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Evolutionarily conserved hedgehog proteins orchestrate the patterning of embryonic tissues, and dysfunctions in their signaling can lead to tumorigenesis. In vertebrates, Sonic hedgehog (Shh) is essential for nervous system development, but the mechanisms underlying its action remain unclear. Early electrical activity is another developmental cue important for proliferation, migration, and differentiation of neurons. Here we demonstrate the interplay between Shh signaling and Ca(2+) dynamics in the developing spinal cord. Ca(2+) imaging of embryonic spinal cells shows that Shh acutely increases Ca(2+) spike activity through activation of the Shh coreceptor Smoothened (Smo) in neurons. Smo recruits a heterotrimeric GTP-binding protein-dependent pathway and engages both intracellular Ca(2+) stores and Ca(2+) influx. The dynamics of this signaling are manifested in synchronous Ca(2+) spikes and inositol triphosphate transients apparent at the neuronal primary cilium. Interaction of Shh and electrical activity modulates neurotransmitter phenotype expression in spinal neurons. These results indicate that electrical activity and second-messenger signaling mediate Shh action in embryonic spinal neurons.
Fig. 1. Spiking cells in the developing neural tube are postmitotic neurons. (A) Ca2+ imaging of the ventral spinal cord of a stage-24 (26-h-postfertilization) embryo for 20 min. Circles identify cells spiking during 20-min recording. Inset shows Ca2+ spike activity for the cell outlined in yellow. (B) (Left) After imaging, the same preparation was whole-mount immunostained for Sox2 and N-β-tubulin. (Right) Immunostaining of a transverse section of the spinal cord from a stage-24 embryo. (C) Ca2+ imaging of an open-book spinal cord preparation. (D) Whole-mount immunostaining of the same preparation for Sox2 and N-β-tubulin. (E) Diagram of the open-book spinal cord preparation shown in C and D. D, dorsal; V, ventral. (Scale bars, 20 μm.)
Fig. 2.
Shh increases Ca2+ spike activity of developing spinal neurons. (A) Lateral view of a developing spinal cord showing higher levels of Ca2+ spike activity in the ventral than in the dorsal neural tube (stage 24). (B) After imaging, the same preparation was whole-mount immunostained for homeodomain protein Hb9, a ventrally expressed neuronal marker, to indicate its dorsoventral orientation. Circles identify cells spiking during 20-min recording, and Insets in A show Ca2+ spike activity for cells outlined in yellow. (C) Incidence of spiking cells per neural tube and frequency of Ca2+ spikes in ventral and dorsal spinal neurons. (D and E) Ventral view of stage-24 developing spinal cord in the absence (D) or presence (E) of N-Shh. Insets show Ca2+ spike activity during 15-min recording from the same cell (outlined in yellow). (F) Doseesponse curve for N-Shhnduced Ca2+ spike activity. Data are mean SEM percent of spiking cells in the presence of N-Shh compared to number of cells spiking before addition of N-Shh (0). (G) Doseesponse curve for cyclopamine blockade of Ca2+ spike activity induced by SAG. Data are mean SEM percent of spiking cells in the presence of SAG and cyclopamine compared to number of cells spiking before addition of cyclopamine (0). (H) Expression of SmoM2 increases Ca2+ spike activity. (H) Electroporation of a stage-19 embryo with SmoM2 demonstrates a higher incidence of Ca2+ spike activity 6 h after electroporation (stage 24) in electroporated cells (red) than in nonelectroporated cells (black). (I) Effective overexpression of SmoM2 was verified by whole-mount immunostaining against Smo after Ca2+ imaging. Circles identify cells spiking during recording. (J) Ca2+ spike activity during 20-min recording for immunonegative and immunopositive cells outlined in yellow in H and I. (K) Bar graphs show mean SEM percent incidence of spiking cells and spike frequency for electroporated (SmoM2) and nonelectroporated (Control) cells. n = 5 stage-24 (26-h postfertilization) embryos per experimental group (C). (L) Endogenous Shh released by the notochord increases Ca2+ spike activity of neurons. (Upper) Dissociated neuron/notochord explant (Not) coculture. (Lower) The imaged field was divided in halves proximal and distal to the notochord explant. Values are mean SEM percent of spiking cells in proximal and distal regions in the absence or presence of cyclopamine (Cyclo). n = 5 independent cultures; *P < 0.05. (Scale bars, 20 μm.)
Fig. 4.
Shh and second-messenger signaling converge at the neuronal primary cilium. (A) Immunostaining of immature spinal neurons grown in vitro for 7 h. Acetylated tubulin staining is shown in green, and DAPI staining is shown in blue. (A) IP3R (red) localize at the base of the primary cilium. (B) TRPC1 (red) localizes to the primary cilium. (C and D) Gαi protein (red) localization at the primary cilium expands when Shh signaling is enhanced. Numbers correspond to the mean SEM percent of acetylated tubulin labeling that overlaps with Gαi staining at the primary cilium in the absence (C) or presence (D) of 100 nM SAG for 4 h. n = 10 cells per condition; *P < 0.005. (E and F) Simultaneous Ca2+ and IP3 imaging reveals synchronous transients. (E) Images correspond to a time before (Left), during (Center), and after (Right) the spike indicated in the trace in F. (F) Traces represent the changes in fluorescence intensity for IP3 and Ca2+ probes in regions of interest (ROI) indicated in E, Right. (G) IP3 transients are apparent at the primary cilium. The cell is the same shown in E, stained with DAPI (blue) and anti-acetylated tubulin (green) and overlapped with IP3 frame (red) corresponding to the peak of the transient shown in E, Center. (Scale bars, 10 μm.) (H) Synchronicity of Ca2+ and IP3 transients. Graph represents onset time of Ca2+ spikes vs. onset time of IP3 transients during simultaneous recordings. Inset represents the histogram of the difference between onset times; Δt = tIP3 − tCa2+.
Fig. 5.
Ca2+ spike activity is necessary for Shh-induced spinal neuron differentiation. (A) (Upper) Immunostaining of transverse sections of the spinal cord from embryos treated with agents indicated in the figure. Cyclo, cyclopamine; d, dorsal; Verat, veratridine; VGC block and VGCbl, voltage-gated Na+ and Ca2+ channel blockers. (Lower) Graph shows mean SEM GABA-immunopositive cells/100 μm of spinal cord. n ≥ 5 stage-34 (45-h postfertilization) embryos per experimental group; *P < 0.05. (Scale bar, 20 μm.) (B) Model of the molecular mechanisms underlying Shh-induced Ca2+ spikes. α, β, γ, subunits of the heterotrimeric G protein; AC, adenylate cyclase; Cav, voltage-gated Ca2+ channels; ER, endoplasmic reticulum; Ptc1, Patched1. Details are given in Discussion.
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