Dendrite patterning by a local cell-autonomous repulsive mechanism

2013-11-05A common feature of many dendritic arbors is evenly spread branches that exhibit very little self-crossing, but maximum coverage of the dendritic field. Such spatial patterning, known as dendrite self-avoidance, involves two key steps: distinguishing between self and non-self, and carrying out an avoidance response. It has recently been suggested that self/non-self recognition is mediated by contact-dependent homophilic interactions of cell surface molecules or capture-and-display of secreted molecules. The molecular mechanism underlying the avoidance response, however, is unknown. Here, we provide evidence to suggest that the repulsive guidance molecule Slit2 and its receptor Robo2 is required for dendritic self-avoidance of cerebellar Purkinje cells (PCs). Both ligand and receptor are highly expressed in PCs during the period of dendritic development and are associated with their dendrites. Using adeno-associated virus (AAV) to deliver Cre recombinase, we show that deletion of Robo2 from PCs leads to excessive crossing of sister dendrites, regardless of arbor size and shape. The self-crosses occur between branches in close proximity to each other, and they occur early in development and persist into adulthood, suggesting the involvement of an active avoidance mechanism that creates space between dendrites during development. Using the same AAV approach, we show that deletion of Slit2 yields the same crossing phenotype, indicating a cell autonomous function of Slit2/Robo2 signaling in mediating self-avoidance. Using a cerebellar explant culture, we show that PC dendrites fail to cross a Slit expressing COS cell boundary, demonstrating that Slits are sufficient to create a boundary to PC dendrite advancement. We also show by way of a rescue experiment that localizing an active fragment of Slit2 to the membrane is sufficient to rescue the self-avoidance defect in Slit2 mutant PCs, suggesting the involvement of local dendrite-dendrite interactions. We also demonstrate the functional consequence of disrupted dendrite self-avoidance by observing minor motor abnormalities in mice with Robo2 deleted in all PCs. Our study has identified a new repulsive function for Slit/Robo signaling in the novel context of dendrite self-avoidance and provided the first evidence for a molecular mechanism underlying the avoidance response during self-avoidance