Lewis BB et al. (2009), Cloning and characterization of voltage-gated c...

XB-ART-40321

Dev Dyn 2009 Nov 01;23811:2891-902. doi: 10.1002/dvdy.22102.

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Cloning and characterization of voltage-gated calcium channel alpha1 subunits in Xenopus laevis during development.

Lewis BB , Wester MR , Miller LE , Nagarkar MD , Johnson MB , Saha MS .

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Voltage-gated calcium channels play a critical role in regulating the Ca2+ activity that mediates many aspects of neural development, including neural induction, neurotransmitter phenotype specification, and neurite outgrowth. Using Xenopus laevis embryos, we describe the spatial and temporal expression patterns during development of the 10 pore-forming alpha1 subunits that define the channels' kinetic properties. In situ hybridization indicates that CaV1.2, CaV2.1, CaV2.2, and CaV3.2 are expressed during neurula stages throughout the neural tube. These, along with CaV1.3 and CaV2.3, beginning at early tail bud stages, and CaV3.1 at late tail bud stages, are detected in complex patterns within the brain and spinal cord through swimming tadpole stages. Additional expression of various alpha1 subunits was observed in the cranial ganglia, retina, olfactory epithelium, pineal gland, and heart. The unique expression patterns for the different alpha1 subunits suggests they are under precise spatial and temporal regulation and are serving specific functions during embryonic development.

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Species referenced: Xenopus laevis
Genes referenced: cacna1a cacna1b cacna1c cacna1d cacna1e cacna1f cacna1g cacna1h cacna1s cacnb3 cav1 cav2 cav3 cav3.2

