Lysyl hydroxylase 3 is required for normal lens capsule formation and maintenance of lens epithelium integrity and fate.

Lens abnormalities are a major cause of reduced vision and blindness. One mechanism that can lead to reduced lens transparency, i.e. cataract, is abnormal behavior of lens epithelial cells (LECs), the precursors of the transparent lens fiber cells. Here we describe a zebrafish mutation causing the embryonic lens epithelium to generate cellular masses comprising partially differentiated lens fiber cells. We identify the mutant gene as plod3, which encodes for Lysyl hydroxylase 3 (Lh3), an enzyme essential for modification of collagens, including Collagen IV, a main component of the lens capsule. We show that plod3-deficient lenses have abnormal lens epithelium from an early developmental stage, as well as abnormal lens capsules. Subsequently, upregulation of TGFß signaling takes place, which drives the formation of lens epithelial cellular masses. We identify a similar phenotype in Collagen IVα5-deficient embryos, suggesting a key role for the defective lens capsule in the pathogenesis. We propose that plod3 and col4a5 mutant zebrafish can serve as useful models for better understanding the biology of LECs during embryonic development and in formation of lens epithelium-derived cataract.

Identification and Ultrastructural Characterization of a Novel Nuclear Degradation Complex in Differentiating Lens Fiber Cells.

An unresolved issue in structural biology is how the encapsulated lens removes membranous organelles to carry out its role as a transparent optical element. In this ultrastructural study, we establish a mechanism for nuclear elimination in the developing chick lens during the formation of the organelle-free zone. Day 12-15 chick embryo lenses were examined by high-resolution confocal light microscopy and thin section transmission electron microscopy (TEM) following fixation in 10% formalin and 4% paraformaldehyde, and then processing for confocal or TEM as described previously. Examination of developing fiber cells revealed normal nuclei with dispersed chromatin and clear nucleoli typical of cells in active ribosome production to support protein synthesis. Early signs of nuclear degradation were observed about 300 µm from the lens capsule in Day 15 lenses where the nuclei display irregular nuclear stain and prominent indentations that sometimes contained a previously undescribed macromolecular aggregate attached to the nuclear envelope. We have termed this novel structure the nuclear excisosome. This complex by confocal is closely adherent to the nuclear envelope and by TEM appears to degrade the outer leaflet of the nuclear envelope, then the inner leaflet up to 500 µm depth. The images suggest that the nuclear excisosome separates nuclear membrane proteins from lipids, which then form multilamellar assemblies that stain intensely in confocal and in TEM have 5 nm spacing consistent with pure lipid bilayers. The denuded nucleoplasm then degrades by condensation and loss of structure in the range 600 to 700 µm depth producing pyknotic nuclear remnants. None of these stages display any classic autophagic vesicles or lysosomes associated with nuclei. Uniquely, the origin of the nuclear excisosome is from filopodial-like projections of adjacent lens fiber cells that initially contact, and then appear to fuse with the outer nuclear membrane. These filopodial-like projections appear to be initiated with a clathrin-like coat and driven by an internal actin network. In summary, a specialized cellular organelle, the nuclear excisosome, generated in part by adjacent fiber cells degrades nuclei during fiber cell differentiation and maturation.

Early stages of induction of anterior head ectodermal properties in Xenopus embryos are mediated by transcriptional cofactor ldb1.

BACKGROUND: Specific molecules involved in early inductive signaling from anterior neural tissue to the placodal ectoderm to establish a lens-forming bias, as well as their regulatory factors, remain largely unknown. In this study, we sought to identify and characterize these molecules. RESULTS: Using an expression cloning strategy to isolate genes with lens-inducing activity, we identified the transcriptional cofactor ldb1. This, together with evidence for its nuclear dependence, suggests its role as a regulatory factor, not a direct signaling molecule. We propose that ldb1 mediates induction of early lens genes in our functional assay by transcriptional activation of lens-inducing signals. Gain-of-function assays demonstrate that the inductive activity of the anterior neural plate on head ectodermal structures can be augmented by ldb1. Loss-of-function assays show that knockdown of ldb1 leads to decreased expression of early lens and retinal markers and subsequently to defects in eye development. CONCLUSIONS: The functional cloning, expression pattern, overexpression, and knockdown data show that an ldb1-regulated mechanism acts as an early signal for Xenopus lens induction.

