Figure 6.
Figure 6. Epithelial movements in response to hypotonic injury are regulated by extracellular NTP hydrolysis. (a, i and ii) Spatial PIV analysis of the indicated number of Tg(krt4:AKT-PH-GFP) larvae subjected to UV laser cut injury in hypotonic medium, with or without translation morpholino-mediated knockdown of entpd3 mRNA (entpd3 MO1; ∼19 ng). (b) Global PIV analysis of the datasets in panel a additionally including morpholino-rescue data (blue curve). (c) Global PIV analysis of MO1 five-nucleotide mismatched control morpholino (entpd3 MO1 5 mm; ∼19 ng). (d) Global PIV analysis of Tg(krt4:AKT-PH-GFP) larvae subjected to mechanical tail fin tip amputation in isotonic solution ± potato apyrase (50 U/ml). After a 10-min preincubation in isotonic mounting agarose with or without apyrase, fish were overlaid with a bolus of hypotonic solution (to initiate the wound response) with or without apyrase. Note that velocimetry analysis does not include the isotonic preincubation period (i.e., t = 0’ in plot is 10’ after injury). (e, i and ii) Spatial PIV analysis of the indicated number of Tg(krt4:AKT-PH-GFP) and Tg(krt4:AKT-PH-mKate2) larvae subjected to UV laser cut injury in hypotonic medium, with or without ATPγS to globally inhibit extracellular NTP hydrolysis. After a 10-min preincubation in isotonic mounting agarose ± 5 mM ATPγS (in the interface drop), fish were overlaid with a bolus of hypotonic solution (to initiate the wound response) ± 5 mM ATPγS. As in d, the velocimetry analysis does not include the isotonic preincubation period. (e, iii) Differential plot derived by subtraction of the indicated velocimetry plots, and t test filtering of statistical significant differences between experimental groups. Statistically significant velocity differences are color-coded (turquoise to pink). Pink, positive values. Turquoise, negative values. (f) Global PIV analysis of the above datasets including data representing the effect of ATPγS after isotonic injury (blue curve). (g) Global PIV analysis of Tg(krt4:AKT-PH-GFP) larvae subjected to UV laser cut injury in isotonic/hypotonic solution ± POM (ENTPD inhibitor, 100 µM). After a 10-min preincubation period in isotonic mounting agarose ± POM, fish were overlaid with a bolus of hypotonic (to initiate the wound response) or isotonic solution ± POM. Note that velocimetry analysis does not include the isotonic preincubation period (i.e., t = 0’ in the plot is 10’ after injury). See also Video 8.

Epithelial movements in response to hypotonic injury are regulated by extracellular NTP hydrolysis. (a, i and ii) Spatial PIV analysis of the indicated number of Tg(krt4:AKT-PH-GFP) larvae subjected to UV laser cut injury in hypotonic medium, with or without translation morpholino-mediated knockdown of entpd3 mRNA (entpd3 MO1; ∼19 ng). (b) Global PIV analysis of the datasets in panel a additionally including morpholino-rescue data (blue curve). (c) Global PIV analysis of MO1 five-nucleotide mismatched control morpholino (entpd3 MO1 5 mm; ∼19 ng). (d) Global PIV analysis of Tg(krt4:AKT-PH-GFP) larvae subjected to mechanical tail fin tip amputation in isotonic solution ± potato apyrase (50 U/ml). After a 10-min preincubation in isotonic mounting agarose with or without apyrase, fish were overlaid with a bolus of hypotonic solution (to initiate the wound response) with or without apyrase. Note that velocimetry analysis does not include the isotonic preincubation period (i.e., t = 0’ in plot is 10’ after injury). (e, i and ii) Spatial PIV analysis of the indicated number of Tg(krt4:AKT-PH-GFP) and Tg(krt4:AKT-PH-mKate2) larvae subjected to UV laser cut injury in hypotonic medium, with or without ATPγS to globally inhibit extracellular NTP hydrolysis. After a 10-min preincubation in isotonic mounting agarose ± 5 mM ATPγS (in the interface drop), fish were overlaid with a bolus of hypotonic solution (to initiate the wound response) ± 5 mM ATPγS. As in d, the velocimetry analysis does not include the isotonic preincubation period. (e, iii) Differential plot derived by subtraction of the indicated velocimetry plots, and t test filtering of statistical significant differences between experimental groups. Statistically significant velocity differences are color-coded (turquoise to pink). Pink, positive values. Turquoise, negative values. (f) Global PIV analysis of the above datasets including data representing the effect of ATPγS after isotonic injury (blue curve). (g) Global PIV analysis of Tg(krt4:AKT-PH-GFP) larvae subjected to UV laser cut injury in isotonic/hypotonic solution ± POM (ENTPD inhibitor, 100 µM). After a 10-min preincubation period in isotonic mounting agarose ± POM, fish were overlaid with a bolus of hypotonic (to initiate the wound response) or isotonic solution ± POM. Note that velocimetry analysis does not include the isotonic preincubation period (i.e., t = 0’ in the plot is 10’ after injury). See also Video 8.

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