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Embryonic expression of epithelial membrane protein 1 in early neurons
Developmental Brain Research 116 Ž1999. 169–180 www.elsevier.comrlocaterbres
Research report
Embryonic expression of epithelial membrane protein 1 in early neurons Philip Wulf, Ueli Suter
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Institute of Cell Biology, Department of Biology, Swiss Federal Institute of Technology, ETH-Honggerberg, CH-8093 Zurich, Switzerland ¨ ¨ Accepted 29 June 1999
Abstract Epithelial membrane protein 1 ŽEMP1. is a member of the peripheral myelin protein 22 ŽPMP22. family. This family is best known for the crucial contribution of PMP22 to the development and maintenance of the peripheral nervous system ŽPNS.. PMP22 is widely expressed, with highest levels in myelinating Schwann cells, and mutations affecting the PMP22 gene lead to PNS-restricted neuropathies. We have investigated the spatio-temporal distribution of EMP1 and compared it to that of PMP22. We found that EMP1 and PMP22 mRNA are most conspicuously expressed in the prenatal mouse brain during neurogenesis. In the developing forebrain, we localized EMP1 mRNA and protein to the first set of neurons that are generated and leave the ventricular zone to form the preplate. Later in development, EMP1 was found in derivatives of the preplate, the marginal zone and the subplate. Reduced expression was observed in the newly generated cortical plate neurons. In other parts of the developing CNS and PNS, EMP1 was also detected in early neurons and along the initial fiber tracts. Furthermore, EMP1 was highly expressed by immature neurons in embryonal dorsal root ganglia-explant cultures and in neuroectodermal differentiated P19 cells. While PMP22 functions mainly in Schwann cell growth and differentiation, the spatio-temporal localization of EMP1 suggests a role in neuronal differentiation and neurite outgrowth. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Preplate; Marginal zone; Subplate; P19 cell line; Neuron; Glia
1. Introduction Epithelial membrane protein 1 ŽEMP1. has previously been identified as a member of the peripheral myelin protein 22 ŽPMP22. gene family w8,25,29,38,40,58x. Currently, five members of this family are known including PMP22, EMP1, epithelial membrane protein 2 ŽEMP2. w8,57x and epithelial membrane protein 3 ŽEMP3. w10,57x, as well as the lens-specific membrane protein 20 ŽMP20. w24,58x. The conserved genomic structures and chromosomal localizations suggest that this family is derived from genomic duplications of an ancestral gene w9,12,25,62,63x. The PMPrEMPrMP20 proteins contain four hydrophobic regions spanning or embedded in the cell membrane and display an amino acid identity of approximately 40% with the exception of the more distantly related MP20, which shares only about 25% identical residues w8,57,58,62x. PMP22 is the best characterized member of this family w35x. It contributes to myelin compaction and mutations
) Corresponding author. Fax: q 41-1-633-1190; E-m ail: [email protected]; Internet address: http:rrwww.cell.biol.ethz.chr
affecting PMP22 are associated with hereditary motor and sensory neuropathies in humans and rodents w54x. Duplications, point mutations, and deletions of the PMP22 gene have been linked to the most common inherited disorder of the peripheral nervous system, Charcot–Marie–Tooth ŽCMT1A. disease, the Dejerine–Sottas syndrome ŽDSS., and to hereditary neuropathy with liability to pressure palsies ŽHNPP. w44,45x. The common feature of these PMP22-based neuropathies is the disturbance in the development and maintenance of myelin and axons. Based on the human pathologies and the phenotypes of naturally occurring and artificially generated rodents mutant for PMP22, it is well established that the PNS is highly sensitive to alterations in PMP22 gene dosage w1,2,20, 21,26,30,43,51,53,55x. On the molecular level, PMP22 and EMP1 are thought to be involved in the regulation of the cell cycle, cell–cell recognition, and cell death w22x. Members of the PMP22 family have been repeatedly found in screens for transcripts whose levels are regulated during the cell-cycle, tissue maturation, or tumorgenesis w 8,14,29,38, 40,41,48,56,59x. EMP1 is upregulated during proliferation of fibroblasts and Schwann cells and this regulation ap-
0165-3806r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 9 . 0 0 0 9 2 - 9
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pears to be inverse to that of PMP22 w16,58x. Furthermore, elevated EMP1 mRNA levels were found in brain tumors and during tumor progression and consequently, the cognate proteins were named ‘‘Tumor-associated Membrane Protein’’ ŽTMP., and ‘‘Progression-associated Protein’’ ŽPAP., respectively w8,40x. In contrast, high-level expression of EMP1 is associated with differentiation and growth arrest in squamous cells and in the haematopoetic system Žcorresponding clones called CL-20 and B4B; w29,38x.. Thus, the correlation between PMP22rEMP1 gene regulation and proliferation or growth arrest may depend on the cellular context. A common feature of PMP22 and EMP1 is the single N-linked carbohydrate chain attached to the protein backbone which carries the HNK-1rL2 carbohydrate epitope w29,46x. This special structure may not only contribute to cell–cell or cell–extracellular matrix recognition functions, but might also affect differentiation, proliferation, and cell growth w39x. The members of the PMP22 gene family are widely expressed, with the exception of MP20, which is specific to the lens. Highest levels of PMP22 are generated by myelinating Schwann cells, in the lung, and in the gastrointestinal tract w47,58x. During embryonic development, PMP22 expression was found in derivatives of all three germ layers w4,25,37x although further studies on PMP22 distribution, and particularly, the analysis of PMP22 mutants suggested that PMP22 is mainly important for the development and the maintenance of the nervous system w2,17,26,36,62x. EMP1 is widely expressed in the adult mouse, with highest levels in the gastrointestinal tract and in the lung w25x. Spatially, EMP1 has been localized to the proliferating and differentiating epithelia of the rat corpus gastricum, of the rabbit tongue, and of the esophagus w29,58x. To extend these studies to the prenatal mouse and to compare the distribution of EMP1 with that previously reported for PMP22 w4,36x, we used in situ hybridization and immunohistochemistry to analyze the expression of EMP1 during embryogenesis. Interestingly, we found most conspicuous expression of EMP1 in the developing CNS and PNS at the onset of neurogenesis.
2.2. DRG-explant cultures DRG of E15.5 mouse embryos were dissected, trypsinized, and cultured in SM medium on coated tissue culture plates w49x. After 5 days in culture, cells were fixed in 4% paraformaldehyde for 15 min and processed for immuncytochemistry. 2.3. P19 cell culture P19 cells were a gift from Dr. R.J. Hardy, Mount Sinai Medical School, New York. Cells were cultured in MEM alpha medium supplemented by 7.5% newborn calf serum, 2.5% fetal calf serum, 2 mM glutamine, 100 Urml penicillin and, 100 mgrml streptomycin w23x. Cultures were maintained at 378C in 5% CO 2 atmosphere. For mesodermal differentiation, monolayer cultures of P19 cells were maintained in the presence of 1 mM retinoic acid ŽRA.. For neural differentiation, P19 cells were aggregated in bacteriological petri dishes at 5 = 10 5 cellsrml in the presence of 1 mM retinoic acid ŽRA. for two days. Subsequently, the medium was replenished and the aggregates incubated for another two days. Aggregates were dissociated by incubation in 0.05% Trypsin, 0.5 mM EDTA, plated on cell culture dishes in P19 medium without RA and after 1 to 7 days processed for immunocytochemistry w18x. 2.4. RNA isolation, Northern blotting, and labeling of nucleic acid probes Total RNA was isolated using a modified acid phenolrguanidinium isothiocyanate method w13x. Ten micrograms of total RNA were separated by electrophoresis
2. Materials and methods
2.1. Animals and tissues Time-mated C57BLr6J mice were obtained from BRL Fullinsdorf, Switzerland, and used in all experiments. The ¨ day of plug detection was considered as E0.5. Mouse embryos were harvested and embedded in O.C.T mounting medium ŽTissue-Tek w ., frozen in isopentane cooled to freezing point by liquid nitrogen, and stored at y808C.
