Understanding the role of growth factors in embryonic development: insights from the lens.

Growth factors play key roles in influencing cell fate and behaviour during development. The epithelial cells and fibre cells that arise from the lens vesicle during lens morphogenesis are bathed by aqueous and vitreous, respectively. Vitreous has been shown to generate a high level of fibroblast growth factor (FGF) signalling that is required for secondary lens fibre differentiation. However, studies also show that FGF signalling is not sufficient and roles have been identified for transforming growth factor-ß and Wnt/Frizzled families in regulating aspects of fibre differentiation. In the case of the epithelium, key roles for Wnt/ß-catenin and Notch signalling have been demonstrated in embryonic development, but it is not known if other factors are required for its formation and maintenance. This review provides an overview of current knowledge about growth factor regulation of differentiation and maintenance of lens cells. It also highlights areas that warrant future study.

The lens epithelium in ocular health and disease.

The lens arises from invagination of head ectoderm during embryonic development and in the adult has a relatively simple structure, comprising just two cell types (epithelial and fibre cells). Its isolation from nerves and blood vessels in the adult make it a tractable model to investigate mechanisms that regulate epithelial cells. A major focus in lens research in the past 50 years has been on the differentiation of fibre cells from epithelial cells. Hence, there has been much interest in the role of signalling systems regulating fibre cell differentiation during development. In contrast, the signalling systems that control the formation and maintenance of the lens epithelium have, until recently, been largely ignored or incidental to studies on differentiation or cataract. One notable example has been the identification of signals that underlie epithelial-mesenchymal transition (EMT) that characterizes anterior subcapsular cataract (ASC) and posterior capsule opacification (PCO). Recent data indicate that normal epithelial phenotype is regulated by several key signalling systems, including receptor tyrosine kinase receptors acting via the MAPK and Akt pathways, Wnt, Notch as well as extracellular matrix cues and possibly the Sal-Warts-Hippo pathway. Here we have shifted emphasis onto molecular mechanisms that regulate the establishment, maintenance and function of the lens epithelium.

Transforming growth factor-beta-induced epithelial-mesenchymal transition in the lens: a model for cataract formation.

The vertebrate lens has a distinct polarity and structure that are regulated by growth factors resident in the ocular media. Fibroblast growth factors, in concert with other growth factors, are key regulators of lens fiber cell differentiation. While members of the transforming growth factor (TGFbeta) superfamily have also been implicated to play a role in lens fiber differentiation, inappropriate TGFbeta signaling in the anterior lens epithelial cells results in an epithelial-mesenchymal transition (EMT) that bears morphological and molecular resemblance to forms of human cataract, including anterior subcapsular (ASC) and posterior capsule opacification (PCO; also known as secondary cataract or after-cataract), which occurs after cataract surgery. Numerous in vitro and in vivo studies indicate that this TGFbeta-induced EMT is part of a wound healing response in lens epithelial cells and is characterized by induced expression of numerous extracellular matrix proteins (laminin, collagens I, III, tenascin, fibronectin, proteoglycans), intermediate filaments (desmin, alpha-smooth muscle actin) and various integrins (alpha2, alpha5, alpha7B), as well as the loss of epithelial genes [Pax6, Cx43, CP49, alpha-crystallin, E-cadherin, zonula occludens-1 protein (ZO-1)]. The signaling pathways involved in initiating the EMT seem to primarily involve the Smad-dependent pathway, whereby TGFbeta binding to specific high affinity cell surface receptors activates the receptor-Smad/Smad4 complex. Recent studies implicate other factors [such as fibroblast growth factor (FGFs), hepatocyte growth factor, integrins], present in the lens and ocular environment, in the pathogenesis of ASC and PCO. For example, FGF signaling can augment many of the effects of TGFbeta, and integrin signaling, possibly via ILK, appears to mediate some of the morphological features of EMT initiated by TGFbeta. Increasing attention is now being directed at the network of signaling pathways that effect the EMT in lens epithelial cells, with the aim of identifying potential therapeutic targets to inhibit cataract, particularly PCO, which remains a significant clinical problem in ophthalmology.

