB2 clearly associated with the denervation state. Interestingly, the NRG1 isoforms were differently regulated depending on the nerve injury type, leading to the hypothesis that both the NRG1α and NRG1β isoforms play a key role in the muscle reaction to injury. Indeed, in vitro experiments with C2C12 atrophic myotubes revealed that both NRG1α and NRG1β treatment influences the best-known atrophic pathways, suggesting that NRG1 might play an anti-atrophic role.

Neuregulin directed molecular mechanisms of visual cortical plasticity.

Experience-dependent critical period (CP) plasticity has been extensively studied in the visual cortex. Monocular deprivation during the CP affects ocular dominance, limits visual performance, and contributes to the pathological etiology of amblyopia. Neuregulin-1 (NRG1) signaling through its tyrosine kinase receptor ErbB4 is essential for the normal development of the nervous system and has been linked to neuropsychiatric disorders such as schizophrenia. We discovered recently that NRG1/ErbB4 signaling in PV neurons is critical for the initiation of CP visual cortical plasticity by controlling excitatory synaptic inputs onto PV neurons and thus PV-cell mediated cortical inhibition that occurs following visual deprivation. Building on this discovery, we review the existing literature of neuregulin signaling in developing and adult cortex and address the implication of NRG/ErbB4 signaling in visual cortical plasticity at the cellular and circuit levels. NRG-directed research may lead to therapeutic approaches to reactivate plasticity in the adult cortex.

Slowing disease progression in the SOD1 mouse model of ALS by blocking neuregulin-induced microglial activation

There are no effective treatments to slow disease progression in ALS. We previously reported that neuregulin (NRG) receptors are constitutively activated on microglia in the ventral horns in both ALS patients and SOD1 mice and in the corticospinal tracts of ALS patients, and that NRG receptor activation occurs prior to significant clinical disease onset in SOD1 mice. Here, we hypothesize that blocking NRG signaling on microglia would slow disease progression in SOD1 mice using a targeted NRG antagonist (HBD-S-H4). Recombinant HBD-S-H4 directly delivered into the central nervous system (CNS) through implanted intracerebroventricular cannulas showed no signs of toxicity and significantly inhibited NRG receptor activation on microglia resulting in reduced microglial activation and motor neuron loss. The treatment also resulted in a delay in disease onset and an increase in survival. The therapeutic effect was dose-dependent that varied as a function of genetic background in two different strains of SOD1 mice. As a complementary drug delivery approach, transgenic mice expressing HBD-S-H4 driven by an astrocytic promoter (GFAP) had slower disease progression in a dose dependent manner, based on the level of HBD-S-H4 expression. These studies provide mechanistic insights into how NRG signaling on microglia may lead to disease progression and demonstrate the utility of a humanized fusion protein that blocks NRG as a novel therapeutic for human ALS.

Crosstalk between developing vasculature and optogenetically engineered skeletal muscle improves muscle contraction and angiogenesis

Capillary networks surrounding skeletal muscle play an important role in not only supplying oxygen and nutrients but also in regulating the myogenesis and repair of skeletal muscle tissues. Herein, we model the early stages of 3D vascularized muscle fiber formation in vitro using a sequential molding technique to investigate interactions between angiogenesis of endothelial cells and myogenesis of skeletal muscle cells. Channelrhodopsin-2 C2C12 muscle fiber bundles and 3D vascular structures (600 μm diameter) were formed at 500 μm intervals in a collagen gel. Endothelial cells exhibited an emergent angiogenic sprouting behavior over several days, which was modulated by the muscle fiber bundle through the secretion of angiopoietin-1. Through a reciprocal response, myogenesis was also upregulated by interactions with the vascular cells, improving muscle contraction via angiopoetin-1/neuregulin-1 signaling. Moreover, continuous training of muscle tissue by optical stimulation induced significantly more angiogenic sprouting. This in vitro model could be used to better understand the formation of vascularized muscle tissues and to test the interactions between muscle growth, repair or training and angiogenesis for applications in tissue engineering and regenerative medicine.

