The second factor is the 2D nature of routine transmission electron microscopy and the uncertainty of identifying a cell profile as a neuron rather than an epithelial cell, a leukocyte or a keratocyte. the cell-cell border between the fused epithelial cell and its neighbor (black arrowheads) (B). Enlargement of the middle inset in panel A reveals a continuation of the double membrane of the nuclear envelope (white arrow), an additional portion of the single membrane of the endoplasmic reticulum (black arrow), as well as the double membrane of a mitochondrion (white arrowhead) with visible internal cristae (C). Enlargement of the bottommost inset in panel A reveals a lack of membrane between the two cells at the site of fusion, a finding common to all serial images KDELC1 antibody of fusion events. If membranes were present, they would be visible as the double membrane of an axonal and epithelial cell border. The slight electron density visible is most likely accounted for by the organized cytoskeleton seen above the hemidesmosomes (Panel A, *) which appears to extend across the fusion site. Scale bars = 500 nm.(TIF) pone.0224434.s003.tif (7.0M) GUID:?DFEFB45E-6F46-4325-88DA-3E73029B8F78 S1 File: Surface-to-volume ratio and basal lamina pore diameter data. (XLSX) pone.0224434.s004.xlsx (23K) GUID:?6833D005-F457-41C7-AAA1-6E8A824DA253 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract The cornea is the most highly innervated tissue in the body. It is generally accepted that corneal stromal nerves penetrate the epithelial basal lamina giving rise to intra-epithelial nerves. During the course of a study wherein we imaged corneal nerves in mice, we observed a novel neuronal-epithelial cell interaction whereby nerves approaching the epithelium in the cornea fused with basal epithelial cells, such that their plasma membranes were continuous and the neuronal axoplasm freely abutted the epithelial cytoplasm. In this study we sought to determine the frequency, distribution, and morphological profile of neuronal-epithelial cell fusion events within the cornea. Serial electron microscopy images were obtained from the anterior stroma in the paralimbus and central cornea of 8C10 week old C57BL/6J mice. We found evidence of a novel alternative behavior involving a neuronal-epithelial interaction whereby 42.8% of central corneal nerve bundles approaching the epithelium contain axons that fuse with basal epithelial cells. The average surface-to-volume ratio of a penetrating nerve was 3.32, while the average fusing nerve was smaller at 1.39 (p 0.0001). Despite this, both neuronal-epithelial cell interactions involve similarly sized discontinuities in the basal lamina. In order to verify the plasma membrane continuity between fused neurons and epithelial cells we used the lipophilic membrane tracer DiI. The majority of corneal nerves were labeled with DiI after application to the trigeminal ganglion and, consistent with our ultrastructural observations, fusion sites recognized Proadifen HCl as DiI-labeled basal epithelial cells were located at points of stromal nerve termination. These studies provide evidence that neuronal-epithelial cell fusion is a cell-cell interaction that occurs primarily in the central cornea, and fusing nerve bundles are morphologically distinct from penetrating nerve bundles. This is, to our knowledge, the first description of neuronal-epithelial cell fusion in the literature adding a new level of complexity to the current understanding of corneal innervation. Introduction The cornea is Proadifen HCl the most highly innervated tissue in the mammalian body [1]. The nerves of the cornea provide autonomic responses such as tearing and blinking and assist in maintaining corneal epithelial homeostasis through the release of trophic factors [2]. Sympathetic innervation comes from nerve fibers originating in the superior cervical ganglion while sensory information is transmitted from the corneal epithelium to cell bodies located in the trigeminal ganglion, [3C6]. It is well-established that corneal stromal nerves enter the cornea in the peripheral stroma and travel horizontally before branching to give rise to vertical axons that penetrate the epithelial basal lamina [7, 8]. Penetrated nerves ramify shortly after entering the corneal epithelium (in a process known as leash formation), and these ramifications constitute the sub-basal plexus. Axons in the sub-basal plexus travel anteriorly and laterally between the wing and superficial-squamous cells of the corneal epithelium, after which they give rise to the epithelial nerve plexus in addition to axon terminals [9, 10]. Corneal innervation is a dynamic process, constantly changing as a result of aging and in response to pathology or injury [11]. The mechanisms by which corneal nerve patterning is regulated are not well established. In addition to data gathered from studies on neurotransmission, our understanding of corneal innervation is largely based on light and electron microscopic imaging. While transmission electron microscopy (TEM) makes it possible to appreciate corneal nerve ultrastructure from single ultrathin sections, it provides only a two-dimensional perspective [12]. For a three-dimensional context, serial sections are needed and while serial sectioning using TEM is possible, Proadifen HCl the technical challenge Proadifen HCl of.