Lewis BB , Miller LE , Herbst WA , Saha MS .

R15 HD077624 NICHD NIH HHS , R15 NS067566 NINDS NIH HHS , R15HD077624-01 NICHD NIH HHS , R15NS067566 NINDS NIH HHS

	Figure 1. Coexpression patterns of xVGlut1 and VGCC α1 subunits in the central nervous system of Xenopus laevis swimming tadpole embryos. VGCC subunit expression is labeled with fluorescein (green), and xVGlut1 expression is labeled with Cy3 (red). Coexpression is indicated by the yellow overlap of both channels. A–E: Cav1.2 coexpression with xVGlut1 in the (A) forebrain, (B) midbrain, (C) hindbrain, (D) anterior spinal cord, (E) and posterior spinal cord. F–J: Cav1.3 coexpression in the (F) forebrain, (G) midbrain, (H) hindbrain, (I) anterior spinal cord, (J) and posterior spinal cord. K–O: Cav2.1 coexpression in the (K) forebrain, (L) midbrain, (M) hindbrain, (N) anterior spinal cord, and (O) posterior spinal cord. P–S: Cav2.2 coexpression in the (P) forebrain, (Q) midbrain, (R) hindbrain, (S) spinal cord. T–AA: Cav3.1 coexpression in the (T) forebrain, (U) midbrain, (V) hindbrain, (W) spinal cord. Cav3.2 coexpression in the (X) forebrain, (Y) midbrain, (Z) hindbrain, and (AA) spinal cord. BB–DD: Cav2.3 coexpression in the (BB) midbrain, (CC) hindbrain, and (DD) spinal cord. For the assistance of color-blind readers, a magenta–green copy of this figure is provided as Supplementary Figure 1. Scale bar = 100 μm in A (applies to A–DD).
	Figure 2. Coexpression patterns of xVGlut1 and VGCC α1 subunits in the retina of Xenopus laevis swimming tadpole embryos. VGCC subunit expression is labeled with fluorescein (green), and xVGlut1 expression is labeled with Cy3 (red). Coexpression is indicated by the yellow overlap of both channels. A–G: xVGlut1 coexpression with (A) Cav1.2, (B) Cav1.3, (C) Cav1.4, (D) Cav2.1, (E) Cav2.2, (F) Cav3.1, and (G) Cav3.2. For the assistance of color-blind readers, a magenta–green copy of this figure is provided as Supplementary Figure 2. Scale bar = 250 μm in A (applies to A–G).
	Coexpression patterns of xVIAAT and VGCC α1 subunits in the central nervous system of Xenopus laevis swimming tadpole embryos. VGCC subunit expression is labeled with fluorescein (green) and xVIAAT expression is labeled with Cy3 (red). Coexpression is indicated by the yellow overlap of both channels. A–D: Cav1.2 coexpression with xVIAAT in the (A) forebrain, (B) midbrain, (C) hindbrain, (D) anterior spinal cord, and (E) posterior spinal cord. F–J: Cav1.3 coexpression in the (F) forebrain, (G) midbrain, (H) hindbrain, (I) anterior spinal cord, and (J) posterior spinal cord. K–O: Cav2.1 coexpression in the (K) forebrain, (L) midbrain, (M) hindbrain, (N) anterior spinal cord, and (O) posterior spinal cord. P–S: Cav2.2 coexpression in the (P) forebrain, (Q) midbrain, (R) hindbrain, and (S) spinal cord. Cav3.1 coexpression in the (T) forebrain, (U) midbrain, (V) hindbrain, and (W) spinal cord. X–AA: Cav3.2 coexpression in the (X) forebrain, (Y) midbrain, (Z) hindbrain, and (AA) spinal cord. BB–DD: Cav2.3 coexpression in the (BB) midbrain, (CC) hindbrain, and (DD) spinal cord. For the assistance of color-blind readers, a magenta–green copy of this figure is provided as Supplementary Figure 4. Scale bar = 100 μm in A (applies to A–DD).