Neuregulin signaling pathway in smoking behavior

Understanding molecular processes that link comorbid traits such as addictions and mental disorders can provide novel therapeutic targets. Neuregulin signaling pathway (NSP) has previously been implicated in schizophrenia, a neurodevelopmental disorder with high comorbidity to smoking. Using a Finnish twin family sample, we have previously detected association between nicotine dependence and ERBB4 (a neuregulin receptor), and linkage for smoking initiation at the ERBB4 locus on 2q33. Further, Neuregulin3 has recently been shown to associate with nicotine withdrawal in a behavioral mouse model. In this study, we scrutinized association and linkage between 15 036 common, low frequency and rare genetic variants in 10 NSP genes and phenotypes encompassing smoking and alcohol use. Using the Finnish twin family sample (N=1998 from 740 families), we detected 66 variants (representing 23 LD blocks) significantly associated (false discovery rate P<0.05) with smoking initiation, nicotine dependence and nicotine withdrawal. We comprehensively annotated the associated variants using expression (eQTL) and methylation quantitative trait loci (meQTL) analyses in a Finnish population sample. Among the 66 variants, we identified 25 eQTLs (in NRG1 and ERBB4), 22 meQTLs (in NRG3, ERBB4 and PSENEN), a missense variant in NRG1 (rs113317778) and a splicing disruption variant in ERBB4 (rs13385826). Majority of the QTLs in blood were replicated in silico using publicly available databases, with additional QTLs observed in brain. In conclusion, our results support the involvement of NSP in smoking behavior but not in alcohol use and abuse, and disclose functional potential for 56 of the 66 associated single-nucleotide polymorphism.

Grb2-associated binder-1 is required for extrafusal and intrafusal muscle fiber development.

The neuregulin-1 (NRG1) signaling pathway plays an important role in the development of the peripheral neuromuscular system, including in muscle spindle and postnatal myelination. We previously showed that NRG1 on the axonal membrane regulates peripheral nerve myelination through Grb2-associated binder 1 (Gab1), a scaffolding mediator of receptor tyrosine kinase signaling. Here, we determined the role of Gab1 in the development of muscles and the muscle spindle using muscle-specific conditional Gab1 knockout mice. The mutant mice showed general retardation in muscular growth and hypotrophy of extrafusal muscle fibers. In addition, the muscle-specific Gab1 knockout mutant exhibited significant underdevelopment of muscle spindles, which are normally regulated by NRG1, and abnormal proprioceptive behavior. Furthermore, the selective knockdown of Gab1 in C2C12 muscle cells reduced NRG1-induced expression of Egr3, a critical transcription factor for muscle spindle development. However, Gab2 knockout mice did not show any defects in the development of muscles or muscle spindles. Our findings suggest that Gab1 is an essential signaling molecule in mediating axonal NRG1 signaling for the development of both extrafusal and intrafusal muscle fibers.

Abstract 2968: Examining the role of breast cancer Neuregulin1 and macrophage ErbB3 in intravasation

