L-type voltage-gated Ca2+ channel CaV1.2 regulates chondrogenesis during limb development

Abstract

All cells, including nonexcitable cells, maintain a discrete transmembrane potential (V_mem), and have the capacity to modulate V_mem and respond to their own and neighbors' changes in V_mem Spatiotemporal variations have been described in developing embryonic tissues and in some cases have been implicated in influencing developmental processes. Yet, how such changes in V_mem are converted into intracellular inputs that in turn regulate developmental gene expression and coordinate patterned tissue formation, has remained elusive. Here we document that the V_mem of limb mesenchyme switches from a hyperpolarized to depolarized state during early chondrocyte differentiation. This change in V_mem increases intracellular Ca²⁺ signaling through Ca²⁺ influx, via Ca_V1.2, 1 of L-type voltage-gated Ca²⁺ channels (VGCCs). We find that Ca_V1.2 activity is essential for chondrogenesis in the developing limbs. Pharmacological inhibition by an L-type VGCC specific blocker, or limb-specific deletion of Ca_V1.2, down-regulates expression of genes essential for chondrocyte differentiation, including Sox9, Col2a1, and Agc1, and thus disturbs proper cartilage formation. The Ca²⁺-dependent transcription factor NFATc1, which is a known major transducer of intracellular Ca²⁺ signaling, partly rescues Sox9 expression. These data reveal instructive roles of Ca_V1.2 in limb development, and more generally expand our understanding of how modulation of membrane potential is used as a mechanism of developmental regulation.

Keywords: calcium channel; chondrogenesis; limb development; membrane potential.

Fig. 1.

Depolarization during chondrogenic differentiation of limb mesenchyme cells. (A and B) Transverse (A) and sagittal sections (B) of mouse hindlimbs at E10.5, E11.5, and E12.5. DiBAC₄(3) (DiBAC) signals are indicated by arrows. (C) Sagittal sections of mouse hindlimbs labeled with immunostaining against Col2a1. (D and E) Limb mesenchyme from Col2a1-tdTomato reporter mice was micromass-cultured in the presence of 4-OHT for 2 d, and stained with DiBAC. (Scale bars: 200 μm in A–C, 1 mm in D, and 100 μm in E.)

Fig. 2.

Ca²⁺ transients in limb mesenchyme are regulated by voltage-gated L-type Ca²⁺ channels. (A–O) Live-imaging analyses of Ca²⁺ transients visualized with GCaMP6s in micromass-cultured chicken limb bud cells. mCherry was co-overexpressed as a control signal. The micromass-cultured cells were treated with K-gluconate (40 mM; C and D), Nifedipine (250 μM; H and I) or both (M and N). At each time point, images were thresholded to visualize GCaMP transients and newly emerged GCaMP signals were counted as GCaMP transients, as marked by yellow arrowheads in (B′, D′, G′, I′, L′, and N′). (E, J, and O) Temporal dynamics of GCaMP transients (n = 3 each). Time-lapse analyses started 5 h after plating. GCaMP flashes were counted every minute for 60 min. (Scale bars: 100 μm in A, F, and K.)

Fig. 3.

Ca²⁺ influx via L-type Ca²⁺ channels regulates chondrogenesis of chicken limb bud mesenchyme in vitro. (A) HH20 chicken limb bud cells were micromass-cultured in the presence of Nifedipine (50 μM or 250 μM) and A23187 (50 nM or 100 nM) for 5 d, and stained with Alcian blue. (B) Quantification of Alcian blue intensity (n = 8 for each group). (C) Sox9, Col2a1, and Ihh mRNA expression levels were measured by qPCR and normalized to β-actin expression (n = 8 each). (D and E) Micromass-cultured chicken cells were treated with DMSO, Nifedipine (250 μM) from days 0 to 2, days 1 to 3, or days 2 to 4, for 5 d, and stained with Alcian blue (n = 5 each). (F) Relative expression levels of Sox9, Col2a1, and Ihh were quantified by qPCR (n = 5 each). Error bars represent SEM. *P < 0.05, **P < 0.01; 1-way ANOVA multicomparison with DMSO as control. (Scale bars: 2 mm in A and D.)

