Spatiotemporal Coordination of Guidance Cues Directs Multipolar Migration During Retinal Lamination

Multipolar migration is a conserved neuronal migration mode in the developing brain, enabling emerging neurons to navigate crowded environments and reach precise laminar positions. Yet how these cells interpret external cues to guide their migration remains unclear. We investigate this question in the developing vertebrate retina using horizontal cells as a model. Combining transcriptomics, targeted CRISPR screening, and live imaging, we reveal the spatiotemporal guidance system underlying horizontal cell lamination: repulsive Slit1b/2–Robo2 signaling in the amacrine cell layer is essential to initiate apical horizontal cell migration, while attractive Neurturin–Gfrα1 signaling from photoreceptors fine-tunes final positioning beneath the photoreceptor layer. Disruption of these pathways causes basal retention of horizontal cells, highlighting the importance of spatially coordinated signaling for proper lamination and functional retinal circuitry. Our results uncover how positional signals and tissue architecture cooperate to achieve neuronal precision, an organizing principle likely relevant across the developing central nervous system.