	Figure 4. Coexpression patterns of xVIAAT and VGCC α1 subunits in the retina of Xenopus laevis swimming tadpole embryos. VGCC subunit expression is labeled with fluorescein (green), and xVIAAT expression is labeled with Cy3 (red). Coexpression is indicated by the yellow overlap of both channels. A–G: xVIAAT coexpression with (A) Cav1.2, (B) Cav1.3, (C) Cav1.4, (D) Cav2.1, (E) Cav2.2, (F) Cav3.1, and (G) Cav3.2. For the assistance of color-blind readers, a magenta–green copy of this figure is provided as Supplementary Figure 3. Scale bar = 250 μm in A (applies to A–G).
	Figure 5. xpression of VGCCs in cultured cells. Expression of the five VGCC α1 subunits detected with FISH in neural cell cultures during early development. Cav1.2 and Cav1.3 are detected at neural plate and neural tube stages, respectively. Cav2.1, Cav2.2, and Cav3.2 are detected throughout the developmental period studied. Cav2.1 and Cav2.2 are expressed at highest levels at the neural fold stage, and Cav3.2 expression is highest at the neural tube stage. At the neural plate stage, n = 280 cells (Cav1.2), 339 cells (Cav2.1), 358 cells (Cav2.2), and 578 cells (Cav3.2). At the neural fold stage, n = 376 cells (Cav2.1), 305 cells (Cav2.2), and 403 cells (Cav3.2). At the neural tube stage, n = 1,843 cells (Cav1.3), 1,227 cells (Cav2.1), 1,197 cells (Cav2.2), and 631 cells (Cav3.2).
	Figure 6. Sample image set from calcium imaging. A: Average brightfield image of cells during calcium imaging. B: Average Fluo4 fluorescence image of cells during calcium imaging. Bright regions indicate cells with high levels of calcium transients. C: Fluorescent image of cells after FISH. Positive cells are circled. D: Sample calcium activity data from a region of interest (ROI).
	Figure 7. Cav2.1 and Cav2.2 activity. A,B: Comparison of calcium activity in cells exhibiting positive expression for Cav2.1 (A) and Cav2.2 (B) with cells displaying no detectable expression for this VGCC (negative cells). Activity for the cells was analyzed over a 2-hour imaging session, and each dissection (st.14, 18, or 22) was divided into three time blocks. A single time block represents 40 minutes of the image, i.e., “a” is t = 0 to t = 40 minutes, “b” is t = 40 minutes to t = 80 minutes, and “c” is t = 80 minutes to t = 120 minutes. Numbers of spikes in positive and negative cells were compared using the Mann–Whitney U-test. Comparisons with P values ≤ 0.05 are marked with an asterisk (*) on the figure. For Cav2.1 experiments, n = 340 cells (neural plate), 294 cells (neural fold), and 1,228 cells (neural tube). For Cav2.2 experiments, n = 357 cells (neural plate), 139 cells (neural fold), and 1,197 cells (neural tube).
