Fig 2: Inhibition of Pak2 activity impairs CM dedifferentiation and proliferation. (A) Schematic of heat shock experiments and FRAX597 treatment for PCNA/Mef2C analyses. Heat shock: 37°C for 2 h daily from 4 to 6 dpa for PCNA/Mef2C assay or over the time period from 4 to 30 dpa for AFOG analysis; treatment: 1 µM FRAX597 or 0.1% DMSO from 4 to 6 dpa. (B‒D) Confocal image analyses displaying PCNA+Mef2C+ cells (arrowheads, B and C) and PCNA-labeled CM proliferation index (D) in Ctrl and Tg(hsp:dnpak2a) hearts at 7 dpa. Insets indicate high-magnification images in dash-lined areas. Data are mean ± SEM from five hearts for each group. Student’s t-test, **P < 0.01. (E‒G) Representative images (E and F) and quantification of regeneration scores (G) of ventricle sections from heat-shocked Ctrl and Tg(hsp70:dnpak2a) animals at 30 dpa and stained with AFOG. Orange: muscle; blue: collagen; red: fibrin. Fisher’s exact test. **P < 0.01. (H‒K) Confocal image analyses displaying PCNA+Mef2C+ cells (arrowheads) and quantification of CM proliferation indices (K) in DMSO-treated (H, Ctrl), Pak2 inhibitor (FRAX597)-treated (I), and FRAX597-treated Tg(hsp:dkk1b) (J) hearts at 7 dpa. Insets indicate high-magnification images of dash-lined area. Data are represented as mean ± SEM from five hearts for each group. Statistical significance was calculated using one-way ANOVA followed by Tukey’s test. n.s., none significance; *P < 0.05, ***P < 0.001. (L‒O) Immunostaining analyses displaying reduction of pS675-β-catenin (L and M) and emCMHC (N and O) at apical myocardial cells of the wounded edge in FRAX597-treated hearts compared to DMSO-treated hearts at 7 dpa. (L′ and M′) High-magnification images of dash-lined windows in L and M, respectively, exhibiting disassembled sarcomeres in DMSO-treated hearts (L′) and relatively normal striated sarcomeres in FRAX597-treated hearts (M′). (P and Q) ISH analysis displaying a reduction of gata4 at the wounded myocardial cell edge in FRAX597-treated hearts compared to DMSO-treated hearts at 7 dpa. Brackets indicate amputation planes. Scale bar, 100 µm (except 10 µm in L′ and M′). (R) A working model for Wnt/Pak2/pS675-β-catenin signaling events regulating heart regeneration. Cardiac injury induces multiple Wnt antagonists that restrain Wnt signaling throughout the wounded heart, enabling CM dedifferentiation and proliferation. In the context of Wnt signaling inhibition, Pak2 is upregulated in cardiac wounds, where it phosphorylates cytoplasmic β-catenin at the Ser 675 residue and increases its stability in disassembled sarcomeres, enhancing CM renewal and heart regeneration.

Fig 3: Müller glia express Sox2 during the regeneration response in gosh mutants. Wild-type and gosh mutant retinas at 5 wpf labeled with anti-Sox2 and anti-PCNA antibodies, and DAPI. Tg(gnat2:GFP) transgene visualizes cones. Wild-type retinas display Sox2 expression in the CMZ and amacrine cells (A,C, dotted boxes show the area magnifies in B,D). Müller glia exhibit faint expression of Sox2. At 5 wpf, gosh mutant retinas show that Sox2 expression is colocalized with PCNA in the INL (D, arrowheads) probably in Müller glia, whereas subsequently differentiating NPCs express only PCNA (D, arrows).

Fig 4: Regulation of injury-induced cardiomyocyte proliferation via c-jun/creb1 signaling. (A,J) Schematic of the experimental plan to analyze PCNA/Mef2c proliferation and AFOG staining after apex amputation. (B–E) AFOG staining assays and quantification showing the apical wound after ventricular resection in wild-type (ctrl), Tg(hsp70l:dn-c-jun), and Tg(hsp70l:dn-creb1) fish at 30 dpa. Red, fibrin; Blue, collagen. HS: heat shock at 37°C for 1 h. (F–H) Confocal microscopy image analyses of PCNA+Mef2C+ cells (arrowheads) in heat-shocked Tg(hsp70l:dn-c-jun), Tg(hsp70l:dn-creb1), and wild-type (ctrl) fish at 7 dpa. Green, PCNA; Red, Mef2C. (I) Bar charts showing the quantification of PCNA-labeled CM proliferation index values for heat-shocked, injured wild-type (ctrl), Tg(hsp70l:dn-c-jun), and Tg(hsp70l:dn-creb1) fish hearts. (K–N) AFOG staining analyses and quantification revealing defective wound healing after ventricular resection in Tg(hsp70l:creb1) and Tg(hsp70l:c-jun) fish compared to that observed in wild-type (ctrl) fish. Red arrow, fibrin; Blue, collagen; HS, heat shock at 37°C. (O–Q) Confocal microscopy image analyses of PCNA+Mef2C+ cells (arrowheads) in Tg(hsp70l:creb), Tg(hsp70l:c-jun), and wild-type (ctrl) fish at 7 dpa. (R) Bar charts showing the quantification of PCNA-labeled CM proliferation index values for control, Tg(hsp70l:creb1), Tg(hsp70l:c-jun) fish at 7 dpa. The data are presented as the means ± SEM from 5 to 7 hearts for each group. For CM proliferation index experiments, proliferation data were collected for 4–6 sections per heart and averaged to generate each data point. Error bars: ± 1 SD. Significance was determined using Student’s t-test: **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar, 100 μm. Ctrl, control. Boxes correspond to the magnified region.

Fig 5: ADMSCs characterization and NFABNS generation. (A) Shows representative images of the immunostaining pattern of stemness markers of the rat ADMSCs. (B) Shows how from a square uncompressed FAH it is possible to fabricate a cylindrical multilayered NFABNS of desire dimensions. (C) Exhibit the histological pattern of NFABNS, ADMSCs distribution with HE and the presence and percentages of proliferating cells (cell proliferation index) detected by immunohistochemistry for PCNA and Ki-67 markers, respectively. (D) Shows the macroscopic and scanning electron microscopy aspect of the NFABNS used to repair the nerve defect in this study. In (E) scanning electron microscopy images show examples of multifasciculated NFABNS that can be generated with this methodology if needed.