Fig. 1. EMP1 and PMP22 transcripts are upregulated in the murine brain during neurogenesis. Ten micrograms total RNA isolated from embryonic or postnatal brains were subjected to Northern blot analysis. Upper panel: Arrows indicate EMP1 transcripts at 2.8 kb and 1.7 kb. Middle panel: Arrow indicates PMP22 transcripts at 1.8 kb. Bottom panel: GAPDH as a loading control. Arrow indicates the GAPDH signal at 1.4 kb.
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in formaldehyde-containing 1.2% agarose gel and capillary blotted onto nylon membranes ŽHybond-N, Amershan.. The membrane was hybridized in 50% formamide buffer at 428C with a 32 P-labeled, randomly primed EMP1 cDNA probe according to the manufacturers instructions ŽPharmacia.. The membrane was washed to a final stringency of 0.1% SSCr0.1% SDS at 558C, and exposed to X-ray film ŽFuji. at y708C. After stripping in 0.1%SDS at 1008C for 5 min, the same membranes were subjected to rehybridization with identically prepared probes for PMP22 and GAPDH.
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2.5. In situ hybridization and nucleic acid riboprobes Serial cryosections of 10 mm thickness from mouse embryos were mounted on SuperFrost Plus slides and postfixed in PBS containing 4% paraformaldehyde for 10 min at room temperature. Slides were prepared for in situ hybridization as described w42x. Digoxigenin ŽDIG. uridinetriphosphate-labeled riboprobes in sense and antisense orientations were synthesized according to the manufacturer’s instructions ŽBoehringer Mannheim.. Detection of alkaline phosphatase coupled to anti-DIG antibody
Fig. 2. EMP1 is conspicuously expressed in the developing nervous system during mid- to late gestation. ŽA, B, C, D. Immunohistochemical EMP-1 staining on sagittal sections. Highest expression of EMP1 was observed in the developing CNS and PNS. ŽA, D. Staining of adjacent sections of E10.5 embryo confirm the specificity of the EMP1 antiserum ŽA. in comparison to preimmune serum ŽD.. ŽE, F. In situ hybridization of serial sections adjacent to ŽC., using a sense ŽE. or antisense ŽF. DIG-labeled EMP1 probe demonstrating specificity of the antisense hybridization. In agreement with the protein distribution, the levels of EMP1 mRNA were highest in the nervous system. Size bar in F s 1 mm for F and E, 0.5 mm for A and D, 0.7 mm for B. Abbreviations: bo, bowel; cn, cranial nerve; drg, dorsal root ganglia; fv, fourth ventricle; ht, heart; li, liver; lv, lateral ventricle; ma, medulla oblongata; mv, mesencephalic vesicle; oe, olfactory epithelium; sc, spinal cord; sn, spinal nerve.
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ŽBoehringer. was carried out with nitroblue tetrazolium ŽNBT., 5-bromo-4-chloro-3-indolyl phosphate ŽBCIP., and levamisole for 2 h at room temperature. An EMP1-sense
control reaction, labeled to comparable activity, was always included in parallel and yielded negligible signals ŽFig. 2F..
Fig. 3. EMP1 is expressed in differentiation fields of the embryonic brain. Serial sagittal sections of E15.5 mouse brain are shown. Dorsal is to the left and rostral at the top. Comparison of EMP1 mRNA and protein distribution suggests neuronal localization in the E15.5 brain. ŽA. In situ hybridization localizes EMP1 transcripts to the neuronal differentiation fields. ŽB. EMP1 immunohistochemistry confirms the EMP1 distribution to the neuronal differentiation fields and shows that EMP1 protein is also present along fiber tracts Žarrows.. Lower levels of EMP1 expression are seen in the proliferative neuroepithelium Žarrowheads.. Size bar in B s 0.5 mm for A and B. Abbreviations: cb, cerebellum; df, dorsal funiculus; is, isthmus; hi, hippocampus; hp, habenulopeduncular tract; hy, hypothalamus; ma, medulla oblongata; nc, neocortex; ob, olfactory bulb; oc, opticchiasma; oe, olfactory epithelium; pa, preoptic area; po, pons; rh, rhinencephalon; sc, spinal cord; sm, stria medullaris; sp, septum; tc, tectum; tg, tegmentum; th, thalamus; to, tongue; vf, ventral funiculus.
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2.6. Immunofluorescence Cells were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 for 15 min. After washing, cells were permeabilized Žfor anti-PMP22 antiserum in 1% Triton X-100; for anti-EMP1 antiserum in 0.2% Tween 20., nonspecific binding was blocked Ž5% normal goat serum in 0.1 M phosphate buffer.. The cells were incubated with the primary antibodies in blocking solution overnight at 48C. Detection was achieved by incubation for 1 h at room temperature with fluorochrome-conjugated species-specific secondary antibodies. All steps were followed by three washes in 0.1 M phosphate buffer.
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goat anti-mouse FITC ŽJackson. were diluted 1:250 in blocking buffer.
3. Results 3.1. Northern blot analysis of EMP1 and PMP22 mRNA in the mouse brain The levels of EMP1 and PMP22 mRNA were assessed by Northern blot analysis in the mouse brain at various stages of development ŽFig. 1.. Maximal expression of both transcripts was detected in late gestation with reduced levels postnatally.
2.7. Antibodies Antisera were raised against synthetic peptides representing the putative extracellular loops of mouse EMP1 w25x: peptide 1, WKNCTGGNCDGSLSYGNEDAIKC and peptide 2, YTHHYAHSEGNFNSSSHQGYC. A C-terminal cysteine residue was added to peptide 1 for coupling purposes. Polyclonal antiserum raised against peptide 2 were superior in immunohistochemistry and used at 1:250 dilution. A polyclonal rabbit antiserum against PMP22 peptide 2 was used at 1:200 dilution w34x. Monoclonal anti-class III beta-tubulin antibody ŽSigma. was used at 1:200 dilution, and monoclonal antibody Rat-401 Žnestin. in a 1:1 dilution in the presence of 0.1% NP40 w19x. The secondary antibodies goat anti-rabbit Cy3 ŽJackson. and
3.2. Spatial analysis of EMP1 expression in the deÕeloping nerÕous system In the first step, we wanted to determine the areas of highest EMP1 expression on sagittal overviews using in situ hybridization and immunohistochemistry. A mouse EMP1 cDNA spanning the complete open-reading frame was used as the template to generate the appropriate riboprobe for mRNA detection w25x. The antisense EMP1probe labeled most conspicuously the developing nervous system ŽFig. 2F., whereas the sense control probe yielded negligible signals ŽFig. 2E.. Next, we tested the suitability of polyclonal anti-EMP1 peptide antisera for detection of the EMP1 protein. Serial sections of mouse embryos were
Fig. 4. EMP1 expression in preplate neurons prior to cortical plate development. Sagittal sections of the developing neocortex, lateral ventricle Žv. to the left and rostral to the top. EMP1 staining was observed in the first neuronal layer of the neocortex and along presumptive radial glia cells Žarrows.. ŽA. EMP1 is expressed along putative radial glia cells at E10.5 Žarrows.. ŽB. Additional EMP1 staining was found in the developed preplate at E13.5. ŽC. The preplate is subdivided by cortical plate neurons into the marginal zone and the subplate at E15.5. Presumptive neurons of the marginal zone and subplate express EMP1, while less immunoreactivity was observed in the cortical plate. Size bar in C s 30 mm for A, B, and C. Abbreviations: cp, cortical plate; iz, intermediate zone; mz, marginal zone; ne, neuroepithelium, pp, preplate Žor primordial plexiform layer.; sp, subplate; v, ventricle; vz ventricular zone.
