The ability of signaling proteins to traverse tissues containing tightly packed cells is of fundamental importance for cell specification and tissue development, however, how this is achieved at a cellular level remains poorly understood1. For over a century, the vertebrate limb bud has served as a paradigm to study cell signaling during embryonic development2. Here we optimize single cell real-time imaging to delineate the cellular mechanisms for how signaling proteins, such as Sonic Hedgehog (Shh), that possess membrane-bound covalent lipid modifications transverse long distances within the limb bud in vivo. By directly imaging Shh ligand production under native regulatory control, our findings show that Shh is unexpectedly produced in the form of a particle that remains associated with the cell via long cytoplasmic extensions that span several cell diameters. We show that these cellular extensions are a specialized class of actin-based filopodia with novel cytoskeletal features that have not been previously described. Strikingly, particles containing Shh traffic along these extensions with a net anterograde movement within the field of Shh cell signaling. We further show that in Shh responding cells specific subsets of Shh co-receptors, including Cdo and Boc, actively distribute and co-localize in specific micro-domains within filopodial extensions, far from the cell body. Stabilized interactions are formed between filopodia containing Shh ligand and those containing co-receptors over a long-range. These results suggest that contact-mediated release propagated by specialized filopodia contributes to the delivery of Shh at a distance. Together, these studies identify an important mode of communication between cells that significantly extends our understanding of ligand movement and reception during vertebrate tissue patterning.

Throughout life, neural stem cells (NSCs) in different domains of the ventricular-subventricular zone (V-SVZ) of the adult rodent brain generate several subtypes of interneurons that regulate the function of the olfactory bulb (OB). The full extent of diversity among adult NSCs and their progeny is not known. Here, we report the generation of at least four previously unknown OB interneuron subtypes that are produced in finely patterned progenitor domains in the anterior ventral V-SVZ of both the neonatal and adult brain. Progenitors of these novel interneurons are responsive to sonic hedgehog (SHH) and are organized into microdomains that correlate with the expression domains of the Nkx6.2 and Zic family of transcription factors. This work reveals an unexpected degree of complexity in the specification and patterning of NSCs in the postnatal mouse brain.

Little is known about how patterns of cell types are organized to form brain structures of appropriate size and shape. To study this process, we employed in vivo electroporation during midbrain development to create ectopic sources of Sonic Hedgehog, a signaling molecule previously shown to specify different neuronal cell types in a concentration-dependent manner in vitro. We provide direct evidence that a Sonic Hedgehog source can control pattern at a distance in brain development and demonstrate that the size, shape, and orientation of the cell populations produced depend on the geometry of the morphogen source. Thus, a single regulatory molecule can coordinate tissue size and shape with cell-type identity in brain development.