Type V Collagen and Bowman's Membrane - The Journal of Biological ...

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Type V Collagen and Bowman's Membrane. QUANTITATION OF mRNA IN CORNEAL EPITHELIUM AND STROMA*. (Received for publication, April 21, 1994, ...
Vol. 269, No. 40, Issue of October 7, pp. 24959-24966, 1994 Printed in U.S.A.

THE JOURNAL OF BIOWGVXL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Type V Collagen and Bowman’s Membrane QUANTITATION OF mRNA IN CORNEAL EPITHELIUM AND STROMA* (Received for publication, April 21, 1994, and in revised form, June 13, 1994)

Marion K. Gordon$, Joseph W. Foley, David E. Birk, John M. Fitch, and Thomas F. Linsenmayer From the Department of Anatomy and Cellular Biology, %fts University Medical School, Boston, Massachusetts 02111

within the type V collagen molecules are rendered sterically Bowman’s membraneis an acellular matrix of the cornea whichlies between the epithelial basal lamina andunavailable to the antibody by type I collagen molecules(5-7). the corneal stroma.By immunoelectron microscopy, we We have observed that thisantigenic masking is characteristic I and V collagen are compo- of almost all type V collagen-containingmatrices (2). In each of have determined that types nents of the collagen fibrils in Bowman’s membrane of the tissues in which type V collagen is antigenically masked, the chick cornea. Although these same components are reactivity with the anti-type V antibodies can be dramatically found in the fibrilsof the stroma, the fibrils of Bowman’s increased by disrupting the structureof the heterotypic fibrils membranearesmaller in diameterand less uniform in a variety of ways, as analyzed by immunofluorescence and than those of the stroma. At early stagesof development, double label immunoelectron microscopy (2, 5, 6,8). In conI and I1 trast, fibril disruption is not required for Bowman’smembrane the corneal epithelium synthesizes the types collagenoftheprimarystroma. We thereforeasked to react with the type V collagen antibodies. This suggests a whether it might also be capable of synthesizing the fundamental difference in the amount and/or arrangement of type V collagen found in Bowman’s membrane at later the type V collagen within this matrix. stages of development. Our results, using competitive Type V collagen, as found in most connectivetissue matrices, polymerase chain reaction to quantitate mRNAfrom ) total fibrillar colconstitutes only a small percentage ( ~ 5 %of avian corneal cells, indicate that the amount of V collagen comlagen content. In corneal stroma, however, type collagen mRNApresent in epithelia, relative a2(I) tocollagen mRNA, is greater than thatin stromal fibroblasts. prises -20% of the fibrillar collagens (9-11). The greater content of type V collagen in stroma is likely to be related to the We postulate that this enables the epithelium to synthesize a higher ratio of type V to type I collagen than the small uniform diameter of its fibrils. We and others have prestroma and that this proportionally higher amount of viously foundusing in vitrofibrillogenesis studies that increasing the proportion of type V collagen in mixtures of type I and type V might account for the ultrastructural appearance of the fibrils inBowman’s membrane. V collagenproduces heterotypic fibrils with progressively smaller diameters (12, 13). In BowII-I~~’s membrane, the diameter of the fibrils is even During the development of the avian cornea, a number of smaller than those of the stroma. Such a difference could arise unique extracellular matrices are synthesized and assembled, if separate cell types produced different ratios of molecular two of which are Bowman’smembrane and the corneal stroma. constituents or assembled them in varying supramolecular orBowman’s membrane is a thin (-4 pm) acellular matrix lying ganizations. For the mature cornea, stromal fibroblasts synthesubjacent to the corneal epithelium and its basement mem- size the stromal type V collagen. ForBowman’s membrane, the is that the brane. The stroma comprises much of the remainder of the source of type V collagen is not known,but it possible cornea. Ultrastructurally, thesematrices are comprised largely distinctive characteristics of this matrix might result from its of striated collagen fibrils of small diameter. Those of the production by the corneal epithelium rather than the stromal fibroblasts. stroma are uniformly about 24 nm, and those ofBowman’s It has been demonstrated previously that thecorneal epithemembrane are thinner (18-22 nm)and more variable in lium of the early chick embryo can produce several collagen diameter ( 1). One striking difference between these matrices is their im- types that are normally thought to be synthesized by mesenmunoreactivity for type V collagen, a member of the fibrillar chymal cells(14, 15).This epithelium is largely responsible for collagen family. Whenmature corneas are reacted with mono- producing the primary corneal stroma, the firststromal matrix clonal antibodies directed against the triple helical domain of t o appear during corneal development. The primary stroma the type V collagen molecule, strong reactivity is observed in contains collagen types I, 11, and E,but it does not contain detectable type V collagen, whichappears only later incorneal Bowman’s membrane, but little if any is seen in the stroma proper (2-4). This “antigenic masking“ within the stroma re- development, after fibroblasts invade the stroma. We have asked whether, in later development, at a time sults from the coassembly of types I and V collagen molecules into heterotypic collagen fibrils, such that helical epitopes when a definitive Bowman’s membrane is being formed, the cells of the corneal epithelium are capable of synthesizing type * This work was supported by National Institutes of Health Grants V collagen. We determined this by quantitating the amount of EY09056 (to M. K. G.),EY05191 (toT.F. L.), and EY05129 (to D. E. B.). aUV) collagen mRNA in corneal epithelia and stromas at 14 The costs of publication of this article were defrayed in part by the days of embryonic development, using competitive reverse be hereby marked payment of page charges. This article must therefore “aduertisement” in accordancewith 18 U.S.C.Section 1734 solely to transcript-polymerase chain reaction (RT-PCR).’ We have observed that these cells do, indeed, synthesize al(V) collagen indicate this fact.