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analysed using the preimmune serum as control. As described for the rat protein before w58x, a polyclonal antiserum raised against the computer-predicted second extracellular loop of the mouse EMP1 protein Žpeptide 2, see Section 2. detected EMP1 efficiently ŽFig. 2A. while the preimmune serum gave no appreciable staining ŽFig. 2D.. Extensive colocalization of EMP1 mRNA and protein confirmed the specificity of both the in situ hybridization probe and the polyclonal antiserum on tissue sections Žcompare Fig. 2C and F.. The highest levels of EMP1 expression were confined to the developing brain, spinal cord, cranial nerves and DRG at E10.5, E13.5 and E15.5 ŽFig. 2A, B and C.. 3.3. Distribution of EMP1 during CNS deÕelopment Since the initial analysis suggested that EMP1 transcript levels are high in the mid- to late gestation embryonic brain, we examined spatial EMP1 expression in the developing CNS in more detail. At E15.5, strong signals were observed in the neuronal differentiating fields of the developing fore-, mid-, and hindbrain ŽFig. 3A, B. w3x. Comparison of the mRNA and protein distribution were consistent with neuronal expression since EMP1 mRNA was mainly seen in areas of differentiating neuronal cell bodies ŽFig.
Fig. 5. EMP1 expression in early neuronal fields of the rhombencephalon. Sagittal section of the thomencephalon at E10.5. Dorsal is to the left and rostral to the top. Predominant EMP1 expression is seen at the ventral side reminiscent of the differentiation fields of the developing rhombencephalon. Size bar s 250 mm.
Fig. 6. In the cerebellum, EMP1 is most prominently expressed in the fiber tracts of the cerebellar vermis. ŽA, B. Sagittal sections of the developing cerebellum, dorsal to the left and anterior fourth ventricle to the bottom. Highest EMP1 expression is seen in the differentiation field of the cerebellum and in the fiber tracts Žcp.. Arrows in A mark the inferior cerebellar peduncle, in ŽB., the superior cerebellar peduncle. Size bar in Bs 40 mm for ŽA and B.. Abbreviations: cp, cerebellar peduncle.
3A., while EMP1 protein was seen in the same areas but also prominently along fiber tracts ŽFig. 3B, arrows.. Lover levels of EMP1 expression were present in the neuroepithelia of the ventricular zones ŽFig. 3B, arrowheads.. In the developing neocortex, EMP1 distribution appears to change as newly generated cells form a laminar structure. At E10.5, EMP1 was expressed in the ventricular zone predominantly in cells at the edge of the ventricle ŽFig. 4a, arrows.. At E13.5, prominent staining of cells of the newly formed preplate became obvious ŽFig. 4B.. At E15.5, EMP1 was localized in the marginal zone and the subplate ŽFig. 4C., with only little staining of the cortical plate. The strongly EMP1-positive cells are likely to correspond to the first postmitotic neurons which leave the ventricular zone to form the preplate and are considered to be pioneer neurons of the newly forming neocortical laminar structure w27,28,33x. Apart from the telencephalon, EMP1 was also found in early-developing neurons in other areas of the nervous system. At E10.5, EMP1 was expressed in the caudal and ventral portions of the developing rhombencephalon which contain many postmitotic neurons w3x ŽFig. 5.. In the developing cerebellum, EMP1 expression is confined to the cerebellar differentiation field and, most conspicu-
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Fig. 7. EMP1 expression in DRG and peripheral nerves. ŽA, B. Cross-sections through the caudal–thoracic region of the spinal cord, dorsal to the top. ŽB. EMP1 mRNA expression is present in developing gray matter of the spinal cord Žmantle layer. and in the DRG at E10.5. ŽA. Immunohistochemistry reveals additional expression in the spinal nerve. ŽC, D. Sagittal sections through the caudal–thoracic region of DRG at E15.5, dorsal to the left. EMP1 mRNA localized most conspicuously to the DRG ŽD., while EMP1 protein was found in the DRG and along nerves ŽC.. Size bar in D s 100 mm for A, B, C and D. Abbreviations: drg, dorsal root ganglion; ml, mantle layer; sc, spinal cord; sn, spinal nerve
ously, along the cerebellar peduncle ŽFig. 6A and B, arrows.. In the spinal cord, EMP1 was found in the developing gray matter Žmantle layer neurons at E10.5; Fig. 7A. and at E15.5, EMP1 protein was present at high levels along the ventral and dorsal columns ŽFig. 3A, B.. This distribution indicates that EMP1 is widely expressed in the differentiating nervous system and mainly localized to the first differentiating neurons. 3.4. Spatio-temporal EMP1 localization during PNS deÕelopment To extend the studies to the PNS, we analyzed EMP1 expression in DRG and along the nerves. EMP1 was present in these structures at all stages examined ŽFigs. 7 and 2A, B, C, F.. High levels of EMP1 were observed in DRG using in situ hybridization or immunohistochemistry at E10.5 and E15.5 ŽFig. 7A, B, C, D., and prominent EMP1 protein distribution along the nerve was detected ŽFig. 7A, C.. Similar to the CNS, the distribution of EMP1 in the PNS was consistent with neuronal expression. To elucidate whether EMP1 expression is restricted to neurons
Fig. 8. EMP1 expression in DRG neurons and gial cells. ŽB. Dissociated embryonic DRG ŽE15.5. show most prominent EMP1 expression in neurons Žlong arrows., but also in glial cells Žshort arrows.. ŽB. Immunostaining for EMP1, ŽA. Phase contrast picture. Size bar in Bs10 mm for A and B.
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or extends also to glial cells, cultures of acutely dissociated E15.5 dorsal root ganglia were immunocytochemically stained for EMP1. High EMP1 levels were observed in neurons but moderate levels were also expressed by glial cells ŽFig. 8.. These findings are consistent with previous reports suggesting that EMP1 mRNA is present in cultures of proliferating rat Schwann cells, derived from postnatal rat sciatic nerves and expanded using mitogens w58x. 3.5. EMP1 and PMP22 are upregulated by retinoic acid treatment of P19 cells Northern blot analysis revealed highest EMP1 and PMP22 transcripts levels during prenatal neurogenesis in the brain ŽFig. 1.. Our data suggested further that EMP1 is mainly expressed in the differentiation fields and appears to be mostly confined to non-proliferative cells while PMP22 has been reported to be concentrated in the neuroepithelial ventricular zone w4,36x. To address this issue closer under defined conditions, we used the embryonal carcinoma P19 cell line as an established in vitro model of neuroectodermal differentiation w32x. Depending on the culture conditions, P19 cells can differentiate into neuroectodermal or the mesoderm cells after retinoic acid treatment w23,31x. Both EMP1 and PMP22 transcript levels
were upregulated in neuroectodermal as well as mesodermal differentiated cultures consistent with the reported high mRNA expression in derivatives of all three germ layers in the adult mouse ŽFig. 9.. Thus, the P19 cell line constitutes an appropriate system to analyze the cellular localization of EMP1 and PMP22 gene expression. For this purpose, P19 cell aggregates were treated with retinoic acid and plated on tissue cultures dishes. After 1 to 7 days, the cultures were immunostained for EMP1 or PMP22 and double-stained with either neuron-specific class III betatubulin as marker for immature neurons w5,6x, or nestin as a marker for neuroepithelial stem cells w19x ŽFig. 10.. Cells with the morphological characteristics of fibroblasts expressed both, PMP22 and EMP1 Ždata not shown.. Cells positive for beta-tubulin isotype III expressed EMP1 ŽFig. 10A–F. while nestin-positive cells were positive for PMP22 ŽFig. 10 G–I.. EMP1 was also found along outgrowing neurites ŽFig. 10A–C., and in further differentiated neurons, most conspicuously in neurite varicosities ŽFig. 10D–F.. These experiments support the notion that EMP1 is expressed by early neurons and accumulates in neurites while PMP22 is expressed in more immature neuroepithelial ventricular stem cells w4,36x.