	Figure 8. Pharmacological disruption of VGCC activity and neurotransmitter phenotype. Embryos were dissected at neural plate, neural fold, and neural tube stages, incubated in either a VGCC antagonist (diltiazem) or a VGCC agonist (BayK 8644), and then assayed for expression of the glutamatergic marker xVGlut1 or the GABAergic marker xGAD67. The number of cells expressing the gene of interest (termed “positives”) and the number of cells not expressing the gene of interest (termed “negatives”) for representative fields of view were recorded. A two-sample z-test was used to compare the percent positive between treatment groups and controls. P ≤ 0.05 was recorded as significant. A: xVGlut1 expression of diltiazem-exposed cells. B: xGAD67 expression of diltiazem-exposed cells. C: xVGlut1 expression of BayK 8644-exposed cells. D: xGAD67 expression of BayK 8644-exposed cells. For diltiazem exposures with xVGlut1 expression, n = 1,398 cells (0 μM, neural plate), 1,069 cells (10 μM, neural plate), 936 cells (100 μM, neural plate), 909 cells (0 μM, neural fold), 1,003 cells (10 μM, neural fold), 1,160 cells (100 μM, neural fold), 2,419 cells (0 μM, neural tube), 1,147 cells (10 μM, neural tube), and 160 cells (100 μM, neural tube). For diltizem exposures with xGAD67 expression, n = 1,259 cells (0 μM, neural plate), 843 cells (10 μM, neural plate), 658 cells (100 μM, neural plate), 576 cells (0 μM, neural fold), 1,523 cells (10 μM, neural fold), 1,441 cells (100 μM, neural fold), 1,486 cells (0 μM, neural tube), 1,509 cells (10 μM, neural tube), and 1,171 cells (100 μM, neural tube). For BayK 8644 exposures with xVGlut1 expression, n = 1,178 cells (0 μM, neural plate), 1,817 cells (10 μM, neural plate), 1,755 cells (100 μM, neural plate), 1,222 cells (0 μM, neural fold), 1,249 cells (10 μM, neural fold), 1,126 cells (100 μM, neural fold), 1,455 cells (0 μM, neural tube), 1,384 cells (10 μM, neural tube), and 1,519 cells (100 μM, neural tube). For BayK 8644 exposures with xGAD67 expression, n = 851 cells (0 μM, neural plate), 1,777 cells (10 μM, neural plate), 1,075 cells (100 μM, neural plate), 1,018 cells (0 μM, neural fold), 674 cells (10 μM, neural fold), 745 cells (100 μM, neural fold), 1,560 cells (0 μM, neural tube), 1,469 cells (10 μM, neural tube), and 1,001 cells (100 μM, neural tube).
	Supplementary Figure 1. (Magenta-green version of Figure 1 for the assistance of color-blind readers.) Coexpression patterns of xVGlut1 and VGCC α1 subunits in the central nervous system of Xenopus laevis swimming tadpole embryos. VGCC subunit expression is labeled with fluorescein (green) and xVGlut1 expression is labeled with Cy3 (red). Coexpression is indicated by the yellow overlap of both channels. Scale bar represents 100 μm. Cav1.2 coexpression with xVGlut1 in the (A) forebrain, (B) midbrain, (C) hindbrain, (D) anterior spinal cord, (E) posterior spinal cord. Cav¬1.3 coexpression in the (F) forebrain, (G) midbrain, (H) hindbrain, (I) anterior spinal cord, (J) posterior spinal cord. Cav2.1 coexpression in the (K) forebrain, (L) midbrain, (M) hindbrain, (N) anterior spinal cord, (O) posterior spinal cord. Cav2.2 coexpression in the (P) forebrain, (Q) midbrain, (R) hindbrain, (S) spinal cord. Cav3.1 coexpression in the (T) forebrain, (U) midbrain, (V) hindbrain, (W) spinal cord. Cav3.2 coexpression in the (X) forebrain, (Y) midbrain, (Z) hindbrain, (AA) spinal cord. Cav2.3 coexpression in the (BB) midbrain, (CC) hindbrain, (DD) spinal cord. 295x420mm (96 x 96 DPI)
