Developmental analysis of SV2 in the embryonic chicken corneal epithelium.

Intraepithelial corneal nerves (ICNs) help protect the cornea as part of the blink reflex and by modulating tear production. ICNs are also thought to regulate the health and homeostasis of the cornea through the release of trophic factors. Disruption to these nerves can lead to vision loss. Despite their importance little is known about how corneal nerves function and even less is known about how the cornea is initially innervated during its embryonic development. Here, we investigated the innervation of the embryonic chicken cornea. Western blot and immunohistochemistry were used to characterize the localization of the synaptic vesicle marker SV2, a molecule thought to be involved in the release of trophic factors from sensory nerves. The data show that both SV2 and synaptotagmin co-localize to ICNs. Nerves in the conjunctiva also contained SV2 and synaptotagmin, but these were localized to below the basal layers of the conjunctiva epithelium. SV2 isolated from corneal epithelium migrates in western blot at a heavier weight than SV2 isolated from brain, which suggests a role in vesicle targeting, as the deglycosylating enzyme PnGase does not affect corneal SV2.

Keratin-14-Positive Precursor Cells Spawn a Population of Migratory Corneal Epithelia that Maintain Tissue Mass throughout Life.

The dynamics of epithelial stem cells (SCs) that contribute to the formation and maintenance of the cornea are poorly understood. Here, we used K14CreERT2-Confetti (Confetti) mice, sophisticated imaging, and computational modeling to trace the origins and fate of these cells during embryogenesis and adult life. We show that keratin-14 (K14+)-expressing progenitors are defined and widely distributed across the E16.5 cornea, after which they undergo cycles of proliferation and dispersal prior to eyelid opening. K14+ clonal patches disappear from the central cornea and are replaced by limbal-derived K14+ streaks, a finding that aligned with bromodeoxyuridine label-retaining studies. We also elucidated the mechanism by which SC clones are lost during life and propose this is due to population asymmetry and neutral drift. Finally, we established that the occurrence of an equatorial migratory mid-line is a consequence of apoptosis in a narrow nasal-temporal region, the site where eyelids meet during blinking.

The Corneal Epithelial Barrier and Its Developmental Role in Isolating Corneal Epithelial and Conjunctival Cells From One Another.

Purpose: During development, the corneal epithelium (CE) and the conjunctiva are derived from the surface ectoderm. Here we have examined how, during development, the cells of these two issues become isolated from each other. Methods: Epithelia from the anterior eyes of chicken embryos were labeled with the fluorescent, lipophilic dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). DiI was placed on the epithelial surface of the developing anterior eye and its diffusion was monitored by fluorescence microscopy. Concomitant morphologic changes in the surface cells of these epithelial were examined by scanning electron microscopy. Immunofluorescence was used to analyze the expression of cytokeratin K3, ZO-1, N-cadherin and Connexin-43 and the function of gap junctions was analyzed using a cut-loading with the fluorescent dye rhodamine-dextran. Results: Prior to embryonic day 8 (E8), DiI placed on the surface of the CE spreads throughout all the epithelial cells of the anterior eye. When older eyes were similarly labeled, dye diffusion was restricted to the CE. Similarly, diffusion of DiI placed on the conjunctival surface after E8 was restricted to the conjunctiva. Scanning electron microscopy showed that developmentally (1) physical separations progressively form between the cells of the CE and those of the conjunctiva, and (2) by E8 these separations form a ring that completely encompasses the cornea. The functional restriction of gap junctions between these tissues did not occur until E14. Conclusions: During ocular development, a barrier to the diffusion of DiI forms between the contiguous CE and conjunctiva prior to the differential expression of gap junctions within these tissues.

Keratin 14 Expression in Epithelial Progenitor Cells of the Developing Human Cornea.

A healthy and transparent cornea is essential for exquisite vision. During adulthood, its epithelium is constantly replenished through the activity of its stem cells (SCs). Precisely when these cells develop and their distribution across the ocular surface remain incompletely characterized in man. We postulated that the human fetal cornea harbors SCs that can be identified with keratin (K) 14 and αv-integrin, two markers we and others previously used to identify their adult counterparts. Immunofluorescence, cell culture, quantitative real-time polymerase chain reaction (qRT-PCR), and colony-forming assays were performed on fetal and adult biomaterial to locate progenitors and establish their phenotypic and functional properties. K14 was used to map the spatiotemporal distribution of precursor cell activity across the developing cornea, divulging a dynamic pattern of vertical and horizontal consolidated expression with increasing gestational age. K14 was coexpressed with αv-integrin in fetal and adult corneas and cultured corneolimbal epithelium, and colony-forming efficiency (an indicator of SC activity) was similar in cells from both sources. Finally, fetal cells were adherent, grew well, and maintained a K14 phenotype on contact lenses, a substrate we previously used to deliver cells to patients with blinding corneal disease. This study provides valuable insights into the development of the cornea, including the formation of the SC repository, the distribution of these cells across the ocular surface, and a preliminary attempt at harnessing, phenotyping, and functionally characterizing these cells. Future studies will focus on isolating fetal SCs to determine their utility as an alternative cell therapy for patients suffering from corneal blindness.