4. Discussion 4.1. Temporal analysis of EMP1 and PMP22 mRNA expression in the mouse brain suggests a function for these molecules during neuronal differentiation Analysis of EMP1 and PMP22 mRNA expression in the developing brain revealed an upregulation in the embryonic brain around E15.5 ŽFig. 1; w25,47x.. The development of the embryonic brain is characterized by three main stages, beginning with the expansion of the proliferative neuroepithelium from E9 to approx. E12. In the second stage, the neuroepithelium remains active, but is coupled with the differentiation of neurons ŽE13 to E16.. At the final prenatal stage, the major brain structures can be identified accompanied by the gradual disappearance of the neuroepithelia w3x. Hence, the observed increased levels of EMP1 and PMP22 transcripts, especially during the second stage of prenatal brain development, are consistent with potential roles of these molecules during neurogenesis.
Fig. 9. EMP1 and PMP22 mRNA are upregulated by retionoic acid-induced differentiation of P19 cells. Northern blot analysis using probes specific for EMP1, PMP22 and GAPDH Žas loading control.. EMP1 and PMP22 were upregulated in differentiating P19 cells. Cell density did not significantly influence EMP1 or PMP22 mRNA expression. Upper panel: EMP1 analysis. Middle panel: PMP22 analysis. Bottom panel GAPDH hybridization confirmed equal loading of P19 cell RNA. Each sample contained 10 mg of total RNA.
4.2. The spatiotemporal expression pattern of EMP1 suggests a role in the first postmitotic neurons and glial cells EMP1 is expressed in most tissues in the adult mouse, with highest levels in the lung and in the gastrointestinal tract w25x. In particular, EMP1 is localized to the highly migratory and differentiating areas of intestinal epithelium. During prenatal development, we found EMP1 mainly
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Fig. 10. Cellular expression of EMP1 and PMP22 in neuroectodermal differentiated P19 cells. ŽA–F. Double-labeling with anti-EMP1 immuneserum and a monoclonal antibody against neuron-specific class III beta-tubulin indicating that early neuronal cells express EMP1. EMP1 localizes to the growth cone ŽB. and to neurite varicosaties ŽE.. ŽG–I. Double-labeling with anti-PMP22 immuneserum and a monoclonal antibody against nestin, a marker for neuroepithelial stem cells, suggesting that P19-derived neuroepithelial stem cells express PMP22. Size bar in I s 20 mm for G–I, 8 mm for A–F.
expressed in the developing nervous system. In all parts of the developing nervous system analyzed, the combination of temporal and spatial expression analysis is consistent with a role of EMP1 in differentiating neurons and during initial tract formation since EMP1 is upregulated in the first differentiation fields of the nervous system. At E10.5, high EMP1 expression is found in the caudal and ventral rhombencephalon and the developing gray matter of the spinal cord, and at E13.5 and E15.5, in the initial fiber tracts of the developing cerebellum. Similarly, EMP1 expression was observed by DRG neurons and distributed along peripheral nerves. In vitro experiments using dissociated DRG cultures confirmed EMP1 expression by neurons and its localization along neurites but revealed also additional low levels of EMP1 in glia cells. The expression of EMP1 in the telencephalon suggests an association with the first postmitotic neurons that leave the ventricular zone to form the preplate. Preplate neurons migrate out of the ventricular zone, form the preplate, and constitute a first primitive cortical organization which is functionally active during early embryonic life w7,50x. By E15.5, the preplate is subdivided by newly generated
cortical plate neurons into the marginal zone and the subplate w27,28x. While the marginal zone and subplate neurons still express EMP1, only reduced expression was observed in the newly generated cortical plate neurons. In the adult mouse, we were unable to detect EMP1 in neocortical layers I and VIb, which are the derivatives of the marginal zone and the subplate Ždata not shown. w52x. Thus, EMP1 may mainly be required during initial events of neurogenesis and the establishment of neural connectivity. Whether the loss of EMP1 in the adult is due to downregulation of expression in maturing cells, or that embryonic EMP1 expression is restricted to a transient subpopulation of cortical neurons remains to be elucidated. 4.3. EMP1 is present in postmitotic neurons while PMP22 is found in neuroepithelial stem cells PMP22 and EMP1 are inversely regulated by the cell cycle in various cell types w22x. The expression of EMP1 by postmitotic neurons and PMP22 by neuroepithelial stem cells indicates an inverse expression pattern also during the second stage of prenatal brain development. It is intriguing
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to notice that EMP1 in the telencephalon is localized to preplate neurons which are thought to be transient w15,60,61x. One might speculate that this observation may be related to previous findings in the haematopoetic system suggesting that EMP1rB4B-positive immature B cell are eliminated before productive VDJC rearrangement of Ig loci w38x and thus, EMP1 appears to be expressed on some transient cell populations in different tissues. Analogies between haematopoesis and neurogenesis have been described within the PMP22 gene family before with regard to EMP3rHNMP-1 which was cloned from a monocyte cell line but shows also abundant expression in the nervous system w10x. Such dual functions of proteins in the nervous and haematopoetic systems are not unusual as exemplified by shared receptor–ligand systems Že.g., the interleukin-6-type cytokine cascade. and adhesion molecules Že.g., HNK-1rL2.. Furthermore, the concept of multipotential stem cell ontogeny is common to both systems. 4.4. Distribution of EMP1 and PMP22 suggests recognition functions during nerÕous system deÕelopment Postnataly, PMP22 is best known for its function during PNS myelination, a highly specialized process of cell growth and extension of Schwann cell plasma membranes. The finding that EMP1 is present in neurons, especially along the newly forming fiber tracts, and in varicosities of P19 cell line-derived neurons, may indicate a similar function for EMP1 in neurite outgrowth which is also a highly specialized process involving cell growth and membrane extension. Besides the putative involvement of EMP1 and PMP22 in cell growth, both gene products have also been implicated in cell–cell recognition. Parmantier and coworkers w36x found PMP22 expression not only in the ventricular zone w4x but also in transverse segments and longitudinal columns of the developing brain defining neuromeric boundaries w11x. A second phase of PMP22 expression started at E18.5 in motor nuclei of the cranial nerves, and postnatally in the spinal cord motoneurons w36,37x. Based on these results, it was speculated that PMP22 might act as an adhesion factor that would provide positional information to adjacent cells andror growing axon pathways. A similar function might be proposed for EMP1 during the initial steps of neurogenesis since EMP1 was localized to the first set of neurons that develop, migrate out, and are thought to form a primary scaffold for the forming neural network by neurite extensions. Similarly, expression of EMP1 by neurons and Schwann cells along the nerve may indicate a function in the critical interplay between glial cells and neurons that regulate cell survival, proliferation, differentiation, and migration. Thus, EMP1 may act as a recognition molecule important in neuronal migration andror neurite outgrowth as suggested by the presence of the HNK-1rL2 carbohydrate chain w29,46x. Moreover, the finding of a PMP22 relative in
zebrafish ŽzPMP22., expressed by sclerotome cells, neural crest cells, and the migratory derivatives of both populations w62x, suggests a conserved role of this protein in migrating cells through evolution. In conclusion, our data demonstrate a correlation between the expression of EMP1 and PMP22 on one side, and cell proliferation, growth arrest, cell migration, and cells relying on extensive adhesive interactions with other cells or the extracellular matrix on the other. These functions may depend on each other in cascades since proteins involved in cell–cell and cell–extracellular matrix interactions are likely to influence also cell growth, differentiation and death.
Acknowledgements We thank Drs. Martin Schwab for suggestions on the developmental aspects of the embryonic distribution of EMP1, Andrew Welcher for anti-EMP1 antisera, and Rebecca Hardy for the gift of P19 cells and advice on their cultivation. We also thank Drs. Ned Mantei for comments and carefully reading the manuscript, Verdon Taylor for advice on PMP22rEMP1 immunodetection and Nadja Mercader for the DRG explants. This work was supported by grants from the Swiss National Science Foundation and the NFP38 Program Žto US..
