Control of tissue growth by Yap relies on cell density and F-actin in zebrafish fin regeneration

Abstract

Caudal fin regeneration is characterized by a proliferation boost in the mesenchymal blastema that is controlled precisely in time and space. This allows a gradual and robust restoration of original fin size. However, how this is established and regulated is not well understood. Here, we report that Yap, the Hippo pathway effector, is a chief player in this process: functionally manipulating Yap during regeneration dramatically affects cell proliferation and expression of key signaling pathways, impacting regenerative growth. The intracellular location of Yap is tightly associated with different cell densities along the blastema proximal-distal axis, which correlate with alterations in cell morphology, cytoskeleton and cell-cell contacts in a gradient-like manner. Importantly, Yap inactivation occurs in high cell density areas, conditional to F-actin distribution and polymerization. We propose that Yap is essential for fin regeneration and that its function is dependent on mechanical tension, conferred by a balancing act of cell density and cytoskeleton activity.

Keywords: Cell density; F-actin; Fin regeneration; Hippo/Yap; Zebrafish.

Fig. 1.

Yap is present and highly dynamic during fin regeneration. (A-F) Representative immunostaining with anti-Yap in caudal fin longitudinal sections at several regenerative stages. (A) Uncut control; (B) 3 hpa; (C) 6 hpa; (D) 24 hpa; (E) 48 hpa; (F) 72 hpa. xz projections of mesenchymal regions highlight Yap intracellular localization. Distal (yellow) and proximal (red) lines at 48 hpa (E), and distal-most (yellow), distal (orange) and proximal (red) lines at 72 hpa (F), correspond to the mesenchymal areas in the medial blastema where xz projections were made (shown below). Dashed lines indicate amputation plane. n=10-15 sections; 5 fish/condition. Scale bars: 50 µm. (G) Quantification of Yap intracellular localization by expressing a ratio between average intensities of nuclear Yap:cytoplasmic Yap of xz projections from blastemas at different time points. Higher ratios correspond to higher intensities of nuclear Yap. P corresponds to xz of proximal (red); D to distal (orange); DM to distal-most regions (yellow). *P<0.05, **P<0.01, ***P<0.001; two-tailed, non-parametric Mann–Whitney test. n=15 sections, 5 fish/condition. Mean±s.d. are shown.

Fig. 2.

Yap influences proliferation in the blastema. (A) Experimental outline of heat-shock protocol used to access Yap functionality during regeneration. After amputations, fish were allowed to regenerate for 24 h, the time at which the first heat-shock was performed. A second heat-shock at 48 hpa was applied and phenotypes were assessed at 60 hpa or 72 hpa. The same protocol was applied to sibling controls. (B) qPCR determination of yap1 expression levels in blastemas of Yap transgenics versus respective siblings upon single heat-shock at 72 hpa. RNA extraction was performed at 2 hpHS. *P<0.01; two-tailed, non-parametric paired Wilcoxon test, logarithmic scale, base 10. (C-F) Representative brightfield images of Yap transgenics and siblings at 72 hpa after the protocol shown in A was performed. (C) CA-yap control; (D) CA-yap positive; (E) DN-yap control; (F) DN-yap positive. n=5 fish/condition. Scale bars: 200 µm. (G-J) Representative immunofluorescence with anti-pH3 in 60 hpa longitudinal sections of double transgenics Ef1α:mag-zGeminin; CA-yap/DN-yap and siblings after the protocol shown in A was performed. (G) CA-yap control; (H) CA-yap positive; (I) DN-yap control; (J) DN-yap positive. (H′,H″,J′,J″) Corresponding transgenic CA-Yap and DN-Yap RFP expression. Siblings do not show RFP expression. Scale bars: 50 µm. (K) Quantification of Yap-RFP intracellular localization in CA-yap and DN-yap transgenics by expressing a ratio between average intensities of nuclear Yap:cytoplasmic Yap of xz projections of respective mesenchymal cells. ***P<0.001; two-tailed, non-parametric Mann–Whitney test. n=16-23 sections, 3 fish/condition. (L,M) Quantification of average proliferation labeled with Geminin and pH3 occurring per 100 µm² in Ef1α:mag-zGeminin; CA-yap/DN-yap and siblings, at 60 hpa. *P<0.05, **P<0.01; two-tailed, non-parametric Mann–Whitney test. n=15 sections, 3 fish/condition. (N) Quantification of average EdU-positive cells occurring per 100 µm² in CA-yap and siblings, at 72 hpa in different times post heat-shock. **P<0.01, ***P<0.001; two-tailed, non-parametric Mann–Whitney test. n=9 sections, 3 fish/condition. (O,P) Representative immunofluorescence with EdU in 72 hpa longitudinal sections of CA-yap and siblings after 12 hpHS. (O) CA-yap control; (P) CA-yap positive. Scale bars: 50 µm. Dashed lines indicate amputation plane. Mean±s.d. are shown.

