Sef and Sprouty expression in the developing ocular lens: Implications for regulating lens cell proliferation and differentiation

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Abstract

In many developmental systems, growth factor signalling must be temporally and spatially regulated, and this is commonly achieved by growth factor antagonists. Here, we describe the expression patterns of newly identified growth factor inhibitors, Sprouty and Sef, in the developing ocular lens. Sprouty and Sef are both expressed in the lens throughout embryogenesis, and become restricted to the lens epithelium, indicating that lens cell proliferation and fibre differentiation may be tightly regulated by such antagonists. Future studies will be aimed at understanding how these negative regulatory molecules modulate growth factor-induced signalling pathways and cellular processes in the lens.

Introduction

The lens of the eye is a relatively simple tissue, comprised of a monolayer of cuboidal epithelial cells that overlies the anterior surface of elongated and precisely aligned fibre cells. The ordered spatial arrangement of these two lens cell types is established during embryogenesis and is maintained as the lens continues to grow throughout life. Lens growth is dependent on proliferation of the epithelial cells and their subsequent differentiation into fibre cells at the lens equator. Above the equator, in the germinative zone, lens epithelial cells divide. Progeny of these divisions are displaced posteriorly, into the transitional zone, where they begin to differentiate into fibre cells. Fibre differentiation is characterized by distinct molecular and morphologic changes such as exit from the cell cycle, cell elongation, loss of organelles and nuclei, as well as the accumulation of fibre-specific crystallin proteins [1], [2], [3]. The normal architecture, and hence transparency of the lens, depends on the maintenance of this distinctive pattern of growth.

It is well established that the distinct patterns of cell behaviour in the lens are primarily regulated by the ocular environment, in particular the ocular fluids that bathe the lens; the aqueous and vitreous humour [3]. Much of the fibre-differentiating activity of the ocular fluid, namely the vitreous, has been shown to be attributed to the presence of FGFs [4]. In vitro studies have led to the identification of members of the FGF family as potent lens fibre-inducing factors [1]. It has also been shown that these FGFs stimulate lens epithelial cells to proliferate and migrate. Significantly, FGF can induce lens epithelial cell proliferation, migration and fibre differentiation in a dose-dependent manner [3], [5]; a low concentration of FGF stimulates only cell division, whereas higher concentrations can induce cell migration and fibre differentiation, respectively. Biologically active FGFs are also present in the ocular media and are expressed in the lens, specifically in regions of high cell proliferation and early fibre differentiation [4], [6], [7], [8]. These findings have led to the hypothesis that in situ, lens growth patterns are regulated by an antero-posterior ‘FGF gradient’, whereby FGF bioavailability is differentially distributed in the eye. In this model, anterior proliferating lens epithelial cells are exposed to a lower level of FGF activity from the aqueous, whereas differentiating fibre cells are exposed to a higher level of FGF activity in the vitreous. This is supported by the distinct expression of FGF receptors in the lens [9] which indicate that FGF influences the spatial patterns of cell proliferation and fibre differentiation. Support for this comes from in vivo studies that have directed the overexpression of different FGFs specifically to the lenses of transgenic mice [10], [11]. In these mice, the anterior lens epithelial cells, now exposed to increased levels of FGF, are induced to differentiate into fibre cells, disrupting the normal polarity of the lens. Moreover, recent studies using conditional gene targeting strategies have generated FGF receptor ‘knockouts’ in the lens. The observation that these mice undergo no fibre differentiation and have impaired lens cell proliferation confirms the requirement for FGF receptor signalling in cell proliferation and fibre differentiation [12]. Taken together, these studies support the model that FGFs are important for determining the spatial patterns of cell differentiation and proliferation in the lens.

Most growth factors mediate cellular responses by binding and activating high affinity cell surface receptor tyrosine kinases (RTKs). This event leads to the activation of several intracellular signalling cascades, including the phosphatidylinositol-3 kinase (PI3-K), phospholipase C gamma (PLC-γ) and Ras to extracellular signal-regulated protein kinase (ERK) pathways; ERKs are a subclass of the mitogen-activated protein kinases (MAPKs). The ERK/MAPKs are the most abundant MAPKs in the lens [13] and their activation plays an important role in regulation of embryonic development, as well as in modulating many cellular events, including cell cycle progression and cell differentiation. Studies by our laboratory and others have recently established an important role for ERK1/2 signalling in growth factor-induced lens cell proliferation and fibre differentiation [14], [15], [16]. Using a lens epithelial explant system, ERK1/2 was differentially activated in response to different concentrations of FGF. Furthermore, inhibition of ERK1/2 activation blocked FGF-induced lens cell proliferation, as well as cell elongation accompanying fibre differentiation [14]. Clearly, FGF signalling in the lens is quite complex, with its activity potentially regulated at multiple levels. Not only do FGFs signal through multiple pathways, there may be cross-talk between these pathways, as well as with different signalling pathways initiated by other growth factors found in the ocular media. As mentioned earlier, FGF signalling in the lens is further complicated by the fact that activation of a specific RTK can result in different cellular responses. In many developmental systems, to ensure a physiologically appropriate cellular response, growth factor signalling must be temporally and spatially regulated, and this can be achieved by a parallel set of inhibitory signals that lead to an accurate and reproducible biological outcome. Antagonists of growth factor signalling pathways can provide such negative signals, and are reported to play important roles in developmental patterning by restricting the range of their cognate inducer [17].