Localization of the extracellular Ca(2+)-sensing receptor in the human placenta.

We have demonstrated using immunohistochemistry and in situ hybridization that the calcium-sensing receptor (CaR) is expressed in both villous and extravillous regions of the human placenta. CaR expression was detected in both first trimester and term placentas. In the villous region of the placenta, the CaR was detected in syncytiotrophoblasts and at lower levels in cytotrophoblasts. Local expression of the CaR in the brush border of syncytiotrophoblasts suggests a role for maternal Ca(2+) concentration in the control of transepithelial transport between the mother and fetus. In the extravillous region of the placenta, the CaR was detected in cells forming trophoblast columns in anchoring villi, in close proximity to maternal blood vessels and in transitional cytotrophoblasts. Given the importance of extravillous cytotrophoblasts in the process of uterine invasion and maintenance of placental immune privilege, the CaR represents a possible target by which the maternal extracellular Ca(2+) concentration could promote or maintain placentation. Thus, the results support hypotheses that the CaR contributes to the local control of transplacental calcium transport and to the regulation of placental development.

Requirement for TGFbeta receptor signaling during terminal lens fiber differentiation.

Several families of growth factors have been identified as regulators of cell fate in the developing lens. Members of the fibroblast growth factor family are potent inducers of lens fiber differentiation. Members of the transforming growth factor beta (TGFbeta) family, particularly bone morphogenetic proteins, have also been implicated in various stages of lens and ocular development, including lens induction and lens placode formation. However, at later stages of lens development, TGFbeta family members have been shown to induce pathological changes in lens epithelial cells similar to those seen in forms of human subcapsular cataract. Previous studies have shown that type I and type II TGFbeta receptors, in addition to being expressed in the epithelium, are also expressed in patterns consistent with a role in lens fiber differentiation. In this study we have investigated the consequences of disrupting TGFbeta signaling during lens fiber differentiation by using the mouse alphaA-crystallin promoter to overexpress mutant (kinase deficient), dominant-negative forms of either type I or type II TGFbeta receptors in the lens fibers of transgenic mice. Mice expressing these transgenes had pronounced bilateral nuclear cataracts. The phenotype was characterized by attenuated lens fiber elongation in the cortex and disruption of fiber differentiation, culminating in fiber cell apoptosis and degeneration in the lens nucleus. Inhibition of TGFbeta signaling resulted in altered expression patterns of the fiber-specific proteins, alpha-crystallin, filensin, phakinin and MIP. In addition, in an in vitro assay of cell migration, explanted lens cells from transgenic mice showed impaired migration on laminin and a lack of actin filament assembly, compared with cells from wild-type mice. These results indicate that TGFbeta signaling is a key event during fiber differentiation and is required for completion of terminal differentiation.

Tgfbeta receptor expression in lens: implications for differentiation and cataractogenesis.

