Unique and overlapping effects of triiodothyronine (T3) and thyroxine (T4) on sensory innervation of the chick cornea.

Multiple aspects of cornea development, including the innervation of the cornea by trigeminal axons, are sensitive to embryonic levels of thyroid hormone (TH). Although previous work showed that increased TH levels could enhance the rate of axonal extension within the cornea in a thyroxine (T4)-dependent manner, details underlying the stimulatory effect of TH on cornea innervation are unclear. Here, by examining the effects throughout all stages of cornea innervation of the two main THs, triiodothyronine (T3) and T4, we provide a more complete characterization of the stimulatory effects of TH on corneal nerves and begin to unravel the underlying molecular mechanisms. During development, trigeminal axons are initially repelled at the corneal periphery and encircle the cornea in a pericorneal nerve ring prior to advancing into the corneal stroma radially from all along the nerve ring. Overall, exogenous T3 led to pleiotropic effects throughout all stages of cornea innervation, whereas the effects of exogenous T4 was confined to timepoints following completion of the nerve ring. Specifically, exogenous T3 accelerated the formation of the pericorneal nerve ring. By utilizing in vitro neuronal explants studies we demonstrated that T3 acts as a trophic factor to directly stimulate trigeminal nerve growth. Further, exogenous T3 caused disorganized and precocious innervation of the cornea, accompanied by the downregulation of inhibitory Robo receptors that normally act to regulate the timing of nerve advancement into the Slit-expressing corneal tissues. Following nerve ring completion, the growth rate and branching behavior of nerves as they advanced into and through the cornea were found to be stimulated equally by T3 or T4. These stimulatory influences of T3/T4 over nerves likely arose as secondary consequences brought on by TH-mediated modulations to the corneal extracellular matrix. Specifically, we found that the levels of nerve-inhibitory keratan- and chondroitin-sulfate containing proteoglycans and associated sulfation enzymes were dramatically altered in the presence of exogenous T3 or T4. Altogether, these findings uncover new roles for TH on corneal development and shed insight into the mechanistic basis of both T3 and T4 on cornea innervation.