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Development of the Anchoring Structures of the Epithelium in Rabbit and Human Fetal Corneas

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Development of the Anchoring Structures of the Epithelium in Rabbit and Human Fetal Corneas Investigative Ophthalmology & Visual Science, Vol. 29, No. 5, May 1988 Copyright © Association for Research in Vision and Ophthalmology Development of the Anchoring Structures of the Epithelium in Rabbit and Human Fetal Corneas Ann S. Tisdale,* Sandra J. Spurr-Michaud,* Merlyn Rodrigues,f Joseph Hackerr,j- Jay Krachmer4 and llene K. Gipson* The sequence of development of the components of the corneal adhesion complex (hemidesmosomes, basal lamina and anchoring fibrils) was studied in rabbit and human fetal corneas using electron microscopy and histochemical localization of type VII (anchoring fibril) collagen. In the rabbit, basal lamina was present at 15 days gestation, followed by hemidesmosomes (HDs) and anchoring fibrils (AFs) at 20 days gestation. Type VII collagen was first localized at 20 days. At 25 days, HDs remained low compared to the adult value. During human corneal development, basal lamina was present at 8 weeks gestation. Through 12 weeks of gestation, no HDs or AFs were discernible nor was there any type VII localization. At 13-19 weeks, HDs and cross-banded AFs were seen, and localization of type VII collagen was first noted. A palisade of filaments extending perpendicularly from the basal lamina into the underlying stroma was discernible from 13 to 27 weeks. A distinct Bowman's layer was present at 19 weeks. By 27 weeks, HDs//xm membrane were > the adult value, and AF penetration into the underlying stroma was also > the adult value. Bowman's layer had not reached adult values by term. These data indicate that after basal lamina deposition, HDs and AFs develop synchronously in both species. In humans the palisade of filaments may be the precursor of Bowman's layer, and the AF network develops within Bowman's layer. Invest Ophthalmol Vis Sci 29:727-736, 1988 Adhesion of stratified epithelia, including corneal The basal lamina lies between basal cells of the epithelium, to the underlying stroma is accomplished epithelium and the anterior stroma with its AF net- by a structural complex, the known components of work. Known components of the corneal epithelial which include hemidesmosomes (HDs), basal lamina basal lamina include laminin and heparan sulfate and anchoring fibrils (AFs). HDs are the junctions proteoglycan." The AF is made up of type VII colla- between the basal cells of the epithelia and the under- gen, molecules of which have a globular and a helical lying basal lamina.1"5 Structurally, the HD consists of domain. Antibodies to the globular domain have an intracellular electron-dense plaque that abuts the been localized to the lamina densa and to anchoring basal cell plasmalemma. Bundles of intermediate fila- plaques,1213 which are patches of electron-dense ex- ments (keratin filaments) insert into this plaque. Ex- tracellular matrix located subjacent to the basal lam- tracellularly, opposite the plaque, fine filaments ina. Helical domains of the molecule associate later- (termed anchoring filaments) extend across the lam- ally to form the cross-banded fibril. The cross-banded ina lucida into the lamina densa. A narrow, electron- fibrils branch and anastomose between the basal lam- dense line courses through these filaments, parallel ina and the anchoring plaques to form the network. and adjacent to the basal cell membrane.6"9 The only Recent data support the hypothesis that HDs are di- known component of the HD is the bullous pemphi- rectly connected to the AF network through the basal goid antigen.10 lamina.91314 Thus, the components of the adhesion complex—the HD, the basal lamina, and the AF net- work—are all interconnected to provide a continu- From the *Eye Research Institute of Retina Foundation and ous linkage between epithelium and stroma. Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, the fNational Eye Institute, National Institutes of Whether there is any relationship between the ad- Health, Bethesda, Maryland, and the ^Department of Ophthalmol- hesion complex and Bowman's layer is not clear. ogy, University of Iowa, Iowa City, Iowa. Bowman's layer in the human comprises randomly Supported by National Institutes of Health grant EY-03306 oriented collagen fibrils of relatively small diame- (I KG). ter.1516 These fibrils form an acellular matrix approx- Submitted for publication: June 15, 1987; accepted October 16, 1987. imately 10 nm thick. By comparison, the AF network Reprint requests: Ann S. Tisdale, Eye Research Institute of Ret- in the anterior Bowman's layer is less than 2 /urn ina Foundation, 20 Staniford Street, Boston, MA 02114. thick. In avian species, Bowman's layer is thought to 727 Downloaded from iovs.arvojournals.org on 09/26/2021 728 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / Moy 1988 Vol. 29 Table 1. Development of