	Supplementary Figure 2. (Magenta-green version of Figure 2 for the assistance of color-blind readers.) Coexpression patterns of xVGlut1 and VGCC α1 subunits in the retina of Xenopus laevis swimming tadpole embryos. VGCC subunit expression is labeled with fluorescein (green) and xVGlut1 expression is labeled with Cy3 (red). Coexpression is indicated by the yellow overlap of both channels. Scale bar represents 250 μm. xVGlut1 coexpression with (A) Cav1.2, (B) Cav1.3, (C) Cav1.4, (D) Cav2.1, (E) Cav2.2, (F) Cav3.1, (G) Cav3.2. A magenta-green copy of this figure is provided as Supplementary Figure 2. 244x124mm (96 x 96 DPI)
	Supplementary Figure 3. (Magenta-green version of Figure 3 for the assistance of color-blind readers.) Coexpression patterns of xVIAAT and VGCC α1 subunits in the central nervous system of Xenopus laevis swimming tadpole embryos. VGCC subunit expression is labeled with fluorescein (green) and xVIAAT expression is labeled with Cy3 (red). Coexpression is indicated by the yellow overlap of both channels. Scale bar represents 100 μm. Cav1.2 coexpression with xVIAAT in the (A) forebrain, (B) midbrain, (C) hindbrain, (D) anterior spinal cord, (E) posterior spinal cord. Cav¬1.3 coexpression in the (F) forebrain, (G) midbrain, (H) hindbrain, (I) anterior spinal cord, (J) posterior spinal cord. Cav2.1 coexpression in the (K) forebrain, (L) midbrain, (M) hindbrain, (N) anterior spinal cord, (O) posterior spinal cord. Cav2.2 coexpression in the (P) forebrain, (Q) midbrain, (R) hindbrain, (S) spinal cord. Cav3.1 coexpression in the (T) forebrain, (U) midbrain, (V) hindbrain, (W) spinal cord. Cav3.2 coexpression in the (X) forebrain, (Y) midbrain, (Z) hindbrain, (AA) spinal cord. Cav2.3 coexpression in the (BB) midbrain, (CC) hindbrain, (DD) spinal cord. A magenta-green copy of this figure is provided as Supplementary Figure 3. 286x412mm (96 x 96 DPI)
	Supplementary Figure 4. (Magenta-green version of Figure 4 for the assistance of color-blind readers.) Coexpression patterns of xVIAAT and VGCC α1 subunits in the retina of Xenopus laevis swimming tadpole embryos. VGCC subunit expression is labeled with fluorescein (green) and xVIAAT expression is labeled with Cy3 (red). Coexpression is indicated by the yellow overlap of both channels. Scale bar represents 250 μm. xVIAAT coexpression with (A) Cav1.2, (B) Cav1.3, (C) Cav1.4, (D) Cav2.1, (E) Cav2.2, (F) Cav3.1, (G) Cav3.2. A magenta-green copy of this figure is provided as Supplementary Figure 4. 244x123mm (96 x 96 DPI)
	Figure 3. Coexpression patterns of xVIAAT and VGCC α1 subunits in the central nervous system of Xenopus laevis swimming tadpole embryos. VGCC subunit expression is labeled with fluorescein (green) and xVIAAT expression is labeled with Cy3 (red). Coexpression is indicated by the yellow overlap of both channels. A–D: Cav1.2 coexpression with xVIAAT in the (A) forebrain, (B) midbrain, (C) hindbrain, (D) anterior spinal cord, and (E) posterior spinal cord. F–J: Cav1.3 coexpression in the (F) forebrain, (G) midbrain, (H) hindbrain, (I) anterior spinal cord, and (J) posterior spinal cord. K–O: Cav2.1 coexpression in the (K) forebrain, (L) midbrain, (M) hindbrain, (N) anterior spinal cord, and (O) posterior spinal cord. P–S: Cav2.2 coexpression in the (P) forebrain, (Q) midbrain, (R) hindbrain, and (S) spinal cord. Cav3.1 coexpression in the (T) forebrain, (U) midbrain, (V) hindbrain, and (W) spinal cord. X–AA: Cav3.2 coexpression in the (X) forebrain, (Y) midbrain, (Z) hindbrain, and (AA) spinal cord. BB–DD: Cav2.3 coexpression in the (BB) midbrain, (CC) hindbrain, and (DD) spinal cord. For the assistance of color-blind readers, a magenta–green copy of this figure is provided as Supplementary Figure 4. Scale bar = 100 μm in A (applies to A–DD).