Wnt/ß-catenin signaling modulates corneal epithelium stratification via inhibition of Bmp4 during mouse development.

The development of organs with an epithelial parenchyma relies on reciprocal mesenchymal-epithelial communication. Mouse corneal epithelium stratification is the consequence of a coordinated developmental process based on mesenchymal-epithelial interactions. The molecular mechanism underlying these interactions remains unclear. The Wnt/ß-catenin signaling pathway is involved in fundamental aspects of development through the regulation of various growth factors. Here, we show that conditional ablation of either ß-catenin (Ctnnb1(cKO)) or co-receptors Lrp5/6 (Lrp5/6(cKO)) in corneal stromal cells results in precocious stratification of the corneal epithelium. By contrast, ectopic expression of a murine Ctnnb1 gain-of-function mutant (Ctnnb1(cGOF)) retards corneal epithelium stratification. We also discovered that Bmp4 is upregulated in the absence of ß-catenin in keratocytes, which further triggers ERK1/2 (Mapk3/1) and Smad1/5 phosphorylation and enhances transcription factor p63 (Trp63) expression in mouse corneal basal epithelial cells and in a human corneal epithelial cell line (HTCE). Interestingly, mouse neonates given a subconjunctival BMP4 injection displayed a phenotype resembling that of Ctnnb1(cKO). Conditional ablation of Bmp4 eradicates the phenotype produced in Ctnnb1(cKO) mice. Furthermore, ChIP and promoter-luciferase assays show that ß-catenin binds to and suppresses Bmp4 promoter activity. These data support the concept that cross-talk between the Wnt/ß-catenin/Bmp4 axis (in the stromal mesenchyme) and Bmp4/p63 signaling (in the epithelium) plays a pivotal role in epithelial stratification during corneal morphogenesis.

Corneal Development: Different Cells from a Common Progenitor.

Development of the vertebrate cornea is a multistep process that involves cellular interactions between various ectodermal-derived tissues. Bilateral interactions between the neural ectoderm-derived optic vesicles and the cranial ectoderm give rise to the presumptive corneal epithelium and other epithelia of the ocular surface. Interactions between the neural tube and the adjacent ectoderm give rise to the neural crest cells, a highly migratory and multipotent cell population. Neural crest cells migrate between the lens and presumptive corneal epithelium to form the corneal endothelium and the stromal keratocytes. The sensory nerves that abundantly innervate the corneal stroma and epithelium originate from the neural crest- and ectodermal placode-derived trigeminal ganglion. Concomitant with corneal innervation is the formation of the limbal vascular plexus and the establishment of corneal avascularity. This review summarizes historical and current research to provide an overview of the genesis of the cellular layers of the cornea, corneal innervation, and avascularity.

Rap1 GTPase is required for mouse lens epithelial maintenance and morphogenesis.

Rap1, a Ras-like small GTPase, plays a crucial role in cell-matrix adhesive interactions, cell-cell junction formation, cell polarity and migration. The role of Rap1 in vertebrate organ development and tissue architecture, however, remains elusive. We addressed this question in a mouse lens model system using a conditional gene targeting approach. While individual germline deficiency of either Rap1a or Rap1b did not cause overt defects in mouse lens, conditional double deficiency (Rap1 cKO) prior to lens placode formation led to an ocular phenotype including microphthalmia and lens opacification in embryonic mice. The embryonic Rap1 cKO mouse lens exhibited striking defects including loss of E-cadherin- and ZO-1-based cell-cell junctions, disruption of paxillin and ß1-integrin-based cell adhesive interactions along with abnormalities in cell shape and apical-basal polarity of epithelium. These epithelial changes were accompanied by increased levels of α-smooth muscle actin, vimentin and N-cadherin, and expression of transcriptional suppressors of E-cadherin (Snai1, Slug and Zeb2), and a mesenchymal metabolic protein (Dihydropyrimidine dehydrogenase). Additionally, while lens differentiation was not overtly affected, increased apoptosis and dysregulated cell cycle progression were noted in epithelium and fibers in Rap1 cKO mice. Collectively these observations uncover a requirement for Rap1 in maintenance of lens epithelial phenotype and morphogenesis.

