title: Heterochronic Pellet Assay to Test Cell-cell Communication in the Mouse Retina.
abstract: All seven retinal cell types that make up the mature retina are generated from a common, multipotent pool of retinal progenitor cells (RPCs) (Wallace, 2011). One way that RPCs know when sufficient numbers of particular cell-types have been generated is through negative feedback signals, which are emitted by differentiated cells and must reach threshold levels to block additional differentiation of that cell type. A key assay to assess whether negative feedback signals are emitted by differentiated cells is a heterochronic pellet assay in which early stage RPCs are dissociated and labeled with BrdU, then mixed with a 20-fold excess of dissociated differentiated cells. The combined cells are then re-aggregated and cultured as a pellet on a membrane for 7-10 daysin vitro.During this time frame, RPCs will differentiate, and the fate of the BrdU+RPCs can be assessed using cell type-specific markers. Investigators who developed this pellet assay initially demonstrated that neonatal RPCs give rise to rods on an accelerated schedule compared to embryonic RPCs when the two cell types are mixed together (Watanabe and Raff, 1990; Watanabeet al., 1997). We have used this assay to demonstrate that sonic hedgehog (Shh), which we found acts as a negative regulator of retinal ganglion cell (RGC) differentiation, promotes RPC proliferation (Jensen and Wallace, 1997; Ringuetteet al., 2014). More recently we modified the heterochronic pellet assay to assess the role of feedback signals for retinal amacrine cells, identifying transforming growth factor β2 (Tgfβ2) as a negative feedback signal, andPtenas a modulator of the Tgfβ2 response (Maet al., 2007; Tachibanaet al., 2016). This assay can be adapted to other lineages and tissues to assess cell-cell interactions between two different cell-types (heterotypic) in either an isochronic or heterochronic manner.
authors.affiliation: Biological Sciences Platform, Sunnybrook Research Institute, University of Toronto, Toronto, Canada; Department of Biochemistry and Molecular Biology, Alberta Children's Hospital Research Institute and Hotchkiss Brain Institute, University of Calgary, Calgary, Canada.
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Tachibana
authors.affiliation: Biological Sciences Platform, Sunnybrook Research Institute, University of Toronto, Toronto, Canada; Department of Biochemistry and Molecular Biology, Alberta Children's Hospital Research Institute and Hotchkiss Brain Institute, University of Calgary, Calgary, Canada.
authors.forename: Dawn
authors.initials: D
authors.lastname: Zinyk
authors.affiliation: Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada.
authors.forename: Randy
authors.initials: R
authors.lastname: Ringuette
authors.affiliation: Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada; Donald K. Johnson Eye Institute, Krembil Research Institute, University Health Network, Toronto, Canada; Department of Ophthalmology and Vision Sciences and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
authors.forename: Valerie
authors.initials: V
authors.lastname: Wallace
authors.affiliation: Biological Sciences Platform, Sunnybrook Research Institute, University of Toronto, Toronto, Canada; Department of Biochemistry and Molecular Biology, Alberta Children's Hospital Research Institute and Hotchkiss Brain Institute, University of Calgary, Calgary, Canada.
authors.forename: Carol
authors.initials: C
authors.lastname: Schuurmans
keywords: Amacrine cell; Heterochronic pellet assay; Heterotypic cell interactions; Negative feedback signaling; Re-aggregation; Retinal differentiation; Retinal progenitor cells