Ординатура / Офтальмология / Английские материалы / Ocular Periphery and Disorders_Dartt, Bex, Amore_2011

development, limiting their use in studying biology of the ocular surface tissues. To circumvent the pitfalls, one of the strategies is conditional gene knock-out that employs Cre–LoxP system to ablate gene in a cell-type-specific manner.

Tissue-specific gene ablation using Cre–LoxP system

The Cre–LoxP system was developed to avoid embryonic lethality as well as to confine the inactivation of the target gene in a cellor tissue-specific manner. Cre is a phage recombinase that specifically deletes any DNA sequence flanked by two LoxP elements. The Cre–LoxP system consists of two mouse lines, one of which uses a tissue-/ cell-type-specific promoter for the expression of Cre. The other mouse line contains a modified genome in which two LoxP elements are inserted in two introns flanking functionally important exon(s) of the targeted gene by gene-targeting techniques. The LoxP-modified (floxed) gene remains fully functional, except in cells expressing Cre that is under the control of a tissue-specific promoter. Thus, the offspring are characterized by the ablation of the gene in a tissue-/cell-type-specific manner. The system allows the inactivation of the target gene in a single cell type and/or a limited number of cell types, depending on the specificity of the promoter, and reduces the probability of embryonic lethality of the experimental mice. Thus, it permits the analysis of physiological and pathophysiological consequences of the genetic alteration in mature animals.

Pitfalls of Cre–LoxP system: Cryptic Cre expression by germ cells during gametogenesis

We have recently discovered that recombination of floxed genes happens during gametogenesis in several Cre/LoxP bitransgenic mouse lines that express tissue-specific Cre outside the gonads, for example, Kera-Cre, Krt12-Cre, and BF1-Cre mice. The frequency of the promiscuous LoxP/ Cre recombination varied in different lines of Cre driver mice and sex of the same driver mice with higher penetrance in male than in female double transgenic mice as well (Table 1). Polymerase chain reaction (PCR) and recombination analysis demonstrate recombination of floxed allele occurs during transition from spermatogonia (diploid) to primary spermatocyte (tetraploid) in testis. Thus, target floxed allele(s) are ubiquitously ablated in Cre/LoxP mice intended for tissue-specific gene deletion. Increasing evidence indicates that many cell-type- specific genes in adults are promiscuously expressed in gonad during gametogenesis. Therefore, the promiscuous expression of Cre recombinase driven by cell-type- specific promoters should always be examined to avoid misinterpretation resulting from haploid deficiency.

Inducible Cre–LoxP system

To overcome the pitfalls of promiscuous Cre expression during gametogenesis, we have developed a doxycyclineinducible tet-ON system to spatially and temporally ablate gene of interest in ocular surface tissues. This tet-ON system employs triple transgenic mice consisting of an ocularsurface tissue-specific promoter rtTA (Kera-rtTA (KR)),

Krt12-rtTA (K12R), and Pax6OS-rtTA (P6R) (Table 2), and tet-O-Cre (TC) transgenes, as well as an Xf/f (a LoxP-

modified gene). Administration of doxycycline will activate rtTA that subsequently turns on the tet-O-Cre transgenes for the synthesis of Cre recombinase, which then excises the Xf/f gene in an ocular-surface tissue-specific manner (as described below).

Strategies of Cornea-Specific Genetic

Modification: Transgenic and Knock-Out/

Knock-In Mice

Identification of Ocular-Surface Tissue-Specific

Promoter

The availability of ocular-surface tissue-specific promoter is a prerequisite for the preparation of experimental animal models in which genetic modifications are made to study the consequence of the loss and/or gain of functions of genes in ocular surface tissues, that is, cornea, conjunctiva, lacrimal glands, and eyelids. To date, our laboratory has identified and characterized two corneaspecific genes: keratin 12 (Krt12) and keratocan (Kera) genes of corneal epithelium and stroma, respectively, and used transgenesis and gene-targeting techniques to create mouse lines in which the gene functions are altered in a corneaspecific manner. However, it should be noted that many systemic genetically modified mouse lines that were created by conventional systemic gene-targeting techniques and/or transgenesis with non-ocular-surface tissue-specific promoters also manifested pathogenesis in ocular surface tissues. Naturally, these mouse lines often exhibited pathogenesis in other tissues than ocular surface tissues as well. To date, only a few promoters that exhibit ocular surface tissue specificity have been identified, for example, keratocan (Kera), keratin 12 (Krt12), and modified Pax6 promoters.

