Morphogen Stimulated Endocytic Recycling Drives Cytoneme Loading for Mammalian Tissue Patterning

During development, morphogens build tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been unfeasible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised Sonic Hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenously-expressed SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor Myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function. RNA-sequencing data of litters of E9.5 control (wild type) and Disp1CS/INDEL embryos generated from Disp1CS/+ females and Disp1INDEL/+ males, and litters of E8.5 control (Myo10+/+) and Myo10m1J/m1J embryos generated from Myo10m1J/+ females and Myo10m1J/+ males.