Sonic hedgehog signaling is decoded by calcium spike activity in the developing spinal cord

Abstract

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) Dose–response curve for N-Shh–induced 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) Dose–response 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–K) 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–K). (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. 3.

Molecular identification of the components linking Shh and Ca²⁺ spike activity. (A–D) Ca²⁺ imaging of the ventral spinal cord. (A) Ca²⁺ influx was blocked by a mixture of Na⁺ and Ca²⁺ voltage-gated channel blockers (VGC block) or by perfusion with a Ca²⁺-free medium (Ca²⁺-free). (B) Gαi was inhibited by 10 mM PTX. Perturbations of PKA activity were implemented by electroporating constitutively active (C_QR) or dominant negative (R_AB) forms of PKA in stage-19 embryos. Ca²⁺ imaging was performed 6 h after electroporation. (C) PLC was inhibited by 10 μM U73122, and IP3R were inhibited by 20 μM 2-aminoethoxydiphenyl borate (2-APB) or 20 μM xestospongin C (XeC). (D) TRPC channels were blocked by 50 μM SKF96365 or by molecular knockdown with xTRPC1 morpholino (MO). Control morpholino (CMO). Values are mean ± SEM percent incidence of spiking cells in the ventral surface of neural tubes compared with control (30-min recording before addition of 100 nM SAG). n = 5 stage-24 (26-h postfertilization) embryos per experimental group; *P < 0.05 (A–D).

Fig. 4.

Shh and second-messenger signaling converge at the neuronal primary cilium. (A–D) 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 VGC_bl, 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.