rad21 Is Involved in Corneal Stroma Development by Regulating Neural Crest Migration.

Previously, we identified RAD21R450C from a peripheral sclerocornea pedigree. Injection of this rad21 variant mRNA into Xenopus laevis embryos disrupted the organization of corneal stroma fibrils. To understand the mechanisms of RAD21-mediated corneal stroma defects, gene expression and chromosome conformation analysis were performed using cells from family members affected by peripheral sclerocornea. Both gene expression and chromosome conformation of cell adhesion genes were affected in cells carrying the heterozygous rad21 variant. Since cell migration is essential in early embryonic development and sclerocornea is a congenital disease, we studied neural crest migration during cornea development in X. laevis embryos. In X. laevis embryos injected with rad21 mutant mRNA, neural crest migration was disrupted, and the number of neural crest-derived periocular mesenchymes decreased significantly in the corneal stroma region. Our data indicate that the RAD21R450C variant contributes to peripheral sclerocornea by modifying chromosome conformation and gene expression, therefore disturbing neural crest cell migration, which suggests RAD21 plays a key role in corneal stroma development.

Identification of a Primary Stroma and Novel Endothelial Cell Projections in the Developing Human Cornea.

Purpose: To investigate the initial events in the development of the human cornea, focusing on cell migration, and extracellular matrix synthesis and organization. To determine whether elastic fibers are present in the extracellular matrix during early human corneal development. Methods: Human corneas were collected from week 7 to week 17 of development. An elastic fiber-enhancing stain, tannic acid-uranyl acetate, was applied to all tissue. Three-dimensional serial block-face scanning electron microscopy combined with conventional transmission electron microscopy was used to analyze the corneal stroma. Results: An acellular collagenous primary stroma with an orthogonal arrangement of fibrils was identified in the central cornea from week 7 of corneal development. At week 7.5, mesenchymal cells migrated toward the central cornea and associated with the acellular collagenous matrix. Novel cell extensions from the endothelium were identified. Elastic fibers were found concentrated in the posterior peripheral corneal stroma from week 12 of corneal development. Conclusions: This study provides novel evidence of an acellular primary stroma in the early development of the embryonic human cornea. Cell extensions exist as part of a communication system and are hypothesized to assist in the migration of the mesenchymal cells and the development of the mature cornea. Elastic fibers identified in early corneal development may play an important role in establishing corneal shape.

Cell-independent matrix configuration in early corneal development.

Mechanisms controlling the spatial configuration of the remarkably ordered collagen-rich extracellular matrix of the transparent cornea remain incompletely understood. We previously described the assembly of the emerging corneal matrix in the mid and late stages of embryogenesis and concluded that collagen fibril organisation was driven by cell-directed mechanisms. Here, the early stages of corneal morphogenesis were examined by serial block face scanning electron microscopy of embryonic chick corneas starting at embryonic day three (E3), followed by a Fourier transform analysis of three-dimensional datasets and theoretical considerations of factors that influence matrix formation. Eyes developing normally and eyes that had the lens surgically removed at E3 were studied. Uniformly thin collagen fibrils are deposited by surface ectoderm-derived corneal epithelium in the primary stroma of the developing chick cornea and form an acellular matrix with a striking micro-lamellar orthogonal arrangement. Fourier transform analysis supported this observation and indicated that adjacent micro-lamellae display a clockwise rotation of fibril orientation, depth-wise below the epithelium. We present a model which attempts to explain how, in the absence of cells in the primary stroma, collagen organisation might be influenced by cell-independent, intrinsic mechanisms, such as fibril axial charge derived from associated proteoglycans. On a supra-lamellar scale, fine cords of non-collagenous filamentous matrix were detected over large tissue volumes. These extend into the developing cornea from the epithelial basal lamina and appear to associate with the neural crest cells that migrate inwardly to form, first the corneal endothelium and then keratocytes which synthesise the mature, secondary corneal stroma. In a small number of experimental specimens, matrix cords were present even when periocular neural crest cell migration and corneal morphogenesis had been perturbed following removal of the lens at E3.

Cell regulation of collagen fibril macrostructure during corneal morphogenesis.