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The nucleotide sequence(s) reported in this paperhas been submitted to the GenBankmIEMBL DataBank with accession number(s) L31917. $ To whom correspondence should be addressed: Dept. of Anatomy and Cellular Biology, Tufts University Schoolof Medicine, 136 Harrison Ave., Boston, MA 02111.

The abbreviationsused are: RT-PCR, reverse transcript-polymerase chain reaction;glyceraldehyde-3-P dehydrogenase, glyceraldehyde-3phosphate dehydrogenase; bp, base pair(s); HPLC, high performance liquid chromatography.

24959

24960

Quantitation of Type V Collagen mRNA

J

FK:.1. Immunoelectron microscopy with gold-labeled anti-typeI and anti-typeV collagen antibodies on Bowman's membrane of 17-day embryonic chick cornea. Bowman's membrane lies subjacent to the basement membrane (indicated by B M ) , which is included in some sections for orientation. Bars designate 100nm. Panel A, single labeling with type I collagen monoclonal antibodies I-BA1 and I-DD4. Panel B, V collagen monoclonal antibody V-DH2. Panel C, six selected areas showing heterotypic fibrils that double are labeled with single labeling with type 5 nm of gold-tagged type V collagen antibody (small arrows). 10 nm of gold-tagged type I collagen antibodies (large arrows) and

mRNA, and, in fact, contain a higher proportion of type V to type I collagen mRNA than do the stromal cells.

tives containing potential al(V) collagen cDNAs. The other a2(I)collagen cDNA, representing the amino endof the polypeptide, was generated by RT-PCR from 17-day embryonicchick tendon RNA. The forward and reverse complement primerscorresponded to nucleotides 173-192 MATERIALS AND METHODS and 374-395, respectively, as numbered in (20). Theamplified product Immunoelectron Microscopy was ligated directly intothe pCRII vector (Invitrogen). ThiscDNA was Corneasweredissected from stagedembryosand processed for subsequently used to make a deletion construct for competitive RTpreembedding immunoelectronmicroscopy as described previously (6). PCR. The following monoclonal antibodies wereused: I-BAl and I-DD4, anti7)pe V Collagen cDNA-An al(V) collagen-specific cDNA was isotype I collagen antibodies (16, 17); and V-DH2, an anti-type V antibody lated a s follows. A AZap I1 cDNA library, made from 13-day embryonic (2). The antibodies were coupled directly to either10- or 5-nm colloidal chick cornea mRNA, was screened for collagen-encoding cDNAs by a gold particles and usedfor both singleand double labeling experiments low stringency hybridization using a fragment of an al(XI1)collagen also as described (6). cDNA a s a generic collagen probe, as described previously (22). Positively hybridizingplaques from this low stringencyscreening were cDNAs cloned. Q p e I collagen cDNAs were eliminated from the candidate pool The identity of all cDNAs listed below was verified by sequencing by high stringency hybridization to al(1) and a2(I) collagen-specific using either a Sequenase(U.S. Biochemical Corp.) or double stranded cDNA probes. One candidatecDNA, designated pMG277, contained an DNA cycle sequencing kit(Life Technologies, Inc.). 840-bp insert that wassubcloned into pBR322 and sequenced. Positive Glyceraldehyde-3-phosphate Dehydrogenase cDNA-A chicken glyc- identification of pMG277 as an al(V) collagen cDNA came from matcheraldehyde-3-phosphate dehydrogenasecDNA was obtainedby random ing amino acid resides 694-713 in the conceptual translation product primed RT-PCR of 17-day embryonic chick tendon mRNA using primers (underlined inFig. 3) with the sequence of an aUV)collagen cyanogen (TTCAT(C/T)GA(T/C)CTG(C/A)ACTACAT) and (CGCTCCTGGAAGAT- bromide peptide(isolationandsequencing described below). The NGTGAT), corresponding to nucleotide positions 159-178 and the repMG277 cDNA encodes amino acid residues 607-886of the 1,018 resiverse complement of nucleotides 270-289 (as in Ref. 18).The product dues in the major triple helical domain of the al(V)collagen chain. was ligated directly into thevector pCRII (Invitrogen). Isolation a n d Sequencing of a l w ) Collagen Peptide Fragments D p e I Collagen cDNAs-An al(1)collagen chain cDNA (in pBR322 vector) was isolatedfrom a tendon cDNA library by screening colonies Collagens were extracted from 17-day chick embryo tendons by treatwith the restriction endonuclease Sau961, as described previously for ment with pepsin and differential salt precipitation (for details seeRef. type XI1 collagen (19). It corresponds to thecarboxyl end of the triple 23). The 1.2 M NaClprecipitatewasdenaturedand applied to a helical domain (aminoacid residues 805-1014), the entire C-propeptide DEAE-52 column run with 25 mM "is-HCI, pH 8.6, 2 M urea and a (amino acid residues Cprol-Cpro272), and 28 nucleotides of the 3'gradient of0-0.1 M NaCI. The eluted al(V) chain was treated with untranslated region(for comparison seeRef. 20). This clone, a s well as cyanogen bromide (24), and fragments were separated by HPLC (model the a2U)collagen cDNA, pYN535 (21), were usedonly to subtractal(1) 334 Beckman chromatograph) usinga reverse phase C18 Vydac Tp 201 and a2(I) collagen cDNAs from the low stringency hybridization posi- column and a 10 mM heptafluorobutyric acid with a gradient of aceto-

Quantitation of Type V Collagen mRNA

2496 1

251-266). a2(I) collagen cDNA primers (as numbered inRef. 20) were: 5' primer, GCAGTAACTTCATACCTAGC(nucleotides 173-192 in exon 1); internal primers, CAGGGCGGAAGGGAGATGGTGAAGACGG (nucleotide residues222-232 linked to292-306) and its reverse complement; 3' primer,AAAGTCAGCCGCTTTAGATGG(reverse complement of exon 6 nucleotides 374-395). al(V) collagen cDNA primers (as numbered in Fig. 3) were: 5' primer, nucleotides427451; internal primers, nucleotide residues581-595 joined to 652-699 and the reversecomplement; 3' primer, reverse complement of nucleotides 739-756. After overlap extensionof the 5' and 3' deletion construct fragments, these competitor PCR products were directly ligated into the pCR I1 vector and used to transform INVaF'cells (Invitrogen). Transformants were analyzed for correct insert sizeby agarose gel electrophoresis and then sequenced. Correct deletion construct plasmids grown werein large scale and used to synthesize competitor RNA by in vitro transcription.