It should be noted that there is no tissue-specific promoter available that can be used to create mouse lines in which genetic modifications are limited to conjunctiva, cornea endothelium, and lacrimal gland.

Stromal keratocyte-specific promoter

Keratan sulfate proteoglycans (KSPGs) play a pivotal role in the development and maintenance of corneal transparency. Keratocan, lumican, and mimecan (osteoglycin) are the major KSPGs in vertebrate corneas. We have cloned both the mouse keratocan gene and its complementary DNA (cDNA). The mouse keratocan gene spans approximately 6.5 kb of the mouse genome and contains three exons and two introns. Northern blotting and in situ hybridization were employed to examine keratocan gene expression during mouse development. Unlike lumican gene, which is expressed by many tissues other than cornea, keratocan messenger RNA (mRNA) is more selectively expressed in the corneal tissue of the adult mouse. During embryonic development, keratocan mRNA was first detected in periocular mesenchymal cells migrating toward developing corneas on embryonic day 13.5 (E13.5). Its expression was then gradually restricted to corneal stromal cells in between E14.5 and E18.5. Interestingly, keratocan mRNA can be detected in scleral cells of E15.5 embryos, but not in E18.5 embryos. In adult eyes, keratocan mRNA can be detected in corneal keratocytes, but not in scleral cells.

To identify and characterize a keratocyte-specific promoter, we have cloned a 3.2-kb genomic DNA fragment 50of the mouse Kera gene, which has promoter activities in driving the expression of reporter genes, for example, b-galactosidase (b-Gal), reverse tetracycline transcription activator (rtTA), Cre recombinase (Cre), by migrating periocular mesenchymal cells of neural crest origin during embryonic development and by cornea stromal keratocytes in adults. For example, in adult Kera-bGal transgenic mice, b-galactosidase activity was detected

only in cornea, not in other tissues (e.g., lens, retina, sclera, lung, heart, liver, diaphragm, kidney, and brain). In contrast, during ocular development, the spatial–temporal expression patterns of bGal reporter gene recapitulated that of endogenous Kera expression in mice. Using X-Gal staining, strong b-galactosidase activity was first detected in periocular tissues of E13.5 embryos, and restricted to corneal keratocytes at E14.5 and thereafter. Interestingly, in addition to cornea, b-galactosidase activity was transiently found in some nonocular tissues, that is, ears, snout, and limbs of embryos of E13.5 and E14.5, but was no longer detected in those tissues of E16.5 embryos. The transient expression of endogenous keratocan in nonocular tissues during embryonic development was confirmed by in situ hybridization. Taken together, the observations suggest that the 3.2-kb Kera promoter contains sufficient cis-regulatory elements to drive heterologous minigene expression in cells expressing keratocan.

Corneal-epithelium-specific promoter

Keratins are a group of water-insoluble proteins that form 10-nm intermediate filaments in all epithelial cells. Approximately 30 different keratin molecules have been identified, which can be divided into acidic and basic neutral subfamilies. In vivo, a basic keratin is usually coexpressed and paired with a particular acidic keratin. The expression of keratin pairs is tissue specific, differentiation dependent, and developmentally regulated. Expression of keratin 3/keratin 12 pair has been found in human, bovine, guinea pig, rabbit, and chicken corneas and is regarded as a marker for corneal-type epithelial differentiation. The expression of keratin 12 is restricted to the corneal epithelium. We have identified and cloned the corneal-epithelium-specific K12 keratin (Krt12) gene. Using gene gun to deliver Krt12-LacZ reporter gene constructs to rabbit corneal epithelium, we have identified the cis-regulatory element that is sufficient and necessary for corneal-epithelium-specific expression of the reporter genes. However, the use of conventional transgenesis techniques failed to identify a functional Krt12 promoter that was capable of driving the expression of reporter genes, for example, b-Gal, chloramphenicol acetyl transferase (CAT), and green fluorescent proteins (GFPs) in corneal epithelium. Transgenesis using lentiviral Krt12lacZ vectors had successfully generated mouse lines that expressed the reporter LacZ gene by corneal epithelium, but multiple insertions by the use of lentivirus vector compromised the efficiency of obtaining stable transgenic mice. To overcome the pitfalls, a gene-targeting construct containing an internal ribosomal entry site–reverse tetracycline transcription activator (IRES-rtTA) cassette was inserted into the Krt12 allele to produce knock-in Krt12-rtTA and Krt12-Cre driver mouse lines through gene-targeting techniques (see below).