Deletion of autophagy-related 5 (Atg5) and Pik3c3 genes in the lens causes cataract independent of programmed organelle degradation.

The lens of the eye is composed of fiber cells, which differentiate from epithelial cells and undergo programmed organelle degradation during terminal differentiation. Although autophagy, a major intracellular degradation system, is constitutively active in these cells, its physiological role has remained unclear. We have previously shown that Atg5-dependent macroautophagy is not necessary for lens organelle degradation, at least during the embryonic period. Here, we generated lens-specific Atg5 knock-out mice and showed that Atg5 is not required for lens organelle degradation at any period of life. However, deletion of Atg5 in the lens results in age-related cataract, which is accompanied by accumulation of polyubiquitinated and oxidized proteins, p62, and insoluble crystallins, suggesting a defect in intracellular quality control. We also produced lens-specific Pik3c3 knock-out mice to elucidate the possible involvement of Atg5-independent alternative autophagy, which is proposed to be dependent on Pik3c3 (also known as Vps34), in lens organelle degradation. Deletion of Pik3c3 in the lens does not affect lens organelle degradation, but it leads to congenital cataract and a defect in lens development after birth likely due to an impairment of the endocytic pathway. Taken together, these results suggest that clearance of lens organelles is independent of macroautophagy. These findings also clarify the physiological role of Atg5 and Pik3c3 in quality control and development of the lens, respectively.

MMP2 activity is critical for TGFß2-induced matrix contraction--implications for fibrosis.

PURPOSE: The fibrotic lens disorder posterior capsule opacification (PCO) develops in millions of patients following cataract surgery. PCO characteristics are extensive extracellular matrix (ECM) production and contraction of the posterior lens capsule, resulting in light-scattering ECM modification (wrinkling). The pro-fibrotic cytokine transforming growth factor beta (TGFß) is central to PCO development. This study aimed to elucidate the role of the ECM modulators matrix metalloproteinases (MMPs) in TGFß-mediated PCO formation. METHODS: The human lens epithelial cell-line FHL-124 and human capsular bag models were employed. Gene expression of MMP family members was determined by oligonucleotide microarray and quantitative real-time RT-PCR. MMP2 and MT1-MMP protein levels were analyzed by ELISA and Western blotting, respectively. Matrix contraction was determined using an FHL-124 patch contraction assay; at end-point, cells were stained with Coomassie brilliant blue and area was determined using image analysis software. Cell coverage and wrinkle formation on the posterior capsule were also assessed using human capsular bag models. RESULTS: Active TGFß2 (10 ng/mL) increased gene and protein levels of MMP2 and MT1-MMP and induced matrix contraction in FHL-124 cells. Specific siRNA inhibition of MT1-MMP did not suppress TGFß2-induced matrix contraction. Active TGFß2-mediated contraction was prevented by broad-spectrum MMP inhibitor GM6001 (25 µM), MMP2 siRNA, and MMP2 neutralizing antibody (4 µg/mL). TGFß2-induced wrinkle formation was attenuated in human capsular bags treated with MMP2 neutralizing antibody (20 µg/mL). CONCLUSIONS: MMP2 plays a critical role in TGFß2-mediated matrix contraction, which appears to be independent of MT1-MMP. MMP2 inhibition provides a novel strategy for the treatment of PCO and potentially other fibrotic disorders.

Integrin-linked kinase deletion in the developing lens leads to capsule rupture, impaired fiber migration and non-apoptotic epithelial cell death.

