Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells’ native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens¹. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.     

Deletion of JAM-A causes morphological defects in the corneal epithelium

Junctional adhesion molecule-A (JAM-A, JAM-1, F11R) is an Ig domain containing transmembrane protein that has been proposed to function in diverse processes including platelet activation and adhesion, leukocyte transmigration, angiogenesis, epithelial cell shape and endothelial cell migration although its function in vivo is less well established. In the mouse eye, JAM-A protein expression is first detected at 12.5 dpc in the blood vessels of the tunica vasculosa, while it is first detected in both the corneal epithelium and lens between 13.5 and 14.5 dpc. In the corneal epithelium, JAM-A levels remain appreciable throughout life, while JAM-A immunostaining becomes stronger in the lens as the animals age. Both the cornea and lens of mice lacking an intact JAM-A gene are transparent until at least a year of age, although the cells of the JAM-A null corneal epithelium are irregularly shaped. In wild-type mice, JAM-A protein is found at the leading edge of repairing corneal epithelial wounds, however, corneal epithelial wound repair was qualitatively normal in JAM-A null animals. In summary, JAM-A is expressed in the corneal epithelium where it appears to regulate cell shape.     

Comparative microscopical study of the gall bladder mucosa.

The gall bladder from 6 Psammophis sibilans, 10 Bufo regularis and 10 Albino mice were extracted and prepared for microscopic examination. It was found that the mucosa of Psammophis sibilans consisted of ovoid and polygonal cells which were occasionally binucleated cells with darkly stained nuclei and occasionally pear-shaped cells with vesicular nuclei and fine processes. These cells were arranged in three layers. Apossible explanation for the different types of cells encountered and their arrangement was given. The gall bladder mucosa of Bufo regularis and Albino mouse were thrown into folds covered with simple columnar epithelium. However, the epithelium of the frog was higher than that of the mouse, with the nuclei situated midway between basement membrane and the lumen. Vacuolated cells were detected in the gall bladder mucosa of the mouse. The significance of the mucosal folds was discussed.     

A cytological and cytoskeletal comparison of Sertoli cells without germ cell and those with germ cells using the W/WV mutant mouse.

The distribution of F-actin and intermediate filaments in the W/WV mouse was investigated by light and transmission electron microscopy, and fluorescence methods. No spermatogenic cells were detected in the seminiferous epithelium of the W/WV mouse. Its seminiferous tubule was one-half the diameter of that in the normal (+/+) mouse. The Sertoli cell which was an only component of the W/WV mouse seminiferous epithelium was decreased in height, but still retained the polarity as evidenced by light microscopy. The Sertoli cell organelles were similar in appearance when normal and mutant mice were compared. F-actin was recognized at ectoplasmic specialization (ES) of the W/WV mouse Sertoli cell and appeared similar to the normal mouse. However, the junction with ES was more extensive compared with that of the normal mouse Vimentin in the W/WV mouse Sertoli cell was distributed around the nucleus and extended towards the tubular lumen similar to the normal mouse. Its extension within the Sertoli cell trunk, however, was restricted to a lesser degree as compared with that in the normal. Thus, the subcellular Sertoli cell and the distribution of F-actin and intermediate filaments (vimentin) in the W/WV mouse Sertoli cell seemed not to be strikingly affected by lack of spermatogenic cells, suggesting minimal influence of germ cells on Sertoli cell cytology and cytoskeleton.     

A role for γS-crystallin in the organization of actin and fiber cell maturation in the mouse lens

γS-crystallin (γS) is a highly conserved component of the eye lens. To gain insights into the functional role(s) of this protein, the mouse gene (Crygs) was deleted. Although mutations in γS can cause severe cataracts, loss of function of γS in knockout (KO) mice produced no obvious lens opacity, but was associated with focusing defects. Electron microscopy showed no major differences in lens cell organization, suggesting that the optical defects are primarily cytoplasmic in origin. KO lenses were also grossly normal by light microscopy but showed evidence of incomplete clearance of cellular organelles in maturing fiber cells. Phalloidin labeling showed an unusual distribution of F-actin in a band of mature fiber cells in KO lenses, suggesting a defect in the organization or processing of the actin cytoskeleton. Indeed, in wild-type lenses, γS and F-actin colocalize along the fiber cell plasma membrane. Relative levels of F-actin and G-actin in wild-type and KO lenses were estimated from fluorescent staining profiles and from isolation of actin fractions from whole lenses. Both methods showed a two-fold reduction in the F-actin/G-actin ratio in KO lenses, whereas no difference in tubulin organization was detected. In vitro experiments showed that recombinant mouse γS can directly stabilize F-actin. This suggests that γS may have a functional role related to actin, perhaps in 'shepherding' filaments to maintain the optical properties of the lens cytoplasm and normal fiber cell maturation.     

