Research ReportDifferential expression of neuregulins and their receptors in the olfactory bulb layers of the developing mouse
Introduction
The neuregulins (NRGs) are a group of glycoproteins that belong to the epidermal growth factor (EGF)-like ligand family with widespread expression during development. At present, four genes encoding NRGs have been identified. The NRG-1–4 subfamily shares high sequence homology in their EGF-like domain, which is required for receptor binding and alternative splicing at the most carboxy-terminal region of the EGF-like domain gives rise to α and β isoforms (Wen et al., 1994). EGF domain β isoforms are prevalently expressed in the nervous system, whereas α isoforms are found in mesenchyme (Meyer and Birchmeier, 1994). Moreover, a region in this sub-domain, the linker, is also highly variable in both length and sequence and gives rise to different isoforms including β1 and β2 isoforms, of respectively intermediate and shorter length.
The neuregulins act through binding members of the EGF receptor (EGF-R) family. This EGF-R family consists of four members: ErbB1 (also called the epidermal growth factor receptor; EGFR), ErbB2/Neu/HER2, ErbB3/HER3 and ErbB4/HER4 (Buonanno and Fischbach, 2001). ErbB proteins are transmembrane receptors with an extracellular-ligand-binding domain, a short transmembrane domain and an intracellular domain that has a tyrosine kinase catalytic activity, with the exception of ErbB3 which has a low functional kinase intracellular domain. ErbB4 has two alternative splice variants, JMa and JMb, which differ in the presence of a 23 or 13 amino acid sequences within the juxtamembrane region respectively. ErbB4-JMa is susceptible to a proteolytic cleavage (Vecchi and Carpenter, 1997) resulting in a fragment of approximately 85 kDa, representing the transmembrane and cytoplasmic domains of the molecule (Vecchi et al., 1996). This 85 kDa fragment can be processed by a second membrane-localized protease and translocated into the nucleus (Ni et al., 2001), where it associates to the transcriptional co-activators YAP-65 (Komuro et al., 2004, Omerovich et al., 2004) and STAT5A (Williams et al., 2004), suggesting possible involvement in transcriptional regulation. Although the EGF-like domains of neuregulins are similar, the binding specificities and affinities are different for the various combinations of ErbB receptors. For example, NRG-1 and NRG-2 bind to ErbB3 and ErbB4 (Carraway et al., 1997), whereas NRG-3 and NRG-4 bind preferentially to ErbB4 (Harari et al., 1999, Zhang et al., 1997). Following activation of the receptors with one of the several EGF family ligands, both homodimeric and heterodimeric combinations of receptors are induced, and their intrinsic catalytic tyrosine kinase activity stimulated (Yarden and Ullrich, 1998).
In the nervous system, NRG-1 activity has been implicated as a modulator of early fate determination, differentiation, migration and survival of glial cells (Buonanno and Fischbach, 2001). A crucial role for NRG-1 signaling has also been demonstrated in the cerebral cortex in establishment, maintenance and maturation of radial glial cells (Schmid et al., 2003). NRG-1 also specifically accelerates oligodendrocytes maturation and myelination, promoting ErbB4 cleavage and its nuclear localization (Lai and Feng, 2004). Neuronal–glia contact in the cerebellum induces morphological differentiation of radial glia via neuregulin-ErbB receptor signaling, and secondarily the ErbB receptor signaling is necessary for neuronal migration (Rio et al., 1997, Patten et al., 2003). Null mutations in ErbB3 result in severe abnormalities of midbrain and hindbrain development (Erickson et al., 1997), whereas ErbB4 deficient mice have abnormal targeting of cranial sensory and motor axons (Burden and Yarden, 1997). Taken together, these data suggest that NRG-1 is critical for the formation of glia in the brain.
