Highlights d Neurons assemble to establish a column d Planar cell polarity regulates the orientation of neurons and columns d Wnt ligands globally regulate neuronal orientation and column arrangement

The brain is organized morphologically and functionally into a columnar structure. According to the radial unit hypothesis, neurons from the same lineage form a radial unit that contributes to column formation. However, the molecular mechanisms that link neuronal lineage and column formation remain elusive. Here, we show that neurons from the same lineage project to different columns under control of Down syndrome cell adhesion molecule (Dscam) in the fly brain. Dscam1 is temporally expressed in newly born neuroblasts and is inherited by their daughter neurons. The transient transcription of Dscam1 in neuroblasts enables the expression of the same Dscam1 splice isoform within cells of the same lineage, causing lineage-dependent repulsion. In the absence of Dscam1 function, neurons from the same lineage project to the same column. When the splice diversity of Dscam1 is reduced, column formation is significantly compromised. Thus, Dscam1 controls column formation through lineage-dependent repulsion.

While Delta non-autonomously activates Notch in neighboring cells, it autonomously inactivates Notch through cis-inhibition, the molecular mechanism and biological roles of which remain elusive. The wave of differentiation in the Drosophila brain, the ‘proneural wave’, is an excellent model for studying Notch signaling in vivo. Here, we show that strong nonlinearity in cis-inhibition reproduces the second peak of Notch activity behind the proneural wave in silico. Based on this, we demonstrate that Delta expression induces a quick degradation of Notch in late endosomes and the formation of the twin peaks of Notch activity in vivo. Indeed, the amount of Notch is upregulated and the twin peaks are fused forming a single peak when the function of Delta or late endosomes is compromised. Additionally, we show that the second Notch peak behind the wavefront controls neurogenesis. Thus, intracellular trafficking of Notch orchestrates the temporal dynamics of Notch activity and the temporal patterning of neurogenesis.

The Drosophila visual center shows columnar structures, basic structural and functional units of the brain, that are shared with the mammalian cerebral cortex. Visual information received in the ommatidia in the compound eye is transmitted to the columns in the brain. However, the developmental mechanisms of column formation are largely unknown. The Irre Cell Recognition Module (IRM) proteins are a family of immunoglobulin cell adhesion molecules. The four Drosophila IRM proteins are localized to the developing columns, the structure of which is affected in IRM mutants, suggesting that IRM proteins are essential for column formation. Since IRM proteins are cell adhesion molecules, they may regulate cell adhesion between columnar neurons. To test this possibility, we specifically knocked down IRM genes in columnar neurons and examined the defects in column formation. We developed a system that automatically extracts the individual column images and quantifies the column shape. Using this system, we demonstrated that IRM genes play critical roles in regulating column shape in a core columnar neuron, Mi1. We also show that their expression in the other columnar neurons, Mi4 and T4/5, is essential, suggesting that the interactions between IRM proteins and multiple neurons shape the columns in the fly brain.

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