Fig. 4

Hh signaling driven Plp1-lineage cells transition is required for the healing of bone injury. a Representative images of tdTomato⁺ cells, CGRP immunostaining in conjunction with in situ hybridization of Shh mRNA in TES from Plp1-creER^T2; tdTomato mice. The Pearson’s correlation coefficient is presented as the quantification of Shh mRNA signals that were colocalized with tdTomato or CGRP (n = 3). Scale bar: 100 μm. b Representative images of tdTomato⁺ cells in conjunction with in situ hybridization of Shh mRNA and quantification of tdTomato⁺ Shh⁺ cells in TES from Plp1-creER^T2; tdTomato mice (n = 6). Scale bar: 100 μm. c Western blot images of Shh expression of FACS-sorted tdTomato⁺ cells from tooth extraction sockets at day 0, 3, and 7 post tooth extraction. d Schematic illustration of FACS assay and purified tdTomato⁺ cells from mandible. Scale bar: 20 μm. e RT-qPCR data of Eng, Gli1, and Mag mRNA expression of SCs treated with recombinant Shh (n = 3). f Shh converts SCs into osteolineage cells as demonstrated by alizarin red staining (ARS) of FACS-sorted tdTomato⁺ cells cultured in regular growth medium (GM) or osteogenic medium (OM). g The quantification of ARS in (f) (n = 3). h Western blot images of Runx2 and COL1 expression of FACS-sorted tdTomato⁺ cells cultured in GM or OM and treated with recombinant Shh. i RT-qPCR data of Runx2 and Alp mRNA expression of SCs cultured in GM or OM and treated with recombinant Shh (n = 3). j Experimental strategy for Hh signaling inhibition in Plp1-creER^T2; tdTomato mice. k H&E staining of tooth sockets from DMSO and GDC mice at day 7 and day 14 post tooth extraction and the quantification analysis (n = 5). Scale bar: 100 μm. l, m Representative images of tdTomato⁺ cells and Gli1/COL1 immunostaining in TES at day 7 post tooth extraction and the relative quantification (n = 5). Scale bar: 100 μm. n Representative images of CD31 and EMCN immunostaining and quantification of CD31⁺EMCN⁺ type H vessels per socket (n = 5). Scale bar: 100 μm. Data were presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001