One of the first identified bona fide feedback regulators of the FGF pathway was Sprouty (Spry), which was initially discovered through a genetic screen in Drosophila and later shown to be important for FGF-induced tracheal branching in this species [18]. Spry proteins are widely conserved, with up to four members identified in mammals (Spry1–4) [19], and are expressed in highly restricted patterns that correlate with known sites of FGF signalling [20], [21]. Spry is recognized in many physiological and developmental processes as an antagonist of RTK signalling pathways, with its overexpression mimicking the functional loss of RTKs, including those activated by FGF [22]. In the chick, overexpression of Spry in the developing limb bud results in inhibition of cell differentiation, displaying a comparable phenotype to that reported in FGF null mutants [22]. Consistent with this, cells overexpressing Spry have also been reported to have a reduced responsiveness to exogenously applied growth factors. Moreover, in Drosophila, Spry null mutants have an ‘overactive’ FGF signalling pathway, inducing increased numbers of cells situated outside the normal range of FGF [18], [23].

Further screening for genes with restricted expression patterns during early development [24] revealed other modulators of the FGF pathway, including Sef (similar expression to fgfs). Sef is a transmembrane protein that unlike Spry, is restricted to vertebrates; however, like Spry, it has been shown to function as an antagonist of FGF signalling during development [25], [26]. Also similar to Spry, overexpression of Sef can inhibit FGF signalling, and impaired Sef expression leads to overstimulation of FGF signalling [17]. The co-expression of Spry and Sef with known sites of RTK signalling during embryogenesis [21] lends credence to the important role they have in cell fate specification; a close spatial and temporal interdependence between RTK signalling (e.g. FGF signalling) and Spry and Sef gene expression has been seen in many mammalian tissues, including brain, muscle, gut, heart and lung [20], [21], [27], [28]. As a means of providing tight autoregulation, the expression of these inhibitors is induced by the pathway they antagonize; Spry and Sef have been shown to be positively regulated by FGF, more specifically FGF-induced ERK1/2 signalling [18], [22], [26], [29]. Overall, given the strong link between FGF signalling with the expression of Spry and Sef in different developmental systems and the important role that FGF plays in lens development, there is a strong rationale for investigating the expression patterns and role of these antagonists in the developing lens.

Section snippets

Methods

All animal procedures were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and the animal care guidelines published by the Institute for Laboratory Animal Research (Guide for the Care and Use of Laboratory Animals). The use of human tissues adhered to the tenets of the Declaration of Helsinki. All studies were approved by the Institutional Ethics Committee of the University of Sydney.

Results

To examine the expression of Sef and Spry in the developing lens, we first applied RT-PCR using specific primers for Spry1, Spry2, Spry4 and Sef, on RNA obtained from postnatal rat lens tissues that were separated into epithelial (lens capsule attached) and fibre cell fractions. Specific amplicons for Spry1, Spry2 and Sef, but not Spry4, were detected in reverse transcribed RNA from both the lens epithelial and fibre cell preparations (Fig. 1). Note that in some cases there was a very weak

Discussion

Much of the lens research carried out to date has focused on identifying the key growth factors, and their respective signalling pathways, that influence the behaviour of lens cells. Aberrant lens cell behaviour, leading to the diseased state, may result from the impaired function of these molecules, and/or the dysregulation of their signalling pathways. As a result, in normal lens development and growth, to ensure a physiologically appropriate cellular response, growth factor signalling events

Acknowledgements

The authors would like to acknowledge the support of the National Health and Medical Research Council (NHMRC) and the Sydney Foundation for Medical Research, Australia as well as NIH, USA (R01 EY0-3177). Much appreciation is also extended to Dr. Michelle Madigan for providing the human foetal lens tissue.

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