Fig. 3.

Transcriptional gene regulation induced by Yap. (A,B) qPCR determination of ctgfa and areg expression levels (relative to sibling controls) in blastemas of CA-yap positive (A) and DN-yap positive (B) transgenics upon single heat-shock at 72 hpa. *P<0.01; two-tailed, non-parametric paired Wilcoxon test, logarithmic scale, base 10. (C-F) Representative ctgfa expression in double transgenics ctgfa:eGFP; CA-yap/DN-yap and siblings upon heat-shock at 72 hpa, at 7 hpHS. (C) CA-yap control; (D) CA-yap positive; (E) DN-yap control; (F) DN-yap positive. n=5 fish/condition. Scale bars: 500 µm. Dashed lines indicate amputation plane. (G,H) Quantification of average eGFP intensity (in arbitrary units, a.u.) of individual rays including blastemas along the PD axis of double transgenics ctgfa:eGFP; CA-yap/DN-yap and siblings. (G) CA-yap positive and siblings; (H) DN-yap+ and siblings. n=80-90 rays, 5 fish/condition; shadows indicate the s.e.m. for each curve. (I,J) qPCR determination of fgf20a, msxb, wnt10a, wnt3a, lef1, dkk1a, dkk1b, shh, bmp2a and bmp2b expression levels (relative to sibling controls) in CA-yap positive (I) and DN-yap positive (J) transgenics upon single heat-shock at 72 hpa. *P<0.05, **P<0.01; two-tailed, non-parametric paired Wilcoxon test, logarithmic scale, base 10. All RNA extractions were performed at 2 hpHS. Mean±s.d. are shown.

Fig. 4.

Mesenchymal cell density and morphology are altered according to the regenerative stage. (A-C) Quantification of average DAPI intensity (in arbitrary units, a.u.) in mesenchymal cells along the PD axis of blastemas at 24 hpa (A), 48 hpa (B) and 72 hpa (C). n=7 sections, 3 fish/condition. (D-F) Representative DAPI-stained longitudinal sections of blastemas at 24 hpa (D), 48 hpa (E) and 72 hpa (F). (G-I) Quantification of average space (a.u.) between mesenchymal cells along the PD axis at 24 hpa (G), 48 hpa (H) and 72 hpa (I). n=9 sections; 3 fish/condition. (J-L) Representative anti-GFP-stained longitudinal sections of ctgfa:eGFP transgenics at 24 hpa (J), 48 hpa (K) and 72 hpa (L). (M) Quantification of average cell aspect ratio of mesenchymal cells at 24, 48 and 72 hpa, in which y is the minor axis of the cell and x the major cell axis. A perfect circular shape corresponds to a ratio between y and x of 1. P corresponds to proximal; D to distal regions. ***P<0.0001; two-tailed, non-parametric Mann–Whitney test. n=45 cells/condition; 5 cells randomly selected/image; 9 sections; 3 fish/condition. (N-R) High magnification images of the boxed areas in J-L highlight cell morphology of blastema cells at 24 hpa (N); 48 hpa proximally (O) and distally (P); and 72 hpa proximally (Q) and distally (R). Dashed lines indicate amputation plane. Scale bars: 50 µm. Medial blastema areas were considered for all measurements: shadows indicate the s.e.m. for each curve.