Ocular-surface epithelium-specific Pax6 promoter (Pax6OS)

Pax6 is a regulatory gene with restricted expression and essential functions in the developing eye and pancreas and distinct domains of the central nervous system (CNS). Three conserved transcription start sites (P0, P1, and a) have been identified in the murine Pax6 locus. Furthermore, the use of transgenic mouse technology has identified the cis-regulatory elements controlling the tissue-specific expression of Pax6. Specifically, a 107-bp enhancer and a 1.1-kb sequence within the 4.6-kb untranslated region upstream of exon 0 are required to mediate Pax6 expression in the lens, cornea, lacrimal gland, conjunctiva, or pancreas. Another 530-bp enhancer fragment located downstream of the Pax6 translational start site is required for expression in the neural retina, the pigment layer of the retina, and the iris. Finally, a 5-kb fragment located between the promoters P0 and P1 can mediate expression into the dorsal telencephalon, the hindbrain, and the spinal cord. The identified Pax6/cis- essential elements are highly conserved in pufferfish, mouse, and human DNA and contain binding sites for several transcription factors indicative of the cascade of control events. Corresponding regulatory elements from pufferfish are able to mimic the reporter expression in transgenic mice. Thus, the results indicate a structural and functional conservation of the Pax6 regulatory elements in the vertebrate genome. This segment of the mouse Pax-6 gene 50 flanking region is necessary and sufficient for reporter construct expression in components of the eye derived from non-neural ectoderm, for example, lens epithelium and ocular surface epithelium. This transcriptional control element has been used to prepare Pax6OS- rtTA (P6R) driver mouse lines for transgene expression.

Ocular Surface Tissue-Specific Driver Mouse

Lines

Corneal stroma-specific mouse lines

The mouse keratocan gene (Kera) expression tracks the corneal morphogenesis during eye development and becomes restricted to keratocytes of the adult, implicating a cornea-specific gene regulation of the mouse Kera. We have identified and cloned a 3.2-kb genomic DNA fragment 50 of the mouse Kera gene, which is capable of driving the expression of LacZ reporter gene that recapitulates the expression patterns of Kera. The keratocan promoter has been used to create transgenic mice, for example, Kera-biglycan, and Kera-Cre and Kera-rtTA driver mouse lines. The Kera-rtTA (KR) mice can be used to create bitransgenic Kera-rtTA/tet-O-reporter construct and tritransgenic KR/TC (tet-O-Cre)/Xf/f (X, gene of interest) mice for keratocyte-specific doxycycline-inducible transgene expression and gene ablation (Table 2).

Corneal-epithelium-specific mouse lines

A similar strategy employing Cre–LoxP and tet-ON systems with keratin 12 promoter (Krt12) would allow us to prepare experimental mouse lines that express transgenes and ablate genes in a corneal-epithelium-specific manner. Thus, for many years we tried to identify and isolate a functional keratin 12 promoter for the preparation of corneal-epithelium-specific transgenic and Cre–LoxP mouse lines without success. To circumvent these difficulties, we have prepared mouse lines carrying IRES-Cre and IRES-rtTA reporter genes into the Krt12 locus through a targeted knock-in strategy.

