Progenitor division and cell autonomous neurosecretion are required for rod photoreceptor sublaminar positioning.

Migration is essential for the laminar stratification and connectivity of neurons in the central nervous system. In the retina, photoreceptors (PRs) migrate to positions according to birthdate, with early-born cells localizing to the basal-most side of the outer nuclear layer. It was proposed that apical progenitor mitoses physically drive these basal translocations non-cell autonomously, but direct evidence is lacking, and whether other mechanisms participate is unknown. Here, combining loss- or gain-of-function assays to manipulate cell cycle regulators (Sonic hedgehog, Cdkn1a/p21) with an in vivo lentiviral labelling strategy, we demonstrate that progenitor division is one of two forces driving basal translocation of rod soma. Indeed, replacing Shh activity rescues abnormal rod translocation in retinal explants. Unexpectedly, we show that rod differentiation also promotes rod soma translocation. While outer segment function or formation is dispensable, Crx and SNARE-dependent synaptic function are essential. Thus, both non-cell and cell autonomous mechanisms underpin PR soma sublaminar positioning in the mammalian retina.

Culturing and Imaging Glioma Stem Cells in 3D Collagen Matrices.

Methods to maintain human glioma stem cells as neurosphere cultures and image their dynamic behavior in 3D collagen matrices are described. Additional approaches to monitor glioma stem cell differentiation into mesenchymal-type cells, along with example data are included. Together, these approaches enable glioma stem cell differentiation to be controlled while maintaining the cells in culture, as well as allowing cell dynamics to be captured and analyzed. These methods should be helpful for those seeking to understand the molecular mechanisms driving the invasion of glioma cells through three-dimensional environments. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Culturing human glioma stem cells as neurospheres Basic Protocol 2: Inducing GSC adherence and monitoring their differentiation into mesenchymal cells Support Protocol 1: Preparing fibronectin-coated dishes for cell microscopy Basic Protocol 3: Embedding GSCs in a 3D collagen matrix to study their invasive behavior Support Protocol 2: Phase-contrast imaging with a tiled matrix to study cell migration in a 3D gel.