Human eye development is characterized by coordinated expression of fibrillin isoforms.

PURPOSE: Mutations in human fibrillin-1 and -2, which are major constituents of tissue microfibrils, can affect multiple ocular components, including the ciliary zonule, lens, drainage apparatus, cornea, and retina. However, the expression pattern of the three human fibrillins and an integral microfibrillar component, MAGP1, during human eye development is not known. METHODS: We analyzed sections from human eyes at gestational weeks (GWs) 6, 8, and 11 and at 1 and 3 years of age with antibodies specific for each human fibrillin isoform or MAGP1, using immunofluorescence microscopy. RESULTS: During embryonic development, each fibrillin isoform was detected in vascular structures bridging the ciliary body and the developing lens, hyaloid vasculature, and retina. In addition, they were present in the developing corneal basement membranes and lens capsule. MAGP1 codistributed with the fibrillin isoforms. In contrast, the juvenile zonule was composed of fibrillin-1 microfibrils containing MAGP1, but fibrillin-2 was absent and fibrillin-3 was only sparsely detected. CONCLUSIONS: Fibrillin-1, -2, and, unique to humans, fibrillin-3 are found in various ocular structures during human embryonic eye development, whereas fibrillin-1 dominates the postnatal zonule. We speculate that vasculature spanning the ciliary body and lens, which elaborates fibrillin-2 and -3, may provide an initial scaffold for fibrillin assembly and zonule formation.

The vertebrate corneal epithelium: from early specification to constant renewal.

BACKGROUND: The cornea is an ectodermal/neural crest derivative formed through a cascade of molecular mechanisms to give rise to the specific optical features necessary for its refractory function. Moreover, during cornea formation and maturation, epithelial stem cells are sequestered to ensure a constant source for renewal in the adult. RESULTS: Recent progress in the molecular and stem cell biology of corneal morphogenesis and renewal shows that it can serves as a paradigm for epithelial /mesenchymal organ biology. This review will synthesize historical knowledge together with recent data to present a consistent overview of cornea specification, formation, maturation, and maintenance. CONCLUSIONS: This should be of interest not only to developmental biologists but also ophthalmologists, as several human vision problems are known to be rooted in defects in corneal development.

Mastermind-like transcriptional co-activator-mediated Notch signaling is indispensable for maintaining conjunctival epithelial identity.

Conjunctival goblet cells primarily synthesize mucins to lubricate the ocular surface, which is essential for normal vision. Notch signaling has been known to associate with goblet cell differentiation in intestinal and respiratory tracts, but its function in ocular surface has yet to be fully characterized. Herein, we demonstrate that conditional inhibition of canonical Notch signaling by expressing dominant negative mastermind-like 1 (dnMaml1) in ocular surface epithelia resulted in complete suppression of goblet cell differentiation during and subsequent to development. When compared with the ocular surface of wild-type mice (OS(Wt)), expression of dnMaml1 at the ocular surface (OS(dnMaml1)) caused conjunctival epithelial hyperplasia, aberrant desquamation, failure of Mucin 5ac (Muc5ac) synthesis, subconjunctival inflammation and epidermal metaplasia in cornea. In addition, conditional deletion of Notch1 from the ocular surface epithelia partially recapitulated OS(dnMaml1) phenotypes. We have demonstrated that N1-ICD (Notch1 intracellular domain) transactivated the mouse Krüppel-like factor 4 (Klf) promoter and that Klf4 directly bound to and significantly potentiated the Muc5ac promoter. By contrast, OS(dnMaml1) dampened Klf4 and Klf5 expression, and diminished Muc5ac synthesis. Collectively, these findings indicated that Maml-mediated Notch signaling plays a pivotal role in the initiation and maintenance of goblet cell differentiation for normal ocular surface morphogenesis and homeostasis through regulation of Klf4 and Klf5.

The corneal epithelium and lens develop independently from a common pool of precursors.

