A fixation method to preserve cultured cell cytonemes facilitates mechanistic interrogation of morphogen transport

Abstract

During development, extracellular cues guiding cell fate determination are provided by morphogens. One mechanism by which morphogens are proposed to traverse extracellular space is by traveling along specialized filopodia called cytonemes. These cellular highways extend between signal producing and receiving cells to enable direct morphogen delivery. Although genetic studies support cytoneme involvement in morphogen transport, mechanistic insight into how they are regulated is limited due to technical challenges associated with performing cell biological analysis of the delicate filopodial structures. Herein, we introduce a fixation method whereby cultured cell cytonemes can be preserved for imaging studies, allowing for interrogation of cytoneme regulation using standard cell biological techniques. Using this method, we examined Hedgehog-containing cytonemes and identified a role for the Hedgehog deployment protein Dispatched in cytoneme stabilization. We demonstrate Hedgehog and Dispatched colocalize in cytonemes, and cholesterol-modified Hedgehog acts through Dispatched to increase cytoneme occurrence. Live imaging suggests this occurs through Dispatched-mediated slowing of cytoneme retraction rates. Dispatched-induced cytoneme modulation was recapitulated in wing imaginal discs of transgenic Drosophila, supporting that cultured cell cytoneme analysis is predictive of in vivo functionality.