Abstract The purpose of our studies is to investigate the role of Neuregulin1 (NRG1) and ErbB3 signaling between breast cancer cells and macrophages in facilitating tumor cell intravasation. The interaction of breast cancer cells with other cell types within the tumor microenvironment plays an important role in metastasis. These interactions are thought to influence tumor cell invasion and intravasation, two important steps in the process of metastasis. Our studies are specifically interested in examining the signaling occurring between tumor cells and macrophages. Previous studies have established the presence of paracrine signaling between breast cancer cells and macrophages, where colony stimulating factor 1 (CSF-1) produced by the tumor cells stimulates the production of epidermal growth factor (EGF) by macrophages, leading to chemotactic invasion of the tumor cells. In addition to this paracrine loop signaling between tumor cells and macrophages, it has been seen that macrophage expression of ErbB3, a member of the EGFR family of receptor tyrosine kinases, may play a role in facilitating tumor cell invasion. In order to examine the effects of signaling between tumor cells and macrophages in intravasation, we utilize an in vitro transendothelial migration (iTEM) assay. This assay uses transwells coated with matrigel and endothelial cells in order to mimic the entry of tumor cells into blood vessels. We show that using an ErbB3 blocking antibody results in a significant reduction of macrophage-induced transendothelial migration of breast cancer cells. Additionally, reduction of expression of the ErbB3 receptor ligand Neuregulin1 in tumor cells yields a similar result. Stimulation of macrophages with NRG1 leads to increased expression of Jagged1 (JAG1), a ligand of the Notch receptor. Activation of the Notch receptor pathway has been shown to be involved in tumor cell invasion. Overall our studies look to further examine the interaction between tumor cells and macrophages, and these observations indicate that ErbB3, NRG1, and JAG1 could all serve as novel targets in metastasis and the tumor microenvironment. Citation Format: Ramon Cabrera, Serena Chiang, Jeffrey Segall. Examining the role of breast cancer Neuregulin1 and macrophage ErbB3 in intravasation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2968. doi:10.1158/1538-7445.AM2017-2968

Multiple-response regression analysis links magnetic resonance imaging features to de-regulated protein expression and pathway activity in lower grade glioma.

Background and PurposeLower grade gliomas (LGGs), lesions of WHO grades II and III, comprise 10-15% of primary brain tumors. In this first-of-a-kind study, we aim to carry out a radioproteomic characterization of LGGs using proteomics data from the TCGA and imaging data from the TCIA cohorts, to obtain an association between tumor MRI characteristics and protein measurements.The availability of linked imaging and molecular data permits the assessment of relationships between tumor genomic/proteomic measurements with phenotypic features.Materials and MethodsMultiple-response regression of the image-derived, radiologist scored features with reverse-phase protein array (RPPA) expression levels generated correlation coefficients for each combination of image-feature and protein or phospho-protein in the RPPA dataset. Significantly-associated proteins for VASARI features were analyzed with Ingenuity Pathway Analysis software. Hierarchical clustering of the results of the pathway analysis was used to determine which feature groups were most strongly correlated with pathway activity and cellular functions.ResultsThe multiple-response regression approach identified multiple proteins associated with each VASARI imaging feature. VASARI features were found to be correlated with expression of IL8, PTEN, PI3K/Akt, Neuregulin, ERK/MAPK, p70S6K and EGF signaling pathways.ConclusionRadioproteomics analysis might enable an insight into the phenotypic consequences of molecular aberrations in LGGs.

Laminin 211 inhibits protein kinase A in Schwann cells to modulate neuregulin 1 type III-driven myelination.

Myelin is required for proper nervous system function. Schwann cells in developing nerves depend on extrinsic signals from the axon and from the extracellular matrix to first sort and ensheathe a single axon and then myelinate it. Neuregulin 1 type III (Nrg1III) and laminin α2β1γ1 (Lm211) are the key axonal and matrix signals, respectively, but how their signaling is integrated and if each molecule controls both axonal sorting and myelination is unclear. Here, we use a series of epistasis experiments to show that Lm211 modulates neuregulin signaling to ensure the correct timing and amount of myelination. Lm211 can inhibit Nrg1III by limiting protein kinase A (PKA) activation, which is required to initiate myelination. We provide evidence that excessive PKA activation amplifies promyelinating signals downstream of neuregulin, including direct activation of the neuregulin receptor ErbB2 and its effector Grb2-Associated Binder-1 (Gab1), thereby elevating the expression of the key transcription factors Oct6 and early growth response protein 2 (Egr2). The inhibitory effect of Lm211 is seen only in fibers of small caliber. These data may explain why hereditary neuropathies associated with decreased laminin function are characterized by focally thick and redundant myelin.

The Scaffolding Protein, Grb2-associated Binder-1, in Skeletal Muscles and Terminal Schwann Cells Regulates Postnatal Neuromuscular Synapse Maturation.

