Tooth replacement is accelerated after plucking in cichlid species with divergent dentitions.

A. Schematic of the modified pulse-chase method using Alizarin and Calcein to quantify tooth replacement. B. Representative fluorescence images used to classify teeth by dye incorporation. Teeth positive for both Alizarin and Calcein were scored as pre-existing old teeth, whereas teeth positive for Calcein only were scored as newly formed teeth. Left, whole jaw from MZ Red, with teeth plucked on the right side and the left side serving as the control; the dotted outline marks the region used for plucking and quantification (Supplemental Figure 2B). Right, example of individually classified teeth (Alizarin, magenta; Calcein, green). C. Pulse-chase reveals an elevated rate of tooth replacement on the plucked side in CA, MZ Red and PT. Tooth gain is shown as the normalized replacement rate (new teeth/total teeth) ***p-value < 0.0001, paired Student’s t-test; CA (n = 8), MZ Red (n = 8) and PT (n = 9). D. Rate of tooth gain is consistent across species after plucking. NS, p-value > 0.05, one-way ANOVA with Tukey-Kramer post-hoc test; CA (n = 8), MZ Red (n = 8), PT (n = 9).

Single-nucleus RNA-seq of cichlid replacement teeth.

A. Schematic of the snRNA-seq experimental and processing workflow. B. Uniform manifold approximation and projection (UMAP) of 27,114 nuclei. Points that are close together represent nuclei with similar gene expression profiles. The color of each point represents the cell type. SI + SR, Stellate reticulum and Stratum intermedium; OEE, Outer Enamel Epithelium; VEE, Ventral Enamel Epithelium; ES-1, Epithelial Supporting Cells 1; ES-2, Epithelial Supporting Cells 2; Pre-AMB, Pre-Ameloblasts; CYC-AMB, Cycling Ameloblasts; M-AMB, Maturing Ameloblasts; PM-AMB, Post Maturation Ameloblasts; PDL, Peridontal Ligament-like; DF, Dental Follicle; DP, Dental Papilla; Pre-ODO, Pre-Odontoblasts; ENDO, Endothelium; NT, Neutrophils; NV, Nerves; MP, Macrophages; Bone-MP, Bone Macrophages; NK/T, Natural Killer and T Lymphocytes; B, B Lymphocytes; OST, Bone; GLIA, Glia; PC, Pericytes; TB, Taste Buds; SM, Smooth Muscle Cells. Canonical marker genes used to identify major cell classes include pitx2 (dental epithelium,) spp1 (bone), pax9 (dental mesenchyme), and aif1 (immune) (Supplementary Table 1). C. Expression of marker genes used for identification of major cell types in (B)

Transcriptional programs of developmental potential in replacement tooth epithelium.

A. CellRank 2 projected velocity fields showing inferred cell–cell transition dynamics in epithelium using CytoTRACE-derived developmental potential. Arrows indicate the predicted direction of state change in the UMAP embedding. B. UMAP overlay with SL (left) and ES (right) cell distribution, identified by co-expression of canonical marker genes curated from the literature. C. The developmental trajectory of dental epithelial cells is analyzed via pseudotime alignment. D. Gene expression dynamics along pseudo-differentiation reveal distinct transcriptional modules, with corresponding GO enrichment for each module. E. Schematic diagram summarizing lineage specification from ES/SL cells.

Transcriptional programs of developmental potential in replacement tooth mesenchyme.

A. CellRank 2 projected velocity fields showing cell–cell transition dynamics in mesenchyme using CytoTRACE-derived developmental potential. Arrows indicate the predicted direction of state change in the UMAP embedding. B. Gene-weighted density estimates overlaid on the UMAP showing expression distributions of twist1, twist2, and dnmt1 across mesenchymal states. C. The developmental trajectory of mesenchymal cells is analyzed via pseudotime alignment. D. Gene expression dynamics along pseudo-differentiation identify distinct transcriptional modules, with corresponding GO enrichment for each module. E. Schematic diagram summarizing lineage specification from mesenchymal progenitor cells.

Gene expression dynamics across cell types following tooth plucking.

A. Differentially expressed genes identified within each cell types, comparing plucked to control across time points. Red and blue dots highlight the top 2-3 up-regulated and down-regulated DEGs, respectively. B. Stage-resolved expression dynamics of DEGs across the time course. The size of each dot is proportional to the percentage of cells expressing DEGs. Purple and green dots indicate DEGs showing higher and lower expression, respectively, in plucked, as compared to control. C. Time-course GO biological processes reveal enriched functional profiling at each developmental stage. The size of each dot is proportional to the number of genes contributing to each enriched term, and the color scale indicates statistical significance.

Cell-cell communication during the time course of accelerated tooth replacement.

A. Heatmap of differential interaction strength among cell populations between plucked and control shows the outgoing and incoming signaling change associated with each cell group. Red (positive values) represents increased signaling in plucked and blue (negative values) represents decreased signaling in plucked, between cell groups, as compared to control. The color scale indicates the magnitude of differential interaction strength.

Signaling pathways and ligand-receptor interactions during accelerated tooth replacement.

A. Signaling pathways between each sender-receiver cell type pair were ranked by differences in communication probability between conditions. Pathways enriched in plucked are shown in red, and pathways enriched in control are shown in grey. B. Up-regulated ligand-receptor interaction network in plucked compared to control. Signaling sources (senders) and targets (receivers) are shown on the bottom and top, respectively. Colored segments indicate their cell identity. Segment size is proportional to the total outgoing or incoming interaction strength associated with each ligand-receptor pair in the corresponding cell sub-population.

Summary of accelerated tooth replacement following plucking.

A. Plucking accelerates tooth replacement, only on the plucked side, with a ∼3-4x increase in replacement rate that is conserved across cichlid species with widely divergent dentitions. B. Conceptual timeline of biological processes activated following tooth removal that accompany accelerated tooth replacement. Early immune activation is associated with increased axonogenesis and vasculogenesis, followed by elevated odontogenesis and bone formation. C. Schematic representation of prominent intercellular communication events and signaling pathways associated with accelerated tooth replacement after plucking.