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).

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