TGFbeta induces changes characteristic of some forms of cataract. However, the responsiveness of lens epithelial cells to TGFbeta is age-dependent; weanling and adult, but not neonatal, lens epithelial cells respond. This study investigated TGFbeta receptor (TbetaRI and TbetaRII) expression during rat lens development and the effects of FGF-2 on TGFbeta responsiveness and TbetaR expression. Immunofluorescence, immunoblotting, RT-PCR and in situ hybridization were used to examine the spatio-temporal expression patterns of TbetaR. Lens explants were used to investigate the effects of FGF-2 on TGFbeta responsiveness and TbetaR expression. In the lens epithelium, little or no immunoreactivity was detected at P3 but at P21 there was distinct reactivity for TbetaRI and TbetaRII. Reactivity for both receptors was also found in the differentiating fibers in the transitional zone and cortex at both ages. Western blotting of lens membrane extracts identified multiple molecular weight forms of TbetaRI (30, 50, 90 kDa) and TbetaRII (70-120 kDa). In situ hybridization with a rat probe for Alk5 (TbetaRI) showed that the lens expresses Alk5 mRNA in epithelium and fibers throughout development. A rat TbetaRII probe revealed distinct expression of a TbetaRII mRNA in lens fibers throughout development and in the lens epithelium at P21 but not at P3. In vitro studies showed that lens epithelial explants from P9 rats did not undergo cataractous changes in response to TGFbeta but P13 explants did. Addition of FGF-2 to P9 explants induced increased TbetaR immunoreactivity and enhanced the competency of lens epithelial cells to respond to TGFbeta. These data indicate that the overall increased expression of TGFbeta receptors in lens epithelium during postnatal development (P3-P21) underlies an age-related change in TGFbeta responsiveness. The results also suggest that lens cells may express multiple forms of TbetaR. Expression of TbetaR in lens fibers throughout lens development and the induction of enhanced TbetaR expression by FGF suggest a role for TGFbeta signaling during FGF-induced responses and fiber differentiation.

Accuracy in gamma camera measurements of point source velocities.

Mucociliary clearance is impaired in many diseases of the respiratory system. We have developed a method for measuring tracheal mucus velocity by the dynamic study of a single point source of radioactivity deposited in the trachea by cricothyroid injection. Preliminary results suggest that patients with airways disease have very low tracheal mucus velocities (<2 mm x min(-1)). The aim of this experiment was to explore the ability of current scintillation detection systems to track a single point as it moves in a dynamic study in small increments and at low velocity (movements of the order of 1 mm). Background noise was estimated to contribute an error in positioning of 0.16 mm (1 standard deviation). Overall errors in velocity were estimated at 0.2 mm x min(-1). This suggests that standard instrumentation in use in most nuclear medicine departments has the capacity to measure accurately velocities as low as 1 mm x min(-1).

Lens development.

This review gives a brief account of the main processes of lens development, including induction, morphogenesis, differentiation and growth. It describes what is known about the molecules and mechanisms that control and regulate these processes. Some of the recent progress made in understanding the molecular basis of lens development is highlighted along with some of the challenging areas for future research.

Differential cataractogenic potency of TGF-beta1, -beta2, and -beta3 and their expression in the postnatal rat eye.

PURPOSE: Transforming growth factor-beta has been shown to induce cataractous changes in rat lenses. This study assesses the relative cataractogenic potential of TGF-beta1, TGF-beta2, and TGF-beta3 and their expression patterns in the rat eye. METHODS: Lens epithelial explants and whole lenses from weanling rats were cultured with TGF-beta1, TGF-beta2, or TGF-beta3 at concentrations ranging from 0.025 ng/ml to 4 ng/ml for 3 to 5 days. Cataractous changes were monitored daily by phase contrast microscopy and by immunofluorescent detection of cataract markers alpha-smooth muscle actin and type I collagen. Expression of TGF-beta was studied by immunofluorescence and in situ hybridization on eye sections from neonatal and weanling rats. RESULTS: All three isoforms induced morphologic changes in lens epithelial explants and cultured lenses that are typically associated with human subcapsular cataract. Transforming growth factor-beta2 and TGF-beta3 were approximately 10 times more potent than TGF-beta1. All three isoforms were expressed in the eye in spatially distinct but overlapping patterns. Transforming growth factor-beta1 and TGF-beta2 and their mRNA were detected in most ocular tissues, including the lens. Although TGF-beta3 was immunolocalized in lens epithelium and fibers and in other ocular tissues, its mRNA was detected only in the retina and choroid. CONCLUSIONS: All three isoforms of TGF-beta are potentially available to lens cells and have the potential to induce cataractous changes. The results suggest that TGF-beta activity is normally tightly regulated in the eye. Activation of TGF-beta in the lens environment, such as may occur during injury, in wound healing, or in pathologic conditions may contribute to cataractogenesis in vivo.

