Complex expression pattern of the SCO-spondin gene in the bovine subcommissural organ: toward an explanation for Reissner's fiber complexity?

Complex expression pattern of the SCO-spondin gene in the bovine subcommissural organ: toward an explanation for Reissner's fiber complexity?
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  Ž . Molecular Brain Research 55 1998 45–53 Research report Complex expression pattern of the SCO-spondin gene in the bovinesubcommissural organ: toward an explanation for Reissner’s fibercomplexity? Isabelle Creveaux, Stephane Gobron, Robert Meiniel, Bernard Dastugue, Annie Meiniel  ) ´  Laboratoire de Biochimie Medicale, INSERM U 384, Faculte de Medecine, 28, place Henri-Dunant, 63001 Clermont-Ferrand Cedex, France ´ ´ ´ Accepted 11 November 1997 Abstract Ž . Bovine SCO-spondin is a glycoprotein secreted by the subcommissural organ SCO , an ependymal derivative located in the roof of the third ventricle. It shows homology with developmental molecules involved in directional axonal growth. Using SCO-spondin cDNAsas probes, we analysed the specific expression of the corresponding gene in the bovine SCO by Northern blot and in situ hybridization Ž . ISH . A strong expression was detected in the secretory ependymal and hypendymal cells of the SCO and the main transcripts showed alarge size 14 kb. A single copy gene was revealed by Southern blot analysis of bovine genomic DNA. The presence of additionaltranscripts suggested a transcriptional regulation of the SCO-spondin gene. A comparative analysis of the results obtained by molecular Ž . and immunological techniques immunoblotting and immunopurification pointed to the presence of several SCO-spondin related proteins Ž . in the SCO encoded by the same gene. The presence in the cerebral hemispheres CH of a 54-kDa glycoprotein with a common epitopeis discussed as a putative cleaved SCO-spondin product carried by the cerebrospinal fluid, that may act on neuronal development. q 1998Elsevier Science B.V. Keywords:  Central nervous system; Subcommissural organ; Reissner’s fiber; SCO-spondin expression; Neuronal development 1. Introduction During early embryogenesis, the secretory ependymaplays a crucial role in the development of the central Ž .  w x nervous system CNS 32 . The most documented secre-tory ependymal differentiation is that of the floor plate,contributing to spinal cord morphogenesis, but the mostobvious ependymal secretion is found in the brain at thelevel of the Sylvian aqueduct, from the so-called subcom- Ž . missural organ SCO .The SCO is a circumventricular organ located caudal tothe pineal organ in the roof of the third ventricle, at theentrance of the Sylvian aqueduct. This particular area isinvolved in the secretion of specific glycoproteins w x 17,18,30,34,35 basally towards the leptomeningeal spacesand apically in the ventricular cavity. When released intothe ventricular cavity, this secretory product aggregates Ž . and forms Reissner’s fiber RF , a thread-like structurerunning along the central canal of the spinal cord. ) Corresponding author. Fax:  q 33-4-73-27-61-32. RF is observed in almost all vertebrates, except in w x humans and the anthropoid apes 28,30 , and in procor- w x dates 29 . In addition, during ontogenesis, the SCO secre-tory product is released early, and RF appears in thecentral canal before the midpoint of embryonic life w x 4,11,22,33 .The exact significance of the SCO secretory activity has Ž . remained a matter of speculation. However, given: i theearly ontogenetic and phylogenetic appearance of the Ž . SCO r RF complex; ii the likely implication of RF in w x w x axial deformities 31 and normal tail regeneration 6 in Ž . several vertebrate species; and iii the in vitro effects of  w x RF on neuronal differentiation 23–25 , a developmentalbiological activity of the SCO r RF complex was postulated w x 15 .Searching for homologies with proteins involved indevelopmental processes is an important step toward as-cribing a putative function to the SCO r RF complex. Re-cently, we characterized cDNA clones related to a secretedcomponent of the SCO r RF complex, which we calledSCO-spondin, and we analysed the corresponding amino 0169-328X 98 19.00 q  1998 Elsevier Science B.V. All ri hts reserved.  ( ) I. Cre Õ eaux et al. r  Molecular Brain Research 55 1998 45–53 46 w x  Ž acid sequence 5,19 . Strong peptidic homologies presence Ž . . of thrombospondin TSP type 1 repeats were found withdevelopmental molecules expressed in the CNS and in-volved in cell adhesivity and directional outgrowth of  w x w x axonal processes such as TSP 1 and 2 27 , F-spondin 12 w x and semaphorins F and G 2 . However, SCO-spondinshows a specific mosaic structure displaying a variety of intermingled conserved domains. Both molecular data andexperimental tests on neuronal cells in vitro argue for adevelopmental activity of SCO-spondin. In addition, the w x molecular homology found between F-spondin 12 se- w x creted by the floor plate and SCO-spondin 5 secreted bythe SCO also suggests that the two types of structure maydisplay an analogous function.To study this new gene, we used the cDNAs previously w x identified 5,19 corresponding to part of the SCO-spondin Ž . encoding sequence as probes to: i analyse the sites and Ž . specificity of expression by in situ hybridization ISH and Ž . by Northern blot; ii determine by Southern blot whether Ž . only one gene encodes SCO-spondin; and iii to compareresults obtained using molecular and immunochemicaltechniques.The results point to a complex expression pattern of theSCO-spondin gene in the bovine SCO. 2. Materials and methods 2.1. Molecular probes A cDNA library was constructed in the vector  l GT11after extraction of mRNA from bovine embryonic SCOs.By subsequent screening of this library we isolated clones w x w x l RF101 19 and  l RF103 5 related to the SCO-spondinencoding gene. These clones were used as probes inNorthern blot, Southern blot and ISH experiments.A cDNA G3PDH probe served as control in the North-ern blot studies. 2.2. In situ hybridization Ž . Adult and embryonic bovine SCOs 40 cm in lengthwere dissected out with the surrounding tissues and in- Ž . cluded in Tissue-Tek OCT Miles Lab. . Cryostat sections Ž . 10- m m thick were collected on gelatine coated slides andfixed for 10 min in 4% paraformaldehyde. The sectionsunderwent different pretreatments: Proteinase K digestion Ž . 2.5  m g r ml Tris–HCl pH 8 for 10 min at 37 8 C and Ž . acetylation in triethanolamine-acetic anhydride 1:250 for Ž . 30 min at room temperature RT .Sense and antisense single stranded  35 S probes weregenerated by in vitro transcription using 100  m Ci  35 S CTPand T3 or T7 RNA polymerases as prescribed by the Ž . supplier Stratagene . Probe length was reduced to about350 nucleotides by alkaline hydrolysis.RNA probes were purified by chromatography on Chro- Ž . maspin-100 Clontech and stored at  y 20 8 C in the pres-ence of 20 mM DTT. Ž A total of 30  m l hybridization buffer 50% formamide,1 =  SSC, 15 mM Na HPO4, 10% dextran sulfate, 1 = 2 Denhardt, 1%  N  -lauroylsarcosine, 0.25 mg r ml tRNA, 0.5 .  6 mg r ml salmon sperm DNA, 10 mM DTT containing 10cpm of RNA probe were applied on each section andhybridization was carried out overnight at 50 8 C in a moistchamber. The sections were washed with 1 = SSC for 1 h Ž . at RT and 0.1 = SSC at 37 8 C. RNase digestion 5  m g r mlwas then performed to eliminate free RNAs. Final washingwas 2 = SSC for 1 h at 55 8 C.After dehydration, the sections were covered with LM1 Ž . nuclear emulsion Amersham .After 8 days of exposure, they were developed, fixedand counterstained with toluidine blue. 2.3. Northern blot analysis Ž . Embryonic bovine SCOs, cerebral hemispheres CHand cerebellum were dissected under a dissecting micro- Ž . q scope and immediately frozen in liquid nitrogen. Poly AmRNAs were isolated using a QuickPrep Micro mRNA Ž . Purification kit Pharmacia . Ž . q Two micrograms of poly A mRNAs was fractionated w x on 1% agarose gels containing 6% formaldehyde 14 and Ž . blotted onto Nytran membranes Schleicher and Schuellovernight. The mRNAs were fixed by baking at 80 8 C for 2h.Filters were hybridized at 65 8 C in: 50% formamide, 125mM NaPO pH 6.8, 250 mM NaCl, 7% SDS, 1 mM 4 EDTA pH 8, 10% PEG 6000, 100  m g r ml sonicatedsalmon DNA and 50  m g r ml yeast tRNA for 16 to 24 h.RNA probes were added at 4.10 6 cpm r ml. Filters werewashed with 0.1 =  SSC, 0.1% SDS at 65 8 C and then70 8 C.A G3PDH cDNA probe was used as a positive control. Ž . After random primed labeling Oncor-Appligene , hy-bridization was performed overnight at 42 8 C in: 50%formamide, 0.75 M NaCl, 1% SDS, 5% dextran sulfate, 50 m g r ml heparin, 10  m g r ml polyA and 50  m g r ml soni-cated salmon sperm DNA. Filters were washed in 0.2 = SSC, 0.1% SDS for 20 min at 42 8 C and exposed at  y 80 8 Cfor autoradiography. 