The expression and function of netrin-4 in murine ocular tissues

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Abstract

Netrin-4, a member of the netrin family, is a potent regulator of embryonic development. It promotes neurite extension and regulates pulmonary airway branching, vasculogenesis patterning, and endothelial proliferation in pathological angiogenesis. The initial characterization of netrin-4 expression was focused on epithelial-derived organs (kidney, lung and salivary gland) and the central nervous system. Ocular development is an ideal system to study netrin-4 expression and function, as it involves both ectodermal (cornea, lens and retina) and mesodermal (sclera and choroid) derivatives and has an extensive and well-characterized angiogenic process. Netrin-4 is expressed in all ocular tissues. It is a prominent component of the basement membranes of the lens and cornea, as well as all three basement membranes of the retina: the inner limiting membrane, vascular basement membranes, and Bruch's membrane. Netrin-4 is differentially deposited in vascular basement membranes, with more intense anti-netrin-4 reactivity on the arterial side. The retinal microcirculation also expresses netrin-4. In order to test the function of netrin-4 in vivo, we generated a conventional mouse lacking Ntn4 expression. Basement membrane formation in the cornea, lens and retina is undisrupted by netrin-4 deletion, demonstrating that netrin-4 is not a major structural component of these basement membranes. In the Ntn4 homozygous null (Ntn4−/−) cornea, the overall morphology of the cornea, as well as the epithelial, stromal and endothelial stratification are normal; however, epithelial cell proliferation is increased. In the Ntn4−/− retina, neurogenesis appears to proceed normally, as does retinal lamination. In the Ntn4−/− retina, retinal ganglion cell targeting is intact, although there are minor defects in axon fasciculation. In the retinal vasculature of the Ntn4−/− retina, the distribution patterns of astrocytes and the vasculature are largely normal, with the possible exception of increased branching in the deep capillary plexus, suggesting that netrin-4 may act as a negative regulator of angiogenesis. These data, taken together, suggest that netrin-4 is a negative regulator of corneal epithelial cell proliferation and retinal vascular branching in vivo, whereas netrin-4 may be redundant with other members of the netrin family in other ocular tissue development. Ntn4−/− mice may serve as a good model in which to study the role of netrins in vivo of the pathobiologic vascular remodeling in the retina and cornea.

Highlights

► Netrin-4 is expressed in corneal stroma and basement membranes. ► Netrin-4 knockout has increased corneal epithelial proliferation. ► Netrin-4 is expressed in retinal vascular basement membrane. ► Netrin-4 knockout has increased deep vascular branching. ► Netrin-4 knockout retina is normal.

Introduction

Ocular development involves neural tube-derived components (retina) and ectoderm-derived components (lens and cornea) (Cvekl and Tamm, 2004). In mouse, retinal ganglion cells (RGCs) are generated prenatally (E11–E19) in an approximately centrifugal order (Drager, 1985; Young, 1985). Their axons exit the eye starting at E11.5 also in a centrifugal order; thus, axons of the later-born RGCs arise from more peripheral regions and travel greater distances before they turn to exit into the optic nerve (Mann et al., 2004). Along their elongation path, RGC axons are in close contact with Müller cell endfeet and with the inner limiting membrane (ILM) (Mann et al., 2004; Stuermer and Bastmeyer, 2000).

The anterior segment follows parallel pathways of development. At E12.5–13.5, cells derived from the neural crest begin to migrate into the space between the anterior epithelium of the lens vesicle and the surface ectoderm. By E14.5–E15.5, the cells closest to the lens form an endothelial monolayer; the surface ectoderm becomes the corneal epithelium; cells between the endothelium and epithelium differentiate into keratocytes of the corneal stroma (Cvekl and Tamm, 2004).

During the development of these ocular structures, many extracellular matrix (ECM) molecules play important roles. For example, netrin-1, laminin α1β1γ1, and ephrin-B are all implicated in RGC axon pathfinding (Hopker et al., 1999; Mann et al., 2004), and ECM molecules are thought to have a role in corneal development and maturation (Kabosova et al., 2007).