PURPOSE: The lens is a powerful model system to study integrin-mediated cell-matrix interaction in an in vivo context, as it is surrounded by a true basement membrane, the lens capsule. To characterize better the function of integrin-linked kinase (ILK), we examined the phenotypic consequences of its deletion in the developing mouse lens. METHODS: ILK was deleted from the embryonic lens either at the time of placode invagination using the Le-Cre line or after initial lens formation using the Nestin-Cre line. RESULTS: Early deletion of ILK leads to defects in extracellular matrix deposition that result in lens capsule rupture at the lens vesicle stage (E13.5). If ILK was deleted at a later time-point after initial establishment of the lens capsule, rupture was prevented. Instead, ILK deletion resulted in secondary fiber migration defects and, most notably, in cell death of the anterior epithelium (E18.5-P0). Remarkably, dying cells did not stain positively for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or activated-caspase 3, suggesting that they were dying from a non-apoptotic mechanism. Moreover, cross to a Bax(fl/fl)/Bak⁻/⁻ mouse line that is resistant to most forms of apoptosis failed to promote cell survival in the ILK-deleted lens epithelium. Electron microscopy revealed the presence of numerous membranous vacuoles containing degrading cellular material. CONCLUSIONS. Our study reveals a role for ILK in extracellular matrix organization, fiber migration, and cell survival. Furthermore, to our knowledge we show for the first time that ILK disruption results in non-apoptotic cell death in vivo.

Nerve repulsion by the lens and cornea during cornea innervation is dependent on Robo-Slit signaling and diminishes with neuron age.

The cornea, the most densely innervated tissue on the surface of the body, becomes innervated in a series of highly coordinated developmental events. During cornea development, chick trigeminal nerve growth cones reach the cornea margin at embryonic day (E)5, where they are initially repelled for days from E5 to E8, instead encircling the corneal periphery in a nerve ring prior to entering on E9. The molecular events coordinating growth cone guidance during cornea development are poorly understood. Here we evaluated a potential role for the Robo-Slit nerve guidance family. We found that Slits 1, 2 and 3 expression in the cornea and lens persisted during all stages of cornea innervation examined. Robo1 expression was developmentally regulated in trigeminal cell bodies, expressed robustly during nerve ring formation (E5-8), then later declining concurrent with projection of growth cones into the cornea. In this study we provide in vivo and in vitro evidence that Robo-Slit signaling guides trigeminal nerves during cornea innervation. Transient, localized inhibition of Robo-Slit signaling, by means of beads loaded with inhibitory Robo-Fc protein implanted into the developing eyefield in vivo, led to disorganized nerve ring formation and premature cornea innervation. Additionally, when trigeminal explants (source of neurons) were oriented adjacent to lens vesicles or corneas (source of repellant molecules) in organotypic tissue culture both lens and cornea tissues strongly repelled E7 trigeminal neurites, except in the presence of inhibitory Robo-Fc protein. In contrast, E10 trigeminal neurites were not as strongly repelled by cornea, and presence of Robo-Slit inhibitory protein had no effect. In full, these findings suggest that nerve repulsion from the lens and cornea during nerve ring formation is mediated by Robo-Slit signaling. Later, a shift in nerve guidance behavior occurs, in part due to molecular changes in trigeminal neurons, including Robo1 downregulation, thus allowing nerves to find the Slit-expressing cornea permissive for growth cones.

Rac1 GTPase-deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival.

Morphogenesis and shape of the ocular lens depend on epithelial cell elongation and differentiation into fiber cells, followed by the symmetric and compact organization of fiber cells within an enclosed extracellular matrix-enriched elastic capsule. The cellular mechanisms orchestrating these different events however, remain obscure. We investigated the role of the Rac1 GTPase in these processes by targeted deletion of expression using the conditional gene knockout (cKO) approach. Rac1 cKO mice were derived from two different Cre (Le-Cre and MLR-10) transgenic mice in which lens-specific Cre expression starts at embryonic day 8.75 and 10.5, respectively, in both the lens epithelium and fiber cells. The Le-Cre/Rac1 cKO mice exhibited an early-onset (E12.5) and severe lens phenotype compared to the MLR-10/Rac1 cKO (E15.5) mice. While the Le-Cre/Rac1 cKO lenses displayed delayed primary fiber cell elongation, lenses from both Rac1 cKO strains were characterized by abnormal shape, impaired secondary fiber cell migration, sutural defects and thinning of the posterior capsule which often led to rupture. Lens fiber cell N-cadherin/ß-catenin/Rap1/Nectin-based cell-cell junction formation and WAVE-2/Abi-2/Nap1-regulated actin polymerization were impaired in the Rac1 deficient mice. Additionally, the Rac1 cKO lenses were characterized by a shortened epithelial sheet, reduced levels of extracellular matrix (ECM) proteins and increased apoptosis. Taken together, these data uncover the essential role of Rac1 GTPase activity in establishment and maintenance of lens shape, suture formation and capsule integrity, and in fiber cell migration, adhesion and survival, via regulation of actin cytoskeletal dynamics, cell adhesive interactions and ECM turnover.