In Vitro Dunscription of Competitor RNA 2 pgof competitor plasmid was linearizedby XhoI digestion thenin vitro transcribed following standardprocedures(28).Theresulting transcripts consistedof the insert plus 120 bases of the vector. Competitor RNA was purified by phenol extraction (twice), ether extraction (twice), and ethanol precipitation (three times). The integrity and quantity of the competitor RNA were determined by gel electrophoretic comparison withknown quantities of rabbit globin a and p chain mRNAs (Life Technologies, Inc.). Concentrations were adjustedto lo4 amovpl; 10-fold and 3.33-fold serialdilutions were prepared.

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Tissue Collection and Isolation of RNA and DNA Corneas without surrounding scleral tissue were removed from 14.5>,,"" * - d day embryonic chicks and placed into a dish containing 50mM EDTA, ; 4 : : % .. plus or minus 0.32% dispase (Boehringer Mannheim) in phosphate," .a, buffered saline, pH7.4, a t 37 "C. After a5-min incubation, the epithelia ., , were gently removed from the underlying stroma. Separated corneal .a": t ,,> , . .,.vfrozen in liquid nitro.-. , ;* j. epithelial and stromal tissues were immediately gen. They were stored a t -80 "C until used. ," . * Several methods were employed to isolate RNA. Two of these also . ...*- 5. allowed for the simultaneous isolationof DNA, which was used to cal: ., .r - , culate cell numbers. Briefly, the isolation methods were: ( a )The Fastw : Track mRNA isolation kit following the manufacturer's protocol (In..-.-e ;%, vitrogen); ( b ) the proteinase K methodfor total nucleic acid extraction, following the procedure in Maniatiset al. (28) up to and including the after which the FIG.2. Immunoelectron microscopyof nondisrupted (paneZA) first phenol extraction and ethanol precipitation steps, in Total RNA was isolatedfrom one half by and partially disrupted (panel B ) fibrils from Bowman's mem- sample was then split half. brane of 17-day embryonicchick cornea with gold-labeled anti- treating it with RNase-free DNase, extracting with phenol, then with an RNase-free Chrotype V collagen antibodies (V-DH2). Temperature manipulation of ether,andfinallyapplyingthesolutionto lathyritic tissues was used to disrupt partially the fibril structure. A maspin-30 column (Clontech); DNA present in the other half of the significant increase in labeling with anti-type Vcollagen antibodies is sample was assayed directlyby fluorometry; (c) the Trizol reagent isoobserved when fibril structure is partially disrupted (panel B ) uersus lation method,following the protocol provided by Life Technologies, Inc. nondisrupted (panel A). This demonstrates that portion a of the type V for the quantitative isolation of both DNA and total RNA (29). collagen is antigenically maskedby the structureof the collagen fibrils. RNA was quantitatedby UV spectroscopy, and DNA was quantitated Bar = 100 nm. by fluorometry of Hoechst dye-reacted samples (30). The DNAcontent of chicken somatic cells is 2.47 pg/cell (31). nitrile. Molecular sizes of eluted peaks werechecked by sodium dodecyl Competitive RT-PCR sulfate-polyacrylamide gel electrophoresis (25). The al(V) cDNA encoded a 36-kDa CNBr fragment, therefore an HPLC fragment of this We employed the technique of Gilliland et al. (32) for competitive size was digested with trypsin. Tryptic peptides were separated by PCR. Experimental determinations consisted of a series of RT-PCR HPLC usinga n aqueous gradientof acetonitrile in 9mM trifluoroacetic reactions containing either 10-fold or 3.33-fold serial dilutions of comacid (26). Peptides were sequenced by automated Edman degradation petitor RNA (ranging between lo3 and amol) plus 0.5 pg of tissue using a n Applied Biosystems 470A peptide microsequenator. The de- RNA (either total or poly(A)+).For first strand cDNA synthesis the rivativeswereanalyzed on-line with an Applied Biosystems 120A RNAs (9 pl) were heated to 65 "C for 5 min, crash cooled on ice, and phenylthiohydantoin derivative analyzer. made 20 p1 in a mixture consisting of 50 mM "is-HCI, pH 8.3, 40 mM KCl, 6 mM MgCI,, 1mM dithiothreitol, 0.1 mg/ml bovine serum albumin, Construction of Competitors 1mM each dNTP, 20 PMrandom hexamers, and 100 units of Superscript For competitive PCR, each reaction mixture must contain a known RNase H- reverse transcriptase (Life Technologies, Inc.). The mixture amount of a competitor RNA that ( a )can be amplified RT-PCR in bythe was incubated a t 37 "C for 1 h; 2 units of ribonuclease H (Life Techsame primer pair as can the endogenous mRNA, and ( b )will produce a nologies, Inc.) were added, andthe 37 "C incubation was continued for PCR amplification,the smaller RT-PCR product, distinguishablefrom that of the endogenous a n additional20min.For first strand mRNA by gel electrophoresis. Such competitors were constructed for competitor/tissue cDNA reactions were heated to 95 "C for 5 min, and 1 glyceraldehyde-3-Pdehydrogenase,a2U)and al(V) collagen chain p1 from each was added to 20-1.11 a reaction mixture consistingof 10 mM cDNAs by deleting approximately 50 bp internal the to 5' and 3' primer Tris-HCI, pH 8.3, 50 mM KCI, 1.5 mM MgCI,, 200 p~ each dNTP, 1 p~ pair used for the PCR. The PCR-based "overlap extension" strategyfor each 5'- and 3"specific primers (listed under"Construction of Competiconstructing such cDNAs has been described by Ho et al. (27). tors"), and 2.5 units of Amplitaq (GeneAmp kit, Perkin Elmer). The Glyceraldehyde-3-P dehydrogenase cDNA primers (as numbered in cycling program consistedof one cycle of 94 "C 1min; 35 cycles of 94 "C Ref. 18) were: 5' primer, GATCTGCACTACATGGT (nucleotides 165for 15 s, 56 "C for 25 s, 72 "C for 1.5 min; and a finalcycle of 72 "C for 181);internal primers, ACATGTTCAAATAGAGAACGGGAAA (nucle- 10 min. otide residues 183-196 linked to 237-248) and its reverse complement; In each experiment, control reactions wereperformed in which either 3' primer, GAGCCCATTGATCACA (reverse complement of positions the competitor or tissue RNA was omitted. For data analysis,electro*. * .c

