Defining compartmentalized stem cell populations with distinct cell division dynamics in the ocular surface epithelium.

Adult tissues contain label-retaining cells (LRCs), which are relatively slow-cycling and considered to represent a property of tissue stem cells (SCs). In the ocular surface epithelium, LRCs are present in the limbus and conjunctival fornix; however, the character of these LRCs remains unclear, owing to lack of appropriate molecular markers. Using three CreER transgenic mouse lines, we demonstrate that the ocular surface epithelium accommodates spatially distinct populations with different cell division dynamics. In the limbus, long-lived Slc1a3CreER-labeled SCs either migrate centripetally toward the central cornea or slowly expand their clones laterally within the limbal region. In the central cornea, non-LRCs labeled with Dlx1CreER and K14CreER behave as short-lived progenitor cells. The conjunctival epithelium in the bulbar, fornix and palpebral compartment is regenerated by regionally unique SC populations. Severe damage to the cornea leads to the cancellation of SC compartments and conjunctivalization, whereas milder limbal injury induces a rapid increase of laterally expanding clones in the limbus. Taken together, our work defines compartmentalized multiple SC/progenitor populations of the mouse eye in homeostasis and their behavioral changes in response to injury.

Biometry of the human cornea and globe: An evaluation by age, gender and population.

The purpose of this study was to examine the effects of age, gender and population origin on human globe and corneal dimensions and to explore the relationships between the dimensions. Human post-mortem eyes were obtained in Hyderabad (n = 223; range, 0-85 years) and Miami (n = 486; range, 6-103 years). The eyes were freed of extraneous tissues and globe antero-posterior length (GAPL), mean globe diameter (MGD) (average of horizontal and vertical), and corneal horizontal (HCD) and vertical (VCD) diameters were measured using digital calipers. The relationships of age, gender and population origin with globe and corneal dimensions and the relationships between the dimensions were assessed by bivalent and multiple regression analyses. Globe and cornea dimensions increase asymptotically with age until around the late teens but do not change thereafter. Bivariate and multivariate regression analyses of the >20-year-old eyes showed that population was significantly correlated with GAPL, MGD, HCD and VCD. Male globes and corneas were larger than those from females, but the difference did not appear to be statistically significant. All Hyderabad dimensions were significantly larger than those from the Miami. Neither GAPL nor MGD were correlated with the corneal dimensions. GAPL was significantly correlated with MGD as was HCD with VCD.

Co-ordinated ocular development from human iPS cells and recovery of corneal function.

The eye is a complex organ with highly specialized constituent tissues derived from different primordial cell lineages. The retina, for example, develops from neuroectoderm via the optic vesicle, the corneal epithelium is descended from surface ectoderm, while the iris and collagen-rich stroma of the cornea have a neural crest origin. Recent work with pluripotent stem cells in culture has revealed a previously under-appreciated level of intrinsic cellular self-organization, with a focus on the retina and retinal cells. Moreover, we and others have demonstrated the in vitro induction of a corneal epithelial cell phenotype from pluripotent stem cells. These studies, however, have a single, tissue-specific focus and fail to reflect the complexity of whole eye development. Here we demonstrate the generation from human induced pluripotent stem cells of a self-formed ectodermal autonomous multi-zone (SEAM) of ocular cells. In some respects the concentric SEAM mimics whole-eye development because cell location within different zones is indicative of lineage, spanning the ocular surface ectoderm, lens, neuro-retina, and retinal pigment epithelium. It thus represents a promising resource for new and ongoing studies of ocular morphogenesis. The approach also has translational potential and to illustrate this we show that cells isolated from the ocular surface ectodermal zone of the SEAM can be sorted and expanded ex vivo to form a corneal epithelium that recovers function in an experimentally induced animal model of corneal blindness.

Exploration of novel candidate genes involved in epidermal keratinocyte differentiation and skin barrier repair in man.

