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Ralph F Nelson, Reena R Abraham, Sara Patterson, Jennifer L Strykowski, Lin Li, Harold A Burgess, Victoria P Connaughton; Zebrafish Transgenic Reports Mushashi1 (Msi1) in Retinal Neurons. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2369.
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Gal4 insertion in the line Et(SCP1:Gal4ff)y245 (y245) is localized to the promoter of msi1, making y245 a candidate reporter for msi1. Here we compare immunoreactivity (IR) of an Msi1 antibody to y245;UAS: kaede fluorescence.
The 14h1 rat Msi1 antibody (MBL), selected because zebrafish Msi1 contains a sequence similar to the 14h1 antigenic peptide, revealed a single 41kDa band on Western blots of 6dpf larval heads. For IR, 6dpf heads were fixed in 4% PFA in PBS, impregnated with 30% sucrose, embedded in OCT, and cryosectioned at 10 µm. After incubation in blocking buffer (2% fetal bovine serum or BSA, 0.2% Triton X-100, 1% DMSO in PBS), sections were incubated overnight (4C) in 14h1 (1:200) in blocking buffer. Goat-anti-rat Alexafluor594 (Abcam, 1:200) secondary antibody and confocal microscopy revealed IR patterns. ERG b2 and PIII waves were isolated from 6dpf eyes perfused with 50µM CNQX or 20mM Na Aspartate (respectively) in 95%O2/5%CO2-saturated MEM (Invitrogen), and recorded with glass microelectrodes (WPI DAM80 amplifier) using a spectral stimulation protocol (Nelson & Singla, 2009).
Western blots of 6dpf heads showed no change of Msi1 expression in mutants (y245+/+). In mutants, hets, and WT eyes, 14h1-IR occurred in perinuclear cytoplasm of ganglion and amacrine cells, inner plexiform layer (IPL), cone synaptic pedicles, and a high band of cone inner segments. Descending cone axons crossed an unreactive dark band between inner segments and pedicles. Bipolar somata labeled faintly. Kaede was brightest in y245;UAS:kaede mutants and localization was identical to 14h1-IR, though in mutants, Müller cells and the dark photoreceptor band were also labeled. Kaede stained cones were previously shown to be red and green types (Cohen et al, 2013). WT retinas displayed regular layering, but y245 mutants often revealed thin and irregular IPL (33% reduction in mean thickness). Mutant 6dpf b2 and PIII spectral sensitivities were depressed.
y245 gene products are found in the same retinal cell types and layers as 14h1-IR suggesting y245 is a complete msi1 reporter. Localization of Msi1, an RNA binding protein, within synaptic layers is of interest for function. y245 mutants show changes in PIII and b2 responsiveness, and altered retinal histology, but no gross reduction in protein expression. This suggests that y245 interferes with the regulation of Msi1 expression.
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