BACKGROUND: The corneal epithelium (CE) overlays a stroma, which is derived from neural crest cells, and appears to be committed during chick development, but appears still labile in adult rabbit. Its specification was hitherto regarded as resolved and dependent upon the lens, although without experimental support. Here, we challenged CE fate by changing its environment at different stages. RESULTS: Recombination with a dermis showed that CE commitment is linked to stroma formation, which results in Pax6 stabilization in both species. Surgical ablation shows that CE specification has already taken place when the lens placode invaginates, while removal of the early lens placode led to lens renewal. To block lens formation, bone morphogenetic protein (BMP) signaling, one of its last inducing factors, was inhibited by over-expression of Gremlin in the ocular ectoderm. This resulted in lens-less embryos which formed a corneal epithelium if they survived 2 weeks. CONCLUSION: The corneal epithelium and lens share a common pool of precursors. The adoption of the CE fate might be dependent on the loss of a lens placode favoring environment. The corneal fate is definitively stabilized by the migration of Gremlin-expressing neural crest cells in the lens peripheral ectoderm.

Wakayama symposium: challenges of future research in ocular surface cell biology.

During embryonic development, surface ectoderm differentiates to form corneal, conjunctival, and eyelid epidermal epithelia, and glandular epithelium (lacrimal and meibomian glands). Periocular mesenchymal cells of neural crest origin migrate and differentiate, leading to the formation of corneal endothelium and the stromas of the cornea, conjunctiva, eyelids, and trabecular meshwork. The formation of functional ocular surface tissues requires coordinated spatial and temporal expression of transcription factors and signaling molecules of various cytokines and signaling pathways, and the synthesis and remodeling of unique extracellular matrix. Although bidirectional interactions and signaling between mesenchyme and epithelium are considered necessary for embryonic formation of ocular surface tissues and homeostasis in adults, the molecular and cellular mechanisms that regulate such processes remain largely unknown. To investigate possible mechanisms, we have developed mouse models in which the gene functions of ocular surface epithelia and stromas can be altered by Doxycycline induction in spatial and temporal specific manners.

Immunolocalization of different collagens in the cornea of human fetal eyes: a developmental approach.

PURPOSE: To study the corneal development in the human fetal eye with particular emphasis on the epithelial basement membrane and Bowman's layer. Thus, immunohistochemical markers supposed to stain this region were employed. MATERIAL AND METHODS: 19 formalin-fixed fetal eyes and a 16-day-old newborn's cornea without any obvious irregularities of the anterior segment were investigated. The age of the fetal eyes ranged from 11 to 38 week of gestation (WoG). The eyes (including the corneal thickness) were measured and, in addition to routine hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) stains, immunohistochemical labeling with antibodies to collagen IV, V, IX, and XVII was performed. RESULTS: Analysis of the H&E stains revealed that measurements of corneal thickness correlated well with corneal development as a basic indicator for maturation. In a more detailed immunohistochemical analysis, collagen IV was expressed in the epithelial basement membrane (BM) of the cornea, conjunctiva, and Descemet's membrane in fetal eyes up to the age of 23 WoG. In fetal eyes older than 23 WoG, staining was confined to the limbal area only. With the antibody against collagen V, the corneal stroma and the BM were intensely stained. Bowman's layer (first detected at 17 WoG by light microscopy) was not labeled. Anti-collagen IX labeled predominantly the conjunctival and corneal epithelium. With anti-collagen XVII, the BM of the cornea and conjunctiva was stained in all fetal eyes, whereas intracellular expression in the epithelium increased with age. CONCLUSION: Our results indicate maturation-associated variations of collagen expression in the human cornea. Measurements of the corneal thickness may serve as an additional parameter to narrow down the developmental age with possible implications for pediatric pathology and forensic issues.

Effect of the synthetic NC-1059 peptide on diffusion of riboflavin across an intact corneal epithelium.

PURPOSE: To investigate the effect of the peptide NC-1059 on riboflavin (RF) diffusion across an intact corneal epithelium into the stroma. METHODS: NC-1059 peptide was synthesized by solid-phase synthesis with 9-fluorenylmethoxycarbonyl chemistry, characterized by reversed-phase HPLC, and matrix-assisted laser desorption ionization time-of-flight mass spectroscopy. The diffusion of RF across embryonic day 18 chick corneal epithelium ex vivo was monitored using confocal microscopy. The depth distributions of RF in the corneal stroma were calculated using a group of linear equations based on the relationship between RF fluorescence intensity and concentration. RESULTS: Data presented in this study demonstrate that the NC-1059 peptide can transiently open the intact epithelial barrier to allow the permeation of RF into the stroma. The effect of NC-1059 peptide on RF diffusion across the corneal epithelium was concentration and time dependent. The amount of RF reaching a 50-µm depth of chick corneal stoma increased dramatically after exposure to NC-1059 for 10 minutes, reaching a plateau by 30 minutes. The concentrations of RF in the presence of NC-1059 at corneal stromal depths of 50, 100, and 150 µm were significantly higher than in the absence of the peptide, and almost as high as in corneas in which the epithelium first had been physically removed. In addition, a cell viability assay indicated that the NC-1059 peptide did not kill corneal epithelial cells. CONCLUSIONS: NC-1059 peptide significantly enhances the diffusion of RF across intact corneal epithelium into the stroma.

