- Title
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Compartmentalization and synergy of osteoblasts drive bone formation in the regenerating fin
- Authors
- Cudak, N., López-Delgado, A.C., Rost, F., Kurth, T., Lesche, M., Reinhardt, S., Dahl, A., Rulands, S., Knopf, F.
- Source
- Full text @ iScience
Experimental approach, the response to repeated amputation, cell clustering, and trajectory analysis (A) 3 dpa fin cryosection of quadruple transgenic reporter zebrafish. BF, bright field, mCh, mCherry, (B) Study design. Reg, regeneration experiment. Blastema, (non-osteoblast) blastema cells, Osteo, Osteoblasts, Basal, BLWE. Spatial reconstruction (pseudospace analysis): distal_r, pseudospace coordinate distal dimension, lateral_r, pseudospace coordinate lateral dimension. AP, alkaline phosphatase coupled antibody, DIG, digoxygenin, NBT, nitro blue tetrazolium, BCIP, 5-bromo-4-chloro-3-indolyl-phosphate. (C) Main clusters identified in the analysis. Basal, BLWE, Blastema, (non-osteoblast) blastema cells, Osteo, osteoblasts. (D) Identified subclusters. (E) PAGA analysis displaying connectivity (reflected by line thickness) between different clusters. (F) Marker gene expression in main clusters. |
Phenotypic diversity and location of (non-osteoblast) blastema cells and osteoblasts (A) (B) (C) (D) (E) (F) (G) (H) Non-exclusive (I) (J) Topology scheme of the 3 dpa regenerate, with vague distinction between Blastema0, 1, and 3. (A)–(I) UMAP, whole-mount RNA ISH (WMISH) and cryosection views. Scale bars whole mounts 100 μm, cryosections 50 μm, insets 10 μm. |
Lineage tracing of (A) Experimental design. 4-OHT, 4-hydroxytamoxifen. Flame icon, heat induction. Camera icon, imaging. (B) Lineage tracing of (C) Fraction of (D) Density of (E) Lineage tracing of (F) UMAP view of (G) Clone size of (H) Variable clone size of (I) Lineage tracing of (J) Magnified view of |
Mixing of distal blastema cells and osteoblasts (A) Overview of TEM of regenerate with positions of cells of interest indicated (1–6). Red dashed line, subepithelial basal lamina. (B) Cells underlying the BLWE with gradual increase of endoplasmic reticulum (er) from distal (1,2) via intermediate (3,4) to proximal positions. More dilated ER in proximal regions with more Golgi complexes (g) and mitochondria (m) suggesting massive protein synthesis. See also (C) UMAP of (D) FACS gates to identify (E) Single and double ISH of (F) Detection of mCherry/GFP protein double+ cells (arrowheads in boxed areas) in 3 dpa fin regenerates of (G) Quantification of mCherry, GFP double+ cells in (H) Double ISH of (I) Detection of mCherry/GFP protein double+ cells in 3 dpa |
Effects of SU5402 on different regenerate domains (A) Treatment regimen. (B) 5 dpa fin regenerates of (C) Quantification of experiment shown in (B). Welch’s t tests. (D) 7 dpa fin regenerates of (E) Quantification of experiment shown in (D). Welch’s t tests. (F) Fluorescence signal intensity of transgenic reporters along fin regenerate. Arrowheads, increased signal intensity. (B), (D) Scale bars 200 μm. (C), (E) Mean, (F) Mean ± SEM. |
Effects of (A) 5 dpa fin regenerates of 3–5 dpa NFP-treated fin regenerates of (B) Quantification of experiment shown in (A). One-way Anova (Tukey), excluding (C) IHC sections of NFP and BrdU treated 5 dpa (D) Quantification of experiment shown in (C). Kruskal-Wallis. Epid, epidermis, Mes, mesenchyme, dist., 0–250 μm from regenerate tip, prox., 250 μm from regenerate tip to amp plane. (E) Recovery treatment regimen. (F) 5 and 7 dpa fin regenerates of 3–5 dpa NFP treated fin regenerates of (G) Increase in regenerate length in (H) DMSO and SU5402 recovery treated 7 dpa fin regenerates of |
Structural integrity and establishment of segment joints are impaired after (A) 7 dpa fin regenerates of 3–5 dpa NFP treated (B) Regenerate length and domain sizes (proximal vs. distal to bifurcation, (C) Regenerate length and domain sizes at 7 dpa, after 2days recovery. Welch’s t tests. p(§, tip region) = 0.0624, p($, dist. (D) 5 dpa fin regenerates of 3–5 dpa DMSO and NFP treated (E) 7 dpa fin regenerates of 3–5 dpa DMSO and NFP treated (F) 5 dpa anti-chondroitin sulfate (CS) and anti-Laminin stained |