1

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1

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I.

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24962

Quantitation of Type V Collagen mRNA

NUC TH AA

1 607

GGG G

NUC TH AA

55 625

GGT CCT ACA GGC CCA CAA GGT CCG ATC GGT CAG CCA GGT CCA GCT GGT GCT GAT G P T G P Q G P I G Q P G P A G A D

NUC TH AA

109 643

GGA G

GAG CCG GGT CCC AGA GGG CAG CAA GGC CTC E P G P R G Q Q G L

NUC TH AA

163 661

GGA G

CCT CGA GGT P R G

NUC TH AA

217 679

GGA CCT G P

NUC TH AA

271 697

GGT CCA CCT GGC

NUC TH AA

325 715

GGG G

NUC TH AA

379 733

GGT GAA TCT GGT GAG CCT GGT CTG CCT GGA GAG GTC GGC CTG CCG GGT CCA AAA G E S G E P G L P G E U G L P G P K

NUC TH AA

433 751

GGT GAA AGA GGT GAA AAG GGT GAA GCA GGT CCC G E R G E K G E A G P

NUC TH AA

487 769

GGC CCT AAA GGT CCA CCA GGT G P K G P P G

NUC TH AA

541 787

GGT TTC CCT GGT GAC CCC G F P G D P

NUC TH A A

595 a05

GGT CCC G P

NUC TH AA

649 a23

GGT G

NUC

703 a41

GGT CCA GCT G P A

TH AA

G

GAG CCT GGC CAG AAG GGC AGC AAG GGT GAC AAA GGG GAG CAG GGG TCA CCA E P G Q K G S K G D K G E Q G S P

E

TTT GGTCAG AAA GGT GAT GAA F G Q K G D E

TTC CCT GGT CCC CCC GGC CCT GTG GGC TTG CAG GGC TTG CCT F P G P P G P U G L Q G L P

CCT GGTGAG AAG GGA GAA ACA GGA GAT GTT GGG CAA ATG GGT CCA CCA P G E K G E T G D U G Q M G E E

E

B

CCC AGA GGC CCT TCC

GGCCCA CCA GGA GCA GAC GGA CCC CAA

P

G

B

G

E

S

E

E

P

A

P

G

E

Q

CCT GCT GGG GGA ATA GGA AAT CCT GGT GCA GTA GGA GAG AAG GGT GAG CCT P A G G I G N P G A U G E K G E P

TCT GGTGCC GCT S G A A

GGT CCA CCT G P P

GAT GAT GGT CCC AAA GGC AGC CCT GGT CCA GTT. D D G P K G S P G P V

GGT CCG CCG GGA GAG CCT GGT CCA GCT GGT CAA GAT G P P G E P G P A G Q D

CCT GGT GAC AAA GGT P G D K G

GAC GAC GGC GAA CCT GGT CAG ACA GGG TCC CCA D D G E P G Q T G S P

CCC ACG GGA GAG CCA GGC CCC TCT GGA CCC CCA GGA AAA AGG GGC CCT CCT P T G E P G P S G P P G K R G P P GGT CCT GAA GGA AGG CAA GGA GAG AAA GGA GCC AAG GGT GAA GCT G P E G R Q G E K G A K G E A

3. Sequence of the partial a 1 0 collagen cDNA and its predicted translation product. The peptide sequence that verified the FIG. identity of the cDNA is underlined. This sequence represents triple helical amino acidresidues 607-886 (where the firstamino acid residue of the major triple helix is denoted as residue 1): phoresis through 3% Metaphor agarose gels (FMC),prepared in TBE, containing 0.5 pg/mlethidium bromide was performed on 10 plaliquots of each PCR reaction. Gels were photographed with positivehegative film (Polaroid type 55); negatives were scanned using a Millipore Bioimage system, and band intensities were quantitated using the “whole band analysis” program. Since the PCR products produced from the tissue-derived RNA and the competitor RNA were of different sizes, a correction factor was applied to compensate for molar differences in the binding of ethidium bromide. For glyceraldehyde-3-Pdehydrogenase cDNA, the competitor product is 60 bp, the endogenous product is 101 bp, therefore, the correction factor is 0.594; for a(I) collagen cDNA, the competitor product is 168 bp, the endogenous productis 233 bp, therefore, the correction factor is 0.721; and for al(V) cDNA, the competitor product is 273bp. the endogenous product is 326bp, therefore the correction factor is 0.83.

freshly isolated from equal numbers of cells (determined by DNA content), we employed competitive RT-PCR to determine the amounts of glyceraldehyde-3-Pdehydrogenase mRNA in each celltype. The results from both the proteinase K and Trizol nucleic acid isolation methods indicated that the glyceraldehyde-3-Pdehydrogenase mRNA content is equivalent in both cornealepithelial and stromal cells (about 2.45 x amol, or 1,500 copiesof mRNA, per cell). Thereafter, 0.5 pgof tissue RNAwas used in each cDNAreaction mix. After performing each RT-PCR using glyceraldehyde-3-P dehydrogenase, a2(I) and al(V) collagen primers, the quantities of a2(I) and al(V) collagen mRNAs were calculated and normalized by expressing each as amol of specific collagen mRNA/amol of glyceraldehyde-3-P dehydrogenase mRNA.