The lens capsule.

The lens capsule is a modified basement membrane that completely surrounds the ocular lens. It is known that this extracellular matrix is important for both the structure and biomechanics of the lens in addition to providing informational cues to maintain lens cell phenotype. This review covers the development and structure of the lens capsule, lens diseases associated with mutations in extracellular matrix genes and the role of the capsule in lens function including those proposed for visual accommodation, selective permeability to infectious agents, and cell signaling.

Laminin beta1 and gamma1 containing laminins are essential for basement membrane integrity in the zebrafish eye.

PURPOSE: In this study recessive zebrafish mutations in the genes encoding laminin beta1 (lamb1) and laminin gamma1 (lamc1) were used to determine the functions of these laminin proteins during ocular basement membrane formation and during zebrafish eye development. METHODS: Ocular defects in lamb1 and lamc1 mutants were characterized by using a combination of histology, immunohistochemistry, in situ hybridization, and transmission electron microscopy. RESULTS: The results demonstrated that zebrafish lamb1 and lamc1 mutants possess defects in two ocular basement membranes--the lens capsule and the inner limiting membrane--whereas Bruch's membrane is largely unaffected. lamb1 and lamc1 mutants possess severe lens dysplasias that result from a compromise in lens capsule integrity. Inner limiting membrane continuity is irregular in these mutants, and these irregularities result in small retinal ectopias that extend from the retina into the interstitial space between the retina and the lens. At late embryonic stages (e.g., 5-7 days after fertilization), retinal lamination defects are also observed in a subset of laminin mutants. CONCLUSIONS: The results demonstrate that laminin beta1 and -gamma1 containing laminins are essential for the integrity of the lens capsule basement membrane and inner limiting membrane in the zebrafish eye.

Vitronectin is present in epithelial cells of the intact lens and promotes epithelial mesenchymal transition in lens epithelial explants.

PURPOSE: Extracellular matrix (ECM) accumulates during the development of posterior capsule opacification (PCO). Vitronectin, an ECM component that is generally prominent in wound healing, has been detected in PCO specimens. Here we set out to investigate the distribution of vitronectin in the lens and determine how it, and other ECM components, influence the lens epithelial phenotype. METHODS: Rat lens epithelial explants were cultured on vitronectin, fibronectin, and laminin substrata. Explants were monitored for cell migration and the appearance of markers for epithelial mesenchymal transition (EMT), using phase contrast microscopy and immunohistochemistry, respectively. Explants were also monitored for evidence of Smad signaling. Vitronectin expression was analyzed in embryonic and postnatal rodent lens development by immunohistochemistry, western blotting, and in situ hybridization. RESULTS: Vitronectin, like fibronectin and laminin, provided a good substratum for cellular attachment and migration. However, in the case of vitronectin and fibronectin, this was accompanied by a major phenotypic change. On either vitronectin or fibronectin, but not laminin, most of the cells became elongated, spindle-shaped and were strongly reactive for filamentous alpha-smooth muscle actin. In these respects this transition was typical of the well known TGFbeta-induced EMT. In explants cultured on vitronectin and fibronectin, but not laminin, cell nuclei showed prominent reactivity for Smad 2/3. Vitronectin was also shown to be expressed during embryonic and postnatal development. Initially mRNA and protein were detected in all lens cells, however as development progressed, expression became restricted to cells of the epithelium and transition zone. CONCLUSIONS: The results clearly show that lens cell engagement with a vitronectin or a fibronectin, but not laminin, substratum has a potent EMT promoting effect and that Smad 2/3 signaling is involved. Thus when considering strategies to slow or prevent PCO, these results highlight the need to take into account ECM molecules such as vitronectin that have the capacity to promote EMT.

