| Optic nerve injuries in adult mammalian vertebrates can result in a permanent loss of vision. This is due to the inability of the central nervous system (CNS) axons to regenerate following injuries. However, CNS axons, like the optic nerve in fish and amphibians, regenerate successfully. Various molecules including growth promoting factors, transcription factors and cell adhesion molecules like cadherins are shown to be crucial for the successful regeneration. Our laboratory previously showed that N-cadherin, also known as Cadherin-2, was not expressed in the adult zebrafish retina, except in germinal zones and outer plexiform layer. N-cadherin (Ncad) expression became greatly increased in the retinal ganglion cell layer (gcl) 2-21 days following an optic nerve crush. To determine Ncad function in the optic nerve regeneration, I interfered with Ncad expression in the fish retina by electroporating a lissamine-tagged Ncad morpholino antisense oligonucleotide (NcadMO) into the eye of adult zebrafish one day after an optic nerve crush, and examined retinal axons regeneration at various time points (e.g. 7, 14 and 21 days). I found that the optic nerve regeneration was severely perturbed in these fish compared to fish with a control MO electroporated into the eye. The vast majority of the regenerating retinal axons in the NcadMO treated fish were found between the optic nerve head region and the crush site (immediately behind the eye) 7 days after the optic nerve crush, while the regenerating retinal axons in the control MO treated fish arrived in the optic tectum, the major target of the retinal axons.;These results suggest that Ncad plays an important role in the fish optic nerve regeneration. As the first step in elucidating the molecular mechanism underlying the Ncad mediated optic nerve regeneration, I performed gene expression analysis, using absolute quantitative-real time PCR and in situ hybridization, on some of the important regeneration associated genes. I found that expression levels of all the genes tested (gap43, a-tubulin , reggie1a, reggie2a, klf7) was increased in 4-day post optic nerve crush retinas from NcadMO treated fish compared to that of the control MO treated fish.;Next, I performed an mRNA expression study for two of the regeneration associated genes: klf7 and klf6a in the adult zebrafish CNS. Both klfs exhibit wide and similar expression in the zebrafish CNS. Brain areas containing strongly labeled cells include the ventricular regions of the dorsomedial telencephalon, the ventromedial telencephalon, periventricular regions of the thalamus and hypothalamus, torus longitudinalis, stratum periventriculare of the optic tectum, granular regions of the cerebellar body and valvula, and superficial layers of the facial and vagal lobes. In the spinal cord, klf7- and klf6a- expressing cells are found in both the dorsal and ventral horns. Numerous sensory structures (e.g. auditory, lateral line, olfactory and visual) and several motor nuclei (e.g. oculomotor, trigeminal, and vagal motor nuclei) contain klf7- and/or klf6a-expressing cells. My results may provide useful information for determining the roles of these Klfs in maintenance and/or function in adult CNS. |