RESULTS Inmunoelectron Microscopy-To localize and assess the To compare the values obtained from the different tissues, it was structural arrangement of type V collagen in Bowman’s memnecessary to normalize the data to somecommon parameter that is brane, a series of immunolocalization experiments was perconstant for both.We determined that expressing the data as“per pg of total RNA” would be misleading, since in four different epithelia and formed. The immunoelectron microscopy data with colloidal fibroblast RNApreparations, the fibroblasts consistently had two to five gold-labeled antibodies (Fig. 1)demonstrate that both types I (panel A ) and V (panel B ) collagen are components of the times more total RNA/cell than the epithelia. We next assayed whether corneal epithelial and stromal cells contain striated collagen fibrils of Bowman’s membrane. Evaluation of the same quantity of mRNA for the “housekeeping” enzyme glyceralde- double labeling experiments was confounded somewhat by the hyde-3-P dehydrogenase. From other work in our laboratory, we knew fact that thefibrils of Bowman’s membrane are very small and that the glyceraldehyde-3-Pdehydrogenase mRNA content per cell is frequently not constant across different cell types; however, the quan- tightly packed, making it difficult to ascertain whether a gold tities in corneal epithelial cells and stromal fibroblasts at 14 days of particle is bound to a particular fibril or to its neighbor (Fig. E ) . We did observe, however,that at least some of the fibrils embryonic development had never been measured. Using total RNA,

Data Analysisand Normalization

Quantitation of Type V Collagen mRNA

Epithelium

24963

Stroma

*E

*C

FIG. 4. Competitive PCRproductsrun on 3fiagarose gels. The gel shown here contains RT-I'CR products ofonc representative experiment. The cDNA is synthesized from a mix of all three competitor KNAs and a fixed quantity of the tissue RNA, then aliquots are taken for PCR. Top panel, PCR using the al(V) collagen cDNA primers. Middle panel, PCR using the n2(I) collagen cDNA primers. Bottom panel, PCR using the glyceraldehyde-3-P dehydrogenase(G3PDH) cDNA primers. For all panels on the the left side, epithelial RNA was used as template in RT-PCR panels on the right side contain stromal RNA as the initial RT-PCR template. The RT-PCR product of the competitor (C) has been designed tobe slightly smaller than that for the endogenous product (E). On each gel, lanes 1-7 contain amplifiedDNA synthesized from a constant amount of epithelial or stromal RNA and decreasing amounts of competitor RNAs. The RNA competitor inthe initial reaction mix was as follows: lane I , lo7 amol each of a2(U and al(V)collagen competitor RNA and IO2amol of glyceraldehyde-3-P dehydrogenase competitor RNA; lane 2,6.7 x 10' amol each of a2(I)and al(V)collagen competitorsand 6.7 x 10'amol of glyceraldehyde-3-P dehydrogenase competitor; lane 3,3.3 x lo2amol each of n2(I) and nl(V)collagen competitor RNA and 3.3 x 10' amol of glyceraldehyde-3-P dehydrogenase competitorRNA; lane 4 , 10' amol each of a2(I) and al(V)collagen competitors and 10' amol of glyceraldehyde-3-P dehydrogenase competitor; lane 5,6.7 x 10'amol each of a2(I) and al(V)collagen competitors and 6.7 x 10"amol of glyceraldehyde-3-P dehydrogenase competitor; lane 6,3.3 x 10' amol each of a2(I)and al(V)collagen competitors and 3.3 x 10' amol of glyceraldehyde-3-P dehydrogenase competitor; lane 7, 10' amol each of a2(I) and al(V)collagen competitorsand 10"amol of glyceraldehyde-3-P dehydrogenase competitor. Theasterisk indicates lanes containing a molecular weight marker (Life Technology, Inc. 100-bp ladder). To the left of the marker lanes, the upper dot is aligned with the 300-bp band and the lower dot with the 200-bp band. are heterotypic structures containing collagen type I (large ar- collagen-specific primers, a second with the a2(I)collagen-sperows) and typeV (small arrows).Temperature manipulationof cific primers, and the third withthe glyceraldehyde-3-P dehylathyritic tissues was used to partially disrupt fibril structure. drogenase-specific primers (which was used as a normalization These experiments showed a significant increase in labeling standard). After PCR, the resultant productsof each reaction with anti-type V collagen antibodies when fibril structure was were separated by agarosegel electrophoresis (Fig.4), and the partially disrupted (Fig. 2). This demonstrated that although equivalencepoint of each was determined by densitometric some of the type V collagen present in fibrils is available with-scanning. The equivalence point (i.e. where the productof the out fibril disruption, a portion of the type V collagen is anti- competitor cDNA equals that of the endogenous cDNA) reflects genically masked by the structure of the collagen fibrils com- the pointat which the initial concentrationof added competitor prising Bowman's membrane. RNA and that of endogenous mRNA are equal. Quantitation of mRNAs for Collagen Qpes I and V-We exFig. 4 shows an ethidium bromide-stained agarose gel patamined whether the type V collagen of Bowman's membrane tern of such an experiment; Fig. 5 shows the data in graphic might be synthesized bythe epithelia, by determiningif these form after densitometric analysis. Since the endogenous tissuecells contain the mRNA for the d ( V ) collagen chain. For this, derived PCR products (upper bands in each lane) are larger we useda cDNA corresponding to the carboxyl end of the triple than those of the competitor, they have an increased molar helical domainof this polypeptide. The sequence of this cDNAs binding of ethidium bromide. To compensate for this, in plotis shown in Fig. 3. Underlined in the predicted translation ting the graphs, the densitometric value of the endogenous product is the sequence of a peptide, isolated from the al(V) tissue-derived bands must be decreased bya molar correction collagen chain, which confirms the identityof the cDNA. factor (see "Materials and Methods"). Each pair of bands in a Epithelial layers were separated from corneal stromas with lane from the agarosegel is expressed as one point ona graph, EDTA, and total RNA and DNA were isolated from each tissue. and, by computer analysis, a best fit line is derived. The point The proportion of mRNAsfor the a2(I) and aUV) collagen at which the ratioof competitor to endogenous PCR product is chains were determinedby competitive RT-PCR. Each quanti- 1 (the equivalence point) provides the value for the quantity of tativedeterminationconsisted of serialdilutions of known specific mRNA species in the sample. quantities of all three competitor RNAs (for a l ( V ) collagen, The amountof mRNAfor a2(I) and al(V) collagen chains and a2(I) collagen, and glyceraldehyde-3-P dehydrogenase) made for glyceraldehyde-3-P dehydrogenase, determined from Fig. 5, by i n vitro transcription, plus a constant amount (0.5 pg) of is summarized in TableIA. In the experiment shown inFig. 4, tissue RNA. This mixture was reverse transcribed intocDNA, the epithelialRNA had been isolated from about 5.5 times more then equal aliquots of each reaction were removed and used forcells than the stromal RNA sample,as determined by glycerthree separate polymerase chain reactions; one with the al(V)aldehyde-3-P dehydrogenase mRNA content. To allow the com-