Severe defects in proliferation and differentiation of lens cells in Foxe3 null mice.

During mouse eye development, the correct formation of the lens occurs as a result of reciprocal interactions between the neuroectoderm that forms the retina and surface ectoderm that forms the lens. Although many transcription factors required for early lens development have been identified, the mechanism and genetic interactions mediated by them remain poorly understood. Foxe3 encodes a winged helix-forkhead transcription factor that is initially expressed in the developing brain and in the lens placode and later restricted exclusively to the anterior lens epithelium. Here, we show that targeted disruption of Foxe3 results in abnormal development of the eye. Cells of the anterior lens epithelium show a decreased rate of proliferation, resulting in a smaller than normal lens. The anterior lens epithelium does not properly separate from the cornea and frequently forms an unusual, multilayered tissue. Because of the abnormal differentiation, lens fiber cells do not form properly, and the morphogenesis of the lens is greatly affected. The abnormally differentiated lens cells remain irregular in shape, and the lens becomes vacuolated. The defects in lens development correlate with changes in the expression of growth and differentiation factor genes, including DNase II-like acid DNase, Prox1, p57, and PDGFalpha receptor. As a result of abnormal lens development, the cornea and the retina are also affected. While Foxe3 is also expressed in a distinct region of the embryonic brain, we have not observed abnormal development of the brain in Foxe3(-/-) animals.

Evo-devo aspects of classical and molecular data in a historical perspective.

We discuss the interplay between evolution and development as reflected in data and concepts since about 1800. Darwin and his "continental apostle" Haeckel put the striking similarity between early vertebrate embryos in an evolutionary context. Haeckel's partly illicit generalizations discredited evolutionary thinking among early experimental embryologists who moreover noted riddles incompatible with contemporary concepts of homology and evolution. Relevant solutions were suggested by the more recent concept of ontogenetic networks that embody complex regulatory properties and genes with partly overlapping functions. Molecular data on development increasingly reveal evolutionary opportunism, for instance when a widespread signaling chain involved in primitive immune defense was apparently recruited later on for dorso-ventral axis determination in some evolutionarily advanced insect groups. Recently, Rickettsia-related bacteria colonizing many arthropod species were found to "manipulate" the first steps of host development to the advantage of their own propagation, but by ways that could also promote host speciation. Molecular genetics can now document evolutionary steps in ontogenetic networks. In the fruit fly for instance, the novel bicoid gene has superseded a crucial patterning function within a pre-existing network--a case of "molecular caenogenesis." The expression patterns of conserved genes that antagonistically determine dorso-ventral polarity support a literal revolution envisioned almost 200 years ago. This is the dorso-ventral inversion of the body plan in some metazoans--ascribed then to the Articulata, now to the Chordata. The final section posits that the opportunistic character of evolutionary innovations is detrimental to parsimony in development.

High exogenous homocysteine modifies eye development in early chick embryos.

BACKGROUND: Homocysteine is a nonessential aminoacid whose increase is related to the appearance of neural tube defects in humans. In chick embryos, high levels of homocysteine produce neural tube defects and alteration of neural crest cell migration. METHODS: In our study, 8 microl of L-homocysteine thiolatone (20 micromol) was added to chick embryos of Stages 3-8/10 (Hamburger and Hamilton, 1951), (1238 hr of incubation). Three days later, 50 embryos, externally normal or carrying isolated spinal neural tube defects, were sectioned and stained by hematoxilin-eosin or anti-fibrillin-1 antibody. RESULTS: The eye showed alterations of the optic cup as microphthalmia, or lens dislocation. In both cases, the incidence of alterations diminished with the age of the homocysteine-increased embryos. Optic cup modifications are probably associated with central nervous system alterations, because most of the affected embryos exhibited isolated spinal neural tube defects and had altered neural crest cells. We have shown for the first time that high exogenous homocysteine during early development could produce a caudally-displaced lens axis before the zonule is formed. Fibrillin-1 is the main component of elastic microfibrils, and in the adult human it is seen as a protein particularly susceptible to homocysteine attack. CONCLUSIONS: Antibody staining against fibrillin-1 showed no evident morphological differences in distribution between experimental and control embryos in the lens, suggesting that fibrillin-1 was not the cause, and malformations may be attributed to other mechanisms.