A proper skin barrier function requires constant formation of stratum corneum, i.e. the outermost layer of epidermis composed of terminally differentiated keratinocytes. The complex process of converting proliferative basal keratinocytes into corneocytes relies on programmed changes in the activity of many well-established genes. Much remains however to be investigated about this process, e.g. in conjunction with epidermal barrier defects due to genetic errors as in ichthyosis. To this end, we re-analyzed two sets of microarray-data comparing altered gene expression in differentiated vs. proliferating keratinocytes and in the skin of patients with autosomal recessive congenital ichthyosis (ARCI) vs. healthy controls, respectively. We thus identified 24 genes to be upregulated in both sets of array and not previously associated with keratinocyte differentiation. For 10 of these genes (AKR1B10, BLNK, ENDOU, GCNT4, GLTP, RHCG, SLC15A1, TMEM45B, TMEM86A and VSNL1), qPCR analysis confirmed the array results and subsequent immunostainings of normal epidermis showed superficial expression of several of the proteins. Furthermore, induction of keratinocyte differentiation using phorbol esters (PMA) resulted in increased expression of eight of the genes, whereas siRNA silencing of PPARδ, a transcription factor supporting differentiation, had the opposite effect. In summary, our results identify ten new candidate genes seemingly involved in human epidermal keratinocyte differentiation and possibly important for epidermal repair in a genetic skin disease characterized by barrier failure.

A comparison of the ultrastructure of the cornea of the pre- and post-metamorphic axolotl (Ambystoma mexicanum, Amphibia).

The corneal ultrastructure of the pre- and post-metamorphic stages of the neotenic axolotl Ambystoma mexicanum is examined using light microscopy and both scanning and transmission electron microscopy to reveal whether there are any morphological changes associated with a switch in lifestyle. Although the complement of corneal layers remains the same, there are significant quantitative changes in corneal, epithelial and stromal thickness, epithelial and endothelial cell size and density, and the thickness of Bowman's layer and Desçemet's membrane. Microholes in the epithelium and vertical sutures within the stroma are predominant features in the pre-metamorphic stage but are rarely seen in the post-metamorphic stage. There are also significant quantitative centro-peripheral differences in the thickness of the whole cornea, primarily due to differences in the thickness of the stroma in both metamorphic stages. These changes may reflect the physiological demands on the cornea as it switches from a purely aquatic to an amphibious lifestyle, which includes venturing onto land.

Relationship of the cornea and globe dimensions to the changes in adult human crystalline lens diameter, thickness and power with age.

It is well known that human crystalline lens shape, dimensions and optical properties change throughout life and influence whole eye refraction. However, it is not clear if lens properties are associated with other ocular parameters. The purpose of the present study was to investigate the relationship of corneal and external globe dimensions with adult lens diameter (LD), lens thickness (LT) and lens power (LP) in order to determine if external factors influence lens properties. Postmortem human eyes (n = 66, age = 20-78 years) were obtained from the Ramayamma International Eye Bank, Hyderabad, India. Globe antero-posterior length (GAPL) and mean (average of horizontal and vertical) diameters of cornea (MCD) and globe (MGD) were measured using digital calipers. Eyes were dissected to produce ocular structures that contain the lens maintained in its accommodating framework, including intact zonules, ciliary body and sections of sclera. Specimens were mounted in a mechanical lens stretching system. LD, LT and LP were measured using high magnification retro-illumination photography, slit illumination photography and Scheiner principle-based optical system respectively in the unstretched (accommodated) state. Relationships between external globe and corneal dimensions and LD, LT or LP were assessed by multiple regression analysis. Age (0.012 ± 0.003 mm/year; p<0.001) and GAPL (0.185 ± 0.045 mm/mm; p<0.001) were significant (p<0.0001) predictors of LD. After adjusting for age-related increases, LD appears to be positively correlated with GAPL. Age (0.010 ± 0.004 mm/year; p = 0.009) and GAPL (-0.143 ± 0.060 mm/mm; p = 0.02) were significant (p = 0.001) predictors of LT. After adjusting for the age-related increase, LT appears to be negatively correlated with GAPL. Only age was a significant predictor of LP (-0.26 ± 0.04 D/year; p<0.001). The results suggest that, apart from aging, lens diameter and thickness are dependent on the anteroposterior length of the eye globe. Lens power is not influenced by globe dimensions.