24964

Quantitation of Qpe V Collagen mRNA

B

A V collagen mRNA

Epithelial type

Stromal type 5

3

c9

a

3 0

0

s

V collagen mRNA

4 -

3

D

C

2 -

k

0 C

c 0

0

100

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3 -

0 ' 0

200

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amol

FIG.5. Graphic analysis of data points calculated from the densitometric scanningof the gels shown in Fig. 3. They axis of the graph represents the ratioof competitor PCR product, C (in E (in amol); amol) to endogenous product, the z axis is the amount of competitor RNA (in amol) addedto the each starting RT-PCR reaction. Panel A , reverse transcription of epithelial RNA and mix of RNA competitors, followed by PCR with d ( V )collagen primers. Panel B , reverse transcription of stromal RNA and mix of RNA competitors, followed by PCR with aUV) collagen primers. Panel C , reverse transcription of epithelial RNA and mix of RNA competitors; a2(U collagen primers used for PCR. Panel D , reverse transcription of stromal RNA and mixof RNAcompetitors; a2(I) collagen primers used for PCR. Panel E , reversetranscription of epithelial RNA and mix of RNA competitors;glyceraldehyde-3-Pdehydrogenase (G3PDH)primers used for PCR. Panel F , reverse transcriptionof stromal RNA and mix of RNA competitors, followed by PCR with glyceraldehyde-3-Pdehydrogenase primers.

300

400

arnol

D

C Epithelial type I collagen mRNA

Stromal type I collagen mRNA

3

I

0

100

0

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0

200

400

800

1000

arnol

amal

E

F Epithelial G3PDH mRNA

Stromal G3PDH mRNA

3r

3

1

OI

3 0

s

800

2

0"

m

c

0

10

20

30

40

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parison of absolute amounts of the different collagen mRNAs in the epithelial and stromalsamples the data are normalized per amol glyceraldehyde-3-P dehydrogenase mRNA (Table IB). As can be seen in thisexperiment, each fibroblast contained about

50

80

70

0

5

10

15

20

25

arnol

five times more a2(I) collagen mRNA than an epithelial cell, but only twice as much d ( V ) collagen mRNA. Thus, although the average stromal cell contains more mRNA for the fibrillar collagens, the average epithelial cell is significantly enriched in

Quantitation of Type V Collagen mRNA TABLE I Quantitation of mRNAs in epithelial and stromal cells A. Calculated from data in Fig. 4 Epithelia

Stromal fibroblasts amol

32 112 145

G3PDH” mRNA a2(I) collagen mRNA al(V) collagen mRNA

5.8 90 52

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TABLE I1 Ratio of alW) to &(I) collagen mRNA Values are means of six epithelial and seven stromal experiments t S.D. Epithelia

