Dataset Information

Induction of Neuronal Morphology in the 661W Cone Photoreceptor Cell Line with Staurosporine.

ABSTRACT:

Purpose

RGC-5 cells undergo differentiation into a neuronal phenotype with low concentrations of staurosporine. Although the RGC-5 cell line was initially thought to be of retinal ganglion cell origin, recent evidence suggests that the RGC-5 line could have been the result of contamination with 661W mouse cone photoreceptor cells. This raised the possibility that a cone photoreceptor cell line could be multipotent and could be differentiated to a neuronal phenotype.

Methods

661W and RGC-5 cells, non-neuronal retinal astrocytes, retinal endothelial cells, retinal pericytes, M21 melanoma cells, K562 chronic myelogenous leukemia cells, and Daudi Burkitt lymphoma cells, were differentiated with staurosporine. The resulting morphology was quantitated using NeuronJ with respect to neurite counts and topology.

Results

Treatment with staurosporine induced similar-appearing morphological differentiation in both 661W and RGC-5 cells. The following measures were not significantly different between 661W and RGC-5 cells: number of neurites per cell, total neurite field length, number of neurite branch points, and cell viability. Neuronal-like differentiation was not observed in the other cell lines tested.

Conclusions

661W and RGC-5 cells have virtually identical and distinctive morphology when differentiated with low concentrations of staurosporine. This result demonstrates that a retinal neuronal precursor cell with cone photoreceptor lineage can be differentiated to express a neuronal morphology.

SUBMITTER: Thompson AF

PROVIDER: S-EPMC4684327 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Publications

Induction of Neuronal Morphology in the 661W Cone Photoreceptor Cell Line with Staurosporine.

Thompson Alex F AF Crowe Megan E ME Lieven Christopher J CJ Levin Leonard A LA

PloS one 20151218 12

<h4>Purpose</h4>RGC-5 cells undergo differentiation into a neuronal phenotype with low concentrations of staurosporine. Although the RGC-5 cell line was initially thought to be of retinal ganglion cell origin, recent evidence suggests that the RGC-5 line could have been the result of contamination with 661W mouse cone photoreceptor cells. This raised the possibility that a cone photoreceptor cell line could be multipotent and could be differentiated to a neuronal phenotype.<h4>Methods</h4>661W a ...[more]

PMID: 26684837

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Project description:Transcription factors have been implicated in the specification and differentiation of all cells in the mammalian retina with several transcription factors controlling the development of multiple neuronal subclasses. Horizontal cells and cone photoreceptors share a particularly intimate functional relationship but have been previously thought to develop through separate and distinct intrinsic molecular processes. We demonstrate that the zinc finger transcription factor Sall3 regulates development of both S-cone photoreceptors and horizontal cells. Our data shows that loss of function of Sall3 down-regulates the horizontal cell specific transcription factor Lhx1 and subsequently causes ectopic formation of horizontal-like wide-field amacrine cells partially phenocopying Lhx1-/- mice. Additionally, horizontal cells which laminate appropriately in Sall3 knockout mice show abnormal horizontal cell gene expression and failures in dendritic arborization. Over-expression of Sall3 also partially re-specifies cells to a horizontal-like wide-field amacrine fate. Intriguingly, Sall3 loss of function experiments also generated a massive reduction in the number of S-cone photoreceptors with remaining S-cones showing deficiencies in S-cone gene expression and morphology. Conversely, over-expression of Sall3 resulted in the ectopic expression of the S-cone specific genes S-opsin (Sop) and cone arrestin (Arr3) in electroporated cells. Our studies reveal that Sall3 regulates aspects of horizontal cell development in two ways: first, by maintaining Lhx1 expression, and second, by directly regulating expression of horizontal cell specific genes. We also show that Sall3 is an essential regulator of S-cone development in the mammalian retina and a potent activator of Sop and Arr3. Sall3 wild-type and knockout retinas used for explant cultures were extracted by microdissection at P0 in sterile PBS. Four cuts were made using micro-dissection scissors and the retinas were transferred to Nucleopore Track-Etch membrane filters (Whatman) and the tissue flattened out evenly. Filters were then floated on DMEM/F12, 10% FBS, 1X penicillin/streptomycin in 12 well plates and incubated at 37oC, 5% CO2 for 7 days. Three retinas (Sall3 knockout or wild-type) were pooled and RNA extracted using the Qiagen RNeasy kit. This was performed in triplicate for both wild-type and Sall3 knockouts retinas. A total of three replicates of Sall3 knockout and Sall3 wild-type RNA were analysed (six total samples).

Dataset Information

Induction of Neuronal Morphology in the 661W Cone Photoreceptor Cell Line with Staurosporine.

Purpose

Methods

Results

Conclusions

Publications

Induction of Neuronal Morphology in the 661W Cone Photoreceptor Cell Line with Staurosporine.

Similar Datasets

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