Netrins are a family of ECM molecules that have homology with the short arm of laminin molecules. Five members of the netrin family have been identified in mammals: netrin-1 (Kennedy et al., 1994), netrin-3 (Van Raay et al., 1997; Wang et al., 1999), netrin-4 (Koch et al., 2000; Yin et al., 2000), netrin-G1 and netrin-G2 (Nakashiba et al., 2000, Nakashiba et al., 2002). All netrin genes encode secreted proteins, but in contrast to netrin-1, -3 and -4, netrin-G1 and -G2 are bound to the membrane via a glycosylphosphatidylinositol (GPI) linkage.

Netrins were first recognized as long range guidance cues regulating the migration of neurons and the behavior of axon growth cones (Barallobre et al., 2005; Kennedy et al., 1994). However, they have wide-spread expression outside the nervous system, and netrins are involved in a wide variety of developmental events, including angiogenesis (Lu et al., 2004; Wilson et al., 2006), cell–cell adhesion (Srinivasan et al., 2003; Yebra et al., 2003), and branching morphogenesis of lung, mammary gland and salivary glands (Liu et al., 2004; Schneiders et al., 2007; Srinivasan et al., 2003).

In the embryo, netrin-1 is deposited at the exit of the optic nerve, and RGC axons express the netrin-1 receptor, DCC (de la Torre et al., 1997; Deiner et al., 1997; Keino-Masu et al., 1996; Livesey and Hunt, 1997) suggesting a role in guidance. In both netrin-1-hypomorphic and DCC-null mouse retinas, RGC axons navigate to the optic disc. Some axons fail to exit into the optic nerve, indicating that netrin-1 is not required for guiding retinal axons to the optic disc, but may be required for proper exit of RGC axons (Deiner et al., 1997). Importantly, DCC-null mice have a more severe phenotype than netrin-1-hypomorphic mice, suggesting there may be additional ligands for the DCC receptor (Fazeli et al., 1997).

We first identified netrin-4 (β-netrin), and demonstrated its expression in multiple tissues (Koch et al., 2000). We showed that, in vitro, netrin-4 promoted the neurite growth from E14 rat olfactory bulb explants, suggesting netrin-4 might be important as a haptotactic factor in promoting axonal elongation or as a substrate for neuronal migration. Netrin-4 has also been shown to regulate epithelial branching and endothelial proliferation (Lejmi et al., 2008; Liu et al., 2004; Park et al., 2004; Schneiders et al., 2007). This study details netrin-4 expression in the mouse eye, provides the first characterization of a Ntn4−/− mouse, and uses that netrin-4 null mouse to define the role of netrin-4 in the development of ocular components in vivo.

Section snippets

Materials and methods

All procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Tufts University and State University of New York – Downstate Medical Center and were in accordance with the National Institute of Health Guide for the Care and Use of Animals.

Netrin-4 expression in the mouse eye

Netrin-4 is first detected in the BM of the presumptive corneal surface epithelium at E13 (Fig. 1; arrowheads). As development proceeds, netrin-4 immunoreactivity (IR) becomes more intense in the corneal epithelial BM, i.e., Bowman's membrane. Concomitant with the migration of mesenchymeal cells, netrin-4 IR is found within the developing corneal stroma beginning at approximately E16. An abrupt boundary in netrin-4 expression at the corneal limbus is evident at this early age. This striking

Conclusions and summary of principle findings

We, and others, have suggested roles for netrin-4 by localization in vivo (Liu et al., 2004), by analyzing effects of netrin-4 on cells in vitro (Lejmi et al., 2008; Park et al., 2004), and by exogenous netrin-4 in vivo (Larrieu-Lahargue et al., 2010). However, this study is the first direct demonstration of a role for netrin-4 in vivo. Although data reported by others have suggested roles for netrin-4 by localization and exogenous application of netrin-4, our results are the first to

Acknowledgments

Portions of this study were completed as part of the doctoral dissertation of Y.N. Li. The authors thank Dale D Hunter, PhD, JD for his careful and insightful editing of the manuscript. Supported by Grants: NS 39502 and EY 12767 (WJB).

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