1.8 f 0.7

Stromal

0.7 f 0.1

Bowman’s are heterotypic structures. However, the significant increase in anti-type V antibody-labeled fibrils after partial Epithelia Stromal fibroblasts fibril disruption indicates that type V collagen also exists in an amol antigenetically masked form. The percentage of anti-type V 3.5 15.5 a2(I) collagen mRNA per labeled fibrils also suggests thatmost if not all fibrils of BOWG3PDH mRNA man’s membrane areheterotypic, containing bothtypes I and V 4.5 9.0 al(V) collagen mRNA collagen. per G3PDH mRNA Despite the possibility that notall of the fibrils of Bowman’s C. Mean RNA contents for corneal epithelial and stromal cellsb membrane areheterotypic, the presence of exposed epitopes for EDithelia Stromal fibroblasts type V collagen throughout Bowman’s membrane suggests that ( a )they may contain a proportionally higher content of type V amol collagen which allows some type V collagen molecules to rea2U) collaeen mRNA Der 2.6 t 0.6 20.1 f 6.2 G3PDHmRNA main on the surface, and ( b ) the collagens comprising these al(V) collagen mRNA 4.5 t 13.3 1.0 f 2.9 fibrils may arise from a different tissue source than those of per G3PDH mRNA stromal fibrils. G3PDH, glyceraldehyde-3-phosphatedehydrogenase. We therefore asked whether the fibrillar collagens of Bow* Values are means 2 S.D.;n = 6 for epithelia; n = 7 for stroma. man’s membrane mightbe synthesized by the overlying corneal epithelium rather than the underlying stromal fibroblasts and that for type V collagen. whether the collagens synthesized by the epithelium are enWhen the averaged data werecompared from six sets of riched in theproportion of type V collagen produced. To deterepithelial andseven sets of stromal experiments,performed on mine whether the corneal epithelium has the potential for such four matched sets of epithelial and stromal RNAs, this trend synthesis we measured the quantities of mRNAs for collagen was confirmed (Table IC). For the averaged data, the a2(I) types I and V in separatedcorneal epithelia and stromas taken collagen mRNA is about eight times greater for stromal cells as at a developmental stage when Bowman’s membrane is being comparedwith epithelial cells, whereasthe al(V) collagen produced (14-day embryos). mRNA is only about three times greater. Consistent with our hypotheses on the origin and structure of Bowman’s membrane, the data presented here demonstrate DISCUSSION that epithelialcells contain mRNA for al(V)collagen and thus By immunoelectron microscopy we have demonstrated that are probably capable of synthesizing typeV collagen. They also, types I and V collagen are components of the striated fibrils of as expected, contain mRNA for the a2(I) collagen chain. The efficiency for Bowman’s membrane. Unlike those of the corneal stroma data also show, assumingequaltranslational proper, however, some of the type V collagen molecules in the these mRNAs, that a corneal epithelial cell is capable of synfibrils of Bowman’s membrane bind antibody in theabsence of thesizing more al(V)polypeptide chains thana2(I) polypeptide chains. Therefore epithelial cells are likely to synthesize type V any unmasking procedure t o disrupt fibrillar structure (see also Ref. 4). The role of type V collagen on the surface of the collagen. Whereas stromal cells contain about two al(V)collafibrils of Bowman’s membrane is unknown. However, it has gen mRNAs for every three a2(I) collagen mRNAs, epithelial been reported that “exposed type V collagen also is found as a cells contain approximately two al(V)collagen mRNAs for evcomponent of the very thin fibrils (-12 nm) found in the sub- ery one a2(I) collagen mRNA (Table 11). To assemble the corresponding types of trimeric collagen epithelial region of the amnion (13,33). Therefore, a possible function of specialized epithelial sites, such Bowman’s as mem- molecules with the proper chain composition, each type V colbrane, may be t o provide a form of type V collagen which assists lagen needs two al(V) chains, whereas each type I collagen in anchoring basement membrane components to the underly- molecule requires only one a2(I) chain. Therefore,to determine the theoretical ratio of types I and V collagen proteins which ing stroma. Since the type V collagen labeling is clearly over fibrils, the can be synthesized, the amountsof al(V)collagen mRNA must availability of type V collagen in this matrix is not due to its be divided by 2. Assuming equal translational efficiency, then assembly into some nonfibrillar form. Both type I and V colla- stromal fibroblasts produce type I and type V collagen molfor antibody binding ecules in a 3:l ratio (ie. 25% is type V collagen). This fits well gen of Bowman’s membrane are available withoutthe need for fibril disruption. Since in heterotypic with observations that corneal stromas (9, lo), as well as culfibrils of the stroma type V collagen is masked by its interaction tures of avian cornealfibroblasts (ll), synthesizeapproxiwith typeI, the unmasked natureof type V collagen in hetero- mately 20% of their fibrillar collagens as typeV collagen.’ Epithelial cells, in contrast, contain about 1.8 times more typic fibrils in Bowman’s membrane may be due to a relative d(V) than a2(I) collagen mRNA (see Table 11). Correcting for paucity of type I collagen in such fibrils. In neither the single nor double label experiments were all chain composition, an epithelial cell has the potential t o synfibrils within a given field labeled with antibody. This is prob- thesize 53% of the fibrillar molecules as type I collagen and 47% ably because of the relatively tortuous arrangement of the as type V collagen. fibrils in Bowman’s membrane, which causes a certain proporThese data were invariant whether EDTA or EDTNdispase tion of the fibrils to dip in and out of the planeof the section and were used to separate the tissues.Also, despite the method of be unavailable for antibody binding. This does, however, preclude a definitive conclusion as to whether all fibrils within D. E. Birk, unpublished observations. B. Normalized data from Fig. 4

