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Naoki Nakaya, Mohor Biplab Sengupta, Stanislav I Tomarev; Vwc2l affects eye development and interacts with olfm1 in zebrafish Vwc2l affects eye development and interacts with olfm1 in zebrafish. Invest. Ophthalmol. Vis. Sci. 2018;59(9):578.
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© ARVO (1962-2015); The Authors (2016-present)
Vwc2l (also known as brorin-like) and olfm1 are components of the ionotropic AMPA-type glutamate receptor complex. Genes encoding these proteins are expressed in developing and adult zebrafish brain and retina. A temporary knockdown of these genes by morpholino oligonucleotide injections results in the disturbance of zebrafish brain and eye development. We investigated physical and functional interactions of mouse and zebrafish vwc2l and olfm1 in vitro and in vivo to assess the function of these proteins in neuronal development.
Mutant zebrafish lines (vwc2lsa16824, olfm1asa221 and olfm1bsa157) were obtained from the zebrafish mutation project at Wellcome Trust Sanger Institute. Vwc2l complete knockout fish were generated using a Crispr/Cas9 method. COS7 cells were transiently transfected with corresponding cDNAs and interactions of mouse and zebrafish vwc2l and olfm1 were analyzed by immunoprecipitation. Larvae were analyzed by immunofluorescence, in situ hybridization, electrophysiology and behavioral analysis.
Mouse and zebrafish vwc2l were co-precipitated with olfm1 from lysates of COS7 cells transiently transfected with corresponding expression constructs. An injection of vwc2l mRNA in zebrafish eggs resulted in severe developmental defects, mainly in the posterior part of larvae. These defects were remarkably reduced when the vwc2l mRNA was injected in olfm1 knockout eggs, indicating that the effects of vwc2l over- or mis-expression may be modulated by olfm1. The homozygous vwc2lsa16824 larvae with a mutation modifying the C-terminus of vwc2l protein demonstrated microphthalmic eyes and reduced brain size. The expression of several retinal patterning genes such as pax6a, pax6b, aldh1a2, aldh1a3, vax1, vax2 and tbx5 were markedly reduced in the vwc2l mutant larval retina. Severe behavioral defects including the absence of spontaneous tail flicks and optokinetic responses were observed in mutant larvae. Nerve growth and innervation of the motor neurons to the muscles was also disturbed in the mutant larvae. We are currently investigating the vwc2l complete knockout larvae and the involvement of the AMPA receptors in the developmental functions of vwc2l and olfm1.
Zebrafish vwc2l interacts with olfm1 in vitro and in vivo. The vwc2l gene may be important for eye, brain and peripheral nerve development.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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