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Interplay between electrical activity and bone morphogenetic protein signaling regulates spinal neuron differentiation - PubMed

  • ️Sun Jan 01 2012

Interplay between electrical activity and bone morphogenetic protein signaling regulates spinal neuron differentiation

Immani Swapna et al. Proc Natl Acad Sci U S A. 2012.

Abstract

A gradient of bone morphogenetic proteins (BMPs) along the dorsoventral axis of the spinal cord is necessary for the specification of dorsal neurons. Concurrently, a gradient of calcium-mediated electrical activity is present in the developing spinal cord but in an opposing ventrodorsal direction. Whether BMPs and electrical activity interact in embryonic spinal neurons remains unknown. We show that BMP decreases electrical activity by enhancing p38 MAPK-mediated negative modulation of voltage-gated sodium channels. In turn, electrical activity affects the phosphorylation status and nuclear level of activated Smads, the canonical components of BMP signaling. This interaction between calcium spike activity and BMP signaling regulates the specification of the dorsal commissural spinal neuron phenotype. The present study identifies an unexpected interplay between BMPs and electrical activity that is critical for decoding the morphogen gradient during spinal neuron differentiation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

BMP signaling decreases Ca2+ spike activity in the developing spinal cord. Neural tubes from stage-24 (26 h postfertilization) embryos were loaded with Fluo4-AM and confocally imaged. (A) Ventral view of a single neural tube imaged consecutively for 20-min intervals under control conditions, in the presence of 100 pg/mL BMP4/7 and a combination of BMP4/7 and 500 ng/mL Noggin. Traces show Ca2+ spike activity for a single cell outlined in yellow. In A and C–F, circles identify cells spiking during recording. (Scale bar, 20 μm.) (B) Dose-dependent effect of BMP4/7 on Ca2+ spike activity. Ca2+ spike incidence and frequency in ventral and dorsal neural tube are 12 ± 2 vs. 6 ± 0.7 spiking cells during 30-min recordings and 7 ± 0.8 vs. 3 ± 0.4 Ca2+ spikes per cell per hour, respectively (20, 21). (C–F) Representative examples of Ca2+-imaged neural tubes from wild-type (C and E) and inducible BMPRIA mutants, Alk3 (D), and tBR (F). Traces show Ca2+ spike activity for cells outlined in yellow. (G) Enhancing or inhibiting BMP signaling exerts opposite effects on Ca2+ spike activity and is translation-independent. Cychex, cycloheximide; BMP4/7, 10 ng/mL; Noggin, 500 ng/mL; Cychex, 10 μg/mL. Results are comparable in dorsal and ventral samples. Data in B and G are mean ± SEM percent of spiking cells and spike frequency compared with control (30-min recording before addition of agents or recording from sibling wild-type embryos, dashed line in G), n ≥ 5 embryos per experimental group, *P < 0.01.

Fig. 3.
Fig. 3.

The interplay between Ca2+ spike activity and BMP signaling regulates differentiation of Lh2A/B-expressing cells. Immunostaining of transverse sections of the spinal cord (outlined) from wild-type embryos or CA-MEK–expressing embryos treated with the indicated agents: VGCblock, voltage-gated Na+ and Ca2+ channel blockers; Verat, veratridine; BMP+verat, BMP4/7 and veratridine; Nog+VGCblock, Noggin and VGCblock; LDN+VGCblock, LDN-193189 and VGCblock. Graphs show mean ± SEM percent of spiking cells and Ca2+ spike frequency in the dorsal and ventral spinal cord compared with control (30-min recording before addition of agents), n ≥ 5 stage-24 (26-h-old) embryos and mean ± SEM of Lh2A/B immunopositive cells/100 μm of dorsal and ventral spinal cord, n ≥ 5 stage-35 (48-h-old) larva per experimental group, *P < 0.05. D, dorsal; V, ventral. (Scale bar, 20 μm.) Concentrations of agents used to impregnate beads are indicated in

SI Materials and Methods

. For Ca2+ imaging experiments: VGCblock, indicated in

SI Materials and Methods

; BMP4/7, 10 ng/mL; Veratridine, 1 μM; Noggin, 1 and 0.5 μg/mL, dorsal and ventral, respectively; LDN-193189, 5 μM.

Fig. 2.
Fig. 2.

The negative modulation of voltage-gated Na+ channel by p38 MAPK participates in the BMP-induced decrease in Ca2+ spike activity. (A) BMP action on Ca2+ spike activity in the presence of kinase inhibitors. Data are mean ± SEM percent of spiking cells and percent of Ca2+ spike frequency in dorsal spinal cord compared with control (30 min prior addition of drug), *P < 0.001. Similar profile of changes in Ca2+ spike activity upon treatments is observed in the ventral spinal cord (

Fig. S2

). (B) BMP4/7 affects phosphorylation status of p38 and Erk1/2 in neural tube explants from stage-24 embryos, as shown in representative Western blots. Graph shows mean ± SEM percent OD, previously normalized to the level of total p38 or Erk1/2, compared with control (0′ BMP4/7), *P < 0.0001. (C) Ca2+ spike activity in dorsal neural tubes from wild-type control, dominant-negative (DN) p38, and CA-MEK–expressing embryos (stage 24). Data are mean ± SEM percent of spiking cells compared with wild-type control, *P < 0.05. (D) Overexpression of mutant Nav1.6 at the p38 phosphorylation site prevents BMP-induced decrease in Ca2+ spikes. Ca2+ spike activity in neural tubes from wild-type (wt) control, wtNav1.6-, mutated (mut)Nav1.6-, and wtCav2.2-overexpressing embryos (stage 24). Data are mean ± SEM percent of spiking cells compared with wt control, *P < 0.01. n = 5 per experimental group, BMP4/7: 10 ng/mL.

Fig. 4.
Fig. 4.

Ca2+ spike activity modulates the phosphorylation status and nuclear levels of activated Smad1/5/8. Western blots of whole-cell, cytosolic, and nuclear extracts from stage-24 neural tube explants incubated with indicated agents for 30 min. Combined treatments consisted of preincubation with veratridine or VGCblock for 30 min before addition of BMP4/7. Graphs show mean ± SEM percent of P-tail-Smad levels normalized to total Smad for whole-cell extracts and to β-tubulin or histone H2B for cytosolic or nuclear fractions, respectively, compared with control, n > 5, *P < 0.001 compared with control or with BMP-treated samples. VGCblock, indicated in

SI Materials and Methods

; BMP4/7, 10 ng/mL; Veratridine, 1 μM.

Fig. 5.
Fig. 5.

Proposed model for the possible molecular interplay between BMP and Ca2+-mediated electrical activity pathways during spinal cord development. Binding of BMP to its receptor activates p38 MAPK, which phosphorylates and negatively modulates Nav activity. This process diminishes the probability of spontaneous Nav-mediated membrane depolarizing events and, hence, prevents further activation of Cav resulting in a decrease in Ca2+ spikes. In turn, Ca2+ spikes activate Erk1/2 and decrease the level of nuclear P-tail-Smad. The interaction between Ca2+ spike activity and BMP signaling regulates the differentiation of the commissural dorsal spinal phenotype.

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