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Research report
Embryonic expression of epithelial membrane protein 1 in early neurons Philip Wulf, Ueli Suter
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Institute of Cell Biology, Department of Biology, Swiss Federal Institute of Technology, ETH-Honggerberg, CH-8093 Zurich, Switzerland ¨ ¨ Accepted 29 June 1999
Abstract Epithelial membrane protein 1 ŽEMP1. is a member of the peripheral myelin protein 22 ŽPMP22. family. This family is best known for the crucial contribution of PMP22 to the development and maintenance of the peripheral nervous system ŽPNS.. PMP22 is widely expressed, with highest levels in myelinating Schwann cells, and mutations affecting the PMP22 gene lead to PNS-restricted neuropathies. We have investigated the spatio-temporal distribution of EMP1 and compared it to that of PMP22. We found that EMP1 and PMP22 mRNA are most conspicuously expressed in the prenatal mouse brain during neurogenesis. In the developing forebrain, we localized EMP1 mRNA and protein to the first set of neurons that are generated and leave the ventricular zone to form the preplate. Later in development, EMP1 was found in derivatives of the preplate, the marginal zone and the subplate. Reduced expression was observed in the newly generated cortical plate neurons. In other parts of the developing CNS and PNS, EMP1 was also detected in early neurons and along the initial fiber tracts. Furthermore, EMP1 was highly expressed by immature neurons in embryonal dorsal root ganglia-explant cultures and in neuroectodermal differentiated P19 cells. While PMP22 functions mainly in Schwann cell growth and differentiation, the spatio-temporal localization of EMP1 suggests a role in neuronal differentiation and neurite outgrowth. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Preplate; Marginal zone; Subplate; P19 cell line; Neuron; Glia
1. Introduction Epithelial membrane protein 1 ŽEMP1. has previously been identified as a member of the peripheral myelin protein 22 ŽPMP22. gene family w8,25,29,38,40,58x. Currently, five members of this family are known including PMP22, EMP1, epithelial membrane protein 2 ŽEMP2. w8,57x and epithelial membrane protein 3 ŽEMP3. w10,57x, as well as the lens-specific membrane protein 20 ŽMP20. w24,58x. The conserved genomic structures and chromosomal localizations suggest that this family is derived from genomic duplications of an ancestral gene w9,12,25,62,63x. The PMPrEMPrMP20 proteins contain four hydrophobic regions spanning or embedded in the cell membrane and display an amino acid identity of approximately 40% with the exception of the more distantly related MP20, which shares only about 25% identical residues w8,57,58,62x. PMP22 is the best characterized member of this family w35x. It contributes to myelin compaction and mutations
) Corresponding author. Fax: q 41-1-633-1190; E-m ail: [email protected]; Internet address: http:rrwww.cell.biol.ethz.chr
affecting PMP22 are associated with hereditary motor and sensory neuropathies in humans and rodents w54x. Duplications, point mutations, and deletions of the PMP22 gene have been linked to the most common inherited disorder of the peripheral nervous system, Charcot–Marie–Tooth ŽCMT1A. disease, the Dejerine–Sottas syndrome ŽDSS., and to hereditary neuropathy with liability to pressure palsies ŽHNPP. w44,45x. The common feature of these PMP22-based neuropathies is the disturbance in the development and maintenance of myelin and axons. Based on the human pathologies and the phenotypes of naturally occurring and artificially generated rodents mutant for PMP22, it is well established that the PNS is highly sensitive to alterations in PMP22 gene dosage w1,2,20, 21,26,30,43,51,53,55x. On the molecular level, PMP22 and EMP1 are thought to be involved in the regulation of the cell cycle, cell–cell recognition, and cell death w22x. Members of the PMP22 family have been repeatedly found in screens for transcripts whose levels are regulated during the cell-cycle, tissue maturation, or tumorgenesis w 8,14,29,38, 40,41,48,56,59x. EMP1 is upregulated during proliferation of fibroblasts and Schwann cells and this regulation ap-
0165-3806r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 9 . 0 0 0 9 2 - 9
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pears to be inverse to that of PMP22 w16,58x. Furthermore, elevated EMP1 mRNA levels were found in brain tumors and during tumor progression and consequently, the cognate proteins were named ‘‘Tumor-associated Membrane Protein’’ ŽTMP., and ‘‘Progression-associated Protein’’ ŽPAP., respectively w8,40x. In contrast, high-level expression of EMP1 is associated with differentiation and growth arrest in squamous cells and in the haematopoetic system Žcorresponding clones called CL-20 and B4B; w29,38x.. Thus, the correlation between PMP22rEMP1 gene regulation and proliferation or growth arrest may depend on the cellular context. A common feature of PMP22 and EMP1 is the single N-linked carbohydrate chain attached to the protein backbone which carries the HNK-1rL2 carbohydrate epitope w29,46x. This special structure may not only contribute to cell–cell or cell–extracellular matrix recognition functions, but might also affect differentiation, proliferation, and cell growth w39x. The members of the PMP22 gene family are widely expressed, with the exception of MP20, which is specific to the lens. Highest levels of PMP22 are generated by myelinating Schwann cells, in the lung, and in the gastrointestinal tract w47,58x. During embryonic development, PMP22 expression was found in derivatives of all three germ layers w4,25,37x although further studies on PMP22 distribution, and particularly, the analysis of PMP22 mutants suggested that PMP22 is mainly important for the development and the maintenance of the nervous system w2,17,26,36,62x. EMP1 is widely expressed in the adult mouse, with highest levels in the gastrointestinal tract and in the lung w25x. Spatially, EMP1 has been localized to the proliferating and differentiating epithelia of the rat corpus gastricum, of the rabbit tongue, and of the esophagus w29,58x. To extend these studies to the prenatal mouse and to compare the distribution of EMP1 with that previously reported for PMP22 w4,36x, we used in situ hybridization and immunohistochemistry to analyze the expression of EMP1 during embryogenesis. Interestingly, we found most conspicuous expression of EMP1 in the developing CNS and PNS at the onset of neurogenesis.
2.2. DRG-explant cultures DRG of E15.5 mouse embryos were dissected, trypsinized, and cultured in SM medium on coated tissue culture plates w49x. After 5 days in culture, cells were fixed in 4% paraformaldehyde for 15 min and processed for immuncytochemistry. 2.3. P19 cell culture P19 cells were a gift from Dr. R.J. Hardy, Mount Sinai Medical School, New York. Cells were cultured in MEM alpha medium supplemented by 7.5% newborn calf serum, 2.5% fetal calf serum, 2 mM glutamine, 100 Urml penicillin and, 100 mgrml streptomycin w23x. Cultures were maintained at 378C in 5% CO 2 atmosphere. For mesodermal differentiation, monolayer cultures of P19 cells were maintained in the presence of 1 mM retinoic acid ŽRA.. For neural differentiation, P19 cells were aggregated in bacteriological petri dishes at 5 = 10 5 cellsrml in the presence of 1 mM retinoic acid ŽRA. for two days. Subsequently, the medium was replenished and the aggregates incubated for another two days. Aggregates were dissociated by incubation in 0.05% Trypsin, 0.5 mM EDTA, plated on cell culture dishes in P19 medium without RA and after 1 to 7 days processed for immunocytochemistry w18x. 2.4. RNA isolation, Northern blotting, and labeling of nucleic acid probes Total RNA was isolated using a modified acid phenolrguanidinium isothiocyanate method w13x. Ten micrograms of total RNA were separated by electrophoresis
2. Materials and methods
2.1. Animals and tissues Time-mated C57BLr6J mice were obtained from BRL Fullinsdorf, Switzerland, and used in all experiments. The ¨ day of plug detection was considered as E0.5. Mouse embryos were harvested and embedded in O.C.T mounting medium ŽTissue-Tek w ., frozen in isopentane cooled to freezing point by liquid nitrogen, and stored at y808C.
