The mechanisms of vertebrate Hedgehog signaling are linked to the biology of the primary cilium, an antenna-like organelle that projects from the surface of most vertebrate cell types. Although the advantages of restricting signal transduction to cilia are often noted, the constraints imposed are less frequently considered, and yet they are central to how Hedgehog signaling operates in developing tissues. In this Review, we synthesize current understanding of Hedgehog signal transduction, ligand secretion and transport, and cilia dynamics to explore the temporal and spatial constraints imposed by the primary cilium on Hedgehog signaling in vivo.

The regulation of proliferation is one of the primary functions of Hedgehog (Hh) signaling in development. Transduction of Hh signaling requires the primary cilium, a microtubule-based organelle that is necessary for several steps in the pathway (

Positional information in developing tissues often takes the form of stripes of gene expression that mark the boundaries of a particular cell type or morphogenetic process. How stripes form is still in many cases poorly understood. Here we use optogenetics and live-cell biosensors to investigate one such pattern: the posterior stripe ofbrachyenteron (byn)expression in the earlyDrosophilaembryo. Thisbynstripe depends on interpretation of an upstream signal — a gradient of ERK kinase activity — and the expression of two target genestailless (tll)andhuckebein (hkb)that exert antagonistic control overbyn. We find that high or low doses of ERK signaling produce either transient or sustainedbynexpression, respectively. These ERK stimuli also regulatetllandhkbexpression with distinct dynamics:tlltranscription is rapidly induced under both low and high stimuli, whereashkbtranscription converts graded ERK inputs into an output switch with a variable time delay. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop architecture, which is sufficient to explain transient or sustainedbyndynamics and adds temporal complexity to the steady-state model ofbynstripe formation. We further show that an all-or-none stimulus can be blurred through intracellular diffusion to non-locally produce a stripe ofbyngene expression. Overall, our study provides a blueprint for using optogenetic inputs to dissect developmental signal interpretation in space and time.

The regulation of proliferation is one of the primary functions of Hedgehog (Hh) signaling in development. Transduction of Hh signaling requires the primary cilium, a microtubule-based organelle that is necessary for several steps in the pathway (Corbit et al., 2005;Huangfu and Anderson, 2005;Huangfu et al., 2003;Liu et al., 2005;Rohatgi et al., 2007). Many cells only build a primary cilium upon cell cycle arrest in G0. In those proliferating cells that do make a cilium, it is a transient organelle, being assembled in G1 and disassembled sometime after, although exactly when is not well-characterized (Ford et al., 2018;Pugacheva et al., 2007;Wang and Dynlacht, 2018). Thus the requirement for primary cilia presents a conundrum: how are proliferative signals conveyed through an organelle that is present for only part of the cell cycle? Here we investigate this question in a mouse medulloblastoma cell line, SMB55, that requires cilium-mediated Hh pathway activity for proliferation (Zhao et al., 2015). We show that SMB55 cells are often ciliated beyond G1 into S phase, and the presence of the cilium determines the periods of Hh pathway activity. Using live imaging over multiple cell cycles, we define two windows of opportunity for Hh pathway activity, either of which is sufficient to effect cell cycle entry. The first is in the ciliated phase of the previous cell cycle, and the second is in G1 of the cell cycle in which the decision is made. We propose that the ability of cells to integrate Hh pathway activity from more than one cell cycle imparts robustness on Hh pathway control of proliferation and may have implications for other Hhmediated events in development.