Differential expression of fibroblast growth factor receptors during rat lens morphogenesis and growth.

PURPOSE: Fibroblast growth factors (FGF) play important roles in the developmental biology of the lens. Recently, it was shown that the expression of one of the FGF receptors, FGFR1 (flg; fibroblast growth factor receptor 1), was closely associated with the onset of lens fiber differentiation. In this study, the expression patterns of three other members of the FGF receptor family were analyzed and compared. METHODS: The expression patterns of FGFR2 (bek and keratinocyte growth factor receptor [KGFR] variants) and FGFR3 were analyzed by in situ hybridization during embryonic and postnatal lens development. RESULTS: In the ocular primordia, both FGFR2 variants were detected on embryonic day 12 (E12) and FGFR3 was detected on E14. From E16 to E20, distinct spatial expression patterns became evident within the lens; FGFR3 showed an anteroposterior increase in expression, with strongest expression in the outer cortical fibers. In contrast, bek showed uniform expression throughout the lens epithelium (including the central and germinative zones) and the transitional zone, with a subsequent decline in maturing fibers. The KGFR variant of FGFR2 showed strongest expression in the early fibers of the transitional zone; its expression in the epithelium was weaker in the germinative zone of embryonic and neonatal rats. There was an age-related decline in expression of FGFRs after birth-an effect that was more marked for FGFR3 than for the FGFR2 variants. CONCLUSIONS: Combined with those in a previous study, these results indicate that the FGFR1, bek, KGFR, and FGFR3 genes exhibit different, yet overlapping, patterns of expression throughout lens development and differentiation. The distinct spatiotemporal patterns of expression of FGF receptors may play an important role in regulating anteroposterior patterns of lens cell behavior.

Expression of FGF-1 and FGF-2 mRNA during lens morphogenesis, differentiation and growth.

PURPOSE: There is now considerable evidence that FGF is involved in lens differentiation and growth throughout life. The aim of this study was to determine potential sites of FGF production in and near the lens during morphogenesis, differentiation and growth. METHODS: The distribution of FGF-1 and FGF-2 mRNAs was analysed in embryonic, weanling and adult rat eyes by in situ hybridization. RESULTS: During lens morphogenesis, there was distinct expression of FGF-1, but not FGF-2, in the lens placode and retinal disc cells. Subsequently, both forms of FGF showed similar expression patterns. During lens differentiation, distinct expression of FGFs was associated with elongating primary fiber cells. From embryonic day 20 onwards, lenses showed strongest expression of FGF mRNAs in the transitional zone, where epithelial cells differentiate into fibers, with weaker expression in the anterior epithelium. Messenger RNAs for both FGFs were also localised in ocular tissues near the lens and bordering the ocular media, particularly the cornea, ciliary body, iris and neural retina. CONCLUSIONS: These findings are consistent with the known distribution of FGF protein in the eye and implicate various ocular tissues as potential sources of FGF that may influence lens cells. Furthermore, the fact that lens cells have the potential for synthesizing FGF, together with evidence from previous studies that lens cells express FGF receptors and respond to lens-derived FGF, raises the possibility that some aspects of lens cell behaviour in situ may be influenced by autocrine mechanism(s) of FGF stimulation.

FGF receptor-1 (flg) expression is correlated with fibre differentiation during rat lens morphogenesis and growth.

