FIGURE 4

FIGURE 4

wnt2bb functions upstream of the c-jun/creb pathway. (A–D) Immunostaining analyses showing jnk1 expression at wounded myocardial cell edges in the control hearts. (A)Jnk1 expression decreased in heat-shocked Tg(hsp70l:dn-wnt2bb) fish hearts at 7 dpa (B) and increased in heat-shocked Tg(hsp70l:wnt2bb) fish hearts. (C)jnk1 expression rescue of Tg(hsp70l:dn-wnt2bb;hsp70l:creb1) double transgenic fish and (D) control fish heat-shocked at 37°C for 1 hr daily from 4 to 6 dpa. (E–H) Confocal image analyses showing CREB1 expression at the wounded myocardial cell edges in control fish hearts (E), elevated CREB expression in injured Tg(hsp70l:wnt2bb) fish hearts (G), decreased CREB expression in Tg(hsp70l:dn-wnt2bb) fish hearts (F), and in rescued Tg(hsp70l:dn-wnt2bb;hsp70l:creb1) double transgenic fish (H). (I–L) Confocal microscopy image analyses showing c-jun expression at wounded myocardial cell edges in control fish hearts (I), elevated c-jun expression in injured Tg(hsp70l:wnt2bb) fish hearts (K), decreased c-jun expression in Tg(hsp70l:dn-wnt2bb) fish hearts (J), and in rescued Tg(hsp70l:dn-wnt2bb;hsp70l:creb1) double transgenic fish (L). (N,O) Confocal microscopy image analyses of PCNA⁺Mef2C⁺ cells (arrowheads) in heat-shocked Tg(hsp70l:dn-wnt2bb;hsp70l:creb1) and wild-type (ctrl) fish at 7 dpa. Green, PCNA; Red, Mef2C. (M) Bar charts showing the quantification of PCNA-labeled CM proliferation index values for heat-shocked, injured wild-type (ctrl), Tg(hsp70l:dn-wnt2bb), Tg(hsp70l:wnt2bb), and Tg(hsp70l:dn-wnt2bb;hsp70l:creb1) fish hearts. Proliferation data were collected for 4–6 sections per heart and averaged to generate each data point. Green, PCNA; Red, Mef2C. (P) Bar charts showing the quantification of relative nkx2.5 expression in heat-shocked, injured wild-type (ctrl), Tg(hsp70l:dn-wnt2bb), Tg(hsp70l:wnt2bb), and Tg(hsp70l:dn-wnt2bb;hsp70l:creb) fish hearts. The data from five hearts in each group are presented. Error bars: ± 1 SD. Significance was determined using a Student’s t-test: **P < 0.01. Tg(hsp70l:wnt2bb), Tg(hsp70l:dn-wnt2bb), Tg(hsp70l:dn-wnt2bb;hsp70l:creb1) and control fish were heat-shocked at 37°C for 1 h daily from 4 to 6 dpa. Brackets indicate the amputation planes. Dotted line brackets indicate the amputation area. Scale bar, 100 μm. Green: jnk1(A–D), c-jun(E–H), and creb1(I–L); Red, MF20. Fluorescence intensities were measured at the injury border zone using ImageJ. Boxes correspond to the magnified region.