14-3-3σ controls corneal epithelium homeostasis and wound healing.

PURPOSE: To investigate the functional role of 14-3-3σ in regulation of the corneal epithelial proliferation, differentiation, and wound-healing response. METHODS: Corneal phenotypes were investigated in heterozygous repeated epilation (Er) mice carrying mutations in the sfn (14-3-3σ) gene. Immunohistochemistry was used to study the corneal morphogenesis of the Er/Er embryos at embryonic day (E)18.5. Corneal homeostasis and the wound-healing response were investigated macroscopically and microscopically in the adult heterozygous Er mice. Corneal epithelial cell proliferation and differentiation were assessed by BrdU incorporation and immunohistochemistry with specific antibodies for differentiation markers. Furthermore, corneal stroma neovascularization and meibomian gland degeneration were examined by immunohistochemistry. The healing of corneal wounds after debridement was monitored and visualized by fluorescent staining. RESULTS: Homozygous mutation of 14-3-3σ led to defects in embryonic corneal epithelial development and differentiation, whereas young heterozygotes showed normal corneal development and homeostasis. However, older heterozygotes displayed a dramatic corneal wound-healing defect characterized by hyperplastic basal progenitor cells (some of which undergo a differentiation switch to express markers of keratinized epidermis); cornea stroma changes including neovascularization; and corneal opacity, leading to plaque formation. Aged heterozygotes also showed meibomian gland atrophy. CONCLUSIONS: 14-3-3σ is essential for corneal epithelium differentiation, and plays an important role in corneal epithelium development and daily renewal of the adult corneal epithelium.

Innervation of the mouse cornea during development.

PURPOSE: Dense innervation of the cornea is important for maintaining its homeostasis and transparency. Although corneal nerves have been well studied in adults, little is known about mammalian corneal innervation during development. This study provides a detailed profile of nerves at various stages of mouse cornea development. METHODS: Mouse heads and corneas were collected at various stages of development including embryonic days (E)12.5 to E16.5, postnatal days (P)0, P10, three weeks after birth, and the adult. Corneas were immunostained with an anti-neuron-specific ß-tubulin antibody (TUJ1). Fluorescently labeled nerves in whole-mount tissues and sections were imaged and analyzed for their axonal projections during eye development. RESULTS: The first nerve bundles appear at the periphery of the anterior portion of the eye by E12.5. Initial projection into the stroma occurs at E13.5 without formation of a pericorneal nerve ring. Between E13.5 and E16.5, nerve bundles project directly into the periphery of the presumptive cornea stroma. They branch repeatedly as they extend toward the cornea center and epithelium. Concomitantly, nerve bundles originating from four quadrants of the eye bifurcate into smaller branches that innervate the entire stroma. The first epithelial innervation occurs at E16.5. Epithelial nerves arrange into patterns that project toward the center subsequently forming a swirl at three weeks after birth, which becomes more pronounced in adults. CONCLUSIONS: Nerve bundles that arise from four quadrants of the eye innervate the mouse cornea. The nerve bundles directly innervate the stroma without forming a pericorneal nerve ring. Radial arrangement of epithelial nerves gradually becomes centrally oriented, subsequently forming a swirl pattern.

Expression of semaphorin 3A and its receptors during mouse corneal development.