Our previous studies indicate an important role for fibroblast growth factor (FGF) in lens development. Here we study the expression of the flg variant of FGF receptor 1 (FGFR1) during lens development by immunohistochemistry and in situ hybridisation. FGFR1 was expressed throughout lens development. Prominent FGFR1 immunoreactivity was associated with cell nuclei, particularly in differentiating lens fibres, suggesting internalisation and nuclear translocation of the receptor. FGFR1 immunoreactivity was also associated with basolateral membranes of cells in the equatorial region and at lens sutures. FGFR1 mRNA was only weakly expressed during early lens morphogenesis but expression increased with the onset of lens fibre differentiation. Once the lens acquired its distinct polarity, an anteroposterior gradient in both protein reactivity and mRNA signal was evident. Anteriorly, central epithelial cells showed weak expression for FGFR1, whereas more posteriorly, in the germinative and transitional zones of the lens where cells maximally proliferate and undergo early stages of fibre differentiation, respectively, expression was significantly stronger. The anteroposterior gradient of increased expression of FGFR1 in the lens coincides with the previously documented anteroposterior gradient of FGF stimulation. In lens epithelial explants, FGF stimulation was found to upregulate FGFR1 expression. Such upregulation may be an important mechanism for generating a high level of FGF stimulation and ensuring a fibre differentiation response. In postnatal rat lenses, there was a significant age-related decline in FGFR1 expression; this correlates with the reduced rate of lens fibre differentiation with age. Overall, these studies support the hypothesis that FGF and FGFR1 are important for regulation of lens fibre differentiation throughout lens development.

Ciliary defects in healthy subjects, bronchiectasis, and primary ciliary dyskinesia.

To develop criteria to aid in the diagnosis of primary ciliary dyskinesia (PCD) we analyzed quantitatively the incidence and the range of ciliary ultrastructural abnormalities in healthy subjects and in patients with respiratory tract disease. The beat frequency and ultrastructure of nasal respiratory tract cilia, including ciliary orientation, were measured in 62 healthy subjects (31 nonsmokers, 20 exsmokers, and 11 smokers), ranging in age from 1 to 76 yr, and in 51 patients with respiratory tract disease. In healthy subjects, ciliary beat frequency (CBF) ranged between 9.6 and 15.3 Hz, the incidence of microtubule defects varied between 0 to 9%, the mean number of inner dynein arms per cilium ranged from 3.0 to 7.1, and the mean number of outer dynein arms per cilium ranged from 7.4 to 9.0. The deviation of cilia in healthy subjects varied between 8 and 29 degrees. By comparing the data for ciliary defects in healthy subjects with the data obtained from patients with respiratory disease, we identified two patient groups: patients with PCD (n = 31) and patients with respiratory tract disease not due to PCD. For comparison with the PCD patients, a group of 20 patients with bronchiectasis was selected and analyzed. Patients with PCD had significantly lower CBF (p < 0.001), significantly higher incidences of peripheral and central tubule defects (p < 0.01), and greater ciliary disorientation (p < 0.005). There was a strong correlation between CBF and the number of outer dynein arm numbers, but not with inner dynein arm numbers, suggesting that inner and outer dynein arms may play different functional roles in producing ciliary motility.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidic and basic FGF in ocular media and lens: implications for lens polarity and growth patterns.

We have shown previously that FGF induces lens epithelial cells in explant culture to proliferate, migrate and differentiate into fibre cells in a progressive concentration-dependent manner. In situ, these processes occur in a distinct anterior-posterior pattern in clearly defined regions of the lens. Thus anterior-posterior differences in the bio-availability of FGF in the lens environment may play a role in determining lens polarity and growth patterns. In this study, using heparin chromatography and western blotting (or ELISA), we established that both acidic and basic FGF are present in the aqueous and vitreous (the ocular media that bathe the anterior and posterior compartments of the lens, respectively). In addition, substantially more FGF was recovered from vitreous than from aqueous. Both forms of FGF were also detected in lens fibre cells and capsule. A truncated form of basic FGF (less than 20 x 10(3) M(r)) predominated in every case with traces of higher M(r) forms in lens cells. For acidic FGF, the classical full-length form (about 20 x 10(3) M(r)) predominated in lens cells and a truncated form was found in vitreous. The capsule contained a higher M(r) form. Using our explant system, we also tested the biological activity of ocular media and FGF fractions obtained from vitreous and lens cells. Vitreous but not aqueous contained fibre-differentiating activity. Furthermore, virtually all the fibre-differentiating activity of vitreous was shown to be FGF-associated, as follows: (a) this activity remained associated with FGF during fractionation of vitreous by heparin and Mono-S chromatography and (b) the activity of the major FGF-containing fraction was blocked by antibodies to acidic and basic FGF. Posterior, but not anterior, capsule was shown to have mitogenic activity, which was neutralised by FGF antibodies and associated only with the cellular surface. These results support our hypothesis that FGF is involved in determining the behaviour of lens cells in situ. In particular, a key role for FGF in determining lens polarity and growth patterns is suggested by the anterior-posterior differences in the bio-availability of FGF in the ocular media and capsule.