While tissue form and function is highly dependent upon tissue-specific collagen composition and organization, little is known of the mechanisms controlling the bundling of collagen fibrils into fibers and larger structural designs that lead to the formation of bones, tendons and other tissues. Using the cornea as a model system, our previous 3 dimensional mapping of collagen fiber organization has demonstrated that macrostructural organization of collagen fibers involving interweaving, branching and anastomosing plays a critical role in controlling mechanical stiffness, corneal shape and refractive power. In this work, the cellular and mechanical mechanisms regulating critical events in the assembly of collagen macrostructure are analysed in the developing chicken cornea. We elucidated the temporal events leading to adult corneal structure and determined the effects of intraocular pressure (IOP) on the organization of the collagen macrostructure. Our findings indicate that the complex adult collagen organization begins to appear on embryonic day 10 (E10) after deposition of the primary stroma and full invasion of keratocytes. Importantly, organizational changes in keratocytes appearing at E9 preceded and predicted later changes in collagen organization. Corneal collagen organization remained unaffected when the development of IOP was blocked at E4. These findings support a primary role for keratocytes in controlling stromal organization, mechanical stiffness and corneal shape that are not regulated by the IOP. Our findings also suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function. STATEMENT OF SIGNIFICANCE: This work by using an ex ovo model system, begins to investigate the potential mechanisms controlling collagen fibril macrostructure. In particular, this work highlights a convergent role for the corneal keratocytes in organizing the complex collagen macrostructure, necessary to support high visual acuity. Our data supports that the intraocular pressure does not influence collagen fibril macrostructure and suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function. Clearly understanding the cellular and molecular mechanisms that underlie collagen fibril macrostructure will be highly beneficial for future tissue engineering and regenerative medicine applications.

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.

Lateral growth limitation of corneal fibrils and their lamellar stacking depend on covalent collagen cross-linking by transglutaminase-2 and lysyl oxidases, respectively.

Corneal stroma contains an extracellular matrix of orthogonal lamellae formed by parallel and equidistant fibrils with a homogeneous diameter of ~35 nm. This is indispensable for corneal transparency and mechanical functions. However, the mechanisms controlling corneal fibrillogenesis are incompletely understood and the conditions required for lamellar stacking are essentially unknown. Under appropriate conditions, chick embryo corneal fibroblasts can produce an extracellular matrix in vitro resembling primary corneal stroma during embryonic development. Among other requirements, cross-links between fibrillar collagens, introduced by tissue transglutaminase-2, are necessary for the self-assembly of uniform, small diameter fibrils but not their lamellar stacking. By contrast, the subsequent lamellar organization into plywood-like stacks depends on lysyl aldehyde-derived cross-links introduced by lysyl oxidase activity, which, in turn, only weakly influences fibril diameters. These cross-links are introduced at early stages of fibrillogenesis. The enzymes are likely to be important for a correct matrix deposition also during repair of the cornea.

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.

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.

Twist2: role in corneal stromal keratocyte proliferation and corneal thickness.

PURPOSE: Twist2 is a member of a family of bHLH transcription factors critical for normal mesenchymal proliferation and differentiation. In this study, the authors analyzed the role of Twist2 in the eye and cornea through examination of a Twist2 loss-of-function mouse mutant. METHODS: Twist2 expression during eye development in the mouse was investigated using RT-PCR and mRNA slide in situ hybridization. Lineage tracing was performed using Cre reporter mice. Morphometric analyses were performed, and cell proliferation and cell death were investigated by immunohistochemistry using Ki67 and cleaved caspase 3 antibodies, respectively. RESULTS: In the mouse, Twist2 is expressed first in the periocular mesenchyme and subsequently in the corneal stroma and endothelium of the developing eye. Loss of Twist2 function leads to corneal thinning and a reduced population of stromal keratocytes. The reduction in the stromal cell population can be traced back to embryonic stages during which the proliferation of stromal progenitor cells is impaired and to the reduced number of proliferating cells in the corneal limbus postnatally. Adult Twist2-null mice display enophthalmia and blepharophimosis. Corneal thinning in mutant mice is not accompanied by glaucoma, an association reported in human patients. CONCLUSIONS: Twist2 is required for normal corneal keratocyte proliferation and eyelid morphogenesis in the mouse. Loss of Twist2 function leads to corneal thinning because of the reduction in stromal keratocyte proliferation.

Differential relative sulfation of Keratan sulfate glycosaminoglycan in the chick cornea during embryonic development.

PURPOSE: To investigate structural remodeling of the developing corneal stroma concomitant with changing sulfation patterns of keratan sulfate (KS) glycosaminoglycan (GAG) epitopes during embryogenesis and the onset of corneal transparency. METHODS: Developing chick corneas were obtained from embryonic day (E)12 to E18 of incubation. Extracellular matrix composition and collagen fibril spacing were evaluated by synchrotron x-ray diffraction, hydroxyproline assay, ELISA (with antibodies against lesser and more highly sulfated KS), and transmission electron microscopy with specific proteoglycan staining. RESULTS: A significant relative increase in highly sulfated KS epitope labeling occurred with respect to hydroxyproline content in the final week of chick development, as mean collagen interfibrillar distance decreased. Small KS PG filaments increased in frequency with development and were predominantly fibril associated. CONCLUSIONS: The accumulation of highly sulfated KS during the E12 to E18 timeframe could serve to fine tune local matrix hydration and collagen fibril spacing during corneal growth, as gross dehydration and compaction of the stroma progress through the action of the nascent endothelial pump.