Role of lumican in the corneal epithelium during wound healing

Lumican regulates collagenous matrix assembly as a keratan sulfate proteoglycan in the cornea and is also present in the connective tissues of other organs and embryonic corneal stroma as a glycoprotein. In normal unwounded cornea, lumican is expressed by stromal keratocytes. Our data show that injured mouse corneal epithelium ectopically and transiently expresses lumican during the early phase of wound healing, suggesting a potential lumican functionality unrelated to regulation of collagen fibrillogenesis, e. g. modulation of epithelial cell adhesion or migration. An anti-lumican antibody was found to retard corneal epithelial wound healing in cultured mouse eyes. Healing of a corneal epithelial injury in Lum(-/-) mice was significantly delayed compared with Lum(+/-) mice. These observations indicate that lumican expressed in injured epithelium may modulate cell behavior such as adhesion or migration, thus contributing to corneal epithelial wound healing.     

Temporal relationships of F-actin bundle formation, collagen and fibronectin matrix assembly, and fibronectin receptor expression to wound contraction

Wound contraction can substantially reduce the amount of new tissue needed to reestablish organ integrity after tissue loss. Fibroblasts, rich in F-actin bundles, generate the force of wound contraction. Fibronectin-containing microfibrils link fibroblasts to each other and to collagen bundles and thereby provide transduction cables across the wound for contraction. The temporal relationships of F-actin bundle formation, collagen and fibronectin matrix assembly, and fibronectin receptor expression to wound contraction have not been determined. To establish these relationships, we used a cutaneous gaping wound model in outbred Yorkshire pigs. Granulation tissue filled approximately 80% of the wound space by day 5 after injury while wound contraction was first apparent at day 10. Neither actin bundles nor fibronectin receptors were observed in 5-d wound fibroblasts. Although fibronectin fibrils were assembled on the surfaces of 5-d fibroblasts, few fibrils coursed between cells. Day-7 fibroblasts stained strongly for nonmuscle-type F-actin bundles consistent with a contractile fibroblast phenotype. These cells expressed fibronectin receptors, were embedded in a fibronectin matrix that appeared to connect fibroblasts to the matrix and to each other, and were coaligned across the wound. Transmission EM confirmed the presence of microfilament bundles, cell-cell and cell-matrix linkages at day 7. Fibroblast coalignment, matrix interconnections, and actin bundles became more pronounced at days 10 and 14 coinciding with tissue contraction. These findings demonstrate that granulation tissue formation, F-actin bundle and fibronectin receptor expression in wound fibroblasts, and fibroblast-matrix linkage precede wound contraction.     

Cultivation of the retinal pigment epithelium in the cavity of the lentectomized eye of newts

A study was made of proliferative activity and transdifferentiation of the cells of retinal pigment epithelium (RPE) cultivated in the cavity of the lensectomized eye of adult newt. Implantation of the newt RPE together with vascular membrane and scleral coat resulted in the regeneration of retina. In this process the character of changes in the proliferative activity of RPE and differentiation of retinal cells were the same as in the regeneration of retina in situ. RPE implanted with the vascular membrane alone, despite a high level of proliferation during the first ten days of cultivation, no differentiated retina was formed. Possible causes of these differences are discussed, and the comparison is made of the data obtained with those on RPE cultivation in vitro. After lens removal, with RPE implants present in the eye cavity, in addition to the regenerated lens, 2-3 extra lenses and retina were formed from the cells of the inner layer of the recipient's dorsal iris. Also some cases were revealed of lens formation from the cells of ventral iris. With a complete detachment of the recipient's retina (an after-effect of transplantation) a second differentiated retina regenerated in situ from the recipient's RPE cells.     