Barbado, Gene regulation by voltage-dependent calcium channels. 2009, Pubmed

Barbado, Gene regulation by voltage-dependent calcium channels. 2009, Pubmed
Borodinsky, Activity-dependent homeostatic specification of transmitter expression in embryonic neurons. 2004, Pubmed , Xenbase
Borodinsky, Dynamic regulation of neurotransmitter specification: relevance to nervous system homeostasis. 2014, Pubmed
Cai, Inhibition of recombinant Ca2+ channels by benzothiazepines and phenylalkylamines: class-specific pharmacology and underlying molecular determinants. 1997, Pubmed
Catterall, Voltage-gated calcium channels. 2011, Pubmed
Catterall, Calcium channels and short-term synaptic plasticity. 2013, Pubmed
Chang, Spontaneous calcium spike activity in embryonic spinal neurons is regulated by developmental expression of the Na+, K+-ATPase beta3 subunit. 2009, Pubmed , Xenbase
Davidson, Neural tube closure in Xenopus laevis involves medial migration, directed protrusive activity, cell intercalation and convergent extension. 1999, Pubmed , Xenbase
De Paoli, Selectivity of different calcium antagonists on T- and L-type calcium currents in guinea-pig ventricular myocytes. 2002, Pubmed
Dolmetsch, Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway. 2001, Pubmed
Dulcis, Illumination controls differentiation of dopamine neurons regulating behaviour. 2008, Pubmed , Xenbase
Dullin, Ptf1a triggers GABAergic neuronal cell fates in the retina. 2007, Pubmed , Xenbase
Fiorio Pla, Canonical transient receptor potential 1 plays a role in basic fibroblast growth factor (bFGF)/FGF receptor-1-induced Ca2+ entry and embryonic rat neural stem cell proliferation. 2005, Pubmed
Gardezi, Long C terminal splice variant CaV2.2 identified in presynaptic membrane by mass spectrometric analysis. 2010, Pubmed
Glaser, Implications of purinergic receptor-mediated intracellular calcium transients in neural differentiation. 2013, Pubmed
Gleason, The vesicular glutamate transporter 1 (xVGlut1) is expressed in discrete regions of the developing Xenopus laevis nervous system. 2003, Pubmed , Xenbase
Gu, Spontaneous neuronal calcium spikes and waves during early differentiation. 1994, Pubmed , Xenbase
Gutiérrez, Activity-dependent expression of simultaneous glutamatergic and GABAergic neurotransmission from the mossy fibers in vitro. 2002, Pubmed
Guzman, Robust pacemaking in substantia nigra dopaminergic neurons. 2009, Pubmed
Hashimoto, Postsynaptic P/Q-type Ca2+ channel in Purkinje cell mediates synaptic competition and elimination in developing cerebellum. 2011, Pubmed
Hirtz, Cav1.3 calcium channels are required for normal development of the auditory brainstem. 2011, Pubmed
Homma, Serotonin induces the increase in intracellular Ca2+ that enhances neurite outgrowth in PC12 cells via activation of 5-HT3 receptors and voltage-gated calcium channels. 2006, Pubmed
Huang, Coexpression of high-voltage-activated ion channels Kv3.4 and Cav1.2 in pioneer axons during pathfinding in the developing rat forebrain. 2012, Pubmed
Jun, Ablation of P/Q-type Ca(2+) channel currents, altered synaptic transmission, and progressive ataxia in mice lacking the alpha(1A)-subunit. 1999, Pubmed
Kasyanov, GABA-mediated giant depolarizing potentials as coincidence detectors for enhancing synaptic efficacy in the developing hippocampus. 2004, Pubmed
Kerstein, Mechanosensitive TRPC1 channels promote calpain proteolysis of talin to regulate spinal axon outgrowth. 2013, Pubmed , Xenbase
Leclerc, Early neural development in vertebrates is also a matter of calcium. 2011, Pubmed
Lee, FGF-activated calcium channels control neural gene expression in Xenopus. 2009, Pubmed , Xenbase
Lepski, cAMP promotes the differentiation of neural progenitor cells in vitro via modulation of voltage-gated calcium channels. 2013, Pubmed
