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. 2013 Apr;123(4):1638-46.
doi: 10.1172/JCI66903. Epub 2013 Mar 15.

Calcium influx through L-type CaV1.2 Ca2+ channels regulates mandibular development

Kapil V Ramachandran  1 , Jessica A Hennessey, Adam S Barnett, Xinhe Yin, Harriett A Stadt, Erika Foster, Raj A Shah, Masayuki Yazawa, Ricardo E Dolmetsch, Margaret L Kirby, Geoffrey S Pitt
Affiliations

Calcium influx through L-type CaV1.2 Ca2+ channels regulates mandibular development

Kapil V Ramachandran et al. J Clin Invest. 2013 Apr.

Abstract

The identification of a gain-of-function mutation in CACNA1C as the cause of Timothy Syndrome (TS), a rare disorder characterized by cardiac arrhythmias and syndactyly, highlighted unexpected roles for the L-type voltage-gated Ca2+ channel CaV1.2 in nonexcitable cells. How abnormal Ca2+ influx through CaV1.2 underlies phenotypes such as the accompanying syndactyly or craniofacial abnormalities in the majority of affected individuals is not readily explained by established CaV1.2 roles. Here, we show that CaV1.2 is expressed in the first and second pharyngeal arches within the subset of cells that give rise to jaw primordia. Gain-of-function and loss-of-function studies in mouse, in concert with knockdown/rescue and pharmacological approaches in zebrafish, demonstrated that Ca2+ influx through CaV1.2 regulates jaw development. Cranial neural crest migration was unaffected by CaV1.2 knockdown, suggesting a role for CaV1.2 later in development. Focusing on the mandible, we observed that cellular hypertrophy and hyperplasia depended upon Ca2+ signals through CaV1.2, including those that activated the calcineurin signaling pathway. Together, these results provide new insights into the role of voltage-gated Ca2+ channels in nonexcitable cells during development.

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Figures

Figure 1
Figure 1. CaV1.2 is expressed in developing jaw.
(A–D) Whole-mount β-gal staining of CaV1.2+/LacZ embryos. CaV1.2 is highly expressed in the developing heart around E9.5 (A). By E11.5 intense staining is present in the first (arrowhead) and second (arrow) pharyngeal arches (B–D). (E and F) β-gal staining of a coronal section through the first mandibular arch. F is a magnified section of the boxed area shown in E.
Figure 2
Figure 2. CaV1.2 expression in pharyngeal arches affects craniofacial development.
(A) X-rays of a Prx-CaV1.2WT skull and a Prx1– littermate skull. Note that the upper jaw extends anteriorly to the mandible (arrows). (B) X-rays of a Prx1-CaV1.2TS skull and a Prx1– littermate skull. When CaV1.2TS is expressed (in the Prx1+ animal), the mandible extends more anteriorly, as is also seen in TS patients. (C) Image showing patient phenotype (arrow points to mandible). Image in C is reproduced with permission from the authors and publisher (1). (D) Mandibles from adult CaV1.2+/LacZ or wild-type (littermates). Note reduced width between coronoid processes in CaV1.2+/LacZ compared with wild-type mice. Scale bars: 5 mm.
Figure 3
Figure 3. Ca2+ influx through CaV1.2 regulates size of mandible in zebrafish.
(A) Alcian blue–stained embryos at 72 hpf. CON, uninjected control; CMO, control morpholino; MO, CaV1.2 morpholinos; MO + WT, CaV1.2 morpholinos coinjected with rabbit CaV1.2WT cRNA; MO + 4EQ, CaV1.2 morpholinos coinjected with rabbit CaV1.2 cRNA with mutations in the 4-pore glutamates (non-Ca2+ permeant); MO + TS, CaV1.2 morpholinos coinjected with rabbit CaV1.2TS cRNA. (B) Mandibular area (as outlined for CON in A normalized to the area in CMO (n = 20 for each). *P < 0.001 versus CMO. (C) Mandibular area normalized to the area in MO (n = 20 for each). *P < 0.001 versus MO. (D) Alcian blue–stained embryos at 72 hpf after treatment with DMSO or nisoldipine. (E) Mandibular area normalized to the area in DMSO (n = 20 for each). *P < 0.001 versus DMSO.
Figure 4
Figure 4. Ca2+ influx through CaV1.2 affects cellular hypertrophy and hyperplasia in the mandible.
(A) Low-power DIC image of flat-mounted, Alcian blue–stained CON embryo. The red box indicates the region from which images in B, D, and F were obtained. (B) Higher magnification of the region shown in A for an embryo treated with DMSO or NIS. (C) Cell size normalized to DMSO (n = 20 for each). *P < 0.001 versus DMSO. (D) Higher magnification of the region shown in A for a CON, CMO, or MO embryo. (E) Cell size normalized to CMO (n = 20 for each). *P < 0.001 versus CMO. (F) Higher magnification of the region shown in A for an MO + WT, MO + TS, or MO + 4EQ embryo. (G) Cell size normalized to MO (n = 20 for each). *P < 0.001 versus MO; **P < 0.001 versus MO + WT or versus MO. (H) BrdU staining and quantification of BrdU+ cells (in GFP+ cells) of sox10+ zebrafish embryos treated with CMO or MO. *P < 0.001. Note that we were unable to use 1-phenyl 2-thiourea, the tyrosinase inhibitor commonly used to block pigmentation and aid visualization (for BrdU staining) in zebrafish because it interferes with neural crest development (32): the larger red nuclei likely denote newly produced melanocytes, which migrate to the same area, and were not counted.
Figure 5
Figure 5. Calcineurin, downstream of CaV1.2, regulates cellular hypertrophy in the developing mandible.
(A) Alcian blue–stained embryo treated with DMSO or a combination of cyclosporine and FK506 (FK/CsA). (B) Mandibular area normalized to the area in DMSO (n = 20 for each). *P < 0.001 versus DMSO. (C) Alcian blue–stained embryo treated with MO or MO plus cRNA for a constitutively active calcineurin (MO + caCN). (D) Mandibular area normalized to the area in MO (n = 20 for each). *P < 0.001 versus MO. (E) Higher magnification of the region shown in Figure 4A for an embryo treated with DMSO or FK/CsA. (F) Cell size normalized to DMSO (n = 20 for each). *P < 0.001 versus DMSO. (G) Higher magnification of the region shown in Figure 4A for an embryo treated with MO or MO + caCN. (H) Cell size normalized to MO (n = 20 for each). *P < 0.001 versus MO. Scale bar: 200 μm.
Figure 6
Figure 6. sox10+ cranial neural crest–derived cells express functional CaV1.2 channels.
(A) GFP images of decapitated sox10-GFP transgenic embryos at approximately 65 hpf of a control or CaV1.2 MO embryo. Arrowhead denotes migrating cranial neural crest. (B) Sample GFP+ cell after single-cell isolation. Image on the right shows a DAPI overlay. Note the GFP– cell. (C) Whole-cell Ca2+ current from a GFP+ cell isolated from an MO or CON embryo. Voltage ramp protocol shown below. (D) Box plot showing current density for MO or CON embryos. *P < 0.01. Scale bar: 5 μm.
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