Actin and type I collagen propeptide distribution in the developing chick cornea.

PURPOSE: To determine the organization of actin filaments and distribution of type I procollagen during the development of the chick corneal stroma. METHODS: Embryonic chicken corneas of ages 6 to 18 days and 18 days posthatch were cryosectioned and fluorescently labeled for filamentous actin with phalloidin and for the N-and C-terminal propeptides of type I procollagen with specific monoclonal antibodies. Tissue sections were examined by fluorescence and confocal microscopy. RESULTS: Prominent actin filament bundles were present at all embryonic stages, arranged in orthogonal arrays. Type I collagen propeptides were also present, with the C-propeptide visible as small foci, often associated with the actin label. The N-propeptide was also detected in the stromal matrix, especially in Bowman's layer. Actin filaments were also prominent in the corneal epithelium, along with collagen propeptide labeling, up to embryonic day 14. CONCLUSIONS: Actin filament bundles are abundant in the stroma, presumably in the keratocytes of the developing chick cornea, and are arranged in an orthogonal manner suggesting a possible role in cell and matrix organization in this tissue. Filament bundles appear to be closely associated with the foci of type I procollagen label, suggesting a possible association between the actin cytoskeleton and the trafficking of collagen. The presence of the N-propeptide of type I collagen in the extracellular matrix and the restricted distribution of the C-propeptide suggest differential processing of these molecules after secretion. The persistence of the N-propeptide implies a role in development, possibly in association with control of collagen fibril diameter and spacing.

Development of the corneal stroma, and the collagen-proteoglycan associations that help define its structure and function.

The cornea of the eye is a unique, transparent connective tissue. It is comprised predominantly of collagen fibrils, remarkably uniform in diameter and regularly spaced, organized into an intricate lamellar array. Its establishment involves a precisely controlled sequence of developmental events in which the embryonic cornea undergoes major structural transformations that ultimately determine tissue form and function. In this article, we will review corneal developmental dynamics from a structural perspective, consider the roles and interrelationships of collagens and proteoglycans, and comment on contemporary concepts and current challenges pertinent to developmental processes that result in an optically clear, mature cornea.

Formation of stromal collagen fibrils and proteoglycans in the developing zebrafish cornea.

PURPOSE: Collagen fibrils and proteoglycans are the main components of the corneal extracellular matrix and corneal transparency depends crucially on their proper arrangement. In the present study, we investigated the formation of collagen fibrils and proteoglycans in the developing cornea of the zebrafish, a model organism used to study vertebrate embryonic development and genetic disease. METHODS: We employed thin-section electron microscopy to investigate the ultrastructure of the zebrafish cornea at different developmental stages. RESULTS: The layering of the zebrafish cornea into an epithelium, a Bowman's layer, stroma and endothelium was observed starting at 72 hr post-fertilization. At this stage, the stroma contained orthogonally arranged collagen fibrils and small proteoglycans. The density of proteoglycans increased gradually throughout subsequent development of the cornea. In the stroma of 2-week-old larvae, the collagen fibrils were organized into thin lamellae and were separated by very large, randomly distributed proteoglycans. At 4 weeks, a regular arrangement of proteoglycans in relation to the collagen fibrils was observed for the first time and the lamellae were also thickened. CONCLUSION: The present study, for the first time, provides ultrastructural details of collagen fibril and proteoglycan development in the zebrafish cornea. Furthermore, it directly correlates the collagen fibril and proteoglycan composition of the zebrafish cornea with that of the human cornea. The similarities between the two species suggest that the zebrafish could serve as a model for investigating the genetics of human corneal development and diseases.

Keratan sulfate glycosaminoglycan and the association with collagen fibrils in rudimentary lamellae in the developing avian cornea.