Decellularization and recellularization of cornea: Progress towards a donor alternative.

The global shortage of donor corneas for transplantation has led to corneal bioengineering being investigated as a method to generate transplantable tissues. Decellularized corneas are among the most promising materials for engineering corneal tissue since they replicate the complex structure and composition of real corneas. Decellularization is a process that aims to remove cells from organs or tissues resulting in a cell-free scaffold consisting of the tissues extracellular matrix. Here different decellularization techniques are described, including physical, chemical and biological methods. Analytical techniques to confirm decellularization efficiency are also discussed. Different cell sources for the recellularization of the three layers of the cornea, recellularization methods used in the literature and techniques used to assess the outcome of the implantation of such scaffolds are examined. Studies involving the application of decellularized corneas in animal models and human clinical studies are discussed. Finally, challenges for this technology are explored involving scalability, automatization and regulatory affairs.

Analysis of Cryopreservation Protocols and Their Harmful Effects on the Endothelial Integrity of Human Corneas.

Corneal cryopreservation can partially solve the worldwide concern regarding donor cornea shortage for keratoplasties. In this study, human corneas were cryopreserved using two standard cryopreservation protocols that are employed in the Tissue Bank of the Teresa Herrera Hospital (Spain) to store corneas for tectonic keratoplasties (TK protocol) and aortic valves (AV protocol), and two vitrification protocols, VS55 and DP6. Endothelial viability and general corneal state were evaluated to determine the protocol that provides the best results. The potential corneal cryopreservation protocol was studied in detail taking into consideration some cryopreservation-related variables and the endothelial integrity and stroma arrangement of the resulting cryopreserved corneas. TK corneas showed mostly viable endothelial cells, while the others showed few (AV) or none (DP6 and VS55). The corneal structure was well maintained in TK and AV corneas. TK corneas showed endothelial acellular areas surrounded by injured cells and a normal-like stromal fiber arrangement. Cryoprotectant solutions of the TK protocol presented an increasing osmolality and a physiological pH value. Cooling temperature rate of TK protocol was of 1 °C/min to -40 °C and 3 °C/min to -120 °C, and almost all of dimethyl sulfoxide left the tissue after washing. Future studies should be done changing cryopreservation-related variables of the TK protocol to store corneas of optical grade.

Can Human Oral Mucosa Stem Cells Differentiate to Corneal Epithelia?

Human oral mucosa stem cells (hOMSCs) arise from the neural crest, they can self-renew, proliferate, and differentiate to several cell lines and could represent a good source for application in tissue engineering. Because of their anatomical location, hOMSCs are easy to isolate, have multilineage differentiation capacity and express embryonic stem cells markers such as-Sox2, Oct3/4 and Nanog. We have used SHEM (supplemented hormonal epithelial medium) media and cultured hOMSCs over human amniotic membrane and determined the cell's capacity to differentiate to an epithelial-like phenotype and to express corneal specific epithelial markers-CK3, CK12, CK19, Pan-cadherin and E-cadherin. Our results showed that hOMSCs possess the capacity to attach to the amniotic membrane and express CK3, CK19, Pan-Cadherin and E-Cadherin without induction with SHEM media and expressed CK12 or changed the expression pattern of E-Cadherin to a punctual-like feature when treated with SHEM media. The results observed in this study show that hOMSCs possess the potential to differentiate toward epithelial cells. In conclusion, our results revealed that hOMSCs readily express markers for corneal determination and could provide the ophthalmology field with a therapeutic alternative for tissue engineering to achieve corneal replacement when compared with other techniques. Nevertheless, further studies are needed to develop a predictable therapeutic alternative for cornea replacement.

