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R. F. Nelson, T. S. Ma, T. Ikenaga, V. P. Connaughton, F. Ono; Forward Transgenic Selectively Labels Amacrine, Ganglion, and Horizontal Cell Types in Zebrafish Retina. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1301.
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© ARVO (1962-2015); The Authors (2016-present)
To identify labeled retinal cell types in the transgenic strain GE4 and its variants.
Using an enhancer trap method, transposase and tol2 eGFP construct were injected into zebrafish eggs at the single cell stage. Larvae were screened for fluorescence, raised to adulthood, and a stable line developed. Eyecups or retinal sections were prepared from adult animals, maintained in L15 medium, and observed by water-immersion confocal microscopy.
In the GE4ab variant scattered cell bodies were labeled in amacrine, ganglion and horizontal cell layers, but not in bipolar or photoreceptor layers. Individual optic-nerve fibers fluoresced, as did a thick bright band in inner plexiform layer (IPL) sublamina b (s5-s7). IPL s4 was unlabeled, while sublamina a (s1-s3) labeled sparsely with puncta. Amacrine cell (AC) bodies were smallest (median area, 25.5 µm2; equivalent diameter, 5.7 µm; N=120). 20% appeared as cell pairs (as if recently divided). The AC mosaic differed from Raleigh (P<.001) with a regularity index of 2.5 and density of 2100 mm-1. In sections, apical dendritic stalks descended from long pyriform ACs to arborize in IPL s6/7, similar to AON-s6 (Connaughton et al, 2004). Short-pyriform and diffuse ACs, most with dendrites stratified in two planes, branched either narrowly or widely in s1-s3, similar to types AOFF-s1/s2, AOFF-s1/s3 and AOFF-s1 (Connaughton and Hsieh, 2008). Ganglion cells (GC) were large bodied (median area, 47 µm2; equivalent diameter 7.7 µm; N=65). The sparse GC mosaic did not differ from Raleigh (P>.05; regularity index, 2.0; density, 270 mm-1). Monostratified dendrites branched in s7. GC morphology resembled the ON-alpha-like type I (Mangrum et al, 2002). Some GCs were displaced. Horizontal cells (HC) bore large irregularly shaped cell bodies with short dendrites and tiny spines ascending to cone pedicles, similar to H1 or H2 (Song et al, 2007). Both smaller and larger bodied HCs were discerned in a single distal layer. Proximal to this layer, small spindle shaped HCs appeared with thick processes curving from each pole. The median area of large HCs was 48 µm2 (N=18, equivalent diameter 7.8 µm). Overall, the median cell body area was 34 µm2 (N=46, equivalent diameter 6.6 µm). The HC mosaic (all cells) was highly regular and non-Raleigh (P<.0001). The regularity index was 4.0 and the density was 3100 mm-1.
The widely distributed yet selective labeling pattern of the GE4ab forward transgenic provides scaffolding for studies of neural circuitry in zebrafish retina, and, potentially, an insight into the inter-relationships between retinal neurons.
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