Fig. 5.

α-Catenin accumulates in distal blastema regions where Yap is mainly cytoplasmic. (A-F) Representative immunofluorescence with anti-Yap and anti-GFP antibodies in 72 hpa longitudinal sections of α-Catenin transgenics. Owing to stronger expression intensity, transgenics were used instead of the α-Catenin antibody. (A) α-Catenin; (B) corresponding Yap expression. (C-F) High magnification images of the boxed areas in A,B showing α-Catenin (C, proximal; D, distal) and Yap (E, proximal; F, distal) expression. (G,H) Corresponding proximal and distal intensity profiles (in arbitrary units, a.u.) of the medial mesenchymal cells shown in C-F showing average intensity of α-Catenin (G) and Yap (H). (I) Quantification of changes in intracellular localization of Yap and α-Catenin across the PD axis by expressing a ratio between average intensities of proximal:distal Yap or α-Catenin of xz projections of respective mesenchymal cells. Mean±s.d. are shown. Dashed lines indicate amputation plane. n=5 sections, 3 fish. Scale bars: 50 µm.

Fig. 6.

Differential PD expression of F-actin associates with Yap intracellular location. (A-F) Representative immunofluorescence with anti-Yap antibody and phalloidin (F-actin) in 72 hpa longitudinal sections. (A) F-actin; (B) corresponding Yap expression. (C-F) High magnification images of the boxed areas in A,B showing F-actin (C,E) and Yap (D,F) localization in proximal (E,F) and distal (C,D) regions. Single color and merged xz projections of distal (C,D) and proximal (E,F) blastemas highlight intracellular localization. (G,H) Corresponding proximal and distal intensity profiles (in arbitrary units, a.u.) of xz projections represented showing average intensity of F-actin (G) and Yap (H). (I) Quantification changes in intracellular localization of Yap and F-actin across the PD axis by expressing a ratio between average intensities of proximal:distal Yap or F-actin of xz projections of respective mesenchymal cells. Mean±s.d. are shown. Dashed lines indicate amputation plane. n=7 sections, 5 fish. Scale bars: 50 µm.

Fig. 7.

F-actin controls Yap intracellular dynamics. (A-D′) Representative immunofluorescence with anti-Yap antibody in 72 hpa longitudinal sections of α-Catenin transgenics injected with jasplakinolide (JASP; B) and DMSO controls (A). (C-D′) High magnification images of the boxed areas in A,B showing Yap and DAPI in DMSO (C,C′) and JASP (D,D′) conditions. Corresponding xz projections of the distal blastemas shown in C,D highlight intracellular localization (DMSO XZ Distal, JASP XZ Distal). (E) Quantification of Yap intracellular localization by expressing a ratio between average intensities of nuclear Yap:cytoplasmic Yap of xz projections from distal blastemas in DMSO or JASP conditions, at 30 min post injection. Higher ratios correspond to higher intensities of nuclear Yap. ***P<0.001, two-tailed, non-parametric Mann–Whitney test. n=8 sections, 4 fish/condition. (F) qPCR determination of ctgfa levels in JASP versus DMSO animals, at 30 min and 2 h post injection, time points when RNA was extracted from blastemas. **P<0.01; two-tailed, non-parametric paired Wilcoxon test, logarithmic scale, base 10. (G-J) α-Catenin (anti-GFP) expression in animals injected with DMSO (G,H) or JASP (I,J). (H,J) High magnification images of the boxed areas in G,I showing α-Catenin in distal blastemas of animals injected with DMSO (H) or JASP (J). Corresponding xz projections of the distal blastemas shown in H,J highlight intracellular localization of α-Catenin and DAPI. Intraperitoneal injections were performed in 72 hpa animals, 30 min pre-fixation of blastemas. n=12 sections, 4 fish/condition. Mean±s.d. are shown. Dashed lines indicate amputation plane. Scale bars: 50 µm.