Fig. 4.

Ca_V1.2 is required for development of skeletal elements in mouse limbs. (A and B) Alcian blue/Alizarin red skeletal preparations of E18.5 Cacna1c^fl/fl (control, A), and Prx1-CreER^+/+; Cacna1c^fl/fl mouse embryos (mutant, B). Tamoxifen injections were performed at E9.5 and E10.5. Forelimbs (FL) and hindlimbs (HL) are separately displayed. (Scale bars: 5 mm in A.) (C and D) Quantification for relative length of the limbs (C) and the number of digits (D) of controls (n = 20 limbs) and mutants (n = 26). Error bars represent SEM. *P < 0.05, **P < 0.01.

Fig. 5.

Increased cell death in Ca_V1.2 mutant limb buds. (A–H) Sagittal sections of fore- or hindlimb buds from E11.5 Control (A, C, E, and G) and mutant mouse embryos (B, D, F, and H) were stained with antibodies for phospho-histone H3 (pH3; A–D) or cleaved-caspase3 (Cas3; E–H). (Scale bars: 200 μm in A and E.) (I and J) Quantification of the number of pH3⁺ or Cas3⁺ cells in the limb buds (n = 12 each for pH3-counting, n = 18 each for Cas3-counting). Error bars represent SEM. **P < 0.01.

Fig. 6.

Requirement of Ca_V1.2 for chondrocyte differentiation in mouse limbs. (A and B) Sagittal sections of E13.5 control (A) and mutant hindlimbs (B) were stained with antibodies for Sox9 (red) and Col2 (cyan). Cartilage elements were marked by anti-Col2 antibodies as indicated by arrowheads. (C and D) Sagittal views of hindlimbs of E11.5 and E13.5 mouse embryos. Chondrocytes were visualized by immunostaining against Sox9. (E) Sox9 and Agc1 expression levels were measured by qPCR and normalized to β-actin expression (n = 7 for each group). (F and G) Immunostaining of phosphorylated Smad1/5 (pSmad) in E11.5 and E13.5 hindlimbs from control (F) and mutant embryos (G). Error bars represent SEM. **P < 0.01. (Scale bars: 200 μm in A–C and F.)

Fig. 7.

NFATc1 is reduced in the Ca_V1.2 cKO cells and is sufficient to up-regulate chondrocyte differentiation in vivo and in vitro. (A) mRNA expression patterns of NFATc1 in E10.5, E11.5, E12.5, and E13.5 mouse limbs. Arrows mark NFATc1 expression at both fore- and hindlimb buds. (B and C) Immunostaining for NFATc1 and Sox9 in cultured mouse limb cells harvested from E11.5 control (B) and mutant limb buds (C). (D) Quantification of the number of NFATc1⁺ or Sox9⁺ cells in the cultured cells (n = 3,436 cells for control, n = 1,585 cells for mutant group). (E) Measurement of fluorescent intensity for NFATc1 and Sox9 signals (n = 270 cells from control group). (F and G) Plasmids carrying ZsGreen1 (control, F) or NFATc1^nuc-IRES-ZsGreen1 (G) were electroporated into chicken forelimbs. Misexpression of NFATc1^nuc resulted in inducing a Sox9⁺ ectopic aggregate (an arrow in G; n = 5 of 15), which was not seen in the controls (n = 0 of 15). (H and I) mCherry or NFATc1^nuc was overexpressed in micromass-cultured cells obtained from E11.5 control and mutant embryos by using lentiviruses (see SI Appendix, Fig. S10 for experimental procedures). (I) Quantification of Alcian blue staining (n = 8 for each group). Error bars represent SEM. **P < 0.01. (Scale bars: 500 μm in A, 100 μm in B, 200 μm in F, and 2 mm in H.)