Abstract
Based on a variety of approaches, evidence suggests that different cell types in the vertebrate retina are generated by multipotential progenitors in response to interactions between cell intrinsic and cell extrinsic factors. The identity of some of the cellular determinants that mediate such interactions has emerged, shedding light on mechanisms underlying cell differentiation. For example, we know now that Notch signaling mediates the influence of the microenvironment on states of commitment of the progenitors by activating transcriptional repressors. Cell intrinsic factors such as the proneural basic helix-loop-helix and homeodomain transcription factors regulate a network of genes necessary for cell differentiation and maturation. What is missing from this picture is the role of developmental chromatin remodeling in coordinating the expression of disparate classes of genes for the differentiation of retinal progenitors. Here we describe the role of Brm, an ATPase in the SWI/SNF chromatin remodeling complex, in the differentiation of retinal progenitors into retinal ganglion cells. Using the perturbation of expression and function analyses, we demonstrate that Brm promotes retinal ganglion cell differentiation by facilitating the expression and function of a key regulator of retinal ganglion cells, Brn3b, and the inhibition of Notch signaling. In addition, we demonstrate that Brm promotes cell cycle exit during retinal ganglion cell differentiation. Together, our results suggest that Brm represents one of the nexus where diverse information of cell differentiation is integrated during cell differentiation.
Highlights
Cell fate specification and subsequent cell differentiation in the nervous system are orchestrated and finessed by interplay between cell intrinsic and cell extrinsic factors
The identification of cell intrinsic and cell extrinsic factors has helped our understanding of the mechanisms that regulate the differentiation of retinal cell types, that of retinal ganglion cells (RGCs), it is not known as to how the remodeling of chromatin, which is necessary for eukaryotic gene expression, is recruited toward the coordinated regulation of genes during RGC differentiation
Brm-mediated Differentiation of Retinal Stem Cells/Progenitors. Both major classes of chromatin remodeling complexes are likely to contribute to developmental processes, including those in the central nervous system (19 –21), evidence is emerging that the SWI/SNF chromatin remodeling complexes play an important role in the differentiation of specific cell types [22]
Summary
Progenitor Cell Culture—Timed-pregnant (E14) SpragueDawley rats were obtained from Sasco (Wilmington, MA). For co-culture, neurospheres were collected, washed extensively to remove BrdUrd, and plated on poly-D-lysine- and laminin-coated glass coverslips with E3 chick/PN1 rat retinal cells in 1% fetal bovine serum. Immunofluorescence Analysis—Immunocytochemical analysis for detection of cell-specific markers and BrdUrd was performed as described previously [35]. Paraformaldehyde-fixed cells/sections were incubated in 1ϫ phosphatebuffered saline containing 5% Normal Goat Serum and 0.2– 0.4% Triton X-100 followed by an overnight incubation in appropriate dilutions of antibodies against Brm (Santa Cruz Biotechnology), Notch (Santa Cruz Biotechnology), Shh (Developmental Studies Hybridoma Bank), Brn3b (Covance), RPF1 [36], and BrdUrd (Accurate Chemical and Scientific Corp.) at 4 °C. The efficiency of electroporation was monitored by green fluorescent protein epifluorescence, and the explants were cultured in RCM containing 5% fetal bovine serum for 4 days.
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