Fig 1: 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 2: Progenitor cells apically polarize WNT inhibitors.(A) Schematic of whole-mount analysis from embryonic skin with examples of optical sections showing interfollicular epidermis (IFE), bud and germ; position of pixel intensity measurement. Z, plane of imaging from the Z-stack. (B) Anti-WIF1 and (C) anti-NOTUM immunofluorescence in placode, bud and germs reveal an apical accumulation of WIF1 and NOTUM. Pixel intensity profiles of basal hair bud progenitors (n = ≥40 WNT signaling progenitors; mean ± SEM; a.u., arbitrary units). Note also absence of WNT inhibitors in upper region of the dermal condensate (encased by yellow dotted line) at this stage of morphogenesis, leaving a WNT inhibitor free zone (yellow arrowheads) for nuclear LEF1 and WNT signaling at the epidermal-dermal boundary (white dashed line) and a WNT inhibitor high zone in suprabasal hair bud cells (arrows). Blue circular dashed lines outline placodes. White dotted lines demarcate epithelial-mesenchymal boundaries. *Denotes magnified cells, shown at right of each frame. Scale bars: 5 μm magnified cells; all others, 20 μm.Figure 4—source data 1.Source data for the graphs shown in Figure 4.
Fig 3: Hair bud progenitors apically polarize WNT inhibitors to protect their own identity and differentially confer WNT signaling to their neighbors.(A) Whole-mount immunofluorescence and quantifications reveal that elevating NOTUM across the epidermal plane results in significantly fewer hair follicles (Mean ± SD; n > 10 mm2 skin analyzed from ≥3 embryos; *p<0.05; **p<0.005; Mann-Whitney test). Scale Bar, 100 μm. OE, overexpression. Insets verify transduced regions. All scale bars for immunofluorescence images are 20 μm. (B) Adding an aquaporin4-tag mispolarizes NOTUM to the basal side of hair bud progenitors. Quantifications reveal that mis-polarizing a WNT inhibitor poses a significant impediment to hair follicle morphogenesis (Mean ± SD; n = 8 mm2 skin analyzed from ≥3 embryos; *p<0.05; **p<0.005; ***p<0.0005; unpaired Student t test). White dotted lines demarcate epithelial-mesenchymal borders throughout. (C) Whole-mount immunofluorescence and quantifications of normalized LEF1 pixel intensities reveals that NOTUM mis-polarization leads to a significant decrease of LEF1 intensity in WNT signaling cells from both the dermal condensate and the hair bud (n ≥ 48 hair follicles from ≥3 embryos each; Mann-Whitney test ***p=0.0002 and unpaired t test **p<0.005; n.s. non-significant; red lines represent the distributions’ median). Yellow boxes show regions magnified at right. Arrowheads show two cells not expressing NOTUM-AQP4-MYC-Tag, which have higher LEF1 signal than their expressing neighbors. (D) Violin Plots show that increasing levels of NOTUM and NOTUM-AQP4 lead to an increase of SOX9 expressing cells (n ≥ 30 developing hair follicle from at least three different embryos; Mann-Whitney test TRE-NOTUM *p=0.0389 and TRE-NOTUM-AQP4 *p=0.0461 n.s. non-significant; red lines represent the distributions’ median).Figure 6—source data 1.Source data for the graphs shown in Figure 6A, B, C, and D.
Fig 4: Evidence of oppositely polarized and short-range action of WNTs and WNT inhibitors in hair bud progenitors that are actively signaling through WNTs.(A) LV-constructs and strategy to monitor WNT inhibitors and WNTs. M, myc-tag. TRE, tetracycline regulatory element. Krt14rtTA is a transgenic mouse line expressing a doxycycline (DOX)-inducible transcriptional activator for TRE. (B, C) Similar to endogenous expression, anti-WIF1 and anti-NOTUM immunofluorescence on MYC-tag transduced skin show apical localization in hair bud progenitors. (D) Anti-MYC-tag immunofluorescence of transduced skins revealing apical polarization of NOTUM and WIF1, but basal polarization of WNT10B. At right are basal-apical MYC-Tag/DAPI pixel intensity profiles of basal hair bud progenitors (n = ≥40 WNT signaling progenitors; mean ± SEM; a.u., arbitrary units). (E) Pixel intensity profile and immunolocalization of endogenous WNT-receptor FRIZZLED 10 shows uniform localization at borders of hair bud and germ WNT signaling cells (n = 37 cells; mean ± SEM; a.u., arbitrary units). *Denotes magnified cells, shown at right of each frame. Scale bars: 5 μm magnified cells; all the others, 20 μm.Figure 5—source data 1.Source data for the graphs shown in Figure 5D and E.
Fig 5: Notum regulates the formation of sharp boundaries between neighboring cell fates.(A) In utero lentiviral delivery strategy to conditionally ablate Notum in R26dtTomato embryos. (B–C) Representative whole-mount immunofluorescence showing LEF1 intensity profile and population size in Notum -/+ and Notum-/- skin. Red boxes denote regions magnified below each image. Note that Notum ablation (but not heterozygous) leads to an increase in LEF1 signal and LEF1+ cell populations in the WNT signaling progenitor cells (n ≥ 75 hair follicles from ≥3 different litters analyzed; Mann-Whitney test *p=0.0332 and ***p=0.0002; n.s. non-significant; red lines represent the distributions’ median).Figure 7—source data 1.Source data for the graphs shown in Figure 7C.
Supplier Page from MilliporeSigma for Anti-NOTUM antibody produced in rabbit