Analysis of lens cell fate and eye morphogenesis in transgenic mice ablated for cells of the lens lineage

Transgenic mice carrying the diphtheria toxin A gene driven by mouse gamma 2-crystallin promoter sequences manifest microphthalmia due to ablation of fiber cells in the ocular lens. Here we map ablation events in the lens by crossing animals hemizygous for the ablation construct with transgenic mice homozygous for the in situ lacZ reporter gene driven by identical gamma 2-crystallin promoter sequences. By comparing the spatial distribution of lacZ-expressing cells and the profile of gamma-crystallin gene expression in the lenses of normal and microphthalmic offspring, the contributions of specific cell types to lens development were examined. The results suggest that phenotypically and developmentally distinct populations of lens fiber cells are able to contribute to the lens nucleus during organogenesis. We also show that dosage of the transgene and its site of integration influence the extent of ablation. In those mice homozygous for the transgene and completely lacking cells of the lens lineage, we show that the sclera, cornea, and ciliary epithelium are reduced in size but, otherwise, reasonably well formed. In contrast, the anterior chamber, iris, and vitreous body are not discernible while the sensory retina is highly convoluted and extensively fills the vitreous chamber.     

Integrin-β4 regulates the dynamic changes of phenotypic characteristics in association with epithelial-mesenchymal transition (EMT) and RhoA activity in airway epithelial cells during injury and repair

Background: In airway disease such as asthma a hyperactive cellular event of epithelial-mesenchymal transition (EMT) is considered as the mechanism of pathological airway tissue remodeling after injury to the airway epithelium. And the initiation of EMT in the airways depends on the epithelial disruption involving dissolution and/or destabilization of the adhesive structures between the cells and ECM. Previously, we have shown that integrin-β4, an epithelial adhesion molecule in bronchial epithelium is an important regulator of cell proliferation and wound repair in human airway epithelial cells. Therefore, in this study we aimed to investigate whether integrin-β4 also regulates EMT phenotypes during injury and repair in airway epithelial cells of both wild type/integrin-β4^-/- mice in vivo and cultured cells treated with integrin-β4/nonsense siRNA in vitro.Methods: We induced injury to the airway epithelial cells by either repeated exposure to ozone and mechanical scratch wound, and subsequently examined the EMT-related phenotypic features in the airway epithelial cells including biomarkers expression, adhesion and cytoskeleton reorganization and cell stiffness.Results: The results show that in response to injury (ozone exposure/scratch wound) and subsequent spontaneous repair (ozone withdrawal/wound healing) both in vivo and in vitro, the airway epithelial cells underwent dynamic changes in the epithelial and mesenchymal biomarkers expression, adhesion and cytoskeleton structures as well as cell stiffness, all together exhibiting enhanced EMT phenotypic features after injury and reversal of the injury-induced effects during repair. Importantly, these injury/repair-associated EMT phenotypic changes in airway epithelial cells appeared to be dependent on integrin-β4 expression. More specifically, when integrin-β4 was deficient in mice (integrin-β4^-/-) the repair of ozone-injured airway epithelium was impaired and the recovery of ozone-enhanced EMT biomarkers expression in the airway epithelium was delayed. Similarly, in the scratch wounded airway epithelial cells with integrin-β4 knockdown, the cells were impaired in all aspects related to EMT during wound and repair including cell proliferation, wound closure rate, adhesion and cytoskeleton protein expression (vinculin and vimentin), mesenchymal-like F-actin reorganization, cell stiffness and RhoA activation.Conclusion: Taken together, these results suggested that integrin-β4 may be essential in regulating the effects of injury and repair on EMT in airway epithelial cells via influencing both the cell adhesion to ECM and cells'' physical phenotypes through RhoA signaling pathway.     

Ultrastructure of traumatic cataractogenesis in the frog: a comparison with mouse and human lens.

Normal and needle-punctured lenses of Rana pipiens were examined with the electron microscope in order to characterize the sequence of ultrastructural changes that follow the injury over a 5-month period. Results were compared with those obtained previously in experimentally injured mouse and accidentally injured human lenses. The normal adult frog lens was found to have a morphology similar to that of mammalian lenses. As in the human, frog lens epithelial cells contained scattered microfilaments and were connected by desmosomes and gap junctions. They differed from mouse cells, which had been shown to lack desmosomes and to have microfilaments organized into dense bundles. These differences are postulated to be related to the degree of accommodative deformation of the lens displayed by these species. After injury, cellular debris and fibrin, accumulated in the wound, were phagocytized by extrinsic cells derived from the blood and ocular tissues. Leucocytes, pigmented cells and fibroblasts remained in the wound for eight weeks, along with epithelial cells which proliferated and migrated from the wound margins.Epithelial cells showed an increase in those organelles associated with protein synthesis and transport, and in microfilaments. In cataractous lenses, epithelial cells showed changes in matrix, and lens fibers became organized into smaller, denser compressed units. At five months, considerable healing had taken place, but localized opacities persisted in many frog lenses.     