Fig. 1. EMP1 and PMP22 transcripts are upregulated in the murine brain during neurogenesis. Ten micrograms total RNA isolated from embryonic or postnatal brains were subjected to Northern blot analysis. Upper panel: Arrows indicate EMP1 transcripts at 2.8 kb and 1.7 kb. Middle panel: Arrow indicates PMP22 transcripts at 1.8 kb. Bottom panel: GAPDH as a loading control. Arrow indicates the GAPDH signal at 1.4 kb.
P. Wulf, U. Suter r DeÕelopmental Brain Research 116 (1999) 169–180
in formaldehyde-containing 1.2% agarose gel and capillary blotted onto nylon membranes ŽHybond-N, Amershan.. The membrane was hybridized in 50% formamide buffer at 428C with a 32 P-labeled, randomly primed EMP1 cDNA probe according to the manufacturers instructions ŽPharmacia.. The membrane was washed to a final stringency of 0.1% SSCr0.1% SDS at 558C, and exposed to X-ray film ŽFuji. at y708C. After stripping in 0.1%SDS at 1008C for 5 min, the same membranes were subjected to rehybridization with identically prepared probes for PMP22 and GAPDH.
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2.5. In situ hybridization and nucleic acid riboprobes Serial cryosections of 10 mm thickness from mouse embryos were mounted on SuperFrost Plus slides and postfixed in PBS containing 4% paraformaldehyde for 10 min at room temperature. Slides were prepared for in situ hybridization as described w42x. Digoxigenin ŽDIG. uridinetriphosphate-labeled riboprobes in sense and antisense orientations were synthesized according to the manufacturer’s instructions ŽBoehringer Mannheim.. Detection of alkaline phosphatase coupled to anti-DIG antibody
Fig. 2. EMP1 is conspicuously expressed in the developing nervous system during mid- to late gestation. ŽA, B, C, D. Immunohistochemical EMP-1 staining on sagittal sections. Highest expression of EMP1 was observed in the developing CNS and PNS. ŽA, D. Staining of adjacent sections of E10.5 embryo confirm the specificity of the EMP1 antiserum ŽA. in comparison to preimmune serum ŽD.. ŽE, F. In situ hybridization of serial sections adjacent to ŽC., using a sense ŽE. or antisense ŽF. DIG-labeled EMP1 probe demonstrating specificity of the antisense hybridization. In agreement with the protein distribution, the levels of EMP1 mRNA were highest in the nervous system. Size bar in F s 1 mm for F and E, 0.5 mm for A and D, 0.7 mm for B. Abbreviations: bo, bowel; cn, cranial nerve; drg, dorsal root ganglia; fv, fourth ventricle; ht, heart; li, liver; lv, lateral ventricle; ma, medulla oblongata; mv, mesencephalic vesicle; oe, olfactory epithelium; sc, spinal cord; sn, spinal nerve.
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ŽBoehringer. was carried out with nitroblue tetrazolium ŽNBT., 5-bromo-4-chloro-3-indolyl phosphate ŽBCIP., and levamisole for 2 h at room temperature. An EMP1-sense
control reaction, labeled to comparable activity, was always included in parallel and yielded negligible signals ŽFig. 2F..
Fig. 3. EMP1 is expressed in differentiation fields of the embryonic brain. Serial sagittal sections of E15.5 mouse brain are shown. Dorsal is to the left and rostral at the top. Comparison of EMP1 mRNA and protein distribution suggests neuronal localization in the E15.5 brain. ŽA. In situ hybridization localizes EMP1 transcripts to the neuronal differentiation fields. ŽB. EMP1 immunohistochemistry confirms the EMP1 distribution to the neuronal differentiation fields and shows that EMP1 protein is also present along fiber tracts Žarrows.. Lower levels of EMP1 expression are seen in the proliferative neuroepithelium Žarrowheads.. Size bar in B s 0.5 mm for A and B. Abbreviations: cb, cerebellum; df, dorsal funiculus; is, isthmus; hi, hippocampus; hp, habenulopeduncular tract; hy, hypothalamus; ma, medulla oblongata; nc, neocortex; ob, olfactory bulb; oc, opticchiasma; oe, olfactory epithelium; pa, preoptic area; po, pons; rh, rhinencephalon; sc, spinal cord; sm, stria medullaris; sp, septum; tc, tectum; tg, tegmentum; th, thalamus; to, tongue; vf, ventral funiculus.
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2.6. Immunofluorescence Cells were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 for 15 min. After washing, cells were permeabilized Žfor anti-PMP22 antiserum in 1% Triton X-100; for anti-EMP1 antiserum in 0.2% Tween 20., nonspecific binding was blocked Ž5% normal goat serum in 0.1 M phosphate buffer.. The cells were incubated with the primary antibodies in blocking solution overnight at 48C. Detection was achieved by incubation for 1 h at room temperature with fluorochrome-conjugated species-specific secondary antibodies. All steps were followed by three washes in 0.1 M phosphate buffer.
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goat anti-mouse FITC ŽJackson. were diluted 1:250 in blocking buffer.
3. Results 3.1. Northern blot analysis of EMP1 and PMP22 mRNA in the mouse brain The levels of EMP1 and PMP22 mRNA were assessed by Northern blot analysis in the mouse brain at various stages of development ŽFig. 1.. Maximal expression of both transcripts was detected in late gestation with reduced levels postnatally.
2.7. Antibodies Antisera were raised against synthetic peptides representing the putative extracellular loops of mouse EMP1 w25x: peptide 1, WKNCTGGNCDGSLSYGNEDAIKC and peptide 2, YTHHYAHSEGNFNSSSHQGYC. A C-terminal cysteine residue was added to peptide 1 for coupling purposes. Polyclonal antiserum raised against peptide 2 were superior in immunohistochemistry and used at 1:250 dilution. A polyclonal rabbit antiserum against PMP22 peptide 2 was used at 1:200 dilution w34x. Monoclonal anti-class III beta-tubulin antibody ŽSigma. was used at 1:200 dilution, and monoclonal antibody Rat-401 Žnestin. in a 1:1 dilution in the presence of 0.1% NP40 w19x. The secondary antibodies goat anti-rabbit Cy3 ŽJackson. and
3.2. Spatial analysis of EMP1 expression in the deÕeloping nerÕous system In the first step, we wanted to determine the areas of highest EMP1 expression on sagittal overviews using in situ hybridization and immunohistochemistry. A mouse EMP1 cDNA spanning the complete open-reading frame was used as the template to generate the appropriate riboprobe for mRNA detection w25x. The antisense EMP1probe labeled most conspicuously the developing nervous system ŽFig. 2F., whereas the sense control probe yielded negligible signals ŽFig. 2E.. Next, we tested the suitability of polyclonal anti-EMP1 peptide antisera for detection of the EMP1 protein. Serial sections of mouse embryos were
Fig. 4. EMP1 expression in preplate neurons prior to cortical plate development. Sagittal sections of the developing neocortex, lateral ventricle Žv. to the left and rostral to the top. EMP1 staining was observed in the first neuronal layer of the neocortex and along presumptive radial glia cells Žarrows.. ŽA. EMP1 is expressed along putative radial glia cells at E10.5 Žarrows.. ŽB. Additional EMP1 staining was found in the developed preplate at E13.5. ŽC. The preplate is subdivided by cortical plate neurons into the marginal zone and the subplate at E15.5. Presumptive neurons of the marginal zone and subplate express EMP1, while less immunoreactivity was observed in the cortical plate. Size bar in C s 30 mm for A, B, and C. Abbreviations: cp, cortical plate; iz, intermediate zone; mz, marginal zone; ne, neuroepithelium, pp, preplate Žor primordial plexiform layer.; sp, subplate; v, ventricle; vz ventricular zone.