The vertebrate neuromuscular junction (NMJ) is considered as a “tripartite synapse” consisting of a motor axon terminal, a muscle endplate, and terminal Schwann cells that envelope the motor axon terminal. The neuregulin 1 (NRG1)-ErbB2 signaling pathway plays an important role in the development of the NMJ. We previously showed that Grb2-associated binder 1 (Gab1), a scaffolding mediator of receptor tyrosine kinase signaling, is required for NRG1-induced peripheral nerve myelination. Here, we determined the role of Gab1 in the development of the NMJ using muscle-specific conditional Gab1 knockout mice. The mutant mice showed delayed postnatal maturation of the NMJ. Furthermore, the selective loss of the gab1 gene in terminal Schwann cells produced delayed synaptic elimination with abnormal morphology of the motor endplate, suggesting that Gab1 in both muscles and terminal Schwann cells is required for proper NMJ development. Gab1 in terminal Schwann cells appeared to regulate the number and process elongation of terminal Schwann cells during synaptic elimination. However, Gab2 knockout mice did not show any defects in the development of the NMJ. Considering the role of Gab1 in postnatal peripheral nerve myelination, our findings suggest that Gab1 is a pleiotropic and important component of NRG1 signals during postnatal development of the peripheral neuromuscular system.

Regulation of cardiomyocyte behavior by Neuregulin signaling

Regulation of cardiomyocyte behavior by Neuregulin signaling

Tenascin C to regulate the function of sHER3, an endogenous inhibitor of neuregulin signaling.

e23002 Background: HER3 expression is associated with poor prognosis in melanoma patients but the mechanistic basis has not been determined. An endogenous secreted isoform of HER3, p85-sHER3, has been shown by us to inhibit neuregulin-1 mediated, Akt-dependent breast and ovarian cancer cell growth in vitro. Here we demonstrate that p85-sHER3 also regulates neuregulin-dependent melanoma cell migration via an Akt- independent, tenascin C-dependent mechanism. Methods: Immunoblot analysis and RT-PCR were used to study HER3 and p85-sHER expression. Cell proliferation and chamber assays were conducted to determine the effect of p85-sHER3 on melanoma cell growth and migration. Mass spectrometry (MS) was performed to identify sHER3-interacting proteins. CRSPR in vitro editing methods were used to knock out tenascin C (TNC) expression. Results: HER3 and p85-sHER3 were expressed in 5 of 6 human melanoma-derived cell lines tested, with exception of SK-MEL-103 which did not express either gene product. Exogenous addition of 10 nM p85-sHER3 inhibited growth in 3 cells lines, although Akt phosphorylation was not reduced. Protein extracts from M14-MEL cell conditioned media were characterized by MS, and identified p85-sHER3 and TNC. Their association was confirmed by co-immunoprecipitation. sHER3 was further shown to inhibit neuregulin stimulated cell migration in 2 cell lines which exhibited high levels of TNC. CRSPR knockout of TNC in these cell lines (but not in control lines) eliminated sHER3 inhibition of neuregulin stimulated melanoma cell migration. Conclusions: Our results suggest that p85-sHER3, predominantly secreted by HER3-expressing melanoma cells, inhibits melanoma cell proliferation and migration via an Akt-independent mechanism, which is likely achieved through interaction(s) with TNC. TNC promotes an intermediate adhesive state favoring cell migration and this state appears to be mediated by Rho-associated kinase signaling. The Rho inhibitor, CCG-203971 has been shown to inhibit melanoma cell migration. Together, these results suggest the existence of a TNC and HER3-dependent signaling pathway in melanoma that regulates cell migration, and therefore may be correlated with poor patient survival.

