Developmental analysis of SV2 in the embryonic chicken corneal epithelium

Highlights

• The secretory vesicle marker SV2 colocalizes with intraepithelial corneal nerves during embryonic development.

• SV2 colocalizes with the calcium sensor Synaptotagmin in corneal nerves, but is absent from the conjunctival epithelium.

• Corneal SV2 is a unique size compared to SV2 isolated from brain and this is not to due to glycosylation.

Abstract

Intraepithelial corneal nerves (ICNs) help protect the cornea as part of the blink reflex and by modulating tear production. ICNs are also thought to regulate the health and homeostasis of the cornea through the release of trophic factors. Disruption to these nerves can lead to vision loss. Despite their importance little is known about how corneal nerves function and even less is known about how the cornea is initially innervated during its embryonic development. Here, we investigated the innervation of the embryonic chicken cornea. Western blot and immunohistochemistry were used to characterize the localization of the synaptic vesicle marker SV2, a molecule thought to be involved in the release of trophic factors from sensory nerves. The data show that both SV2 and synaptotagmin co-localize to ICNs. Nerves in the conjunctiva also contained SV2 and synaptotagmin, but these were localized to below the basal layers of the conjunctiva epithelium. SV2 isolated from corneal epithelium migrates in western blot at a heavier weight than SV2 isolated from brain, which suggests a role in vesicle targeting, as the deglycosylating enzyme PnGase does not affect corneal SV2.

Introduction

Intraepithelial corneal nerves (ICNs) protect the cornea through several mechanisms. These include participating as the afferent limb in both the blink and tear reflex (Muller et al., 2003). It is also believed that ICNs help maintain the homeostasis of the corneal epithelium through the release of trophic factors (Garcia-Hirschfeld et al., 1994; Marfurt et al., 2001). The role of nerves in corneal epithelial homeostasis is supported by in vitro studies where co-culture with neurons or conditioned media increased proliferation or migration of cultured corneal epithelial cells (Garcia-Hirschfeld et al., 1994). There is less support in vivo, but, in conditions where corneal innervation is lost like corneal neuropathy resulting from diabetes mellitus, corneal epithelial homeostasis can be disrupted (Markoulli et al., 2017). Also, studies in other tissues that are rich in innervation, for example the intestine, support a functional role for nerves in maintaining associated epithelial cells through the release of trophic factors (Bulut et al., 2008).

The factors thought to be released from ICNs include substance P and CGRP and are stored in vesicles, either small clear-cored vesicles or larger dense cored vesicles prior to their release (Ueda et al., 1989). SV2 is a glycoprotein that was first characterized (Buckley and Kelly, 1985) as a novel type of transmembrane transporter that localized predominately to synaptic vesicles in neurons and secretory vesicles in endocrine cells (Bindra et al., 1993; Feany et al., 1992). SV2 is required for normal Ca²⁺ evoked secretion from neurons (Wan et al., 2010; Xu and Bajjalieh, 2001). This function in neurosecretion is dependent on an interaction with synaptotagmin, which has not previously been described in the cornea, and acts as a Ca²⁺ sensor (Schivell et al., 1996, 2005). Functionally, SV2 and synaptotagmin are necessary for the release of synaptic vesicles from presynaptic axon terminals at synapses. In the cornea, ICNs do not form synapses, and instead terminate as “free nerve endings” (Marfurt et al., 2001; Ueda et al., 1989). These, based on ultra-structural electron microscopy studies, have been described as prototypic, simplified endings without the complicated interactions between the nerves and target cells observed in endings such as Pacinian and Meissners corpuscles (Messlinger, 1996). The exact mechanisms for how factors like substance P and CGRP are released from sensory in the cornea, as well as other tissues, are unclear.

We have previously shown that an interaction between ICNs and corneal epithelial cells begins during the developmental innervation of the cornea. During these studies we identified small clear-cored vesicles in the endings of ICNs by transmission electron microscopy (Kubilus and Linsenmayer, 2010a). This led us to hypothesize that these vesicles are involved in the regulation of corneal epithelial homeostasis by ICNs. Here, we have investigated the developmental appearance of these vesicles using the synaptic vesicle markers SV2 and synaptotagmin in the embryonic chicken cornea.

These studies demonstrate that developmentally, SV2 is observed in the corneal epithelium concomitantly with the appearance of ICNs. By immunohistochemistry, punctate SV2 labeling was most dense in a zone apical to the basal nerve plexus. This region was also labeled by synaptotagmin. Labeling was also observed in the apical layers of the epithelium as well. As both SV2 and synaptotagmin play role in the calcium-evoked release of vesicles from neurons, the localization of these to the corneal epithelium suggests this is the area of regulated, active release. Also, in comparison to SV2 isolated from another source of synaptic vesicles, the brain, corneal SV2 was a unique size, and unlike brain SV2, the migration of SV2 isolated from cornea was unaffected by deglycosylation in western blot analyses.