Since the days of Hans Spemann, the ocular lens has served as one of the most important developmental systems for elucidating the fundamental processes of induction and differentiation. Lens is an important source of signals that influence the eye development and a variety of genes expressed by the lens have been identified. The identification of additional molecule(s), especially secreted ones that might mediate signals, will extend our knowledge of the molecular mechanisms of eye and lens development. Here, we will introduce a soluble molecule, Equarin, and discuss its vital role in multiple aspects of lens development.
Normal development of an organism requires elaborate control over cell proliferation, migration and differentiation. Owing to its simple structure and ease of manipulation, the ocular lens has been focused on by developmental biologists and has been considered for many decades to be an excellent tissue in which to uncover the general mechanisms underlying embryonic developmental processes. To function properly, lenses depend on transparency and refractive properties that directly relate to cellular features that arise during fiber cell differentiation. Therefore, detailed knowledge of the differentiation process is the central to understanding the fundamental optical properties of the lens as well as the etiology of certain types of cataracts. Over 20 years of evidence accumulated using several different vertebrate species has suggested that fibroblast growth factors (FGFs) play a key role in lens development. However, this signaling is only part of the story. Identification of additional molecule(s), especially secreted ones that possibly mediate signal transductions, will extend our knowledge of the molecular mechanisms of eye and lens development. In this review, we introduce a soluble molecule, Equarin, which was isolated from chick E6 lens and is expressed in the lens equatorial region. We focus on the role of Equarin in different aspects of lens development and what questions remain to be answered.
Overview of lens development
During embryogenesis, the lens arises from a region of head ectoderm that lies adjacent to the region of the neural plate from which the optic vesicle will form. As the development proceeds, the region of presumptive lens ectoderm becomes closely associated with the optic vesicle. The presumptive lens thickens to form the placode and invaginates together with the optic vesicle to form the lens vesicle and optic cup, respectively. Subsequently, the cells located anteriorly in the lens vesicle form an epithelial monolayer that covers the anterior surface, and cells in the posterior half of the vesicle elongate and differentiate to form primary fibers (Reza & Yasuda 2004; Lovicu & McAvoy 2005; Robinson 2006). In this way, the lens acquires its distinctive polarity. All further growth of the lens is due to the proliferation of epithelial cells and their subsequent differentiation into secondary fiber cells at the equatorial region of the organ (reviewed in Piatigorsky 1981; Wride 1996). How the two types of lens cells differentiate is a major focus of ocular developmental biology.
Fibroblast growth factor signaling
Ocular medium is a rich source of growth factors, and the lens itself expresses members of the major growth factor families and a variety of growth factor receptors. More than 20 years of evidence accumulated from several different vertebrate species suggested that FGFs and FGF receptors (FGFRs) play key roles in lens development. FGF signaling has been implicated in lens induction, lens cell proliferation and survival, lens fiber differentiation and lens regeneration (Robinson et al. 1995; Lovicu & Overbeek 1998; Robinson et al. 1998; Le & Musil 2001; Robinson 2006). Essentially, studies have shown that FGF1 or FGF2 promoted morphological and molecular changes in lens epithelial explants that are characteristic of secondary fiber differentiation.
Indications that other factors besides FGF are required for lens development have come from two main lines of investigation. First, FGF1- and FGF2-deficient mice exhibited no lens development defects (Dono et al. 1998; Miller et al. 2000). Second, other factors, including insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), (Wang et al. 2010), bone morphogenetic proteins (BMPs) (Boswell et al. 2008a,b; Rajagopal et al. 2008, 2009) and Wnt (Stump et al. 2003; Lyu & Joo 2004; de Iongh et al. 2006), are required to coordinate with FGF for proper fiber differentiation and maintenance of lens epithelium. In recent years, particular attention has been paid to identifying and investigating novel factors that maintain normal lens structures and functions.
