(A) Genetic recombination systems. The initial column lists various reporter alleles, while the subsequent second and third columns present the corresponding reporter constructs and the recombinases utilized. The final column provides the outcomes associated with the respective reporter alleles. Traditional reporter mechanisms comprise both single-color and multicolor systems (not shown in the figure), created through a singular recombination event. Advanced dual-reporter configurations encompass intersectional reporters, exclusive reporters, and nested reporters. (B) The craniofacial hard tissue, including maxilla and mandible of transgenic mice with multiple DNA recombinases-based genetic lineage tracing system, were obtained for advanced imaging technologies. (C-a) The tissue deep clearing procedure based on SUMIC, which needed to go through 4% PFA fixation, 0.5M EDTA decalcification, different concentration of methanol dehydration, 5% H2O2 bleaching, ECI tissue clearing, and finally used the light-sheet system to directly image the whole tissue. (C-b) The traditional frozen section technology needs fixation, decalcification, dehydration and other steps, which takes about two weeks.

The whole-mount and high-speed imaging of (A) mouse molar with a tiling light-sheet microscope. (B) Schematic illustration of lineaging tracing in Pdgfr-αCreER; Nfatc1DreER; LGRT mice. The mice were administrated with tamoxifen at D1 and D3, and sacrificed at D5. (C) The contoured M1 of maxilla, including pulp and PDL with virtual dentin shell (white) in buccal view (scale bar = 300 μm). (D) Image stack was displayed in buccal view, coronal view, and radicular view of pulp and PDL, respectively (scale bar = 300 μm). (E) An optical slice was acquired on the X-Z (scale bar = 300 μm) and X-Y direction (scale bar = 400 μm) to display the pulp and PDL.

Using traditional serial section for imaging of maxilla of Pdgfr-αCreER; Nfatc1DreER; LGRT mice (pulse). (A) Operation process of frozen section. Using this procedure, a total of 121 slices were collected in this sample. (B) Representative images acquired by confocal microscopy (scale bar = 200 μm). Box 1: coronal pulp, Box 2: root pulp, Box 3: PDL, scale bar = 100 μm. (C) Maxilla M1 after 3D reconstruction by imaris, including pulp and PDL with virtual dentin shell (white) in buccal view (scale bar = 300 μm). (D) Image stack was displayed in buccal view, coronal view, and radicular view of pulp and PDL, respectively (scale bar = 300 μm).

Using traditional serial section for imaging of mandible of PdgfrαCreER; Nfatc1DreER; LGRT mice (pulse). (A) Schematic illustration of lineaging tracing in Pdgfr-αCreER; Nfatc1DreER; LGRT mice. (B) Operation process of frozen section. Using this procedure, a total of 88 slices were collected in this sample. (C) Mandible M1 after 3D reconstruction by Imaris, including pulp and PDL with virtual dentin shell (white) in buccal view, coronal view and radicular view (scale bar = 200 μm). (E) 3D reconstruction of mandible M1 by DICOM-3D; in PDL, ZsGreen+ cells in green, tdTomato+ cells in rose red; in pulp, ZsGreen+ cells in purple, tdTomato+ cells in blue. The image stack was also displayed in buccal view, coronal view, and radicular view of pulp and PDL, respectively. (scale bar = 200 μm).

The observation and analysis of lineage tracing of PDGFR-α+ & NFATc1+ cells in M1 by whole-mount and high-speed imaging. (A) The flowchart of tracing. The mice were administrated with tamoxifen at D1 and D3, and sacrificed at D11. (B) The 3D images of contoured M1 of maxilla, including pulp and PDL with virtual dentin shell (white) in buccal view (scale bar = 300 μm). (D) Image stack was displayed in buccal view, coronal view, and radicular view of pulp and PDL, respectively. (E) An optical slice was acquired on the X-Z (scale bar = 400 μm) and X-Y direction to display the pulp and PDL (scale bar = 300 μm).

Using traditional serial section for imaging of maxilla of Pdgfr-αCreER; Nfatc1DreER; LGRT mice (tracing 11 days). (A) Using this procedure, a total of 117 slices were collected in this sample. (B) Representative images acquired by confocal microscopy (scale bar = 200 μm). Box 1: coronal pulp; Box 2: root pulp; Box 3: PDL (scale bar = 50 μm). (C) Maxilla M1 after 3D reconstruction by imaris, including pulp and PDL with virtual dentin shell (white) in buccal view (scale bar = 300 μm). (D) Image stack was displayed in buccal view, coronal view, and radicular view of pulp and PDL, respectively (scale bar = 300 μm).

H11-LGRT tracing distinct cell populations simultaneously in the pulp and PDL. (A) Schematic illustration of pulse and lineaging tracing in Pdgfr-αCreER; Nfatc1DreER; LGRT. (B) 3D reconstruction of mandible M1 using Imaris, including pulp and PDL with virtual dentin shell (white) (scale bar = 200 μm). The image stack was displayed in buccal view, coronal view, and radicular view of pulp and PDL, respectively. (C) 3D reconstruction of mandible M1 using DICOM-3D, in which the distribution of NFATc1+ cells in pulp and PDL tissues can be more precisely perceive (scale bar = 200 μm).

IR1 tracing distinct cell populations simultaneously in the pulp and PDL. (A) Schematic illustration of lineaging tracing in Pdgfr-αCreER; IR1, Nfatc1DreER; IR1 and Pdgfr-αCreER; Nfatc1DreER; IR1 mice. (B) Schematic diagram of the IR1 working principle. (C) 3D reconstruction of maxilla M1 using Imaris, including pulp and PDL with virtual dentin shell (white) (scale bar = 200 μm). The image stack was displayed in buccal view, coronal view, and radicular view of PDL (D) and pulp (E), respectively.

Ablation of PDGFR-α+ Cells Disrupts the morphology of dental pulp and periodontal ligament tissues. A) Schematics of tamoxifen induction. B) Schematic diagram of the DTA working principle. C, E) Representative H&E images of pulp C) and PDL E) of mandible M1 in Pdgfr-aCreER; DTA and control mice. D, F) Masson trichrome staining of pulp D) and PDL F) of mandible M1 in Pdgfr-aCreER; DTA and control mice. Arrows C, D) indicate the odontoblast cell layer. Dotted lines E, F) outline ROI of the PDL. D: dentin; AB: alveolar bone. Scale bar: 50 μm.