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	Figure 1. Spatial expression patterns of VGCC alpha 1 subunits in stage 20-21 (neurula) Xenopus laevis embryos using whole-mount in situ hybridization. Embryos were viewed dorsally, with anterior to the left. A,A prime : CaV1.1 expression. B,B prime : CaV1.2 expression. C,C prime : CaV2.1 expression. D,D prime : CaV2.2 expression. E,E prime : CaV3.2 expression. Arrowheads indicate regions of expression. nt, neural tube; so, somite; VII, cranial placode VII, geniculate (facialis) placode. Scale bar = 1.0 mm in A-E; 0.25 mm in A prime -E prime .
	Figure 2. Spatial and temporal expression patterns of VGCC alpha 1 subunits in developing X. laevis embryos using whole-mount in situ hybridization. Embryos were viewed laterally, with anterior to the left and dorsal to the top. A,D,G,J,M,P,S,V,Y: Stages 24-26 (early tail bud). B,E,H,K,N,Q,T,W,Z: Stages 29-31 (late tail bud). C,F,I,L,O,R,U,X,AA: Stages 34-36 (swimming tadpole). A-C: CaV1.1 expression. D-F: CaV1.2 expression. G-I: CaV1.3 expression. J-L: CaV1.4 expression. M-O: CaV2.1 expression. P-R: CaV2.2 expression. S-U: CaV2.3 expression. V-X: CaV3.1 expression. Y-AA: CaV3.2 expression. D prime ,F prime ,S prime ,T prime ,W prime ,Y prime : Magnified images of portions of the embryos indicated by the hollow boxes. AA prime : Magnified image of the tail of an embryo at swimming tadpole stage showing CaV3.2 expression. Arrowheads indicate regions of expression. fb, forebrain; e, eye; hb, hindbrain; ht, heart; mb, midbrain; oe, olfactory epithelium; ot, otic vesicle; pa, pharyngeal arches; pg, pineal gland; sc, spinal cord; so, somite; t, tail bud; V, cranial ganglion V, trigeminal ganglion; VII, cranial ganglion VII, geniculate (facialis) ganglion; IX, cranial ganglion IX, glossopharyngeal ganglion; X, cranial ganglion X, vagal ganglion. Scale bars = 1.0 mm in A-AA, 0.4 mm in AA prime ; 0.2 mm in D prime ,F prime ,S prime ,T prime ,W prime ,Y prime .
	Figure 3. Forebrain histological analysis of voltage-gated calcium channel (VGCC) alpha 1 subunit expression in stages 35-37 (swimming tadpole) Xenopus laevis using whole-mount in situ hybridization. A-J: Transverse 18- mu m sections within the forebrain region of the anterior-posterior axis, with dorsal to the top. A: CaV1.1 expression. B: CaV1.2 expression. C: CaV1.3 expression. D: CaV1.4 expression. E: CaV2.1 expression. F: CaV2.2 expression. G,H: CaV3.1 expression. I,J: CaV3.2 expression. Arrowheads indicate regions of expression. fb, forebrain; e, eye; ht, heart; oe, olfactory epithelium; pa, pharyngeal arches; pg, pineal gland. Scale bar = 0.25 mm.
	Figure 4. Midbrain histological analysis of voltage-gated calcium channel (VGCC) alpha 1 subunit expression in stages 35-37 (swimming tadpole) X. laevis using whole-mount in situ hybridization. A-J: Transverse 18- mu m sections within the midbrain region of the anterior-posterior axis, with dorsal to the top. A,B: CaV1.2 expression. C: CaV1.3 expression. D,E: CaV2.1 expression. F,G: CaV2.2 expression. H,I: CaV3.1 expression. J: CaV3.2 expression. Arrowheads indicate regions of expression. mb, midbrain; e, eye; ht, heart; V, cranial ganglion V, trigeminal ganglion; VII, cranial ganglion VII, geniculate (facialis) ganglion. Scale bar = 0.25 mm.
	Figure 5. Hindbrain histological analysis of voltage-gated calcium channel (VGCC) alpha 1 subunit expression in (A-G,J,K) stage 35-37 (swimming tadpole) or (I) stage 30 (late tail bud) X. laevis using whole-mount in situ hybridization. A-K: Transverse 18- mu m sections of embryos within the hindbrain region of the anterior-posterior axis, with dorsal to the top. A: CaV1.1 expression. B,C: CaV1.2 expression. D: CaV1.3 expression. E,F: CaV2.1 expression. G,H: CaV2.2 expression. I: CaV2.3 expression. J: CaV3.1 expression. K: CaV3.2 expression. Arrowheads indicate regions of expression. hb, hindbrain; ot, otic vesicle; so, somite; VIII, cranial ganglion VIII, acousticus ganglion; IX, cranial ganglion IX, glossopharyngeal ganglion; X, cranial ganglion X, vagal ganglion. Scale bar represents 0.25 mm.
	Figure 6. Spinal cord histological analysis of voltage-gated calcium channel (VGCC) alpha 1 subunit expression in stages 35-37 (swimming tadpole) Xenopus laevis using whole-mount in situ hybridization. A-G: Transverse 18- mu m sections within the spinal cord region of the anterior-posterior axis, with dorsal to the top. A: CaV1.1 expression. B: CaV1.2 expression. C: CaV1.3 expression. D: CaV2.1 expression. E: CaV2.2 expression. F: CaV3.1 expression. G,H: CaV3.2 expression. Arrowheads indicate regions of expression. Abbreviations: sc, spinal cord; so, somite. Scale bar = 0.25 mm.
	Figure 7. Retina histological analysis of voltage-gated calcium channel (VGCC) alpha 1 subunit expression in stage 40 (swimming tadpole) X. laevis using whole-mount in situ hybridization. A-G: Transverse 18- mu m sections of the retina, with dorsal to the top. A: CaV1.2 expression. B: CaV1.3 expression. C: CaV1.4 expression. D: CaV2.1 expression. E: CaV2.2 expression. F: CaV3.1 expression. G: CaV3.2 expression. Brackets indicate retinal layers with expression. GCL, ganglion cell layer; INL, inner nuclear layer; L, lens; PR, photoreceptor cells. Scale bar = 0.1 mm.



	cacna1a ( calcium channel, voltage-dependent, P/Q type, alpha 1A subunit ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.

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