|
Fig. 12
Overexpression of cx43 rescues esco2 knockdown phenotypes. A: Figure depicting the timeline of fin amputation, morpholino injection and electroporation, heat shock, and data analysis. The 3 groups included in this experiment were Tg+HS- (transgenic hsp70:miR-133sp pd48-positive, heat shock), Tg-HS + (transgenic hsp70:miR-133sp pd48-negative, heat shock) and Tg+HS- (transgenic hsp70:miR-133sp pd48-positive, not heat shocked). Briefly, fins from all three groups were amputated at 50% level and allowed to regenerate for 3 days. At 3 dpa, morpholino was injected to one half of the regenerating fin tissue, immediately followed by electroporation on both sides. After an interval of 4 hr fish receiving heat shock were shifted to 37°C for 1 hr. Induction of the transgene expression upon heat shock was confirmed after 24 hr by screening for GFP-positive fins in the Tg+HS + group. The control groups (Tg-HS + and Tg+HS-) were negative for GFP expression. For ISH experiments, the fins were harvested at 1 dpe/4 dpa. For measurement of regenerate length and segment length, fins were calcein stained at 4 dpe/7 dpa. For each experiment, at least 6–8 fish were used per trial and at least 3 independent trials were performed. B: Graph reveals significant (*) rescue of esco2-dependent segment length defects in heat shock miR-133sp pd48 -positive esco2-knockdown fins (Tg+HS+) compared both with heat shock transgene-negative (Tg-HS+) and with miR-133sp pd48 -positive with no heat shock (Tg+HS-). Measurements from the injected side and the uninjected side of the same fin were used to calculate percent similarity and the average was calculated for each group. Note that a percent similarity of 100% indicates no difference between the knockdown side and the control sides of the fin. Student's t-test was used for determining statistical significance where P < 0.05. Standard error is represented by error bars. (C) Graph reveals significant (*) rescue of esco2-dependent regenerate length defects in heat shocked miR-133sp pd48 -positive esco2-knockdown fins (Tg+HS+) compared both with heat shocked transgene-negative (Tg-HS+) and with miR-133sp pd48 -positive in the absence of heat shock (Tg+HS-). Measurements from the injected side and the uninjected side of the same fin were used to calculate percent similarity and the average was calculated for each group. Note that a percent similarity of 100% indicates no difference between the knockdown side and the control sides of the fin. Student's t-test was used for determining statistical significance where P < 0.05. Standard error is represented by error bars.