Expression of the matricellular protein SPARC in murine lens: SPARC is necessary for the structural integrity of the capsular basement membrane.

SPARC (Secreted Protein, Acidic and Rich in Cysteine) is a matricellular glycoprotein that modulates cell proliferation, adhesion, migration, and extracellular matrix (ECM) production. Although SPARC is generally abundant in embryonic tissues and is diminished in adults, we have found that the expression of SPARC in murine lens persists throughout embryogenesis and adulthood. Our previous studies showed that targeted ablation of the SPARC gene in mice results in cataract formation, a pathology attributed partially to an abnormal lens capsule. Here we provide evidence that SPARC is not a structural component of the lens capsule. In contrast, SPARC is abundant in lens epithelial cells, and newly differentiated fiber cells, with stable expression in wild-type mice up to 2 years of age. Pertubation of the lens capsule in animals lacking SPARC appears to be a consequence of the invasion of the lens cells situated beneath the capsule. Immunoreactivity for SPARC in the lens cells was uneven, with minimal reactivity in the epithelial cells immediately anterior to the equator. These epithelial cells appeared essentially noninvasive in SPARC-null mice, in comparison to the centrally located anterior epithelial cells, in which strong labeling by anti-SPARC IgG was observed. The posterior lens fibers exhibited cytoplasmic extensions into the posterior lens capsule, which was severely damaged in SPARC-null lenses. The expression of SPARC in wild-type lens cells, together with the abnormal lens capsule in SPARC-null mice, indicated that the structural integrity of the lens capsule is dependent on the matricellular protein SPARC. The effects of SPARC in the lens appear to involve regulation of lens epithelial and fiber cell morphology and functions rather than deposition as a structural component of the lens capsule.

Chondroitin sulphate proteoglycan is involved in lens vesicle morphogenesis in chick embryos.

Proteoglycans have been implicated in the invagination and formation of various embryonal cavitied primordia. In this paper the expression of chondroitin sulphate proteoglycan (CSPG) is analysed in the lens primordium during lens vesicle formation, and demonstrate that this proteoglycan has a specific distribution pattern with regard to invagination and fusion processes in the transformation of placode into lens vesicle. More specifically, CSPG was detected in: (1) the apical surface of lens epithelial cells, where early CSPG expression was observed in the whole of the lens placode whilst in the vesicle phase it was restricted to the posterior epithelium; (2) intense CSPG expression in the basal lamina, which remained constant for the entire period under study; (3) CSPG expression in the intercellular spaces of the lens primordium epithelium, which increased during the invagination of the primordium and which at the vesicle stage was more evident in the posterior epithelium; and (4) CSPG expression on the edges of the lens placode both prior to and during fusion. Treatment with beta- D -xyloside causes significant CSPG depletion in the lens primordium together with severe alterations in the invagination and fusion of the lens vesicle; this leads to the formation of lens primordia which in some cases remain practically flat or show partial invagination defects or fusion disruption. Similar results were obtained by enzyme digestion with chondroitinase AC but not with type II heparinase, which indicates that alterations induced by beta- D -xyloside were due to interference in CSPG synthesis. The findings demonstrate that CSPG is a common component of the lens primordium at the earliest developmental stages during which it undergoes specific modifications. It also includes experimental evidence to show that 'in vivo' CSPG plays an important role in the invagination and fusion processes of the lens primordium.

Beta 1 integrins in epithelial tissues: a unique distribution in the lens.