Unlike prokaryotes, most mammalian mRNAs are monocistronic (i.e., one message encodes one protein). However, some viral mRNAs and/or translationally regulated mRNAs (e.g., fibroblast growth factor 2 (FGF-2) and

c-myc) are bicistronic in that they have IRESs in the mRNA that allows a second initiation of translation after the stop codon of the first reading frame for the synthesis of a second protein from the mRNA. Inclusion of such IRES elements in the reporter gene constructs has allowed the generation of transgenic mouse lines that express two proteins encoded by a single transgene.

To create corneal-epithelium-specific gene ablation in mice, a targeting construct containing intron 2 to exon 8 of Krt12 gene was prepared in which an IRES-Cre and the phosphoglycerate kinase-neomyocin resistance gene (PGK-Neo) reporter genes were inserted right after the stop codon within exon 8 as shown in Figure 1. Germ line chimera mice were obtained through conventional gene-targeting techniques using embryonic stem cells. The K12-Cre mice were crossed with reporter

(b)

(c)5 probe

Tg(CAG-Bgeo/ALPP)1Lbe (ZAP) mice that harbor a transgene containing a chicken b-actin promoter, a floxed LacZ gene (two LoxP elements flanked at 50 and 30 ends of LacZ), and followed by alkaline phosphatase (AP) gene in tandem. The Cre activity was assessed by the detection of LacZ and expression of AP in corneas of the offspring as shown in Figure 2. Thus, the K12-Cre knock-in mice can be used to create mouse lines in which any floxed genes are ablated in corneal epithelium. Thus, it will allow us to investigate the role of a gene in corneal morphogenesis and homeostasis through the loss of function without threatening the life of the experimental animals. Using a similar strategy, a gene-targeting construct containing an IRES-rtTA cassette was inserted into the Krt12 allele to

produce a knock-in Krt12rtTA/w mouse line through genetargeting techniques (Figure 1). The Krt12rtTA/w knock-in

mice were bred with tet-O-LacZ reporter mice to obtain Krt12rtTA/w/tet-O-LacZ bitransgenic mice. The corneal-

epithelium-specific expression of the LacZ gene was induced in bitransgenic mice by administration of doxycycline in the drinking water and chow as shown in Figure 3.

To further expand the usefulness of Krt12-rtTA knock-in mice, a tritransgenic Krt12rtTA/w/tet-O-Cre/ZEG mouse

line was prepared in which the expression of enhanced green fluorescence protein (EGFP) was activated by feeding mice doxycycline that subsequently excised the LacZ gene from the ZEG allele and allowed the expression of EGFP. Figure 4 shows the doxycycline-induced

expression of EGFP by corneal epithelial cells of Krt12rtTA/w/TC/ZEG mice. ZEG is a transgenic mouse

line that has dual reporter genes of a LacZ flanked by LoxP, followed by EGFP driven by a chicken actin promoter. Chicken actin promoter drives the expression of LacZ in all cells except those of which express Cre and show green fluorescence due to the excision of LacZ and expression of EGFP. Using the strategies, we have prepared several mouse lines that can be used to overexpress transgenes and ablate genes of interest in a cornea-specific manner as summarized in Table 2.

Ocular surface tissue-specific Pax6OS-rtTA mouse line

We have recently prepared an ocular surface epitheliumrtTA (Pax6OS-rtTA) driver mouse line with cis-regulatory elements of the 4.6-kb un-translated region upstream of exon 0 of Pax6, which mediate Pax6 expression in the lens, cornea, lacrimal gland, conjunctiva, or pancreas. The

bitransgenic Pax6OS-rtTA/Tet-O-EGFP mice express EGFP upon doxycycline induction (data not shown). This Pax6OS-rtTA mouse line is useful for generating experimental mice of overexpression of tet-O-transgene

and ablation of floxed gene of tritransgenic (Pax6OS-rtTA/ tet-O-Cre/X f/f) mice.