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analysed using the preimmune serum as control. As described for the rat protein before w58x, a polyclonal antiserum raised against the computer-predicted second extracellular loop of the mouse EMP1 protein Žpeptide 2, see Section 2. detected EMP1 efficiently ŽFig. 2A. while the preimmune serum gave no appreciable staining ŽFig. 2D.. Extensive colocalization of EMP1 mRNA and protein confirmed the specificity of both the in situ hybridization probe and the polyclonal antiserum on tissue sections Žcompare Fig. 2C and F.. The highest levels of EMP1 expression were confined to the developing brain, spinal cord, cranial nerves and DRG at E10.5, E13.5 and E15.5 ŽFig. 2A, B and C.. 3.3. Distribution of EMP1 during CNS deÕelopment Since the initial analysis suggested that EMP1 transcript levels are high in the mid- to late gestation embryonic brain, we examined spatial EMP1 expression in the developing CNS in more detail. At E15.5, strong signals were observed in the neuronal differentiating fields of the developing fore-, mid-, and hindbrain ŽFig. 3A, B. w3x. Comparison of the mRNA and protein distribution were consistent with neuronal expression since EMP1 mRNA was mainly seen in areas of differentiating neuronal cell bodies ŽFig.
Fig. 5. EMP1 expression in early neuronal fields of the rhombencephalon. Sagittal section of the thomencephalon at E10.5. Dorsal is to the left and rostral to the top. Predominant EMP1 expression is seen at the ventral side reminiscent of the differentiation fields of the developing rhombencephalon. Size bar s 250 mm.
Fig. 6. In the cerebellum, EMP1 is most prominently expressed in the fiber tracts of the cerebellar vermis. ŽA, B. Sagittal sections of the developing cerebellum, dorsal to the left and anterior fourth ventricle to the bottom. Highest EMP1 expression is seen in the differentiation field of the cerebellum and in the fiber tracts Žcp.. Arrows in A mark the inferior cerebellar peduncle, in ŽB., the superior cerebellar peduncle. Size bar in Bs 40 mm for ŽA and B.. Abbreviations: cp, cerebellar peduncle.
3A., while EMP1 protein was seen in the same areas but also prominently along fiber tracts ŽFig. 3B, arrows.. Lover levels of EMP1 expression were present in the neuroepithelia of the ventricular zones ŽFig. 3B, arrowheads.. In the developing neocortex, EMP1 distribution appears to change as newly generated cells form a laminar structure. At E10.5, EMP1 was expressed in the ventricular zone predominantly in cells at the edge of the ventricle ŽFig. 4a, arrows.. At E13.5, prominent staining of cells of the newly formed preplate became obvious ŽFig. 4B.. At E15.5, EMP1 was localized in the marginal zone and the subplate ŽFig. 4C., with only little staining of the cortical plate. The strongly EMP1-positive cells are likely to correspond to the first postmitotic neurons which leave the ventricular zone to form the preplate and are considered to be pioneer neurons of the newly forming neocortical laminar structure w27,28,33x. Apart from the telencephalon, EMP1 was also found in early-developing neurons in other areas of the nervous system. At E10.5, EMP1 was expressed in the caudal and ventral portions of the developing rhombencephalon which contain many postmitotic neurons w3x ŽFig. 5.. In the developing cerebellum, EMP1 expression is confined to the cerebellar differentiation field and, most conspicu-
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Fig. 7. EMP1 expression in DRG and peripheral nerves. ŽA, B. Cross-sections through the caudal–thoracic region of the spinal cord, dorsal to the top. ŽB. EMP1 mRNA expression is present in developing gray matter of the spinal cord Žmantle layer. and in the DRG at E10.5. ŽA. Immunohistochemistry reveals additional expression in the spinal nerve. ŽC, D. Sagittal sections through the caudal–thoracic region of DRG at E15.5, dorsal to the left. EMP1 mRNA localized most conspicuously to the DRG ŽD., while EMP1 protein was found in the DRG and along nerves ŽC.. Size bar in D s 100 mm for A, B, C and D. Abbreviations: drg, dorsal root ganglion; ml, mantle layer; sc, spinal cord; sn, spinal nerve
ously, along the cerebellar peduncle ŽFig. 6A and B, arrows.. In the spinal cord, EMP1 was found in the developing gray matter Žmantle layer neurons at E10.5; Fig. 7A. and at E15.5, EMP1 protein was present at high levels along the ventral and dorsal columns ŽFig. 3A, B.. This distribution indicates that EMP1 is widely expressed in the differentiating nervous system and mainly localized to the first differentiating neurons. 3.4. Spatio-temporal EMP1 localization during PNS deÕelopment To extend the studies to the PNS, we analyzed EMP1 expression in DRG and along the nerves. EMP1 was present in these structures at all stages examined ŽFigs. 7 and 2A, B, C, F.. High levels of EMP1 were observed in DRG using in situ hybridization or immunohistochemistry at E10.5 and E15.5 ŽFig. 7A, B, C, D., and prominent EMP1 protein distribution along the nerve was detected ŽFig. 7A, C.. Similar to the CNS, the distribution of EMP1 in the PNS was consistent with neuronal expression. To elucidate whether EMP1 expression is restricted to neurons
Fig. 8. EMP1 expression in DRG neurons and gial cells. ŽB. Dissociated embryonic DRG ŽE15.5. show most prominent EMP1 expression in neurons Žlong arrows., but also in glial cells Žshort arrows.. ŽB. Immunostaining for EMP1, ŽA. Phase contrast picture. Size bar in Bs10 mm for A and B.
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or extends also to glial cells, cultures of acutely dissociated E15.5 dorsal root ganglia were immunocytochemically stained for EMP1. High EMP1 levels were observed in neurons but moderate levels were also expressed by glial cells ŽFig. 8.. These findings are consistent with previous reports suggesting that EMP1 mRNA is present in cultures of proliferating rat Schwann cells, derived from postnatal rat sciatic nerves and expanded using mitogens w58x. 3.5. EMP1 and PMP22 are upregulated by retinoic acid treatment of P19 cells Northern blot analysis revealed highest EMP1 and PMP22 transcripts levels during prenatal neurogenesis in the brain ŽFig. 1.. Our data suggested further that EMP1 is mainly expressed in the differentiation fields and appears to be mostly confined to non-proliferative cells while PMP22 has been reported to be concentrated in the neuroepithelial ventricular zone w4,36x. To address this issue closer under defined conditions, we used the embryonal carcinoma P19 cell line as an established in vitro model of neuroectodermal differentiation w32x. Depending on the culture conditions, P19 cells can differentiate into neuroectodermal or the mesoderm cells after retinoic acid treatment w23,31x. Both EMP1 and PMP22 transcript levels
were upregulated in neuroectodermal as well as mesodermal differentiated cultures consistent with the reported high mRNA expression in derivatives of all three germ layers in the adult mouse ŽFig. 9.. Thus, the P19 cell line constitutes an appropriate system to analyze the cellular localization of EMP1 and PMP22 gene expression. For this purpose, P19 cell aggregates were treated with retinoic acid and plated on tissue cultures dishes. After 1 to 7 days, the cultures were immunostained for EMP1 or PMP22 and double-stained with either neuron-specific class III betatubulin as marker for immature neurons w5,6x, or nestin as a marker for neuroepithelial stem cells w19x ŽFig. 10.. Cells with the morphological characteristics of fibroblasts expressed both, PMP22 and EMP1 Ždata not shown.. Cells positive for beta-tubulin isotype III expressed EMP1 ŽFig. 10A–F. while nestin-positive cells were positive for PMP22 ŽFig. 10 G–I.. EMP1 was also found along outgrowing neurites ŽFig. 10A–C., and in further differentiated neurons, most conspicuously in neurite varicosities ŽFig. 10D–F.. These experiments support the notion that EMP1 is expressed by early neurons and accumulates in neurites while PMP22 is expressed in more immature neuroepithelial ventricular stem cells w4,36x.