Lessons learned from Equarin studies on lens development
Identification and characterization of Equarin
Tissue interactions that take place in early embryos are required for normal eye development (Spemann 1901; Lewis 1904). Without a lens placode, the optic vesicle remains rudimentary, and the optic cup with well-defined neuroretinal and pigmented layers is not formed (Hyer et al. 1998). Ablation of the developing lens using a toxin transgene inhibits the formation of the iris, the ciliary body, the vitreous body, and the retina (Breitman et al. 1989; Harrington et al. 1991). Therefore, the lens is an important source of signals that influence eye development, and a variety of genes expressed by the lens have been identified. Mu et al. 2003; used signal sequence trap screens (Klein et al. 1996) to isolate molecule(s) from a chick E6 lens cDNA library. A novel clone was found in the lens equatorial region and was therefore designated ‘Equarin’.
Two forms of Equarin were isolated from a chick E6 lens cDNA library. Equarin-L consists of 592 amino acid residues, the N-terminal regions of which are identical to those of Equarin-S as well as an extra 366 amino acid residues in the C-terminal region. In mammals, this gene and its encoded protein have been renamed CCDC80 (coiled-coil domain containing 80, also known as DRO1 and URB). Murine and human CCDC80 have high homology (murine 65.6% and human 66.4%) to Equarin (Aoki et al. 2002; Mu et al. 2003). Rat, murine and human CCDC80 have a wide tissue distribution (e.g., fat, lung, ovary, uterus, mammary gland, testis, liver, spleen, pancreas, kidney, heart, stomach, bladder, skeletal muscle, skin, and brain) (Aoki et al. 2002; Okada et al. 2008; Tremblay et al. 2009). Three repeat regions of Equarin-L and mouse CCDC80 display similarity to a C-terminal region of SRPX (sushi-repeat-containing protein encoded on the X chromosome)/drs (downregulated by Src) and SRPX2 (sushi-repeat protein upregulated in leukemia)/SRPUL proteins (Dry et al. 1995; Meindl et al. 1995; Pan et al. 1996; Inoue et al. 1998; Kurosawa et al. 1999; Marcantonio et al. 2001; Mu et al. 2003; Liu et al. 2004) (Fig. 1A). Pawłowski et al. has described not only vertebrate examples of this domain, which was named DUDES (DRO1-URB-DRS-Equarin-SRPUL) (Bommer et al. 2005), but also the many prokaryotic homologues. Therefore, they decided to rename the domain to P-DUDES (Prokaryotes-DUDES) (Pawłowski et al. 2010). Although the sequence similarity of the DUDES domains in CCDC80, SRPX and SRPX2 has been discussed on many occasions, the functional relativity between them has been rarely discussed. Roles for CCDC80 in tumor suppression, energy metabolism, embryonic development and skeletal formation have been suggested. However, the role of CCDC80 and its regulation at the cellular level are only just beginning to be understood.
Developmental expression of Equarin
During chick eye development, Equarin mRNAs are expressed exclusively in the lens until postnatal day 2. Equarin is first detected in the lens placode at HH 13 and in the proximal side of the lens vesicle at HH 20. As lens development proceeds, Equarin is strongly expressed in the lens equatorial region with a high-dorsal-to-low-ventral gradient (Fig. 1B,C). Two circles are visible in the lens, which correspond to the annular pad and the newly formed fiber cells around the equatorial region (Mu et al. 2003; Song et al. 2012). This expression pattern in chick lens is similar to that observed in mouse lens using murine CCDC80 antisense probe (Song et al. 2012). The specific expression pattern of Equarin was comparative with the lens fiber marker β-crystallin and the lens epithelium cell marker Pax6 (Mu et al. 2003). Protein distribution coincides with expression at the mRNA level. Equarin protein is also strongly expressed in the lens capsule and distributed to the peripheral retina in a high-dorsal-to-low-ventral manner. No Equarin protein was detected in the central retina. Because microinjection of Equarin mRNA into Xenopus embryos causes ventral eye malformation (Mu et al. 2003), the protein distribution in peripheral retina suggests that Equarin plays a role in retinal polarity.
In addition to expression in the lens, Equarin mRNAs are expressed at HH 19 in the isthmus, the cranial neural tube (especially rhombomeres r4 and r6), the dermatome, the dorsal region of the neural tube, the vitelline vein, and the anterior pore of the neural tube.