Lewis, Cloning and characterization of voltage-gated calcium channel alpha1 subunits in Xenopus laevis during development. 2009, Pubmed , Xenbase
Li, The role of early lineage in GABAergic and glutamatergic cell fate determination in Xenopus laevis. 2006, Pubmed , Xenbase
Lipscombe, Alternative splicing: functional diversity among voltage-gated calcium channels and behavioral consequences. 2013, Pubmed
Lu, Numb-mediated neurite outgrowth is isoform-dependent, and requires activation of voltage-dependent calcium channels. 2009, Pubmed
Marek, cJun integrates calcium activity and tlx3 expression to regulate neurotransmitter specification. 2010, Pubmed , Xenbase
Massé, Purines as potential morphogens during embryonic development. 2012, Pubmed , Xenbase
McDonough, Dissection, culture, and analysis of Xenopus laevis embryonic retinal tissue. 2012, Pubmed , Xenbase
Mochida, Regulation of presynaptic Ca(V)2.1 channels by Ca2+ sensor proteins mediates short-term synaptic plasticity. 2008, Pubmed
Moreau, Increased internal Ca2+ mediates neural induction in the amphibian embryo. 1994, Pubmed
Morton, Characterization of L-type voltage-gated Ca(2+) channel expression and function in developing CA3 pyramidal neurons. 2013, Pubmed
Nishiyama, Semaphorin 3A induces CaV2.3 channel-dependent conversion of axons to dendrites. 2011, Pubmed , Xenbase
Papanayotou, Calfacilitin is a calcium channel modulator essential for initiation of neural plate development. 2013, Pubmed
Rajapaksha, Novel splice variants of rat CaV2.1 that lack much of the synaptic protein interaction site are expressed in neuroendocrine cells. 2008, Pubmed
Roberts, A functional scaffold of CNS neurons for the vertebrates: the developing Xenopus laevis spinal cord. 2012, Pubmed , Xenbase
Rosenberg, Calcium signaling in neuronal development. 2011, Pubmed
Sagala, Neurochemical characterization of the striatum and the nucleus accumbens in L-type Ca(v)1.3 channels knockout mice. 2012, Pubmed
Sanhueza, Expression of voltage-activated calcium channels in the early zebrafish embryo. 2009, Pubmed
Shim, XTRPC1-dependent chemotropic guidance of neuronal growth cones. 2005, Pubmed , Xenbase
Spitzer, Activity-dependent neurotransmitter respecification. 2012, Pubmed
Spitzer, Homeostatic activity-dependent paradigm for neurotransmitter specification. 2005, Pubmed
Spitzer, Outside and in: development of neuronal excitability. 2002, Pubmed
Takahashi, Pathway interactions and synaptic plasticity in the dendritic tuft regions of CA1 pyramidal neurons. 2009, Pubmed
Takano, Development of Ca²(+) signaling mechanisms and cell motility in presumptive ectodermal cells during amphibian gastrulation. 2011, Pubmed
Tan, Alternative splicing at C terminus of Ca(V)1.4 calcium channel modulates calcium-dependent inactivation, activation potential, and current density. 2012, Pubmed
Tuluc, Divergent biophysical properties, gating mechanisms, and possible functions of the two skeletal muscle Ca(V)1.1 calcium channel splice variants. 2011, Pubmed
Turner, Signaling complexes of voltage-gated calcium channels. 2011, Pubmed
Vandael, Cav1.3 and Cav1.2 channels of adrenal chromaffin cells: emerging views on cAMP/cGMP-mediated phosphorylation and role in pacemaking. 2013, Pubmed
Watt, Specific frequencies of spontaneous Ca2+ transients upregulate GAD 67 transcripts in embryonic spinal neurons. 2000, Pubmed , Xenbase
Wester, Expression patterns of glycine transporters (xGlyT1, xGlyT2, and xVIAAT) in Xenopus laevis during early development. 2008, Pubmed , Xenbase
Wheeler, CaMKII locally encodes L-type channel activity to signal to nuclear CREB in excitation-transcription coupling. 2008, Pubmed
Zhang, Alternative splicing modulates diltiazem sensitivity of cardiac and vascular smooth muscle Ca(v)1.2 calcium channels. 2010, Pubmed
Zhou, Identification and expression of voltage-gated calcium channel beta subunits in Zebrafish. 2008, Pubmed