Genetic Analysis of the Organization, Development, and Plasticity of Corneal Innervation in Mice.

The cornea has the densest sensory innervation of the body, originating primarily from neurons in the trigeminal ganglion. The basic principles of cornea nerve patterning have been established many years ago using classic neuroanatomical methods, such as immunocytochemistry and electrophysiology. Our understanding of the morphology and distribution of the sensory nerves in the skin has considerably progressed over the past few years through the generation and analysis of a variety of genetically modified mouse lines. Surprisingly, these lines were not used to study corneal axons. Here, we have screened a collection of transgenic and knockin mice (of both sexes) to select lines allowing the visualization and genetic manipulation of corneal nerves. We identified multiple lines, including some in which different types of corneal axons can be simultaneously observed with fluorescent proteins expressed in a combinatorial manner. We also provide the first description of the morphology and arborization of single corneal axons and identify three main types of branching pattern. We applied this genetic strategy to the analysis of corneal nerve development and plasticity. We provide direct evidence for a progressive reduction of the density of corneal innervation during aging. We also show that the semaphorin receptor neuropilin-1 acts cell-autonomously to control the development of corneal axons and that early axon guidance defects have long-term consequences on corneal innervation.SIGNIFICANCE STATEMENT We have screened a collection of transgenic and knockin mice and identify lines allowing the visualization and genetic manipulation of corneal nerves. We provide the first description of the arborization pattern of single corneal axons. We also present applications of this genetic strategy to the analysis of corneal nerve development and remodeling during aging.

Using BXD mouse strains in vision research: A systems genetics approach.

We illustrate the growing power of the BXD family of mice (recombinant inbred strains from a cross of C57BL/6J and DBA/2J mice) and companion bioinformatic tools to study complex genome-phenome relations related to glaucoma. Over the past 16 years, our group has integrated powerful murine resources and web-accessible tools to identify networks modulating visual system traits-from photoreceptors to the visual cortex. Recent studies focused on retinal ganglion cells and glaucoma risk factors, including intraocular pressure (IOP), central corneal thickness (CCT), and susceptibility of cellular stress. The BXD family was exploited to define key gene variants and then establish linkage to glaucoma in human cohorts. The power of this experimental approach to precision medicine is highlighted by recent studies that defined cadherin 11 (Cdh11) and a calcium channel (Cacna2d1) as genes modulating IOP, Pou6f2 as a genetic link between CCT and retinal ganglion cell (RGC) death, and Aldh7a1 as a gene that modulates the susceptibility of RGCs to death after elevated IOP. The role of three of these gene variants in glaucoma is discussed, along with the pathways activated in the disease process.

Keratin expression by corneal and limbal stem cells during development.

Keratins are the forming units of intermediate filaments (IF) that provide mechanical support, and formation of desmosomes between cells and hemi desmosomes with basement membranes for epithelium integrity. Keratin IF are polymers of obligate heterodimer consisting one type I keratin and one type II keratin molecules. There are 54 functional keratin genes in human genome, which are classified into three major groups, i.e., epithelial keratins, hair follicle cell-specific epithelial keratins and hair keratins. Their expression is cell type-specific and developmentally regulated. Corneal epithelium expresses a subgroup of keratins similar to those of epidermal epithelium. Limbal basal stem cells express K5/K14, and K8/K18 and K8/K19 IF suggesting that there probably are two populations of limbal stem cells (LSCs). In human, LSCs at limbal basal layer can directly stratify and differentiate to limbal suprabasal cells that express K3/K12 IF, or centripetally migrate then differentiate to corneal basal transient amplifying cells (TAC) that co-express both K3/K12 and K5/K14 prior to moving upward and assuming suprabasal cells phenotype of only K3/K12 expression that signifies corneal type epithelium differentiation. In rodent, the differentiated cornea epithelial cells express K5/K12 in lieu of K3/K12, because K3 allele exists as a pseudogene and does not encode a functional K3 protein. The basal corneal cells of new-born mice originate from surface ectoderm during embryonic development slowly commit to differentiation of becoming TAC co-expressing K5/K12 and K5/K14 IF. However, the centripetal migration may still occur at a slower rate in young mice, which is accelerated during wound healing. In this review, we will discuss and compare the cornea-specific keratins expression patterns between corneal and epidermal epithelial cells during mouse development, and between human and mouse during development and homeostasis in adult, and pathology caused by a mutation of keratins.