Roles of Growth Factors on Ocular Surface Tissue Morphogenesis during Development and Wound Healing Elucidated from Transgenic Mice

Corneal morphogenesis during eye development of vertebrates involves the differentiation of cells from surface ectoderm and the migration of periocular mesenchymal cells of neural crest origin. The differentiation of surface ectoderm gives rise to corneal and conjunctival epithelia of the ocular surface as well as to glandular epithelium,

+Dox

Day 0

Day 2

Day 4

Day 7

Day 14

−Dox

for example, lacrimal and meibomian glands. The mesenchymal cells of neural crest origin become corneal endothelial cells and keratocytes, and the stromal cells of other ocular surface tissues, that is, eyelids, iris, ciliary body, and trabecular meshworks.

The corneal epithelium synthesizes components of extracellular matrix (ECM) for the formation of primary stroma when the lens detaches from the ectoderm during development. In vertebrate corneal development, the first wave of mesenchymal cells that migrate underneath the primary stroma forms the endothelium. The mesenchymal cells of the second wave invade the primary stroma and become keratocytes, which are responsible for

the formation of secondary stroma of adult vertebrates. The third waves of mesenchymal cells contribute to the stromas of eyelids, iris, ciliary body, and trabecular meshworks. This orderly cellular migration and differentiation are controlled by cues from various cytokines and the components of ECM, which are under constant remodeling during embryonic development. For example, members of the transforming growth factor beta (TGFb) superfamily play pivotal roles in embryonic development. However, the precise cytokines and their functions that modulate corneal morphogenesis remain unknown. It is very likely that cytokine signaling may modulate the expression of specific transcription factors that contribute

to this orderly corneal morphogenesis during development or vice versa.

TGFb Receptor Signaling Pathways during Development and Corneal Wound Healing

Role of TGFb2 on development

TGFb has a pivotal role in embryonic development. In mammals, three isoforms of TGFb (b1, -2, and -3) are known. Members of TGFb family are multifunctional cytokines involved in development, tissue repair, and other physiological or pathologic processes. Among the knock-out mice of the three Tgfb isoforms, only Tgfb2–/– mice exhibit ocular pathology of thin corneal stroma, absence of corneal endothelium, fusion of cornea to lens, a phenotype resembling Peter’s and Axenfeld anomaly in humans, and accumulation of hyaline cells in vitreous. Delayed appearance of macrophages in ocular tissues was observed in Tgfb2–/–mice. Malfunctioning macrophages may account for accumulation of cell mass in vitreous of Tgfb2-null mice. InTgfb2–/–mice, fewer keratocytes were found in stroma that have a decreased accumulation of ECM; for example, lumican, keratocan, and collagen I were greatly diminished. The thinner stroma resulting from decreased ECM synthesis may account for the decreased cell number in the stroma of Tgfb2-null mice. The absence of TGFb2 did not compromise corneal epithelial cell proliferation, nor enhance apoptosis. Keratin 12 expression was not altered in Tgfb2–/–mice, implicating that TGFb signaling is not essential for cornea-type epithelium differentiation. This suggestion is further supported by the

observation that ablation of TGFb type II receptor in Krt12Cre/Cre/Tbr2f/f mice does not cause corneal epithelium

anomaly (our unpublished observation).

Role of TGF-b signaling in wound healing of corneal epithelium

Corneal epithelial defects must be rapidly resurfaced to avoid microbial infection and further damage to the underlying stroma. Epithelial healing is achieved by migration of the epithelial sheet to cover the denuded surface and enhanced cell proliferation to reestablish the epithelial stratification quickly after resurfacing. It is of interest to note that in the early phase of healing only one of the two cellular responses, cell migration, takes place, whereas cell proliferation is suppressed. Although cell migration promotes rapid re-epithelialization, the cessation of cell proliferation may impede healing if such cessation is prolonged.

Various growth factors, including TGFb, orchestrate the behavior of healing corneal epithelium: for example, cell migration and/or proliferation, cell death, and protein synthesis. It has been demonstrated that the TGFb isoforms and their receptors are present in corneal and limbal epithelia and other supporting tissues (e.g., conjunctiva and tear fluid). Therefore, it has long been

speculated that the TGFb isoforms play pivotal roles in maintaining corneal homeostasis in a paracrine and autocrine fashion as TGFb inhibits cell proliferation of cultured keratinocytes and corneal epithelial cells in vitro, thus it may suppress corneal epithelial cell proliferation in vivo. This notion is further supported by the observation in which the administration of anti-TGFb-neutralizing antibodies reduces scar tissue formation in injured corneas. Recently, it was shown that epithelial debridement causes an upregulation of TGFb receptor expression on migrating corneal epithelial cells, suggesting that this ligand may have a pivotal role in modulation of functions of migrating corneal epithelial cells during wound healing.

