Abstract
Purpose::
In embryonic chicks, nerve axons extend from the trigeminal ganglion to the edge of the cornea by embryonic day [E]6, surround the cornea from E7-E10, and enter the corneal stroma from all sides on E10. Factors controlling this nerve axon entry are unknown. Thyroxine [T4] injected in ovo on E9 induces precocious transparency by E12. The present study asks whether T4 treatment of E8 embryos influences corneal innervation by E11, expressions of T4 nuclear and non-nuclear receptors, and expressions of nerve-growth-related genes.
Methods::
E8 eggs received 2.5 or 5 µg T4, 5 or 10 mg 2-Thiouracil (2-TU), 5 µg T4+5 mg 2-TU, or buffer. Corneas were dissected on E11. Nerves were visualized immunohistochemically. Whole-cell RNA was isolated with RNeasy, cDNA synthesized with iScript, and mRNA expression assessed by real-time-PCR using iQSupermix.
Results::
Normally, growth cones enter the corneal stroma on E10, extend about 30% of a radius toward the center by E11, about 60% by E12, about 75% by E13, and reach the cornea center by E14. Corneal nerve density increases until hatching. E8-administered T4 stimulates increased innervation by E11 in a dose-dependent fashion. 2-TU inhibits T4 nuclear receptor function, but does not affect nerve extension from E8 to E11. Membrane T4/rT3 receptor Integrin αV mRNA expression is unchanged on E18 compared to E9, whereas Integrin ß3 expression declines ~7.5-fold, nerve-specific tubulin ß2 declines ~10-fold, Schwann-cell marker Substance P declines ~2-fold, and Ephrin B1 declines ~8-fold. In contrast, expression of myelin protein P0 rises ~7.5-fold, brain-derived neurotrophic factor (BDNF) rises ~7.5-fold, tyrosine kinase receptor B rises ~10-fold, nerve growth factor low affinity receptor rises ~15-fold, and neuregulin 1 & 2 isoforms rise ~7.5-10-fold. Precocious T4 treatment does not alter expressions of αV or ß3 integrins by E12, but does down-regulate expression of Substance P. Expressions of other nerve-related genes are unaffected by precocious T4 stimulation.
Conclusions::
Precocious T4 stimulates embryonic chick corneal innervention in a thyroxine-nuclear-receptor independent manner. Corneal cells express a membrane T4 receptor integrin. Many nerve-related genes are expressed in the cornea; Substance P expression is repressed by T4, but no nerve-related gene expressions are stimulated by T4. Results suggest that T4 influences corneal nerve growth by non-genetic mechanisms, perhaps facilitated by its integrin receptor.
Keywords: cornea: basic science • innervation: neural regulation