Pigment Epithelium-Derived Factor (PEDF) Fragments Prevent Mouse Cone Photoreceptor Cell Loss Induced by Focal Phototoxicity In Vivo.

Here, we evaluated the effects of PEDF (pigment epithelium-derived factor) and PEDF peptides on cone-photoreceptor cell damage in a mouse model of focal LED-induced phototoxicity (LIP) in vivo. Swiss mice were dark-adapted overnight, anesthetized, and their left eyes were exposed to a blue LED placed over the cornea. Immediately after, intravitreal injection of PEDF, PEDF-peptide fragments 17-mer, 17-mer[H105A] or 17-mer[R99A] (all at 10 pmol) were administered into the left eye of each animal. BDNF (92 pmol) and bFGF (27 pmol) injections were positive controls, and vehicle negative control. After 7 days, LIP resulted in a consistent circular lesion located in the supratemporal quadrant and the number of S-cones were counted within an area centered on the lesion. Retinas treated with effectors had significantly greater S-cone numbers (PEDF (60%), 17-mer (56%), 17-mer [H105A] (57%), BDNF (64%) or bFGF (60%)) relative to their corresponding vehicle groups (≈42%). The 17-mer[R99A] with no PEDF receptor binding and no neurotrophic activity, PEDF combined with a molar excess of the PEDF receptor blocker P1 peptide, or with a PEDF-R enzymatic inhibitor had undetectable effects in S-cone survival. The findings demonstrated that the cone survival effects were mediated via interactions between the 17-mer region of the PEDF molecule and its PEDF-R receptor.

Role of α1-adrenoceptor subtypes on corneal epithelial thickness and cell proliferation in mice.

Adrenergic stimuli are important for corneal epithelial structure and healing. The purpose of the present study was to examine the hypothesis that the lack of a single α1-adrenoceptor (α1-AR) subtype affects corneal epithelial thickness and cell proliferation. Expression levels of α1-AR mRNA were determined in mouse cornea using real-time PCR. In mice devoid of one of the three α1-AR subtypes (α1A-AR-/-, α1B-AR-/-, α1D-AR-/-) and in wild-type controls, thickness of individual corneal layers, the number of epithelial cell layers, and average epithelial cell size were determined in cryosections. Endothelial cell density and morphology were calculated in corneal explants, and epithelial cell proliferation rate was determined with immunofluorescence microscopy. Moreover, the ultrastructure of the corneal epithelium was examined by transmission electron microscopy. Messenger RNA for all three α1-AR subtypes was expressed in whole cornea and in corneal epithelium from wild-type mice with a rank order of abundance of α1A ≥ α1B > α1D. In contrast, no α1-AR mRNA was detected in the stroma, and only α1B-AR mRNA was found in the Descemet endothelial complex. Remarkably, corneal epithelial thickness and mean epithelial cell size were reduced in α1A-AR-/- mice. Our findings suggest that the α1A-AR exerts growth effects in mouse corneal epithelial cells.

Autocrine IL-1ß mediates the promotion of corneal neovascularization by senescent fibroblasts.