adhesion complex (n = 5) corneas were fixed in either half-strength in rabbit corneal epithelium Karnovsky's fixative or 3.75% OsO4 in 0.2 M s-colli- dine buffer, pH 7.4 (to optimize AF visualization).13 No. of Presence of Type VI1 cell HDs/nm cross-banded antibody All tissues were processed routinely for TEM, sec- Stage* layers membrane AFs localization tioned, and photographed on a Philips 410 electron microscope (Lico, Inc., Bedford, MA). Days of gestation 15 2-3 0 - - 17 2-3 0 - - Hemidesmosome and Anchoring Fibril 20 3 0.84 ± 0.05 + + Morphometric Analysis 22 3-4 0.79 ± 0.08 + + 25 4-5 0.82 ± 0.07 + + A Zeiss Videoplan Image Analysis system (Rainin 5-7 Adult 2.48 ±0.10 + + Instruments, Woburn, MA) was used to count * n = 3 for each development stage. HDs/jum membrane on each of eight to ten micro- graphs/specimen as previously described.9 The depth of penetration of the five longest AFs into the stroma be a remnant of the primary stroma deposited by the 15 on each of eight to ten micrographs of the adult rabbit embryonic epithelia. There is no evidence of epithe- 13 and human fetal corneas was also measured. lial origin in other species. Comparing the components of the adhesion com- Immunohistochemical Localization of Type VII plex of the corneal epithelium during development in Collagen species without (rabbit) and with (human) Bowman's membrane may provide information about the as- Rabbit: Fetal and adult rabbit corneas were frozen sembly of the components and their relationship to for cryostat sectioning in OCT. Six-micrometer sec- tions were taken and processed for immunofluores- Bowman's layer. Thus we have correlated the time of 13 appearance of HDs and AFs at the electron micro- cence microscopy. The primary antibody, a mouse scopic level and compared these data with the appear- monoclonal to human type VII collagen, was gener- ously supplied by Dr. R. Burgeson (Shriners Hospital ance of Bowman's layer in human tissue. To verify 12 electron microscopic observations of AFs, we local- for Crippled Children, Portland, OR). The second- ized type VII collagen immunohistochemically. ary antibody (fluorescein isothiocyanate-conjugated goat anti-mouse IgG) was obtained from Cooper Bio- medical (Malvern, PA). Negative control tissue sec- Materials and Methods tions (primary antibody omitted) were run with every Corneas were excised from adult (~2.5 kg) New antibody-binding study. The sections were viewed Zealand white rabbits and fetuses at 15, 17, 20, 22 and photographed using a Zeiss photomicroscope III and 25 days gestation. The adult rabbits were sacri- equipped for epi-illumination. ficed with an intravenous overdose of sodium pento- Human: For human fetal and adult corneas, type barbital. All investigations involving animals con- VII collagen was localized using the avidin-biotin- formed to the ARVO Resolution on the Use of Ani- peroxidase (ABC) procedure on cryostat sections.17 mals in Research. Corneas from human fetal eyes (8, Frozen sections were fixed in acetone for 5 min, 9,11,13,15,19,27,36 and 40 weeks gestation, and 3 transferred to tris-buffered saline (0.05 M, pH 7.4), days and 9 months post-term) were obtained through and incubated with the primary antibody for 30 min. the University of Iowa Eye Bank and Gynecology The slides were then washed in tris-buffered saline Clinic. Adult human donor corneas were obtained and incubated with the secondary antibody, biotin- through the New England Eye Bank. conjugated goat anti-mouse IgG (Vector Labs, Bur- lingame, CA), for 30 min. After washing in tris-buf- Transmission Electron Microscopy (TEM) fered saline, ABC complex (Vector Labs) was applied Excised fetal rabbit corneas (n = 3 for each devel- for 45 min. The slides were washed and incubated in opmental stage) were fixed in half-strength Kar- diaminobenzidine for 5 min and mounted. novsky's fixative (2% paraformaldehyde, 2.5% glu- Results taraldehyde in 0.1 M cacodylate buffer, pH 7.4), Rabbit postfixed in 1% buffered OsO4, dehydrated in ace- tones, and embedded in epon-araldite. Human fetal The development of the adhesion complex in the corneas (n = 1 for each stage) were fixed in 2.5% rabbit corneal epithelium involves an increase from phosphate-buffered glutaraldehyde, pH 7.4, and two to three cell layers at 15 days of gestation to five postfixed in 1% OsO4, dehydrated in alcohols, and to seven cell layers in the adult (Table 1). At 15 and embedded in epon. Adult rabbit (n = 3) and human 17 days of gestation (Fig. 1), a basal lamina was Downloaded from iovs.arvojournals.org on 09/26/2021 No. 5 DEVELOPMENT OF ANCHORING STRUCTURES OF EPITHELIUM / Tisdole er ol. 729 Fifj. 1. (A) Electron micrograph or a cross-section of 15-day fetal rabbit cornea demonstrating two to three epithelial cell layers and the presence of a continuous basal lamina (arrow) (X3000). Developing adhesion zone of (B) 15-day and (C) 17-day fetal rabbit corneas (both X31.000K Basal lamina is seen, but no hemidesmosomes or anchoring fibrils.
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