Fig 2: Recombination by the Lyve1 Cre driver. (a–f) Double immunostaining of Iba1 and the tdTomato in Lyve1 Cre/+ ; tdTomato reporter mice at E10.5 (a,b), E12.5 (c,d) and E16.5 (e,f). Recombination in the CNS parenchyma starts to be detected at E10.5 as Iba1‐tdTomato double‐positive cells (arrowheads). Such cells dramatically increase at E12.5 (c,d). At E16.5, most Iba1+ cells still express tdTomato (white arrowheads) with few cells that express tdTomato but not Iba1 (yellow arrowheads in h). (g–j) Conditional deletion of Sall1 using the Lyve1 Cre driver. In wild type brains, SALL1 is expressed in PU.1+ immune cells in the brain parenchyma (arrowheads in g,h), but not in Sall1 conditional knockout mice. PU.1+ cells (arrowheads in j) do not express SALL1 (i). (k,l) Very few cells express LYVE1 in the brain parenchyma in the neocortex at E12.5 (arrowheads in k). For (c–l), a high‐magnification image is shown (e.g., c′ for c). The location of each high‐magnification image is shown as a yellow box in each panel. nCx, neocortex; Thal, thalamus; MGE, medial ganglionic eminence; LGE, lateral ganglionic eminence.

Fig 3: Detailed analysis of mesenchymal branching point and odontoblast lineage.a Selection of non-mature subpopulation. An unbiased cluster of cells that do not represent mature pulp populations was selected (left, upper), and preodontoblasts (left, lower), were excluded from it, resulting in non-mature subpopulation (right). b Analysis of ICs stability reveals five biologically driven aspects of heterogeneity of non-mature subpopulation. Average correlation of ICs to the most similar ICs across 100 runs of subsamplings of 70% of cells (right) reveals 5 out of 20 ICs with stability substantially higher than expected from shuffled control (left). Stability of all ICs of control shuffled matrix and 15 ICs of original matrix are around 0.4 indicating background expectations of spurious components. c Five ICs of non-mature subpopulation reveal processes related to (from left to right) apical gradient (IC 1), cell cycle (IC 2), Fgf3-mediated (IC 3), and Foxd1-mediated (IC 5) heterogeneity restricted to the progenitor states. Colors show intensities of identified ICs. Genes that have the highest (red) and lowest (blue) associations with corresponding IC are shown. d Graph showing cells expressing Foxd1 in comparison to cell-cycle score. e Transcriptional events during pulp differentiation trajectories. Each trajectory encompasses cell states from progenitor, as identified from analysis of preodontoblasts, to mature states with cells arranged by pseudotime reflecting maturation process. The black cells belong to the trajectory being shown, while other cells are shown in light gray (upper panels). Heatmap shows smoothed gene expression profiles with cells arranged by pseudotime and genes (rows) arranged by pseudotime of maximal expression (lower panels). f–i Expression analysis of the selected genes determining odontoblasts (Col1a1, Dmp1, and Dspp) and together with previously unrecognized identified odontoblast marker genes (Notum and Sall1). Notum expression is visualized on t-SNE embedding of the pulp dataset and immunohistochemistry proves NOTUM to be expressed in odontoblasts (g). Sall1 expression is visualized on t-SNE embeddings of the both pulp (h) and complete incisor dataset (i). Scale bars: 50 µm.

Fig 4: Fetal metanephric mesencyme in interspecific chimeras between mouse and rat. a Representative photomicrographs of contribution of ESCs (pseudocolored green) to Sall1-positive cells (red) at E11.5 in interspecific chimera by injection of rat ESCs into wildtype mouse blastocysts and at E13.5 in that of mouse ESCs into wildtype rat blastocysts. The dotted line indicates the metanephric mesenchyme. Chimeric mice were generated by microinjecting tdTomato-labeled rat ESCs, into mouse blastocysts. Chimeric rats were generated by microinjecting GFP-labeled mouse ESCs into rat blastocysts. mBL mouse blastocyst, rBL rat blastocyst, mESCs mouse embryonic stem cells, rESCs rat embryonic stem cells. Scale bar: 100 μm. b Quantification of the contribution of Sall1-positive cells in a. Horizontal bars represent the mean percentage. All data were obtained from 2 independent experiments. *p < 0.05 by two-tailed Student’s t-test analysis. Source data are provided as a Source Data file

Fig 5: Developmental dynamics of dental mesenchyme.a Analysis of mouse-incisor dental mesenchymal cells isolated for separate analysis from general dataset. Colors show unbiased clusters. The principal tree correctly captures positions of mature mesenchymal derivative and progenitor populations. b Analysis of RNA velocity shows major directions of cell progression in the transcriptional space. The arrow start- and endpoints indicate current and predicted future cell states. c Model of stem-cell dynamics in mesenchymal compartment with relation to dental follicle. d Prediction and validation of spatially restricted Foxd1+ stem cells. The Foxd1-associated axis was selected for validation, and is shown on t-SNE (genes with the strongest positive and negative associations are shown in red and blue respectively). Foxd1+ cells (in situ hybridization) are located in the mesenchyme surrounding the labial cervical loop. e Lineage tracing of FoxD1CreERT2/R26tdTomato strain confirmed the predicted stem-cell nature of Foxd1+ mesenchymal cells. In short-term tracing (5days) tdTomato+ cells are predominantly around LaCL in contrast to long-term (1-month-long and 3 months) tracing where pulp and odontoblast progeny are observed. Importantly, tdTomato+ cells are maintained in their original position in the long-term manner (3 months) and at the same time point tdTomato+ distally located odontoblasts can be observed supporting the theory of Foxd1+ cells being a long-living mesenchymal stem cells. f Nature of Foxd1+ cells progeny confirmed by a combination of FoxD1CreERT2/R26tdTomato tracing and SALL1 and SOX9 immunohistochemical stainings. FoxD1CreERT2/R26tdTomato-traced cells contribute to both the SALL1+ odontoblasts (arrows) and SOX9+ pulp cells (arrowheads). Asterisks show the of subodontoblast layer FoxD1CreERT2/R26tdTomato traced cells. g Variability of cells assigned to a branch leading to odontoblasts (inset) was reanalysed using principal component analysis. Colors mark five clusters obtained by unbiased hierarchical clustering. Left-right axis reflects developmental stages of odontoblasts. h Gradual odontoblast differentiation (suggested in g) from near-CL area into fully differentiated odontoblasts. Left: expression pattern acquired from scRNA-seq, right: in situ hybridization-based histological validations of the proximal part of the mouse incisor proving suggested gradual transition. i Spatial pattern of a discovered (pre)odontoblast transcription factor—SALL1 (Immunohistochemistry). (LaCL Labial Cervical Loop, pre-od. preodontoblasts, Od. Odontoblasts, Am. Ameloblasts). Scare bars: 50 µm.