Our previous study has confirmed that senescent fibroblasts promote corneal neovascularization (CNV) partially via the enhanced secretion of matrix metalloproteases (MMPs). However, the regulation of MMP expression in senescent fibroblasts remained unclear. In this study, we identified that the expression and secretion levels of interleukin-1ß (IL-1ß) were significantly upregulated in senescent human corneal fibroblasts than that in normal fibroblasts. Moreover, compared with vehicle-pretreated senescent fibroblasts, IL-1ß pretreatment enhanced the expression of angiogenic factors but reduced the expression of angiostatic factors in senescent fibroblasts. When cocultured with human umbilical vein endothelial cells, IL-1ß-pretreated senescent fibroblasts more strongly promoted their proliferation, migration, and tube-formation capacities than the vehicle-controlled senescent fibroblasts. In addition, either interleukin-1 receptor antagonist or anti-IL-1ß neutralization completely inhibited the promotion of senescent fibroblasts in vascular tube formation in vitro and CNV in vivo. Therefore, we concluded that autocrine IL-1ß mediated the promotion of senescent fibroblasts on corneal neovascularization.

Postnatal periodontal ligament as a novel adult stem cell source for regenerative corneal cell therapy.

Corneal opacities are a leading cause of global blindness. They are conventionally treated by the transplantation of donor corneal tissue, which is, restricted by a worldwide donor material shortage and allograft rejection. Autologous adult stem cells with a potential to differentiate into corneal stromal keratocytes (CSKs) could offer a suitable choice of cells for regenerative cell therapy. Postnatal periodontal ligament (PDL) contains a population of adult stem cells, which has a similar embryological origin as CSK, that is cranial neural crest. We harvested PDL cells from young adult teeth extracted because of non-functional or orthodontic reason and differentiated them towards CSK phenotype using a two-step protocol with spheroid formation followed by growth factor and cytokine induction in a stromal environment (human amnion stroma and porcine corneal stroma). Our results showed that the PDL-differentiated CSK-like cells expressed CSK markers (CD34, ALDH3A1, keratocan, lumican, CHST6, B3GNT7 and Col8A2) and had minimal expression of genes related to fibrosis and other lineages (vasculogenesis, adipogenesis, myogenesis, epitheliogenesis, neurogenesis and hematogenesis). Introduction of PDL spheroids into the stroma of porcine corneas resulted in extensive migration of cells inside the host stroma after 14-day organ culture. Their quiescent nature and uniform cell distribution resembled to that of mature CSKs inside the native stroma. Our results demonstrated the potential translation of PDL cells for regenerative corneal cell therapy for corneal opacities.

Neonatal and Juvenile Ocular Development in Sprague-Dawley Rats: A Histomorphological and Immunohistochemical Study.

As in many altricial species, rats are born with fused eyelids and markedly underdeveloped eyes. While the normal histology of the eyes of mature rats is known, the histomorphological changes occurring during postnatal eye development in this species remain incompletely characterized. This study was conducted to describe the postnatal development of ocular structures in Sprague-Dawley (SD) rats during the first month of age using histology and immunohistochemistry (IHC). Both eyes were collected from 51 SD rats at 13 time points between postnatal day (PND)1 and PND30. Histologic examination of hematoxylin and eosin-stained sections was performed, as well as IHC for cleaved-caspase-3 and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) to evaluate apoptosis, and IHC for Ki-67 and phospho-histone-H3 to evaluate cell proliferation. Extensive ocular tissue remodeling occurred prior to the eyelid opening around PND14 and reflected the interplay between apoptosis and cell proliferation. Apoptosis was particularly remarkable in the maturing subcapsular anterior epithelium of the lens, the inner nuclear and ganglion cell layers of the developing retina, and the Harderian gland, and was involved in the regression of the hyaloid vasculature. Nuclear degradation in the newly formed secondary lens fibers was noteworthy after birth and was associated with TUNEL-positive nuclear remnants lining the lens organelle-free zone. Cell proliferation was marked in the developing retina, cornea, iris, ciliary body and Harderian gland. The rat eye reached histomorphological maturity at PND21 after a rapid phase of morphological changes characterized by the coexistence of cell death and proliferation.

