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K. J. Wahlin, R. Adler; Neuroligin Expression Patterns and Spliced Isoforms in the Developing and Mature Chick Retina. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1283.
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© ARVO (1962-2015); The Authors (2016-present)
Cell replacement therapy is an enticing approach for treatment of blinding retinal diseases; however, lack of synapse formation between graft and host tissues remains a major obstacle. Overcoming this obstacle requires a better understanding of the molecules involved in synaptogenesis. We have therefore concentrated on the neuroligins, whose role in other regions of the CNS is well known. We have cloned several neuroligin (NLGN) isoforms from the chick retina, analyzed their sequences, determined their developmental patterns of expression, and investigated their differential distribution in different nuclear layers of the retina.
Retinas from either embryonic or post hatched chicks were lysed, RNA purified and cDNA synthesized by oligo-(dT) primed reverse transcription. Sequences from published EST’s, published genomic data and products derived from RACE-PCR (rapid amplification of cDNA ends) were assembled as a contig and used to design oligonucleotides designed to amplify coding regions and isoform specific regions spanning conserved ‘A’ and ‘B’ sites. Laser capture microdissection (LCM) was used to collect specific cell layers for RNA isolation, cDNA synthesis, and PCR analysis.
Despite repeated attempts we were unable to identify sequences corresponding to NLGN-2. On the other hand, NLGN -1, -3 and -4 coding sequences were cloned and their sequences compared with that of several mammalian counterparts. Elucidation of the full length NLGN-3 gene was accomplished only after repeated RACE-PCR using conditions appropriate for -GC rich templates. Both NLGN-1 and -3 exhibited splice variation at conserved ‘A’ and ‘B’ sites, previously described in mammals. Expression of the isoforms appeared developmentally regulated, since NLGN-1 and -3 each exhibited two ‘A’ site variants at earlier stages and three ‘A’ site variants at later stages of development. NLGN-4 did not appear to exhibit any isoform variation at either the ‘A’ or ‘B’ sites. Analysis of LCM captured samples also demonstrated layer-specific differences in the profiles of NLGN-1 and -3 spliced isoforms.
We have identified and cloned in the chick retina several trans-synaptic cell adhesion molecules that are linked to synapse formation elsewhere in the CNS. The rich variability in splicing of neuroligin gene family members, particularly in their heterogeneous distribution, may play a role in the diversity of synaptic connections in the eye. We are currently in the process of evaluating the functionality of these genes in vivo and in vitro.
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