Mechanism of repair of lenticular wounds in Rana pipiens. I. Role of cell migration

The earliest visible changes that occur in the normal organization of the lens epithelium after a penetrating wound in the lens suggest that passage of an injury stimulus outward from the wound occurs within the first half day after injury: changes in normal tissue architecture appear near the wound at six hours and move outward to involve the proliferative zone by 12 hours. This is followed by migration of cells toward the wound. There is a slight increase in cell number in the proliferative zone within the first day, followed at later intervals by a decrease there and a concomitant increase in cell number adjacent to the wound. After a pre-injury injection of H³-TdR (or I¹²⁵-UdR), labeled cells that had incorporated the precursor in the normal proliferative zone were found progressively closer to the wound with increasing time. Only the cells which incorporated the radioactive tracer could be followed, but it is likely that cells in the central areas also migrated toward the wound since they showed spindling and superimposition. Migration of cells into the wound margins is an important phase of wound closure which begins long before the major productions of new cells by mitosis.     

The pulling,pushing and fusing of lens fibers

Lens development and differentiation are intricate and complex processes characterized by distinct molecular and morphological changes. The growth of a transparent lens involves proliferation of the epithelial cells and their subsequent differentiation into secondary fiber cells. Prior to differentiation, epithelial cells at the lens equator exit from the cell cycle and elongate into long, ribbon-like cells. Fiber cell elongation takes place bidirectionally as fiber tips migrate both anteriorly and posteriorly along the apical surface of the epithelium and inner surface of the capsule, respectively. The differentiating fiber cells move inward from the periphery to the center of the lens on a continuous basis as the lens grows throughout life. Finally, when fiber cells reach the center or suture line, their basal and apical tips detach from the epithelium and capsule, respectively, and interlock with cells from the opposite direction of the lens and form the suture line. Further, symmetric packing of fiber cells and degradation of most of the cellular organelle during fiber cell terminal differentiation are crucial for lens transparency. These sequential events are presumed to depend on cytoskeletal dynamics and cell adhesive interactions; however, our knowledge of regulation of lens fiber cell cytosketal reorganization, cell adhesive interactions and mechanotransduction, and their role in lens morphogenesis and function is limited at present. Recent biochemical and molecular studies have targeted cytoskeletal signaling proteins, including Rho GTPases, Abl kinase interacting proteins, cell adhesion molecules, myosin II, Src kinase and phosphoinositide 3-kinase in the developing chicken and mouse lens and characterized components of the fiber cell basal membrane complex. These studies have begun to unravel the vital role of cytoskeletal proteins and their regulatory pathways in control of lens morphogenesis, fiber cell elongation, migration, differentiation, survival and mechanical properties.     

Integration of Single and Multicellular Wound Responses

Single cells and multicellular tissues rapidly heal wounds. These processes are considered distinct, but one mode of healing—Rho GTPase-dependent formation and closure of a purse string of actin filaments (F-actin) and myosin-2 around wounds—occurs in single cells [1] and [2] and in epithelia [3], [4], [5], [6], [7], [8], [9] and [10]. Here, we show that wounding of one cell in Xenopus embryos elicits Rho GTPase activation around the wound and at the nearest cell-cell junctions in the neighbor cells. F-actin and myosin-2 accumulate at the junctions and around the wound itself, and as the resultant actomyosin array closes over the wound site, junctional F-actin and myosin-2 become mechanically integrated with the actin and myosin-2 around the wound, forming a hybrid purse string. When cells are ablated rather than wounded, Rho GTPase activation and F-actin accumulation occur at cell-cell junctions surrounding the ablated cell, and the purse string closes the hole in the epithelium. Elevation of intracellular free calcium, an essential upstream signal for the single-cell wound response [2] and [11], also occurs at the cell-cell contacts and in neighbor cells. Thus, the single and multicellular purse string wound responses represent points on a signaling and mechanical continuum that are integrated by cell-cell junctions.     

