Thyroxine increases the rate but does not alter the pattern of innervation during embryonic chick corneal development.
OBJECTIVE
Embryonic chick corneal nerves reach limbal mesenchyme by embryonic day (E)5, encircle the cornea in several days, then defasciculate into the stroma simultaneously from all sides, while extracellular keratan sulfate proteoglycan (KSPG) accumulates from posterior to anterior stroma. Precocious thyroxine (T4)-induced increases in corneal thinning/transparency are blocked by 2-thiouracil (2-TU) inhibition of T3 synthesis. The hypothesis for this study was that precocious T4 exposure increases corneal innervation similarly.


METHODS
E8 embryos received T4, 2-TU, T4+2-TU, or buffer; corneas were harvested on E12. Corneal nerves were stained with neuronal beta-tubulin-specific TuJ1 antibody or chick nerve-specific CN antibody. Corneal thickness was determined from cryostat sections, and mRNA expression was measured by real-time PCR.


RESULTS
Nerves avoided the cornea until E9, then entered the anterior stroma, extended toward and reached the cornea center by E14, and never invaded posterior stroma. E7 to E18 corneal expressions of nerve growth factor and neurotrophin-3 genes were unchanged; receptor gene expressions rose. E7 to E12 semaphorin 3A and 3F and ephrin A2 and A5 expressions did not change significantly; semaphorin and ephrin/eph expressions increased from E9 to E18. E8 T4 administration increased nerve extension by E11, but did not alter circumferential penetration, anterior-only penetration, or neurotrophin expressions. 2-TU prevented T4-induced precocious corneal thinning, but augmented T4 nerve stimulation.


CONCLUSIONS
No changes in corneal neurotrophin or nerve pathfinding gene expressions accompany corneal transition to nerve growth cone permissiveness. T4 increases corneal nerve penetration rates by a non-T3-dependent mechanism. Results are consistent with possible roles for corneal KSPGs in regulating corneal nerve growth.
Conrad
Abigail H
AH
Division of Biology, Kansas State University, Manhattan, KS 66506, USA. aconrad@ksu.edu
Strafuss
Jessica M
JM
Wittman
Maria D
MD
Conway
Sabrina
S
Conrad
Gary W
GW
D000818:Animals; D002642:Chick Embryo / Q000187:drug effects*; D003315:Cornea / Q000196:embryology* / Q000294:innervation* / Q000378:metabolism; D047108:Embryonic Development / Q000187:drug effects; D036383:Ephrin-A2 / Q000235:genetics; D036386:Ephrin-A5 / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D020932:Nerve Growth Factor / Q000235:genetics; D020933:Neurotrophin 3 / Q000235:genetics; D009882:Ophthalmic Nerve / Q000196:embryology* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D039961:Semaphorins / Q000235:genetics; D013889:Thiouracil / Q000494:pharmacology; D013974:Thyroxine / Q000494:pharmacology*
D036383:Ephrin-A2; D036386:Ephrin-A5; D020933:Neurotrophin 3; D012333:RNA, Messenger; D039961:Semaphorins; D013889:Thiouracil; D020932:Nerve Growth Factor; D013974:Thyroxine