The olfactory bulb (OB) is a well-organized structure with cell types and synaptic connections segregated into discernable anatomical layers (for review, Shipley et al., 2004). The OB undergoes considerable postnatal maturation with the bulk of interneurons generated postnatally (Bayer and Altman, 1975) and migrating into the bulb along the rostral migratory stream and through the granule cell layer (Lois and Alvarez-Buylla, 1994, Luskin, 1993). During the first postnatal week, there is also extensive reorganization of radial glia into astrocytes (Bailey et al., 1999, Puche and Shipley, 2001) and the formation of the unique glial tubes in the rostral migratory stream (Alves et al., 2002, Peretto et al., 2005). Anton et al. (2004) recently analyzed the expression of ErbB receptors and their ligands in the developing rat olfactory system. In situ hybridization showed that at postnatal day 11 (P11) ErbB4 is expressed at high levels in the rostral migratory stream and remains detectable in these cells as they migrate into the glomerular layer (GL) and granule cell layer (GCL) (Anton et al., 2004, Perroteau et al., 1998, Pollock et al., 1999). In adult, expression persists in granule neurons in the mature OB, but at reduced levels. In rat, ErbB3 expression at P11, and in adult at reduced levels, is prominent in ensheathing cells of the olfactory nerve layer but is almost absent in the internal layers (Anton et al., 2004, Perroteau et al., 1998, Pollock et al., 1999). However, little is known about the developmental expression profiles of the other ErbBs or any of the ErbB splice variants in mouse. Neuregulin-1 is expressed in the GCL, GL and in the mitral cell layer (MCL); NRG-2 in the GCL, GL and in the external plexiform layer (EPL); NRG-3 in the MCL and GL of P11 and adult rat (Anton et al., 2004, Longart et al., 2004). Other reports have examined NRG 1–3 in the OB as part of expression pattern profiles in the entire mouse brain (Chen et al., 1994, Corfas et al., 1995, Longart et al., 2004, Meyer et al., 1997); however, comparative studies across the different OB layers and of the NRG splice variants during development have yet to be performed. Understanding the developmental expression patterns of the different splicing isoforms may contribute to understand their role in OB development.
Taking advantage of the laminar organization of the OB, we microdissected each layer and compared the developmental expression by parallel analysis of mRNA and protein from the same sample using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Unlike in situ hybridization, RT-PCR on single OB layers does not provide information on the expression at the cellular level, but allows detection and analysis, in one sample, of the relative abundance of several isoforms of the same molecule providing complementary information with respect to the cellular localization. We found a strong correlation between the temporal profiles of ErbB4 and NRG-1 in all layers, confirming our previous immunohistochemical findings in the adult deafferented OB (Oberto et al., 2001). In addition, we show that the NRG-1 isoform β2 and ErbB4-JMa, which are absent in the adult OB, are highly expressed in the prenatal granule cell layer. Other members of the NRG/ErbB family were also differentially expressed during development and across the bulb layers with less dramatic changes than NRG-1 and ErbB4.
Section snippets
Results
The olfactory bulb is a laminated structure with a highly organized distribution of different cell types to particular layers. Taking advantage of this laminar organization, we microdissected each layer and compared the expression of ErbB receptors and neuregulins by RT-PCR and immunoblotting. The olfactory nerve layer (ONL), glomerular layer (GL), mitral cell layer (MCL) and granule cell layer (GCL) were microdissected from E18, P0, P2, P4, P8, P16 and adult mouse olfactory bulbs. Due to size
Discussion
Neuregulins (NRGs) and their receptors, the ErbBs, play important roles in the development of the central nervous system by regulating both neuronal and glial precursor proliferation, migration, differentiation and survival (Burden and Yarden, 1997, Gassmann and Lemke, 1997, Lemke, 1996, Longart et al., 2004). Understanding the functional roles of these molecules in the nervous system is complicated by the existence of numerous splicing variants whose specific temporal and spatial expression
Animals
CD-1 adult mice (25–30 g body weight) and neonates (Charles River) were maintained under a 12-h light, 12-h dark cycle. Animals were anesthetized by 100 mg/kg sodium pentobarbital for all procedures. Animal care was in accordance with D.L. 116/92 from the Italian Government and received the approval of the animal ethics committee of the University of Turin and of the University of Maryland Institutional Animal Care Usage Committee (IACUC).
Isolation of olfactory bulb layers
Microdissections of individual olfactory bulb layers
Acknowledgments
This work was supported by FIRB PRONEURO (FIRB RBNE01WY7P), FIRB fund, grant number RBAU01BJ95 and National Institute of Health NIDCD DC05739 to A.C. P.S. Bovetti is a recipient of a fellowship sponsored by Fondazione Cassa di Risparmio di Cuneo. The authors wish to thank Dr. Claudio Dati (Department Human and Animal Biology, University of Turin, Italy) for providing the NRG-3 PCR primers.
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