Using genetically engineered mice for radiation research

The laboratory mouse has been used for many decades as a model system for radiation research. Recent advances in genetic engineering now allow scientists to delete genes in specific cell types at different stages of development. The ability to manipulate genes in the mouse with spatial and temporal control opens new opportunities to investigate the role of genes in regulating the response of normal tissues and tumors to radiation. Currently, we are using the Cre-loxP system to delete genes, such as p53, in a cell-type specific manner in mice to study mechanisms of acute radiation injury and late effects of radiation. Our results demonstrate that p53 is required in the gastrointestinal (GI) epithelium to prevent radiation-induced GI syndrome and in endothelial and/or hematopoietic cells to prevent late effects of radiation. We have also used these genetic tools to generate primary tumors in mice to study tumor response to radiation therapy. These advances in genetic engineering provide a powerful model system to dissect both the mechanisms of normal tissue injury after irradiation and the mechanisms by which radiation cures cancer.     

Plasma membrane-cytoskeleton damage in eye lenses of transgenic mice expressing desmin

Immunocytochemistry of eye lens cells from transgenic mice coexpressing desmin and vimentin reveals that the transgenic desmin expression is not uniform. In the same lens, some epithelial and fiber cells overexpress desmin, while in others the desmin gene seems to be silent. Conversely, the endogenous vimentin is always expressed. The concomitant expression of vimentin and desmin results in the assembly of hybrid intermediate filaments (IFs). Moreover, the overexpression of the transgene generates pleomorphic IF assembly and leads to intermingled filamentous whorls and to accumulation of amorphous desmin. The abnormalities of IF assembly induced by the genetic manipulation are correlated with perturbation of the enucleation process in the lens fibers, changes in cell shape, fiber fusion and extensive internalization of the general plasma membrane and junctional domains. The alterations of lens cells described in this study were observed in all transgenic mice examined. The level of expression of the transgene was paralleled by the degree of damage. Our results indicate that proper expression, assembly and membrane interaction of IFs play an important role in the terminal differentiation of the lenticular epithelium into fiber cells. We anticipate that alterations during these processes may initiate cataract formation.     

Evidence for gelsolin as a corneal crystallin in zebrafish

We have shown that gelsolin is one of the most prevalent water-soluble proteins in the transparent cornea of zebrafish. There are also significant amounts of actin. In contrast to actin, gelsolin is barely detectable in other eye tissues (iris, lens, and remaining eye) of the zebrafish. Gelsolin cDNA hybridized intensely in Northern blots to RNA from the cornea but not from the lens, brain, or headless body. The deduced zebrafish gelsolin is approximately 60% identical to mammalian cytosolic gelsolin and has the characteristic six segmental repeats as well as the binding sites for actin, calcium, and phosphatidylinositides. In situ hybridization tests showed that gelsolin mRNA is concentrated in the zebrafish corneal epithelium. The zebrafish corneal epithelium stains very weakly with rhodamine-phalloidin, indicating little F-actin in the cytoplasm. In contrast, the mouse corneal epithelium contains relatively little gelsolin and stains intensely with rhodamine-phalloidin, as does the zebrafish extraocular muscle. We propose, by analogy with the diverse crystallins of the eye lens and with the putative enzyme-crystallins (aldehyde dehydrogenase class 3 and other enzymes) of the mammalian cornea, that gelsolin and actin-gelsolin complexes act as water-soluble crystallins in the zebrafish cornea and contribute to its optical properties.     

Thrombospondin: biosynthesis, distribution, and changes associated with wound repair in corneal endothelium

