DEV02812 Supplementary Material

Files in this Data Supplement:

• Supplemental Table 1 - Adobe PDF

• Supplemental Table 2 - Adobe PDF

• Supplemental Figure 1 -

  Fig. S1. Increase in DiBAC₄(3) intensity with depolarization. Cells were depolarized using very high concentrations of Nigericin (Sigma) (Horiuchi and Iwata, 1983) and carbonyl cyanide M-chlorophenylhydrazone (CCCP; Sigma) (Holoubek et al., 2003). Tails were imaged with epifluorescence on the Olympus BX61 (12-bit images). Untreated tails were imaged once, then the coverslip was removed and the ionophores were added, and a new coverslip was put on. Images were collected at various time points afterward, then analyzed using IPLab software; images were background-corrected, then a clearly fluorescent region of interest (ROI) was circled on the first image, and the mean and maximum pixel values were recorded (only pixels with values from 1 to 4094 were included because underexposed and overexposed pixels can artificially inflate the number of 0 and 4095 pixels, respectively). The ROI was then transposed to the other images, adjusted spatially to account for movement of the tail, and quantified in the same way. A control for the physical disturbance of adding solution showed no effect on intensity. Three different measures of intensity are graphed: the change in the value of the minimum intensity, the change in the mean intensity, and the change in the maximum intensity. All three measures show intensity increasing with time as the cells become depolarized by the ionophores.

• Supplemental Figure 2 -

  Fig. S2. Depolarization of cells using K-gluconate. For this graph, membrane voltage was estimated using the Goldman equation with known or published values of ion concentration and permeability. Wherever possible, data for Xenopus cells were used; if Xenopus values were not available, values measured in other animals were used. The voltages predicted using this assortment of values came out remarkably close to values measured directly in large cells of the early embryo. As predicted, both the mean and the maximum intensity of pixels (values 1 to 254) increased as external K⁺ increased (by addition of K⁺-gluconate).

• Supplemental Figure 3 -

  Fig. S3. DiSBAC₄(2) imaging to confirm the DiBAC images using a dye with an alternate, complimentary, means of reporting membrane voltage. DiSBAC is cationic, whereas DiBAC is anionic. Thus, DiBAC intensity increases as the membrane potential becomes more positive; DiSBAC intensity, by contrast, increases as the membrane voltage becomes more negative. Thus, DiSBAC images should be inverted relative to DiBAC images. Shown are DiBAC and DiSBAC images from regenerating tails. Superimposed on the micrographs are circles indicating the three ROIs used for these images; the graph shows the summed intensities of these particular images, normalized to the mean intensity of WT trunk (as in Fig. 3E). The blue line on the graph shows the characteristic concave shape of regenerating tails (brightest at the shoulder, less bright in the bud and trunk). The shape of the DiSBAC curve is predicted to be convex. In this example, the overall intensity of the bud is not higher than the shoulder; however, this is well within the variation we saw with all treatments. Moreover, looking at the image shows that if the brightest pixels (red) were looked at alone, it is clear that most of the strongly polarized cells are in the bud and trunk.

• Supplemental Figure 4 -

  Fig. S4. Time-course of V-ATPase expression. In situ hybridization with a probe to V-ATPase subunit c demonstrates that zygotic gene expression is strong in the bud at 24 hpa, and is detectable in the new growth until 72 hpa. Red arrows indicate expression; white arrow indicates lack of expression.

• Supplemental Figure 5 -

  Fig. S5. Western blot of KCNK1 antibody. The antibody used to identify the presence of the KCNK1 protein marker identifies a single band of the predicted size on tissue from Xenopus larvae. Negative controls resulted in no detectable bands. This result was obtained using a polyclonal antibody from rabbit, raised against a peptide in the predicted extracellular loop between TM1 and P1 (C-EDLLRQELRKLKRRFLEEHECLSE) by S. A. Goldstein and D. Bockenhauer.

  References

  Holoubek, A., Vecer, J., Opekarova, M. and Sigler, K. (2003). Ratiometric fluorescence measurements of membrane potential generated by yeast plasma membrane H(+)-ATPase reconstituted into vesicles. Biochim. Biophys. Acta 1609, 71-79.

  Horiuchi, M. and Iwata, S. (1983). Effect of nigericin on distribution of sodium, potassium and calcium ion in rabbit lens. Exp. Eye Res. 37, 439-445.