Integrins have been shown to play a role in directing and maintaining cell differentiation and polarization. The embryonic lens provides a good system in which to examine their role in epithelial cell differentiation, because all stages of lens development are represented in an individual embryonic lens. Therefore, we examined the expression and distribution of beta 1 integrin heterodimers in both the lens epithelium and the differentiated lens fiber cells. In lens epithelial cells beta 1 integrin was found to be localized to all membrane surfaces. Lens fiber cells contained beta 1 integrin all along their lateral borders as well as at the site of their attachment to the lens capsule and at their interface with lens epithelial cells. The distribution of beta 1 integrin in the lens was distinct from that observed in simple epithelia, the retinal pigment epithelium (RPE), kidney, and intestine, where it was limited to a basal lateral localization. We examined the specific beta 1 integrin heterodimers expressed in the lens by Western blot analysis for the integrin alpha subunits following beta 1 immunoprecipitation and compared them with those expressed in RPE cells. In the lens we detected alpha 3 and alpha 6 subunits but not alpha 1, alpha 5, or alpha v. When the lens was separated into epithelial and fiber cells, we found that alpha 3 was expressed at a higher level in the epithelial cells, while alpha 6 was primarily associated with the fiber cells. In the RPE the primary beta 1 integrin detected was alpha 3. Unlike in lens and kidney, alpha 6 beta 1 integrin in RPE cells was expressed only at a low level. alpha v was also expressed in RPE cells but not as a beta 1 heterodimer. As in the lens, neither alpha 5 beta 1 nor alpha 1 beta 1 integrin was detected in RPE. Both lens and RPE cells express a specific subset of beta 1 integrin heterodimers which are likely to be important to the initiation and maintenance of their differentiated phenotype.

Early morphogenesis of persistent hyperplastic tunica vasculosa lentis and primary vitreous (PHTVL/PHPV). Scanning electron microscopic observations.

This study provides scanning electron microscopic observations on the early morphogenesis of persistent hyperplastic tunica vasculosa lentis and primary vitreous (PHTVL/PHPV) in canine fetuses at days 28 35 postcoitum (D28 and D35). From previous studies regarding PHTVL/PHPV it is known that a retrolental plaque of fibrovascular tissue is present in eyes of affected canine fetuses from the D33 stage. The contribution of vitreous cells to the formation of the plaque is supported by the results of this study. The lens capsules at the stages described were not found to contain abnormalities such as transparent (thinner) parts or rents, as have been described for postnatal cases of PHTVL/PHPV. These findings support the hypothesis that the capsular anomalies observed in postnatal patients are secondary entities.

The lens capsule and zonulae.

The embryology and natural history of the lens capsule and the zonular apparatus have been described according to the present knowledge of the subject. Clinical evidence pointing towards an active turnover of lens capsule material is presented.

Distribution of acidic and basic fibroblast growth factors (FGF) in the foetal rat eye: implications for lens development.

Previously we reported that, in vitro, lens cells proliferate, migrate or differentiate in response to low, medium and high concentrations of FGF respectively. To examine further the role of FGF in lens development we used immunohistochemistry to study the distribution of aFGF and bFGF in the eye of the 20 day rat foetus. Strong aFGF-like reactivity was localised in a band of cells near the lens equator which included the germinative zone where most cell proliferation occurs and the transitional zone where epithelial cells differentiate into fibres. The closely apposed inner epithelial layer of the ciliary and iridial retina also reacted strongly. Reactivity for aFGF was also found in the epidermis and in the corneal and conjunctival epithelia. In the neural retina, reactivity was found in the nerve fibre layer and in isolated cells of the inner plexiform layer. bFGF-like reactivity was found in the retinal ganglion cell layer, extra-ocular muscles and associated with endothelial cells of the hyaloid, lenticular and choroid vasculatures. Pre-digestion of sections with hyaluronidase caused loss of cell-associated reactivity but revealed strong bFGF-like reactivity in ocular basement membranes, in particular, the lens capsule. The sensitivity of this capsular bFGF localisation to heparinase indicates that bFGF in the extracellular matrix is complexed with heparan sulphate proteoglycans. The results of this study are consistent with the hypothesis that FGF plays an important role in lens development via both autocrine and paracrine mechanisms.