Interaction between prenatal pesticide exposure and a common polymorphism in the PON1 gene on DNA methylation in genes associated with cardio-metabolic disease risk\u2014an exploratory\xa0study

BackgroundPrenatal environmental conditions may influence disease risk in later life. We previously found a gene-environment interaction between the paraoxonase 1 (PON1) Q192R genotype and prenatal pesticide exposure leading to an adverse cardio-metabolic risk profile at school age. However, the molecular mechanisms involved have not yet been resolved. It was hypothesized that epigenetics might be involved. The aim of the present study was therefore to investigate whether DNA methylation patterns in blood cells were related to prenatal pesticide exposure level, PON1 Q192R genotype, and associated metabolic effects observed in the children.MethodsWhole blood DNA methylation patterns in 48 children (6–11 years of age), whose mothers were occupationally unexposed or exposed to pesticides early in pregnancy, were determined by Illumina 450 K methylation arrays.ResultsA specific methylation profile was observed in prenatally pesticide exposed children carrying the PON1 192R-allele. Differentially methylated genes were enriched in several neuroendocrine signaling pathways including dopamine-DARPP32 feedback (appetite, reward pathways), corticotrophin releasing hormone signaling, nNOS, neuregulin signaling, mTOR signaling, and type II diabetes mellitus signaling. Furthermore, we were able to identify possible candidate genes which mediated the associations between pesticide exposure and increased leptin level, body fat percentage, and difference in BMI Z score between birth and school age.ConclusionsDNA methylation may be an underlying mechanism explaining an adverse cardio-metabolic health profile in children carrying the PON1 192R-allele and prenatally exposed to pesticides.

Epidermal Growth Factor Receptor Family Signaling in the Regenerating Axolotl Lung

Efforts to elucidate novel mechanisms of lung regeneration have the potential to change lives and transform health care. The axolotl salamander, Ambystoma mexicanum, has long been held as a model organism of vertebrate regeneration, particularly in limbs. It is thought that they regenerate all of their tissues, but exploration of lung regeneration has not been previously characterized. In this study, we have conducted experiments demonstrating that axolotls are capable of significant lung regeneration after amputation of the distal third of the lung. We have shown the importance of ErbB signaling in the lung tissue, and the potential of neuregulin signaling in the induction of new lung cells. Proliferation was observed to be a global response throughout the lung tissue of the axolotl after lung injury. This would suggest the regeneration utilizes a compensatory mechanism. ErbB4 mRNA was found to be upregulated at one week post-amputation in the axolotl lung above other EGF family receptors. Injected neuregulin-1 was mitogenic to lung tissue and upregulated ErbB4 as seen in lung injury. When we inhibited ErbB2, we found that lung regeneration was halted. Epidermal growth factor signaling is crucial for regeneration to take place, specifically through the ErbB2:ErbB4 epidermal growth factor family receptor heterodimer. Neuregulin-1 can induce proliferation in the lung, and is a likely candidate to exert molecular control over lung regeneration. Support or Funding InformationNSF Grant #1558017 to JRM

Neuregulin1 fine-tunes pre-, post-, and perisynaptic neuromuscular junction development.

Neuromuscular junction (NMJ) development is a multistep process mediated by coordinated interactions between the nerve terminal, target muscle, and perisynaptic Schwann cell that require constant back-and-forth communication. Retrograde and anterograde growth and differentiation factors have been postulated to participate in this communication. While neuregulin1 (NRG1) has been shown to be potent anterograde signal that activates acetylcholine receptor (AChR) transcription and clustering in vitro, its roles in NMJ development in vivo remain elusive. Using the model of chicken embryo, we measured the effects of NRG1 signaling during NMJ development in ovo using quantitative, sequential measures of AChR cluster size and density, pre- and postsynaptic apposition, and the alignment of perisynaptic Schwann cells. Using in ovo electroporation at early stages and a targeted soluble neuregulin antagonist through all developmental stages, we found soluble NRG1 regulates AChR cluster density and size at the earliest stage prior to nerve-AChR cluster contact. Once the nerve contacts with muscle AChRs, NRG1 has pronounced effects on presynaptic specialization and on the alignment of perisynaptic Schwann cells at endplates. These findings suggest that, while NRG1 may not be critical for overall development, it appears to be important in fine-tuning pre-, post-, and perisynaptic development of the NMJ. Developmental Dynamics 246:368-380, 2017. © 2017 Wiley Periodicals, Inc.