4. Discussion 4.1. Temporal analysis of EMP1 and PMP22 mRNA expression in the mouse brain suggests a function for these molecules during neuronal differentiation Analysis of EMP1 and PMP22 mRNA expression in the developing brain revealed an upregulation in the embryonic brain around E15.5 ŽFig. 1; w25,47x.. The development of the embryonic brain is characterized by three main stages, beginning with the expansion of the proliferative neuroepithelium from E9 to approx. E12. In the second stage, the neuroepithelium remains active, but is coupled with the differentiation of neurons ŽE13 to E16.. At the final prenatal stage, the major brain structures can be identified accompanied by the gradual disappearance of the neuroepithelia w3x. Hence, the observed increased levels of EMP1 and PMP22 transcripts, especially during the second stage of prenatal brain development, are consistent with potential roles of these molecules during neurogenesis.
Fig. 9. EMP1 and PMP22 mRNA are upregulated by retionoic acid-induced differentiation of P19 cells. Northern blot analysis using probes specific for EMP1, PMP22 and GAPDH Žas loading control.. EMP1 and PMP22 were upregulated in differentiating P19 cells. Cell density did not significantly influence EMP1 or PMP22 mRNA expression. Upper panel: EMP1 analysis. Middle panel: PMP22 analysis. Bottom panel GAPDH hybridization confirmed equal loading of P19 cell RNA. Each sample contained 10 mg of total RNA.
4.2. The spatiotemporal expression pattern of EMP1 suggests a role in the first postmitotic neurons and glial cells EMP1 is expressed in most tissues in the adult mouse, with highest levels in the lung and in the gastrointestinal tract w25x. In particular, EMP1 is localized to the highly migratory and differentiating areas of intestinal epithelium. During prenatal development, we found EMP1 mainly
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Fig. 10. Cellular expression of EMP1 and PMP22 in neuroectodermal differentiated P19 cells. ŽA–F. Double-labeling with anti-EMP1 immuneserum and a monoclonal antibody against neuron-specific class III beta-tubulin indicating that early neuronal cells express EMP1. EMP1 localizes to the growth cone ŽB. and to neurite varicosaties ŽE.. ŽG–I. Double-labeling with anti-PMP22 immuneserum and a monoclonal antibody against nestin, a marker for neuroepithelial stem cells, suggesting that P19-derived neuroepithelial stem cells express PMP22. Size bar in I s 20 mm for G–I, 8 mm for A–F.
expressed in the developing nervous system. In all parts of the developing nervous system analyzed, the combination of temporal and spatial expression analysis is consistent with a role of EMP1 in differentiating neurons and during initial tract formation since EMP1 is upregulated in the first differentiation fields of the nervous system. At E10.5, high EMP1 expression is found in the caudal and ventral rhombencephalon and the developing gray matter of the spinal cord, and at E13.5 and E15.5, in the initial fiber tracts of the developing cerebellum. Similarly, EMP1 expression was observed by DRG neurons and distributed along peripheral nerves. In vitro experiments using dissociated DRG cultures confirmed EMP1 expression by neurons and its localization along neurites but revealed also additional low levels of EMP1 in glia cells. The expression of EMP1 in the telencephalon suggests an association with the first postmitotic neurons that leave the ventricular zone to form the preplate. Preplate neurons migrate out of the ventricular zone, form the preplate, and constitute a first primitive cortical organization which is functionally active during early embryonic life w7,50x. By E15.5, the preplate is subdivided by newly generated
cortical plate neurons into the marginal zone and the subplate w27,28x. While the marginal zone and subplate neurons still express EMP1, only reduced expression was observed in the newly generated cortical plate neurons. In the adult mouse, we were unable to detect EMP1 in neocortical layers I and VIb, which are the derivatives of the marginal zone and the subplate Ždata not shown. w52x. Thus, EMP1 may mainly be required during initial events of neurogenesis and the establishment of neural connectivity. Whether the loss of EMP1 in the adult is due to downregulation of expression in maturing cells, or that embryonic EMP1 expression is restricted to a transient subpopulation of cortical neurons remains to be elucidated. 4.3. EMP1 is present in postmitotic neurons while PMP22 is found in neuroepithelial stem cells PMP22 and EMP1 are inversely regulated by the cell cycle in various cell types w22x. The expression of EMP1 by postmitotic neurons and PMP22 by neuroepithelial stem cells indicates an inverse expression pattern also during the second stage of prenatal brain development. It is intriguing
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to notice that EMP1 in the telencephalon is localized to preplate neurons which are thought to be transient w15,60,61x. One might speculate that this observation may be related to previous findings in the haematopoetic system suggesting that EMP1rB4B-positive immature B cell are eliminated before productive VDJC rearrangement of Ig loci w38x and thus, EMP1 appears to be expressed on some transient cell populations in different tissues. Analogies between haematopoesis and neurogenesis have been described within the PMP22 gene family before with regard to EMP3rHNMP-1 which was cloned from a monocyte cell line but shows also abundant expression in the nervous system w10x. Such dual functions of proteins in the nervous and haematopoetic systems are not unusual as exemplified by shared receptor–ligand systems Že.g., the interleukin-6-type cytokine cascade. and adhesion molecules Že.g., HNK-1rL2.. Furthermore, the concept of multipotential stem cell ontogeny is common to both systems. 4.4. Distribution of EMP1 and PMP22 suggests recognition functions during nerÕous system deÕelopment Postnataly, PMP22 is best known for its function during PNS myelination, a highly specialized process of cell growth and extension of Schwann cell plasma membranes. The finding that EMP1 is present in neurons, especially along the newly forming fiber tracts, and in varicosities of P19 cell line-derived neurons, may indicate a similar function for EMP1 in neurite outgrowth which is also a highly specialized process involving cell growth and membrane extension. Besides the putative involvement of EMP1 and PMP22 in cell growth, both gene products have also been implicated in cell–cell recognition. Parmantier and coworkers w36x found PMP22 expression not only in the ventricular zone w4x but also in transverse segments and longitudinal columns of the developing brain defining neuromeric boundaries w11x. A second phase of PMP22 expression started at E18.5 in motor nuclei of the cranial nerves, and postnatally in the spinal cord motoneurons w36,37x. Based on these results, it was speculated that PMP22 might act as an adhesion factor that would provide positional information to adjacent cells andror growing axon pathways. A similar function might be proposed for EMP1 during the initial steps of neurogenesis since EMP1 was localized to the first set of neurons that develop, migrate out, and are thought to form a primary scaffold for the forming neural network by neurite extensions. Similarly, expression of EMP1 by neurons and Schwann cells along the nerve may indicate a function in the critical interplay between glial cells and neurons that regulate cell survival, proliferation, differentiation, and migration. Thus, EMP1 may act as a recognition molecule important in neuronal migration andror neurite outgrowth as suggested by the presence of the HNK-1rL2 carbohydrate chain w29,46x. Moreover, the finding of a PMP22 relative in
zebrafish ŽzPMP22., expressed by sclerotome cells, neural crest cells, and the migratory derivatives of both populations w62x, suggests a conserved role of this protein in migrating cells through evolution. In conclusion, our data demonstrate a correlation between the expression of EMP1 and PMP22 on one side, and cell proliferation, growth arrest, cell migration, and cells relying on extensive adhesive interactions with other cells or the extracellular matrix on the other. These functions may depend on each other in cascades since proteins involved in cell–cell and cell–extracellular matrix interactions are likely to influence also cell growth, differentiation and death.
Acknowledgements We thank Drs. Martin Schwab for suggestions on the developmental aspects of the embryonic distribution of EMP1, Andrew Welcher for anti-EMP1 antisera, and Rebecca Hardy for the gift of P19 cells and advice on their cultivation. We also thank Drs. Ned Mantei for comments and carefully reading the manuscript, Verdon Taylor for advice on PMP22rEMP1 immunodetection and Nadja Mercader for the DRG explants. This work was supported by grants from the Swiss National Science Foundation and the NFP38 Program Žto US..
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