Semaphorin 3A (Sema3A) functions to guide the growth of neurons during development, with its effects being mediated by receptor complexes composed of neuropilin (Npn) and plexin (Plx) proteins. We have now examined the expression of Sema3A and its receptor components Npn1 and PlxA during development of the mouse cornea. Sema3A and Npn1 showed similar patterns of expression by immunohistofluorescence analysis, with such expression being prominent in the corneal epithelium during both embryonic and postnatal development. In contrast, PlxA was not expressed in the corneal epithelium until after eye opening between postnatal days 12 and 14. Laser capture microdissection followed by reverse transcription and polymerase chain reaction analysis also showed that the abundance of PlxA mRNA in corneal epithelial cells increased significantly during postnatal development, again in association with eye opening. Given that atmospheric oxygen is thought to play a role in corneal epithelial differentiation and maintenance, our results suggest that the up-regulation of PlxA expression in the corneal epithelium during postnatal development is triggered by exposure of the cornea to the atmosphere. Furthermore, the newly expressed PlxA may contribute to the differentiation of corneal epithelial cells by mediating Sema3A signaling.

Developmental corneal innervation: interactions between nerves and specialized apical corneal epithelial cells.

PURPOSE: The corneal epithelium is one of the most highly innervated structures in the body, and proper innervation is necessary for corneal maintenance and sensation. However, little is known about how these nerves function and how innervation occurs developmentally. The authors have examined certain aspects of corneal innervation in the developing chicken embryo. METHODS: DiI was used to determine the source of the neurons responsible for innervating the cornea. Immunohistochemistry, electron microscopy, and immunoelectron microscopy were used to examine corneal innervation and the relationships that develop between nerves and corneal epithelial cells. RESULTS: Corneal nerves in the embryonic chicken originate entirely from the ophthalmic lobe of the trigeminal ganglion. Within the cornea the nerves interact with apical corneal epithelial (ACE) cells to form specialized structures that are synapse-like because they contain accumulations of vesicles and have the SV2 synaptic vesicle protein. These ACE cells themselves have unique characteristics, including transient expression of the neuronal isoform of class III beta-tubulin and formation of extensive intercellular channels and clefts that contain these specialized synapse-like structures and nerves; in addition, they are mitotically active. Given that these ACE cells react with a monoclonal antibody against this neuronal isoform of beta-tubulin (the TuJ-1 antibody), we have termed them TuJ-1(+)ACE cells. CONCLUSIONS: During avian corneal development the nerves make close associations with a specialized type of ACE cell. There they form synapse-like structures, suggesting that not all nerves within the CE terminate as free nerve endings.

Targeted deletion of Dicer disrupts lens morphogenesis, corneal epithelium stratification, and whole eye development.

Dicer, a ribonuclease essential for miRNA processing, is expressed abundantly in developing mouse cornea and lens. We studied the roles of Dicer and miRNAs in eye development by conditionally deleting the Dicer gene in the mouse lens and corneal epithelium. Adult Dicer conditional null (DicerCN) mice had severe microphthalmia with no discernible lens and a poorly stratified corneal epithelium. Targeted deletion of Dicer effectively inhibited miRNA processing in the developing lens at 12.5 day of embryogenesis (E12.5). Lens development initiated normally but underwent progressive dystrophy between E14.5 and E18.5. Microarray analysis revealed activation of P53 signaling in DicerCN lenses at E13.5, consistent with increased apoptosis and reduced cell proliferation between E12.5 and E14.5. Expression of Pax6 and other lens developmental transcription factors were not greatly affected between E12.5 and E14.5 but decreased as the lens degenerated. Our data indicated an indispensible role for Dicer and miRNAs in lens and corneal development.

Developmental regulation of the nuclear ferritoid-ferritin complex of avian corneal epithelial cells: roles of systemic factors and thyroxine.

Previously we observed that avian corneal epithelial cells protect their DNA from oxidative damage by having the iron-sequestering molecule ferritin - normally cytoplasmic - in a nuclear location. This localization involves a developmentally-regulated ferritin-like protein - ferritoid - that initially serves as the nuclear transporter, and then as a component of a ferritoid-ferritin complex that is half the size of a typical ferritin and binds to DNA. We also observed that developmentally, the synthesis of ferritin and ferritoid are regulated coordinately - with ferritin being predominantly translational and ferritoid transcriptional. In the present study we examined whether the mechanism(s) involved in this regulation reside within the cornea itself, or alternatively involve a systemic factor(s). For this, we explanted embryonic corneas of one age to the chorioallantoic membrane (CAM) of host embryos of a different age - all prior to the initiation of ferritin synthesis. Consistent with systemic regulation, the explants initiated the synthesis of both ferritin and ferritoid in concert with that of the host. We then examined whether this systemic regulation might involve thyroxine - a hormone with broad developmental effects. Employing corneal organ cultures, we observed that thyroxine initiated the synthesis of both components in a manner similar to that which occurs in vivo (i.e. ferritin was translational and ferritoid transcriptional).