Purpose NOGO-A is a myelin associated protein best known for its ability to inhibit CNS regeneration in adults and may be involved in the maintenance of established neural pathways. Recent experiments have identified NOGO-A during development, when regeneration is less relevant. Thus, its role during development is unclear. We hypothesize that NOGO-A has a role in neural tube induction or early neuronal differentiation.
Methods To test our hypothesis we isolated chicken NOGO-A using RT-PCR. We utilized a protocol for difficult templates to isolate and replicate the GC rich NOGO-A transcript. A 219 base pair probe was synthesized and used to perform whole mount in situ hybridization (WISH) to localize NOGO-A expression in Hamburger and Hamilton (HH) stages 4-17 chick embryos. Regulation and induction of NOGO-A was tested by Shh, Fgf8b and RA bead implantation in HH4 embryos. These were harvested after 4 and 8 hours and processed by WISH.
Results We identified two different isoforms of NOGO-A in the chick embryo; 3.8KB and 4.3KB isoforms that we have sequenced. WISH showed initial NOGO-A expression in the neural plate at induction (HH4). Expression then followed formation of the neural tube (HH8), primary brain vesicles (HH12) and somites (HH17). Ectopic Shh increased local NOGO-A expression at 4 hours, Fgf8b showed increased local expression at 8 hours, and RA showed no increased expression.
Conclusions Our WISH experiments demonstrated that NOGO-A expression begins early in development and is induced by Shh and Fgf-8, known mediators of neural tube development. Additionally, we identified two isoforms of NOGO-A in the chicken embryo. It remains to be determined if these two isoforms are splice variants, and if they have distinct roles. Our results support our initial hypothesis that NOGO-A has a role beyond inhibition of CNS regeneration and is involved in CNS development.
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