We recently examined the roles of TGF-b signaling pathways in regulating cell migration and proliferation of the healing of corneal epithelium debridement. TGF-b type II receptor (Tbr2) floxed mice were bred with Krt12-Cre mice to generate bitransgenic mice in which the Tbr2 gene was disrupted selectively in the corneal epithelial cells. Corneal epithelial debridement

(2 mm in diameter) was created in 2-month-old bitransgenic Krt12Cre/Cre/Tbr2 f/f mice and their littermates as controls Krt12Cre/Cre/Tbr2f/w and Krt12Cre/Cre/Tbr2w/w.

Our results indicated that corneal epithelium of Krt12Cre/ Cre/Tbr2 f/f mice exhibited delayed healing of debridement in comparison to that of control littermates that were

heterozygous floxed and wild-type Tbr2. The naive uninjured corneal epithelium of Krt12Cre/Cre/Tbr2 f/f mice

exhibited higher cell proliferative activities than controls as determined by BrdU incorporation. It is of interest to note that corneal epithelium debridement caused cessation of epithelial cell proliferation of all experimental mice in 6–12 h, irrespective of whether the Tbr2 was ablated or not.

Immunohistochemistry using anti-phospho-p38 mitogen-activated protein kinase (MAPK) revealed, following epithelium debridement, that the activation of p38MAPK was seen in 6 h of injury in control mice. In contrast, phosphorylation and nuclear translocation of p38MAPK were markedly delayed in mice lacking Tbr2 in corneal epithelium compared to control mice. The observation is consistent with results of our previous studies, in which we demonstrated that addition of p38MAPK inhibitors blocked cell migration more markedly than neutralizing anti-TGFb antibody and enhanced cell proliferation in the injured corneal epithelium. The observation suggested that p38MAPK, but not the mothers against decapentaplegic (Smad, another signaling cascade active by TGFb) cascade, plays a major role in promoting cell migration and in suppressing cell proliferation in migrating epithelium.

Role of FGF7 in Maintenance of Corneal

Homeostasis

During mammalian embryogenesis, epithelial– mesenchymal interactions play a determining role in

normal tissue patterning and development. FGF7 (also known as keratinocyte growth factor, KGF), a member of the FGF family, is a mesenchymally derived mitogen for epithelial cells in regulating epithelial cell behavior, as the FGF7 receptor is expressed by epithelial cells. Overexpression of human FGF7 by a crystalline promoter in the eye caused hyperproliferation of embryonic corneal epithelial cells and their subsequent differentiation into functional lacrimal gland-like tissues. This indicates that stimulation of the FGF7 receptor early in development, in surface ectoderm normally destined to form corneal epithelium, is sufficient to alter the fate of these cells. This further suggests that the correct spatial and temporal expression of FGFs plays a critical role in normal lacrimal

gland induction. It is of interest to note that overexpression of FGF7 in Krt12rtTA/wt/tetO-FGF7 bitransgenic mice

by doxycycline induction during embryonic development resulted in the formation of vascularized cornea with epithelium hyperplasia, resembling human ocular surface squamous neoplasia (OSSN) as shown in Figure 5. The phenotype variations of the two mouse models can be explained by the fact that a-crystalline expression by lens commences at E10–E11.5, whereas the expression of keratin by corneal epithelium begins at E14.5.