Thrombospondin is a cell adhesion molecule which interacts via specific domains with a wide array of extracellular matrix components, including fibrinogen, fibrin, fibronectin, collagen, and heparan sulfate proteoglycan. Although this protein has been localized in several human tissues, its presence in corneal tissues had not been previously established. In the present study, we have demonstrated that cultured bovine corneal endothelial cells synthesize thrombospondin and incorporate it into their extracellular matrix. We have also shown immunofluorescently the presence and distribution of thrombospondin in these cultured cells and in the noninjured and injured corneal endothelium in situ. Ultrastructural immunoperoxidase cytochemistry revealed that thrombospondin could be displaced from the cell surface by heparin, but not by keratan sulfate. Confluent cultures of corneal endothelium synthesize and secrete the three cell adhesion proteins laminin, thrombospondin, and fibronectin in the ratios 1:8.2:51.8. Only the laminin B chains were detected in immunoprecipitates. Immunofluorescent studies of these cultured cells, using a polyclonal antiserum raised against purified thrombospondin, revealed a low level of fluorescence associated with the cell layer but a punctate fluorescent pattern at the level of the extracellular matrix. Noninjured corneal endothelium in situ also demonstrated a low level of fluorescence throughout the cell layer. However, this dramatically changed after a circular freeze injury to the tissue. By 24 h after wounding, cells surrounding the injury zone displayed a prominent fluorescence that was still observed at 48 h post-injury. In addition to its increased intracellular fluorescence, thrombospondin was also localized as migration tracks, oriented in the direction of cellular migration into the wound site. Thus, in corneal endothelium, thrombospondin appears to play a major role in injury-induced cell migration in situ along a natural basement membrane.     

A Native-Like Corneal Construct Using Donor Corneal Stroma for Tissue Engineering

Tissue engineering holds great promise for corneal transplantation to treat blinding diseases. This study was to explore the use of natural corneal stroma as an optimal substrate to construct a native like corneal equivalent. Human corneal epithelium was cultivated from donor limbal explants on corneal stromal discs prepared by FDA approved Horizon Epikeratome system. The morphology, phenotype, regenerative capacity and transplantation potential were evaluated by hematoxylin eosin and immunofluorescent staining, a wound healing model, and the xeno-transplantation of the corneal constructs to nude mice. An optically transparent and stratified epithelium was rapidly generated on donor corneal stromal substrate and displayed native-like morphology and structure. The cells were polygonal in the basal layer and became flattened in superficial layers. The epithelium displayed a phenotype similar to human corneal epithelium in vivo. The differentiation markers, keratin 3, involucrin and connexin 43, were expressed in full or superficial layers. Interestingly, certain basal cells were immunopositive to antibodies against limbal stem/progenitor cell markers ABCG2 and p63, which are usually negative in corneal epithelium in vivo. It suggests that this bioengineered corneal epithelium shared some characteristics of human limbal epithelium in vivo. This engineered epithelium was able to regenerate in 4 days following from a 4mm-diameter wound created by a filter paper soaked with 1 N NaOH. This corneal construct survived well after xeno-transplantation to the back of a nude mouse. The transplanted epithelium remained multilayer and became thicker with a phenotype similar to human corneal epithelium. Our findings demonstrate that natural corneal stroma is an optimal substrate for tissue bioengineering, and a native-like corneal construct has been created with epithelium containing limbal stem cells. This construct may have great potential for clinical use in corneal reconstruction.     

Differential expression of the HMGN family of chromatin proteins during ocular development

The HMGN proteins are a group of non-histone nuclear proteins that associate with the core nucleosome and alter the structure of the chromatin fiber. We investigated the distribution of the three best characterized HMGN family members, HMGN1, HMGN2 and HMGN3 during mouse eye development. HMGN1 protein is evenly distributed in all ocular structures of 10.5 days post-coitum (dpc) mouse embryos however, by 13.5dpc, relatively less HMGN1 is detected in the newly formed lens fiber cells compared to other cell types. In the adult, HMGN1 is detected throughout the retina and lens, although in the cornea, HMGN1 protein is predominately located in the epithelium. HMGN2 is also abundant in all ocular structures of mouse embryos, however, unlike HMGN1, intense immunolabeling is maintained in the lens fiber cells at 13.5dpc. In the adult eye, HMGN2 protein is still found in all lens nuclei while in the cornea, HMGN2 protein is mostly restricted to the epithelium. In contrast, the first detection of HMGN3 in the eye is in the presumptive corneal epithelium and lens fiber cells at 13.5dpc. In the lens, HMGN3 remained lens fiber cell preferred into adulthood. In the cornea, HMGN3 is transiently upregulated in the stroma and endothelium at birth while its expression is restricted to the corneal epithelium in adulthood. In the retina, HMGN3 upregulates around 2 weeks of age and is found at relatively high levels in the inner nuclear and ganglion cell layers of the adult retina. RT-PCR analysis determined that the predominant HMGN3 splice form found in ocular tissues is HMGN3b which lacks the chromatin unfolding domain although HMGN3a mRNA is also detected. These results demonstrate that the HMGN class of chromatin proteins has a dynamic expression pattern in the developing eye.