Role of Equarin in lens differentiation
Equarin is strongly expressed early on, and overexpression of Equarin didn't show an effect on morphology or lens induction. However, β-crystallin is upregulated by Equarin, suggesting a promotion of lens fiber differentiation by Equarin. Additionally, autonomous lens fiber differentiation was markedly diminished when endogenous Equarin was blocked by ZFN (Zinc-finger nuclease) technology. Gain-of-function using in ovo electroporation and loss-of-function assays using ZFN technology provide convincing evidence that Equarin serves as a bona fide differentiation factor during chick lens development (Song et al. 2012).
Equarin protein is strongly distributed to the posterior lens capsule (Song et al. 2012). FGF1 and FGF2 are secreted from various ocular sites into the capsule, where they bind to heparan sulfate proteoglycans (Schulz et al. 1997) and specific FGF receptors (de Iongh et al. 1997). Direct interactions between Equarin and FGF1 and FGF2 have been demonstrated by immunoprecipitation assays although the specific binding domain remains to be identified (Song et al. 2012). In vitro studies using rat lens explants suggest a potential role of MAPK/ERK1/2 in the regulation of FGF-induced lens proliferation and fiber differentiation (Lovicu & McAvoy 2001). We proposed that Equarin exerts its function by modulating FGF signaling. The following observations support this conclusion: (i) overexpression of Equarin upregulated the expression of ERK-P, the downstream effector of FGF-MAPK, both in vivo and in vitro; (ii) activation of ERK was diminished by the presence of the FGFR inhibitor PD173074; (iii) a minimal level of Equarin and FGF upregulated fiber differentiation; and (iv) loss of endogenous Equarin by ZFNs diminished the upregulation of fiber markers by FGF.
Tyrosine kinase Janus kinase 2 (JAK2) is activated by many cytokine receptors, including growth factor receptors, and promotes the growth, proliferation, and/or differentiation of many cell types (Aaronson & Horvath 2002; Hou et al. 2002). In the lens, STAT proteins involved in the JAK signal transduction pathway are constitutively expressed and activated by FGF1, FGF2, IGF-1 and PDGF (Potts et al. 1998; Ebong et al. 2004a,b). Interestingly, a recent study found that CCDC80 binds to the active, tyrosylphosphorylated form of JAK2. The DUDES domains of CCDC80 are required for binding to JAK2 (O`Leary et al. 2013). CCDC80 elevates the phosphorylation of Stat5 and Stat3 and therefore stimulates a specific subset of signaling molecules in the JAK2 network. DUDES protein is characterized in a novel class of proteins that bind activated JAK2. Therefore, Equarin most likely participates in the regulation of the JAK pathway during lens development, although there is no direct evidence for this hypothesis.
Equarin protein contains a signal peptide, which suggests that Equarin may localize to and possibly move between multiple cellular compartments. Mu et al. 2003; demonstrated the presence of Equarin protein in both supernatant and whole-cell lysate in transient-expressing COS-7 cells. Additionally, O`Leary et al. 2013; further confirmed the subcellular localization of CCDC80, suggesting the function of CCDC80 in the intracellular compartment. Consequently, we propose that Equarin plays important roles in multiple cellular compartments. On the cell surface, Equarin, cell-surface heparan sulfate proteoglycans, and FGF most likely form a ternary complex that effectively increases the local concentration of FGF in proximity to the FGFR, upregulating FGF signaling (Fig. 2). Subcellularly, Equarin binds JAK2 and elevates the phosphorylation of Stat3 and Stat5 (Fig. 2). In addition to FGF, other signaling pathways may also participate in the function of Equarin. This is because (i) both MAPK and JAK pathways mediate not only FGF but also EGF, IGF, vascular endothelial growth factor (VEGF) and integrin receptor signaling pathways and (ii) FGFR inhibitor could not completely inhibit the activation of ERK that was induced by Equarin (Song et al. 2012).