EGFR/EGF, TGFa Signaling Pathways on Eyelids Morphogenesis

Transgenic Kera-Bgn mice overexpressing biglycan, driven by keratocan promoter under the keratocan promoter, exhibit exposure keratitis and premature eye opening from noninfectious eyelid ulceration due to perturbation

of eyelid muscle formation and the failure of meibomiangland formation. In addition, in vitro analysis revealed that biglycan binds to TGFa, thus interrupting epidermal growth factor receptor (EGFR) signaling pathways essential for mesenchymal cell migration induced by eyelid epithelium. The defects of TGFa signaling by excess biglycan were further augmented by the interruption of the autocrine or paracrine loop of the EGFR signaling pathway of heparin-binding (HB)-EGF expression elicited by TGFa.10 These results are consistent with the notion that under physiological conditions, biglycan secreted by mesenchymal cells serves as a regulatory molecule for the formation of a TGFa gradient serving as a morphogen of eyelid morphogenesis (Figure 6).

MEK kinase 1 (MEKK1) is an MAPK originally identified as an upstream activator for several MAPK pathways. During mouse embryogenesis, MEKK1 controls cell shape changes and formation of actin stress fibers that are required for sealing epidermis in the embryos in a process known as eyelid closure. MEKK1-null mice display eyeopen at birth (EOB), a phenotype found also in mice impaired in activin, a subgroup of the TGFb family, or in EGFR or its ligand TGFa, or in transcription factor c-Jun. Molecular analyses have revealed at least two signaling mechanisms in the control of eyelid closure. One is originated from the activins and is transduced through MEKK1, leading to transcription-independent actin stress fiber formation and transcription-dependent keratinocyte migration. Another is the TGFa/EGFR signal that is transduced through an MEKK1-independent pathway to the activation of the extracellular signal-regulated kinase (ERK) MAPK, which also leads to keratinocyte migration.

c-Jun might serve as a connection between the two pathways. As embryonic eyelid closure is a specific morphogenetic process that is easily detectable, genetic mutant mice with EOB will be ideal models to understand the signaling mechanisms in the control of epithelial cell migration and the morphogenetic process of epithelial sheet movement.

Thus, the observation supports the hypothesis that tissue morphogenesis during development is regulated by growth factors and cytokines, and is characterized by constant remodeling of ECM in response to signaling molecules, for example, growth factors, cytokines, and so forth. Proteoglycans that bind growth factors are potential regulators of tissue morphogenesis during embryonic development.

Conclusion: The Clinical Relevance of

Tet-ON Mouse Models in Elucidating

Pathophysiology of Ocular Surfaces

Diseases

Many transgenic and knock-out mice exhibit pathogenesis resembling human ocular surface diseases. Thus, the clinical manifestations of mouse lines can be used as clues for identifying inherited human disease of unknown etiology. However, embryonic lethality and congenital defects of the mouse lines do not allow further examination of the effects of altered genetic functions on pathophysiology of acquired diseases in adults. The difficulties can be overcome by preparing mouse lines of inducible transgene

expression, tissue-specific gene ablation, and inducible tissue-specific gene ablation. The conditional transgenic mouse lines will live normally until the administration of doxycycline, which induces expression of the transgene and/or ablation of gene of interest. Use of these genetically modified mouse lines can simulate the pathophysiology of ocular surface diseases, for example, wound healing, tumorigenesis, and irregular hormone and cytokine signaling that offsets homeostasis in adults.

Acknowledgments

This work was supported by NIH grants EY 10556, EY 11845, and EY 13755, Challenge Grant for Research to Prevent Blindness, Inc., and Unrestricted grant from Ohio Lion Eye Research Foundation.

See also: Conjunctival Goblet Cells; Cornea Overview; Corneal Epithelium: Cell Biology and Basic Science; Corneal Epithelium: Wound Healing Junctions, Attachment to Stroma Receptors, Matrix Metalloproteinases, Intracellular Communications; Gene Therapy for the Cornea, Conjunctiva, and Lacrimal Gland; Lacrimal Gland Hormone Regulation; Lacrimal Gland Overview; Lacrimal Gland Signaling: Neural; Lids: Anatomy, Pathophysiology, Mucocutaneous Junction; Overview of Electrolyte and Fluid Transport Across the Conjunctiva; The Surgical Treatment for Corneal Epithelial Stem Cell Deficiency, Corneal Epithelial Defect, and Peripheral Corneal Ulcer.