2.4. Southern blot analysis Total cellular DNA was extracted from bovine blood by w x the method of Bowtell 3 .A total of 10  m g of genomic DNA was digested to Ž completion with several restriction enzymes  Eco RI, .  Hin dIII,  Nco I,  Bam HI,  Pst  I,  Taq I,  Hin cII,  Rsa I andsubjected to electrophoresis on 0.8% agarose gel. DNAfragments were then transferred to a Hybond N q  nylon  ( ) I. Cre Õ eaux et al. r  Molecular Brain Research 55 1998 45–53  47 Ž . filter Amersham . Prehybridization was performed at 65 8 Cfor 1 h in: 40% formamide, 0.75 M NaCl, 5% dextransulfate, 50  m g r ml heparin, 1% SDS, 10  m g r ml polyAand 50  m g r ml salmon sperm DNA. Hybridization wascarried out overnight at 42 8 C in the same solution contain-ing 2.10 6 cpm r ml random primed cDNA probe. Washeswere performed at 42 8 C as follows: 2 = SSC, 20 min; 2 = SSC, 20 min; 0.2 = SSC, 0.1% SDS 20 min. Autoradio-graphic exposure was usually for 4 days using one intensi-fying screen at  y 80 8 C. 2.5. Immunoblotting An anti-RF antibody was raised in the rabbit usingbovine RF extracted from the central canal of the spinal w x cord as antigen 15 .The SCOs from bovine adults were dissected out from Ž . the adjacent posterior commissure PC . In parallel, theclassical ependyma and CH that served as control werealso dissected. These tissues were immediately frozen inliquid nitrogen. They were thawed in 3 vol of phosphatebuffered saline pH 7.4, supplemented with a mixture of  Ž protease inhibitors 1 mM EDTA, 1 mM EGTA, 10  m g r mlleupeptine, 10  m g r ml pepstatine, 10  m g r ml chymosta- . tine; Sigma . After sonication and centrifugation at Ž . 100,000 = g , 1 vol of soluble extracts 50  m g proteins Ž were diluted in 1 vol of sample buffer 62.5 mM Tris–HCl . pH 6.8; 3.75%  b  -SH; 10% glycerol and 1.5% SDS and Ž . loaded onto SDS-gradient gels 8–18% . Electrophoresis w x was carried out according to Laemmli 13 . Electrotransfer Ž . Western blot was performed on nitrocellulose sheets Ž .  w x Millipore HA 0.45  m m according to Towbin et al. 36 .The blots were treated with 0.1% Tween 20 in PBS for 1 hat 37 8 C and then with an anti-RF antibody diluted to1 r 400 for 2 h at 37 8 C. Immunoreactive bands were re- Ž vealed using the Vectastain ABC kit Vector Lab, . Ž Burlingame, CA . Protein standards high chain myosin:200 kDa; galactosidase: 116.25 kDa;  b  -phosphorylase:92.5 kDa; serum albumin: 66 kDa; ovalbumin: 45 kDa;carbonic anhydrase: 31 kDa; trypsin inhibitor: 21.5 kDa . and lysozyme: 14.4 kDa: Bio-Rad served to determine Ž . protein molecular weight MW . 2.6. Immunoaffinity chromatography Immunoaffinity chromatography was performed using a w x monoclonal antibody, C1B8A8 20,21 directed against theSCO r RF secretory product. C1B8A8 IgGs were purifiedfrom ascites fluid on protein A with a purification kit Ž . Pierce . IgGs were then immobilized by their carbo- Ž . hydrate residues on two separate columns Pierce eachcontaining 2 ml of gel with 1.2 mg of immobilized C1B8A8IgG.The SCOs from bovine embryos were dissected outfrom the adjacent PC and the CH were removed. Thetissues were immediately frozen in liquid nitrogen. Theywere thawed in 3 vol of phosphate buffered saline pH 7.4, Ž supplemented with a mixture of protease inhibitors 1 mMEDTA, 1 mM EGTA, 10  m g r ml leupeptine, 10  m g r ml . Ž . pepstatine, 10  m g r ml chymostatine Sigma . After soni-cation and centrifugation at 100,000 = g , soluble extracts Ž . 1 ml f 2 mg proteins were respectively loaded onto thecolumns containing 2 ml of immunoadsorbent and incu-bated for 2 h at room temperature. After extensive washingwith 1 M NaCl, the bound fraction was eluted with Pierceelution buffer.Samples of the soluble extracts, and the bound andunbound fractions underwent SDS polyacrylamide gel Ž .  w x 10% electrophoresis 13 , run at 30 mA r gel in a minigel Ž . electrophoresis system Touzart et Matignon, Paris . After Ž . electrotransfer on nitrocellulose Millipore, 0.45  m m ,glycoproteins were revealed using concanavaline A-per- Ž . oxidase 50  m g r ml and treated with the avidin–biotin– Fig. 1. The schematic representation of the SCO-spondin cDNA probes used for in situ hybridization, Northern blot and Southern blot studies. Boxesrepresent the sequences encoding amino acidic conserved domains.  l RF101 is located in a region without sequence conserved domains compared with l RF103.  ( ) I. Cre Õ eaux et al. r  Molecular Brain Research 55 1998 45–53 48 Ž . Ž . Fig. 2. The specific expression of the SCO-spondin in the SCO of a 5-month old 40 cm bovine embryo. A Brightfield image of a sagittal section after insitu hybridization with the  35 S-labeled  l RF103 antisense riboprobe. Strong labeling is present only in the SCO epithelium, showing the presence of  Ž . Ž . Ž . specific transcripts in the ependyma E and hypendyma H . A few cells arrow are also labeled in the PC. Toluidine blue counterstaining. Bar: 500  m m. Ž . Same results were obtained with the  l RF101 probe. B High magnification showing strong radioactive labeling in the cytoplasm of the secretory Ž . Ž . Ž .  35 ependymal cells arrow lining the ventricular cavity V and in the hypendymal cells arrow head .  l RF103 S antisense riboprobe. Toluidine blue Ž . counterstaining. Bar: 100  m m. Same results were obtained with  l RF101 probe. C Brightfield photomicrograph of a neighboring section after in situhybridization with the  35 S-labeled  l RF103 sense strand probe. No labeling can be seen in the different parts of the section. Nuclei are stained with Ž . Ž . toluidine blue. SCO: subcommissural organ with its two layers, ependyma E and hypendyma H . Same results were obtained with  l RF101 sense strandprobe.  ( ) I. Cre Õ eaux et al. r  Molecular Brain Research 55 1998 45–53  49Fig. 3. The Northern blot analysis of SCO-spondin mRNA in the embry-onic bovine brain. Two micrograms of polyA q  RNA isolated from SCOand control nervous tissues were separated by agarose formaldehyde gelelectrophoresis. After blotting on nylon sheet, the filter was hybridized 32 Ž . with a P-labeled  l RF103 antisense riboprobe upper lane . Hybridiza- Ž . tion with a G3PDH cDNA probe lower lane confirmed that comparableamounts of RNA were loaded in each lane. Sizes are indicated in kb. Ž . Same profile was observed with a  l RF101 antisense probe. 1 Cerebral Ž . Ž . hemispheres. 2 Subcommissural organ. 3 Cerebellum. Ž . peroxidase complex as prescribed by the supplier Vector .The peroxidase activity was revealed using DAB. 3. Results 3.1. Main characteristics of the SCO-spondin cDNAs Fig. 1 shows the respective positions and the main characteristics of   l RF101 and  l RF103 cDNAs used as probesin this study. l RF101 was isolated by immunoscreening of the bovineSCO cDNA library, using an antibody raised against bovine w x RF 19 . This clone contains a 420-pb insert showing nohomology with known sequences in the databases. l RF103 is an overlapping clone containing a 2.6-kbinsert. In the corresponding amino acidic sequence several Ž . TSP type 1 repeats and low density lipoprotein LDL Ž . receptor type A repeats were identified Fig. 1 . Other Ž features potent sites of   N  -glycosylation, potent cytokine . binding sites, EGF-like domains are detailed by Gobron et w x al. 5 .Using both inserts, the specificity of their expressionwas analysed by Northern blot and ISH experiments. 3.2. In situ hybridization Using both antisense  l RF101 and  l RF103 RNA probes,a strong radioactive signal was revealed in the SCO, in thesecretory ependymal cells lining the ventricular cavity, and Ž . in the underlying hypendymal cells Fig. 2A and B . Cellsarranged in rays in the PC also expressed correspondingtranscripts. In the nervous tissue of the PC, no labelingwas observed. This confirmed that the SCO-spondin tran-scripts are strongly and specifically expressed in the secre-tory ependymal cells of the SCO.No signal was detected in the SCO with the correspond- Ž . ing sense RNA probes used as negative controls Fig. 2C . 3.3. Northern analysis Ž . q Northern analysis of embryonic SCO poly A mRNAs Ž showed the presence of a major transcript of 14 kb Fig. . 3 . In addition, three minor transcripts could be detected at10 kb, 7 kb and 4.9 kb in the SCO polyA q  extracts. No Ž . q signal was observed in the lanes corresponding to poly AmRNAs extracted from the CH or cerebellum. The same Fig. 4. Bovine genomic Southern. Five micrograms of bovine DNA were Ž . Ž . Ž . Ž . Ž . digested with  Eco RI 1 ,  Hin dIII 2 ,  Nco I 3 ,  Bam HI 4 ,  Pst  I 5 , Ž . Ž . Ž . Taq I 6 ,  Hin cII 7 and  Rsa I 8 . After electrophoresis and transfer onnylon membrane, hybridization was carried out with the  32 P-dCTP-labeled l RF103 insert probe. Single restriction fragments are present in lanes 1and 2.
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