Role of Equarin in lens adhesion
Cell adhesion is responsible for assembling cells together and is fundamental for both development and differentiation. Lens epithelial cells at the equator migrate posteriorly, contacting factors in the vitreous humor and initiating differentiation. Elongation of the differentiating fiber cells is coupled with directed migration posteriorly along the capsule and anteriorly along the fiber cell-epithelial interface, which generates a symmetrically organized fiber cell mass with aligned suture planes. To make these movements, cells systematically create and dissolve cell–cell and cell–matrix adhesions and form connections between these adhesions and the cytoskeleton, generating contractile force. Interaction between lens cells and specific extracellular matrix (ECM) components is central to the directed migration that fiber cells undergo at their basal ends to form posterior sutures (Piatigorsky 1981; Taylor et al. 1996; Bassnett et al. 1999; Kuszak et al. 2004; Zelenka 2004). Mouse CCDC80 is involved in cell adhesion and migration (Manabe et al. 2008). Equarin protein is localized to the extracellular region of lens cells and cell–cell borders. This localization pattern coincides with the fact that many ECM molecules are secreted and immobilized outside the cells. Meanwhile, Equarin protein is strongly localized to the lens posterior capsule. By in vitro attachment, Equarin promoted lens cell adhesion in a dose-dependent manner through heparan sulfate proteoglycan (Song et al. 2013). Several receptors that contain heparan sulfate chains are expressed on the cell surface, including the syndecan family of cell surface proteoglycans, phosphatidylinositol-linked glypican, and the part-time proteoglycans beta glycan and CD44E (Carey 1997; Bernfield et al. 1999). Previous studies demonstrated that syndecan receptors are involved in the regulation of FGF signaling and cell–matrix adhesion (Carey 1997; Iba et al. 2000; Rapraeger 2001). Their heparan sulfate chains bind to a large number of molecules, including ECM components (e.g., Fibronectin and collagen) and heparan-binding growth factors (e.g., FGFs). In the chick lens, only syndecan-3 is expressed by the lens fiber cells after the head ectoderm has differentiated into the lens placode (Gloud et al. 1995). By the co-precipitation assay, it has been demonstrated that Equarin binds to syndecan-3 but not syndecan-2. Therefore, these data suggest that syndecan-3 may provide the heparan sulfate chain used by Equarin to mediate cell adhesion (Song et al. 2013).
Integrins and heparan sulfate proteoglycans are the primary ECM adhesion receptors that coordinate signaling events and determine signaling outcomes (Iba et al. 2000; Kim et al. 2011). Using a tumor cell line, RD cells, we found that cell adhesion activity mediated by Equarin was blocked by heparin, rather than function blocking mABs to β1 integrin (AIIB2), RGD peptide, and RGE peptide. Strikingly, when cells were treated with function blocking monoclonal antibodies (mABs) to β1 integrin (AIIB2), cells attached but no longer spread on Equarin (Song unpubl. data, 2013). Similar results were obtained using H1080 cells. Therefore, we may conclude that Equarin use heparan sulfate proteoglycan as the initial receptor for Equarin-mediated cell adhesion and use β1 integrin to initiate cell spreading in tumor cells. Further research is still needed to show involvement of integrin receptors in the Equarin-mediated lens cell adhesion response and the associated signaling pathways. If this is true in lens cells, another question is how the heparin sulfate proteoglycan works in concert with β1 integrin. One possibility is Equarin binds heparin sulfate proteoglycan and provides signals that activate integrin. The binding of Equarin to integrin and their functional relations remain to be identified.
Equarin and its homologue CCDC80 are expressed in a large number of tissues in both embryos and adults and have been implicated in a wide range of functions. In the lens, Equarin is expressed exclusively in the equatorial region and plays important roles in lens cell differentiation, adhesion and migration (Fig. 2). Clarifying the roles of Equarin in lens development will provide a foundation to better understand its roles in other tissues.
We thank all collaborators and members of our laboratories for their valuable help. This work was supported by MEXT KAKENHI Grant Number 22122009 (K.O.), JSPS KAKENHI Grant Number 20370089 (K.O.), and the Global COE Program (Cell Fate Regulation Research and Education Unit) (X.S. and H.T.).