Osteoblast differentiation was regulated by autophagy in zebrafish larvae. (A–E) Embryos or transgenic Tg (-2.2col10a1a:GFP) embryos were injected with 5MO and MisMO at the 1–4 cell stage, and larvae were collected at 6 dpf for transcriptom sequencing, qPCR, western blot and Alizarin Red staining, transgenic larvae were collected at 4 dpf for image capture. (A) Heat map showed osteoblast-related genes mRNA level, data were from transcriptome sequencing. (B) Transcriptional level of osteoblast genes were detected by qPCR. (C) Col10a1a expression pattern in transgenic zebrafish larvae at 4 dpf. (D) Osteoblast proteins SP7 and BMP2 were detected by western blot. (E) Alizarin Red staining showed the mineralization of osteoblast at larvae den and rib. 5MO-II:abnormal phenotype. (F–H) Larvae were exposed to 50 µM RAPA at 3 dpf, larvae were collected at 6 dpf for western blot and staining assay separately. (F,G) Western blot assay showed autophagy proteins and osteoblast proteins were changed by RAPA. (H) Alizarin Red staining showed osteoblast mineralization. Black circle, dentary (den); blue rectangle, rib; blue circle, opercle (op); green circle, ceratobranchial 5 (cb5). The means and s.d. were derived from triple duplicates, *P<0.05, **P<0.01, ***P<0.001 versus WT groups (one-way ANOVA). The data of western blots were from three independent experiments. Scale bars: 50 µm.

Reduction of Hh signaling inhibited osteoblast differentiation. Larvae were exposed to 10 µM and 50 µM cyA at 3 dpf for 3 days, and were collected to measure osteoblast-related gene mRNA, protein level and mineralization of osteoblasts with DMSO treated as a control. (A) qPCR detected osteoblast genes in transcription level. (B) BMP2 and SP7 proteins were detected by western blot. (C) Alizarin Red staining showed mineralization of osteoblasts. Note that the DMSO panel on the right is reproduced from middle WT panel in Fig. 4E. (D) Transgene larvae Tg (-2.2col10a1a:GFP) were treated with 10 µM and 50 µM cyA at 3 dpf for 1 day, images were obtained by fluorescence microscope at 4 dpf. Embryos were injected with SHHMO, Gli2MO and Gli2Mis at the 1–4 cell stage. Lateral view. (E–H) At 6 dpf, larvae were collected for qPCR, western blot and Alizarin Red staining. (E) Osteoblast genes bmp2, sp7, col10a1, runx2 and alp mRNA were detected by qPCR. (F) Osteoblast-related proteins BMP2 and SP7 were tested by western blot. (G) Alizarin Red staining showed mineralization of osteoblasts including nt (notochord tip, blue arrow), rib (blue rectangle), hm (hyomandibular, yellow circle) and regions delayed in SHHMO- and Gli2MO-injected groups. Green circle, bop (basioccipital articulatory process). The difference in osteoblast name and abbreviation were derived from previous sources (Laue et al., 2008; Aceto et al., 2015). (H) In Tg (-2.2col10a1a:GFP) transgene zebrafish, col10a1a signal was shown. White circle, cb (ceratobranchial 5); red arrow, br; white arrow, op. *P<0.05, **P<0.01, ***P<0.001 versus untreated groups (one-way ANOVA). The data were from three independent assays. Scale bars: 50 µm.

Increasing Hh signaling by Ptch1 knockdown might be beneficial for osteoblast development. Embryos were injected with Ptch1MO and Ptch1Mis at the 1–4 cell stage, at 6 dpf, larvae were collected for qPCR, western blot and Alizarin Red staining respectively. (A) QPCR showed osteoblast genes mRNA level. (B) Western blot results showed BMP2 and SP7 protein level. (C) Alizarin Red staining showed bone mineralization; blue rectangle, rib. (D) Col10a1a protein expression in cb5 (ceratobranchial 5, white circle) region was observed in Ptch1MO-injected group. *P<0.05, **P<0.01, ***P<0.001 versus the untreated groups (one-way ANOVA). The data were from three independent assays.

Autophagy inducer RAPA and block of Hh signaling co-inhibited osteoblast mineralization. (A) Larvae at 3 dpf were exposed to 10 µM cyA, 10 µM RAPA or cyA plus RAPA, with larvae collected for Alizarin Red staining at 6 dpf. Blue circle, op; white circle, cb. (B–D) Embryos were injected with 50 µM SHHMO, Gli2MO, Gli2Mis, PTCH1MO and PTCH1Mis at the 1–4 cell stage, DMSO control group and injected group larvae were treated with 10 µM RAPA, with larvae collected at 6 dpf for Alizarin Red staining. Yellow circle, bop; green circle, hm; blue rectangle, rib. Scale bars: 50 µm.