Limited versus total epithelial debridement ocular surface injury: Live fluorescence imaging of hemangiogenesis and lymphangiogenesis in Prox1-GFP/Flk1::Myr-mCherry mice.

BACKGROUND: Immunohistochemical staining experiments have shown that both hemangiogenesis and lymphangiogenesis occur following severe corneal and conjunctival injury and that the neovascularization of the cornea often has severe visual consequences. To better understand how hemangiogenesis and lymphangiogenesis are induced by different degrees of ocular injury, we investigated patterns of injury-induced corneal neovascularization in live Prox1-GFP/Flk1::myr-mCherry mice, in which blood and lymphatic vessels can be imaged simultaneously in vivo. METHODS: The eyes of Prox1-GFP/Flk1::myr-mCherry mice were injured according to four models based on epithelial debridement of the: A) central cornea (a 1.5-mm-diameter circle of tissue over the corneal apex), B) total cornea, C) bulbar conjunctiva, and D) cornea+bulbar conjunctiva. Corneal blood and lymphatic vessels were imaged on days 0, 3, 7, and 10 post-injury, and the percentages of the cornea containing blood and lymphatic vessels were calculated. RESULTS: Neither central corneal nor bulbar conjunctival debridement resulted in significant vessel growth in the mouse cornea, whereas total corneal and corneal+bulbar conjunctival debridement did. On day 10 in the central cornea, total cornea, bulbar conjunctiva, and corneal+bulbar conjunctival epithelial debridement models, the percentage of the corneal surface that was occupied by blood vessels (hemangiogenesis) was 1.9±0.8%, 7.14±2.4%, 2.29±1%, and 15.05±2.14%, respectively, and the percentage of the corneal surface that was occupied by lymphatic vessels (lymphangiogenesis) was 2.45±1.51%, 4.85±0.95%, 2.95±1.27%, and 4.15±3.85%, respectively. CONCLUSIONS: Substantial corneal debridement was required to induce corneal neovascularization in the mouse cornea, and the corneal epithelium may therefore be partially responsible for maintaining corneal avascularity. GENERAL SIGNIFICANCE: Our study demonstrates that GFP/Flk1::myr-mCherry mice are a useful model for studying coordinated hemangiogenic and lymphangiogenic responses.

Defective eyelid leading edge cell migration in C57BL/6-corneal opacity mice with an "eye open at birth" phenotype.

Development of the eyelid requires coordination of the cellular processes involved in proliferation, cell size alteration, migration, and cell death. C57BL/6J-corneal opacity (B6-Co) mice are mutant mice generated by the administration of N-ethyl-N-nitrosourea (100 mg/kg). They exhibit the eyelids open at birth phenotype, abnormal round cell shape from tightened F-actin bundles in leading edge keratinocytes at E16.5, and gradual corneal opacity with neovessels. The tip of the leading edge in B6-Co mice did not move forward, and demonstrated a sharp peak shape without obvious directionality. Analysis of the biological characteristics of B6-Co mice demonstrated that abnormal migration of keratinocytes could affect eyelid development, but proliferation and apoptosis in B6-Co mice had no effect. Mutant gene mapping and sequence analysis demonstrated that in B6-Co mice, adenosine was inserted into the untranslated regions, between 3030 and 3031, in the mRNA 3'-terminal of Fgf10. In addition, guanine 7112 was substituted by adenine in the Mtap1B mRNA, and an A2333T mutation was identified in Mtap1B. Quantitative real-time polymerase chain reaction analysis showed that expression of the Hbegf gene was significantly down-regulated in the eyelids of B6- Co mice at E16.5, compared to B6 mice. However, the expression of Rock1, Map3k1, and Jnk1 genes did not show any significant changes. Abnormal keratinocyte migration and down-regulated expression of the Hbegf gene might be associated with impaired eyelid development in B6-Co mice.