Highlights d Stochastic filopodia dynamics are required for robust synapse formation in fly brains d Only 1-2 filopodia at a time contain synaptic seeding factors and are synaptogenic d 4D tracking and computational modeling support a serial synapse formation model

In the fly optic lobe, $800 highly stereotypical columnar microcircuits are arranged retinotopically to process visual information. Differences in cellular composition and synaptic connectivity within functionally specialized columns remain largely unknown. Here, we describe the cellular and synaptic architecture in medulla columns located downstream of photoreceptors in the dorsal rim area (DRA), where linearly polarized skylight is detected for guiding orientation responses. We show that only in DRA medulla columns both R7 and R8 photoreceptors target to the bona fide R7 target layer where they form connections with previously uncharacterized, modality-specific Dm neurons: two morphologically distinct DRA-specific cell types (termed Dm-DRA1 and Dm-DRA2) stratify in separate sublayers and exclusively contact polarization-sensitive DRA inputs, while avoiding overlaps with color-sensitive Dm8 cells. Using the activity-dependent GRASP and trans-Tango techniques, we confirm that DRA R7 cells are synaptically connected to Dm-DRA1, whereas DRA R8 form synapses with Dm-DRA2. Finally, using live imaging of ingrowing pupal photoreceptor axons, we show that DRA R7 and R8 termini reach layer M6 sequentially, thus separating the establishment of different synaptic connectivity in time. We propose that a duplication of R7/Dm circuitry in DRA ommatidia serves as an ideal adaptation for detecting linearly polarized skylight using orthogonal e-vector analyzers.

In the fly optic lobe ~800 highly stereotypical columnar microcircuits are arranged retinotopically to process visual information. Differences in cellular composition and synaptic connectivity within functionally specialized columns remains largely unknown. Here we describe the cellular and synaptic architecture in medulla columns located downstream of photoreceptors in the 'dorsal rim area' (DRA), where linearly polarized skylight is detected for guiding orientation responses.

Following axon pathfinding, growth cones transition from stochastic filopodial exploration to the formation of a limited number of synapses. How the interplay of filopodia and synapse assembly ensures robust connectivity in the brain has remained a challenging problem. Here, we developed a new 4D analysis method for filopodial dynamics and a data-driven computational model of synapse formation for R7 photoreceptor axons in developing Drosophila brains. Our live data support a 'serial synapse formation' model, where at any time point only a single 'synaptogenic' filopodium suppresses the synaptic competence of other filopodia through competition for synaptic seeding factors. Loss of the synaptic seeding factors Syd-1 and Liprin-α leads to a loss of this suppression, filopodial destabilization and reduced synapse formation, which is sufficient to cause the destabilization of entire axon terminals. Our model provides a filopodial 'winner-takes-all' mechanism that ensures the formation of an appropriate number of synapses.

The amyloid precursor protein (APP) is a structurally and functionally conserved transmembrane protein whose physiological role in adult brain function and health is still unclear. Because mutations in APP cause familial Alzheimer’s disease (fAD), most research focuses on this aspect of APP biology. We investigated the physiological function of APP in the adult brain using the fruit fly Drosophila melanogaster, which harbors a single APP homologue called APP Like (APPL). Previous studies have provided evidence for the implication of APPL in neuronal wiring and axonal growth through the Wnt signaling pathway during development. However, like APP, APPL continues to be expressed in all neurons of the adult brain where its functions and their molecular and cellular underpinnings are unknown. We report that APPL loss of function (LOF) results in the dysregulation of endolysosomal function in neurons, with a notable enlargement of early endosomal compartments followed by neuronal cell death and the accumulation of dead neurons in the brain during a critical period at a young age. These defects can be rescued by reduction in the levels of the early endosomal regulator Rab5, indicating a causal role of endosomal function for cell death. Finally, we show that the secreted extracellular domain of APPL interacts with glia and regulates the size of their endosomes, the expression of the Draper engulfment receptor, and the clearance of neuronal debris in an axotomy model. We propose that APP proteins represent a novel family of neuroglial signaling factors required for adult brain homeostasis.