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Graduate School of Biomedical Sciences

The James Kubilus Lab

Sensory Innervation in the Developing Cornea

The focus of my laboratory is on the development of the peripheral nervous system of the cornea. Specifically, we are investigating the molecular factors that regulate the growth of sensory nerves into the cornea during embryonic development and also the functional interactions between the nerves and the epithelial cells of the cornea. The cornea is located at the front of the eye and provides a clear window for light to pass through on its way to the photoreceptors at the back of the eye. It is necessary for the cornea to remain clear for normal vision. The shape of the cornea is also fundamental to proper vision because the cornea is responsible for approximately two-thirds of the focal power of the eye. Therefore, anything that damages the cornea, affects its clarity or alters its shape can potentially hinder normal vision. To prevent damage, the cornea is one of the most densely innervated structures in the body. These sensory nerves serve to protect the cornea from damage and maintain its normal function.

Sema3A AND SLIT2 Guide Corneal Innervation

Recently, work from our lab and others has shown that the axon guidance molecules semaphorin 3A (Sema3A) and SLIT2 regulate the spatiotemporal growth of nerves into the cornea. During embryonic development of the eye the cornea is free, initially, of nerves. Expression of Sema3A within the early cornea prevents the ingrowth of nerves. Sema3A instead causes the nerves to grow around the cornea, forming a peri-corneal ring. As this ring is completed the expression of Sema3A significantly decreases, allowing the nerves to radially enter the cornea. We believe this mechanism partially accounts for the even spatial distribution of nerves within the cornea. Another mechanism that ensures that the cornea is properly innervated is through regulation by SLIT2. In these studies we used a function blocking antibody approach to demonstrate that the branching pattern of corneal nerves is controlled by the SLIT2. In these studies, when we blocked SLIT2 in ovo, the branching of corneal nerves within the epithelium was significantly decreased when visualized by confocal microscopy.

Kubilus Fig 1

Figure 1. Whole mount immunohistochemistry with the TuJ-1 antibody shows the growth of nerves towards the eye and formation of the peri-corneal ring in the developing embryo.

Kubilus Fig 2

Figure 2. Blocking of SLIT2 function causes a significant decrease in the branching of the corneal nerves compared to IgG control.

Interaction between Sensory Nerves and Corneal Epithelial Cells

We have also been investigating how the sensory nerves within the cornea function, and how this is affected by their interaction with the cells of the corneal epithelium. Sensory nerves within the cornea are thought to be primarily sensory nociceptors, meaning they transduce any mechanical, thermal or chemical stimuli as sensations of pain. It has also been shown that there is reciprocal interaction between the corneal nerves and epithelial cells where they support each other through the release of trophic factors. Loss of this functional relationship, such as when corneal innervation is impaired, can lead to damage and deterioration of the cornea. However, the mechanisms involved in forming and regulating this interaction are unclear. Our research has shown that during development the corneal nerves form complex interactions with the apical layer of squamous epithelial cells. We have isolated these cells and compared the RNA expression profiles at different time points during the developmental innervation of the cornea. In these studies we have observed a significant change in many genes that may be involved in either the formation of the interaction or its maintenance, or possibly both. We are now using a number of techniques including siRNA silencing to test the function of these genes and their role in corneal innervation.

Kubilus Fig 3

Figure 3. Immunohistochemistry on a corneal section shows a nerve (arrow) reaching the apical squamous layer the corneal epithelium.

Kubilus Fig 4

Figure 4. Transmission electron microscopy of the interaction between the apical epithelial cells and the nerves shows the formation of complex structures.