MEGF9: a novel transmembrane protein with a strong and developmentally regulated expression in the nervous system

Abstract

MEGF9 [multiple EGF (epidermal growth factor)-like-domains 9], a novel transmembrane protein with multiple EGF-like repeats, is predominantly expressed in the developing and adult CNS (central nervous system) and PNS (peripheral nervous system). The domain structure of MEGF9 consists of an N-terminal region with several potential O-glycosylation sites followed by five EGF-like domains, which are highly homologous with the short arms of laminins. Following one single pass transmembrane domain, a highly conserved short intracellular domain with potential phosphorylation sites is present. The protein was recombinantly expressed and characterized as a tissue component. To study the expression pattern further, immunohistochemistry was performed and staining was detected in Purkinje cells of the cerebellum and in glial cells of the PNS. Additional expression was observed in the epidermal layer of skin, papillae of the tongue and the epithelium of the gastrointestinal tract. By immunoelectron microscopy, MEGF9 was detected in glial cells of the sciatic nerve facing the basement membrane. MEGF9 represents a novel putative receptor, expressed in neuronal and non-neuronal tissues, that is regulated during development and could function as a guidance or signalling molecule.

Figure 1. Exon/intron organization of the human and murine MEGF9 genes and domain structures of the MEGF9 proteins

The genes are approx. 110 kb long and consist of six exons. Potential N-glycosylation sites are marked by asterisks, whereas predicted O-glycosylation sites are marked by dotted lines. SP, signal peptide; TM, transmembrane; CT, cytoplasmatic tail.

Figure 2. Homology of the mouse and human MEGF9 amino acid sequences

The signal peptide (SP) is underlined, the N-terminal region is shown in dotted boxes, the EGF-like domains are in closed boxes, and the transmembrane region is in a dashed box. Identical amino acid residues are marked in grey. m, mouse; h, human.

Figure 3. Northern-blot and RT–PCR analyses

(A) Northern-blot analysis of murine tissues detects MEGF9 mRNA as a single band at approx. 7.4 kb in brain. Weaker signals at the same size are present in non-neuronal tissues such as heart, spleen, liver, kidney and testis. (B) mRNA levels are regulated during development. (C) Semi-quantitative RT–PCR analysis depicts different levels of mRNA in neuronal tissues and in skin.

Figure 4. Structural analysis of MEGF9 protein

(A) Rod-like particles representing the recombinant MEGF9–EGF protein were detected by rotary shadowing electron microscopy. (B) The affinity-purified rabbit anti-MEGF9 antibody KR18 recognizes the recombinant protein at approx. 75 kDa in SDS/PAGE immunoblot. PNGase F digestion results in a mass shift of approx. 30 kDa. (C) MEGF9 is detected as a single approx. 180 kDa band in brain extract that shifts to approx. 150 kDa upon PNGase F digestion. The electrophoretic mobility is analysed under reduced or non-reduced condition in (D) recombinant MEGF9–EGF and (E) a brain tissue extract detected with the KR18 antibody by immunoblotting.

Figure 5. Protein expression in the CNS

MEGF9 expression is detected in all structures of (A) embryo (E18.5) and (B) newborn brain (P1). The strongest staining is in (C) cerebellum (E18.5), mesencephalon, hindbrain, and in the hippocampus (E: adult). (D) In the adult cerebellum expression is restricted to Purkinje cells (D; adult). Facial nuclei, dorsal medulla oblongata, trigeminal ganglia and nerves also express MEGF9 (A and B respectively, see arrows), as do cells of the olfactory bulb (F: adult) and olfactory epithelium (G: P1). Further signals are found in the developing eye. (H) At stage E12.5 protein expression can be detected in the anterior lens and by P1, throughout the lens and inner retinal layers (I). (J) In adult eyes, the staining is restricted to specific cell layers, e.g. the inner and outer plexiform layer, the photoreceptor layer and Bruchs membrane. (K) MEGF9 is expressed in cells of the spinal cord, the dorsal horn, and the ventral root (E19.5). Additional staining is detected in dorsal root ganglia (L), thoracic sympathetic chain ganglia of E19.5 embryos (M), and dorsal root ganglia of adult animals (N). Co, cortex; Po, posteriour thalamic nuclei group; DpMe, deep mesencephalic nuclei; Pn, pontine nuclei; Fn, facial nuclei; TriN, trigeminal nerve; TriG, trigeminal ganglia; hip, hippocampus; LPG, lateral paragigantocellular nuclei; Mo, medulla oblongata; Sp5C, spinal 5 nuclei caudal part; Cpu, caudate putamen striatum; DG, dentate gyrus of hippocampus; SC, superior colliculus; IC, inferior colliculus; C, cerebellum; PC, Purkinje cells of the cerebellum; CA1 and CA2, field CA1 and CA2 of hippocampus; Mi, mitral cell layer of olfactory bulb; Gro, granular cell layer of the olfactory bulb; EPI, external plexiform layer of olfactory bulb; RGC, retinal ganglion cell layer; IPL, INL, inner plexiform and nuclear layers; OPL, ONL, outer plexiform and nuclear layers; PRl, photoreceptor layer; BM, Bruchs membrane; PE, pigment epithelium, SC, spinal cord; DHSC, dorsal horn of spinal cord; VHSC, ventral horn of spinal cord; DRG, dorsal root ganglion; VR, ventral root; SCG, sympathetic chain ganglion; TSG, thoracal sympathetic ganglia.

Figure 6. MEGF9 protein expression in the PNS

MEGF9 (red) staining is (A) present in satellite glia cells and (B) around dorsal root nerves co-stained with IB4 (green), a marker for non-myelinated fibres. (C) MEGF9 expression overlaps with that for laminins containing the γ1 chain present in the basement membranes of Schwann cells. (D, E) Labelling of MEGF9 and neurofilament 200 kDa (green) in sciatic nerves shows that MEGF9 is expressed in myelinating Schwann cells. MEGF9 and (F) GFAP, expressed by subsets of Schwann cells, also show a limited overlap. In cell culture, (G, H) both RN2 schwannoma cells (H: co-stain for F-actin) and (I) C6 oligodendroglia cell lines are both positive for MEGF9. (J) MEGF9 expression in peripheral nerves is detected in whole mount preparations of trigeminal nerves. (K, L) Electron microscopy images of immuno-gold-labelled sciatic nerve sections. MEGF9 is present close to the basement membrane of myelinating Schwann cells.

Figure 7. MEGF9 protein expression in skin and other keratinized tissues

On cryosections of embryonic and newborn tissues, MEGF9 is detected in (A–C) skin and around hair follicles (P1), (D, E) in keratinocytes of the tongue (P1), (F) oesophagus (E16.5) and (G) intestine (E16.5). TriG, trigeminal ganglia; TriN, trigeminal nerve; Wh, whiskers; Hf, hair follicle; olfE, olfactory epithelium; K, keratinocytes.