The role of fibroblast growth factor in eye lens development.

In this review we have presented evidence that FGF plays an important role in inducing events in lens morphogenesis and growth. Our studies show that FGF stimulates lens epithelial cells in explants to proliferate, migrate, and differentiate into fibers at low, medium, and high concentrations, respectively. This has some important implications for understanding the behavior of lens cells in the eye. The fact that aFGF is detected in the equatorial region of the lens where cells are actively proliferating, possibly migrating, and differentiating into fibers suggests that these events may be under autocrine control in vivo, at least to some extent. Because FGF is also present in the ciliary and iridial region of retina and in the vitreous, paracrine control may also be involved. Cell proliferation, fiber differentiation, and (possibly) cell migration occur in characteristic spatial patterns that are related to distinct compartments of the lens. We suggest that cells in the germinative zone receive only a low level of FGF stimulation arising from the cells themselves and possibly also from the ciliary and iridial regions of the retina but, whatever the source, this is only sufficient to stimulate proliferation. Lens epithelial cells that migrate or are displaced into the transitional zone below the lens equator receive some FGF from these sources but in addition receive a strong stimulus from the high level of FGF in the vitreous; thus, fiber differentiation is induced. Cells at the junction between these two zones may receive an intermediate level of FGF stimulation, sufficient to induce cell migration. In essence, we are proposing that, in the eye, FGF acts as a lens morphogen in the sense that different levels of FGF stimulation elicit different lens cell responses. Hence its characteristic distribution in the eye establishes lens polarity and growth patterns. Since FGF has an inductive effect on lens cells from mature age animals, we also propose that this specific distribution of FGF in the eye is also important for maintenance of a normal lens throughout life. Finally the synergistic effects of insulin/IGF on the FGF-induced responses highlight the importance of considering the distribution of members of the insulin/IGF family of molecules in vivo. Mechanisms that control levels of both the FGF and insulin/IGF families of factors in the eye are probably of crucial importance in the formation and maintenance of a normal lens.

A quantitative ultrastructural study of motor and sensory lumbosacral nerve roots in the thalidomide-treated rabbit fetus.

A quantitative electron microscopic study was conducted on the dorsal and ventral nerve roots (L7-S1) supplying the hindlimbs of control and thalidomide-treated rabbit fetuses. The ventral roots at segmental levels L7 and S1 of treated nondeformed fetuses (TND) and deformed fetuses, demonstrated significant reductions (20-30%) of four of the parameters measured; total fascicular area (TFA), myelinated axon number (AN), Schwann cell counts (SCC) and axoplasmic area. In the dorsal roots of treated animals, thalidomide affected primarily segmental level S2, where significant reductions (15-30%) were observed in three parameters (TFA, AN, SCC). The occurrence of significant reductions in these measures in TND fetuses may indicate a neurotoxic action for thalidomide in the embryo. Despite apparent reductions in Schwann cell numbers in dorsal and ventral nerve roots, analyses of axon caliber and myelin sheath thickness indicated no evidence for any effects of thalidomide on the myelination of either sensory or motor axons. The segmental distribution of the axonal lesion, being maximal at segmental levels L7 and S1 in the ventral roots and at segmental level S2 in the dorsal roots, indicates a discrete period during development when nervous tissue is susceptible to the effects of thalidomide. The results of this study suggest thalidomide may have effects on neural development as well as on limb development.