PURPOSE: Keratan sulfate (KS), through its association with fibrillar collagen as KS-substituted proteoglycan (KS PG), is thought to be instrumental in the structural development of the corneal stroma. The authors used two different sulfate motif-specific antibodies to identify the sequence of appearance, and the association with collagen, of sulfated KS during avian corneal morphogenesis. METHODS: Corneas from chicken embryos throughout the developmental period, from day 8 through day 18 of incubation, were examined by immunofluorescence and immunoelectron microscopy using monoclonal antibodies 5D4 and 1B4, which react with high- and low-sulfated epitopes on KS, respectively. RESULTS: KS was identified as punctate labeling at incubation day 8, the earliest stage examined, suggesting a cell-associated distribution. By day 10, labeling was more homogeneous, indicating that KS sulfation motifs were present in the stromal extracellular matrix. At day 12 through day 14, immunopositive sites were concentrated primarily in the anterior stroma but became more uniform throughout the full stromal thickness by day 18. From day 10 on, electron microscopy revealed a high-sulfated KS epitope closely associated with bundles of regularly arranged collagen fibrils, initially near cell surfaces in rudimentary lamellae. Individual cells, associated with collagen bundles with different fibril orientations, imply the potential for simultaneous deposition of multiple lamellae. CONCLUSIONS: During chick corneal morphogenesis, significant matrix deposition of high-sulfated KS epitope occurs by day 10, with accumulation subsequently proceeding in an anterior-to-posterior manner. High-sulfated KS likely serves to help define the regular spatial organization of collagen fibrils in bundles newly extruded into the extracellular milieu.

Developmentally regulated expression of Sp1 in the mouse cornea.

PURPOSE: To examine the expression of transcription factor Sp1 in the cornea of the mouse eye throughout developmental stages. The environmental effect of light on Sp1 expression was also assessed. METHODS: C57BL/6 mice were set up for timed mating. Embryos on embryonic day (E)10.5, E12.5, E15.5, and E18.5 and eyes from mice on postnatal day (P)0, P7, P11, P15, P30, and P60 were collected for immunohistochemical staining and in situ hybridization. One group of mice was bred strictly in the dark between E18.5 and P15, and the eyes were collected at P0, P7, P11, and P15 time points. RESULTS: Sp1 expression was observed in the ectoderm and lens vesicle as early as E10.5. Both Sp1 protein and mRNA were abundant in the corneal basal epithelium and keratocytes until P11. Their levels were markedly reduced at P15, right after eyelid opening, and declined further between P15 and P60. In those mice bred in the dark, Sp1 was evident in the cornea at P0. The Sp1 level gradually increased until P11 and was decreased at P15. This expression pattern was nearly identical in mice bred either in a light/dark cycle or in the dark. The Sp1 level in the central lens epithelium was much lower than that in the cornea from E15.5 to late stages. CONCLUSIONS: The present study indicates that Sp1 expression is developmentally regulated, providing a basis for further investigations on the regulation of the Sp1 gene during the course of corneal development and in diseases such as keratoconus.

[Corneal anomalies in murine trisomy 16]. / Minoranomalien der Hornhaut bei der murinen Trisomie 16.

BACKGROUND: The prevalence of human Down's syndrome is about 1:700. Investigations using animal models are therefore of clinical relevance for understanding its etiopathogenesis. No corneal changes have been reported with transgenic murine trisomy 16. METHODS: A total of 20 fetal mice (n=40 eyes) with experimentally induced trisomy 16 were investigated from day 18 of pregnancy in order to determine whether visible developmental disorders of the cornea occur. All specimen were investigated microscopically in serial sections. RESULTS: In addition to disturbances in systemic development, the transgenic mouse fetuses showed high rates of malformation of the eyes. Developmental and differentiation disorders of the corneal epithelial cell layers and structural disturbances of the corneal parenchyma were found. Our findings are the first demonstration of developmental disorders of the cornea in mouse fetuses with trisomy 16. These minor anomalies of the cornea could well have resulted in keratoconus if the animals had survived. CONCLUSIONS: Our findings in transgenic mouse fetuses with trisomy 16 correspond to the clinical pattern of Down's syndrome in humans. Disturbed development of lids and lenses have a high prevalence, whereas corneal hypoplasia is found less often.

Cellular invasion of the chicken corneal stroma during development: regulation by multiple matrix metalloproteases and the lens.

Avian corneal development requires cellular invasion into the acellular matrix of the primary stroma. Previous results show that this invasion is preceded by the removal of the fibril-associated type IX collagen, which possibly stabilizes matrices through interfibrillar cross-bridges secured by covalent crosslinks. In the present study, we provide evidence for the expression of three matrix metalloproteinases (MMPs) in early corneas, two of which act cooperatively to selectively remove type IX collagen in situ. In organ cultures, MMP inhibitors (either TIMP-2 or a synthetic inhibitor) resulted in arrested development, in which collagen IX persisted, and the stroma remained compact and acellular. We also show that blocking covalent crosslinking of collagen allows for cellular invasion to occur, even when the removal of type IX collagen is prevented. Thus, one factor regulating corneal invasion is the physical structure of the matrix, which can be modified by either selective proteolysis or reducing interfibrillar cross-bridges. We also detected another level of regulation of cellular invasion involving inhibition by the underlying lens. This block, which seems to influence invasive behavior independently of matrix modification, is a transient event that is released in ovo just before invasion proceeds.