~

Quantitation of Q p e V Collagen mRNA

24966

RNA isolation employed, competitive RT-PCR consistently gave the same relative ratios of a2(I) andal(V) collagen mRNAs. In addition, any possible reverse transcriptase bias was eliminated by adding themix of competitor RNAs to the tissueRNA prior to cDNA synthesis. Thus,both the endogenous tissue and competitor RNAs were subjected t o exactly the same manipulations. The competitive RT-PCR method also allowed us toovercome obstacles encountered in other methods that result from the small amount of tissue in the cornea and the difficulty in obtaining large amounts of isolated epithelial and stromal layers. A typical experiment used epithelia and stromal layers from only two to three dozen chick embryos. The elevated amounts of type V collagen mRNA in both corneal tissues are consistent with the molecule being greatly enriched in this structure ascompared with other structures. That the epithelialcells have the potentialfor making proportionally more type V collagen than the stromalcells is consistent with the hypothesis that thefibrils of Bowman’s membrane are epithelialproducts enriched in thismolecule. It also eliminates any possibility that themRNAs detected are due tocontamination of the epithelial preparationsby a few errant stromal cells. In addition, this relativeenrichment of type V collagen mRNA in epithelial cells compared with those of the stroma isconsistent with a role for this molecule in regulating the extreme small diameter of the fibrils found in Bowman’s membrane. Acknowledgments-We thank Elida Rodriguez, Bernard Dublet, and Michel van der Rest for the chicken type V collagen peptide sequencing; Yoshifumi Ninomiya for the (r2(I) collagen cDNA, pYN535; as well as Joanne Babiarz and EmanuelZycband for providing excellent technical and photographic assistance. We also thank Jeffrey Marchant, who prepared the chick cornea cDNA library. REFERENCES 1. Hay, E. D., and Revel, J. P. (1969) in Monographs in Developmental Biology (Wolsky,A,, and Chen, P. S., eds) pp. 1-144, Karger, Base1 2. Linsenmayer, T.F., Fitch, J. M., Schmid, T. M., Zak,N. B., Gibney, E., Sanderson, R. D., and Mayne, R. (1983) J. Cell Bid. 96,124-132 3. Linsenmayer, T. F., Fitch, J. M., and Mayne, R. (1984) Invest. Ophthalrnol. & Visual Sei. 2 6 , 4 1 4 7 4. Birk, D. E., Fitch, J. M., and Linsenmayer, T.F. (1986) Znuest. Ophthalmol. &

Visual Sei. 27, 1470-1477 5. Fitch, J. M., Birk, D. E., Mentzer, A,, Hasty, K A,, Mainardi, C., and Linsenmayer, T.F. (1988) Znuest. Ophthalrnol. & Visual Sci. 29, 1125-1136 6. Birk, D. E.,Fitch, J. M., Babiarz, J. I?, and Linsenmayer, T.F. (1988) J. Cell B i d . 106,999-1008 7. Linsenmayer, T. F., Gibney, E., Igoe, F., Gordon, M. K, Fitch, J. M., Fessler, L. I., and Birk, D. E.(1993) J. Cell Biol. 121, 1181-1189 8. Fitch, J. M., Gross, J., Mayne, R., Johnson Wint, B., and Linsenmayer, T.F. (1984) Proc. Natl. Acad. Sci. U. S. A . 81, 2791-2795 9. Tseng, S. C. G., Smuckler, D., and Stem, R. (1982)J. Biol. Chem. 267,26272633 10. von der Mark, K., and Ocalan, M. (1982) Collagen Relat. Res. 2, 541-555 11. McLaughlin, J. S., Linsenmayer, T. E , and Birk, D. E. (1989) J. Cell Sci. 94, 371379 12. Birk, D. E., Fitch, J. M., Babiarz, J. P., Doane, K. J., and Linsenmayer, T. F. (1990) J . Cell Sci. 96,649-657 13. Adachi, E., and Hayashi, T. (1986) Connect. Tissue Res. 14,257-266 14. Linsenmayer, T. F., Smith, G. N., Jr., and Hay, E. D. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 39-43 15. Linsenmayer, T. F., Gibney, E., Gordon, M. K , Marchant, J. K , Hayashi, M., and Fitch, J. M. (1990) Inuest. Ophthalrnol. & Visual Sci. 31, 1271-1276 16. Linsenmayer, T.F., Hendrix, M. J., and Little, C. D. (1979) Proc. Natl. Acad. Sci. U. S. A . 76,3703-3707 17. Swasdison, S., Mayne, P.M., Wright, D. W., Accavitti, M. A,, Fitch, J. M., Linsenmayer, T. F., and Mayne, R. (1992)Matrix 12,5&65 18. Panabieres, F., Piechaczyk, M., Rainer, B., Dani, C., Fort, P., Riaad, S., Marty, L., Imbach, J. L., Jeanteur, P., and Blanchard, J. M. (1984) Biochern. Biophys. Res. Comrnun. 118,767-773 19. Gordon, M. K., Gerecke, D. R., and Olsen, B. R. (1987)Proc. Natl. Acad. Sci. U. S. A. 84,6040-6044 20. Boedtker, H., Finer, M., and Aho, S. (1985) Ann. N. Y Acad. Sci. 460,85116 21. Ninomiya, Y.,and Olsen, B. R. (1984)P m . Natl. Acad. Sci. U. S. A. 81,30143018 22. Marchant, J. K , Linsenmayer, T. F., and Gordon, M. K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1560-1564 23. Dublet, B., and Van der Rest, M. (1991) J. Biol. Chern. 266, 6853-6858 24. Sugrue, S. P., Gordon, M. K., Seyer,J., Dublet, B., Van der Rest, M., and Olsen, B. R. (1989) J. Cell Bid. 109,93%945 25. Laemmli, U. K. (1970) Nature 227,680-685 26. Van der Rest, M., and Fietzek, P. P. (1982) EUI:J. Biochern. 126,491-496 27. Ho, S. N., Hunt, H. D., Horton, R. M., F’ullen, J. K, and Pease, L. R. (1989) Gene (Amst.) 77,51-59 28. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1989) Molecular Cloning.’ A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, Nv _. 29. Chomczynski, P. (1993) BioTechniques 16, 532-534, 536-537 30. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A,, and Struhl, K. (1993) Current Protocols in Molecular Biology, John Wiley & Sons, New York 31. Mirsky, A. E., and Ris, H. (1949) Nature 163,66&667 32. Gilliland, G., Pemn, S., Blanchard, K., and Bunn, H. F. (1990) Proc. Natl. Acad. Sci. U.S. A . 87,2725-2729 33. Modesti,A,, Kalebic, T., Scarpa, S., Togo, S., Grotendorst, G., Liotta, L. A., and Triche, J. (1984) Eur. J. Cell Bid. 36, 24G255

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