Dynamic integration of morphogen and juxtacrine signaling specifies cell fate at single-cell resolution.

Morphogen gradients guide tissue patterning but do not act in isolation. To address how they integrate with other signaling modalities and how such integrations influence pattern resolution and robustness, we focused on the Drosophila lamina, where columns of precursors are patterned with single-cell resolution into distinct motion-processing neurons. Although a photoreceptor-derived Hedgehog gradient diversifies cell fates, it cannot account for all fates specified. Combining experiments and theory, we show that glial-induced extracellular signal-regulated kinase (ERK) activity drives Delta expression in lamina precursors, generating graded Notch activity. This subdivides Hedgehog-responsive domains into subdomains that map to distinct fates. We identify a Hedgehog morphogen relay between photoreceptors and the lamina and show that Notch restricts this relay, enhancing positional information. Glia act as timekeepers, scheduling ERK-driven differentiation to coincide with stable Hedgehog and Notch activity patterns. Thus, Notch and ERK dynamically integrate with Hedgehog to encode positional information, enabling precise and reproducible cell fate patterning.