Decomposing a deterministic path to mesenchymal niche formation by two intersecting morphogen gradients

Abstract

Organ formation requires integrating signals to coordinate proliferation, specify cell fates, and shape tissue. Tracing these events and signals remains a challenge, as intermediate states across many critical transitions are unresolvable over real time and space. Here, we designed a unique computational approach to decompose a non-linear differentiation process into key components to resolve the signals and cell behaviors that drive a rapid transition, using the hair follicle dermal condensate as a model. Combining scRNA sequencing with genetic perturbation, we reveal that proliferative Dkk1+ progenitors transiently amplify to become quiescent dermal condensate cells by the mere spatiotemporal patterning of Wnt/β-catenin and SHH signaling gradients. Together, they deterministically coordinate a rapid transition from proliferation to quiescence, cell fate specification, and morphogenesis. Moreover, genetically repatterning these gradients reproduces these events autonomously in "slow motion" across more intermediates that resolve the process. This analysis unravels two morphogen gradients that intersect to coordinate events of organogenesis.

Keywords: Wnt; dermal condensate; dermis; development; hair follicle; morphogen; niche; single-cell RNA-seq; sonic hedgehog.

Figure 1:. A selectively proliferative population transitions over a short timeframe into quiescent DC cells

(A) HF development over time. (B) UMAP of dermal scRNA-seq data with Sox2 (left), UMAP showing ordering based on distance from DC state in transcriptome space (middle), G1/G0 fraction or gene levels across pseudo-order (right). (C) Sox2+ cell number per DC over time. (D) %EdU+ of DC population after E13.75 or E14.5 EdU pulse. (E) Expression level of indicated genes or fraction G1/G0 over pseudo-order (dashed line, highly proliferative state). (F) FISH showing Dkk1, Ccna2, or Cdkn1a with EdU (arrowheads indicating corner region of peri-DC). (G) 3D views and quantification of Tom+ Dkk1-lineage traced cells at indicated times in peri-DC or DC population; n=4. (H) Cartoon of spatial progression of DC differentiation. Data as mean± SEM (n=8 per time); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA; ns, not significant. Scale bars, 50 µm. Related to Figure S1.

Figure 2:. Decomposing a non-linear differentiation process into key components

(A) Lef1 and Dkk1 levels by pseudo-order; dashed line, highly proliferative pre-DC state. (B, C) FISH and quantification of Lef1 transcript levels across space (n=4) with schematic of Lef1 levels over space. (D) Cartoon of predicted diffusion map by Wnt and DC components. (E) Diffusion map defined by eigenvectors that most correlate with Lef1 and Sox2. (F) Pseudo-order by Wnt component; dashed line, when Sox2 and Lef1 covary. (G) Correlation plot showing other Wnt targets and DC genes correlate with the two components. Related Figure S2.

Figure 3:. Unbiased identification of the placode factor essential for the transition to the DC component

(A) UMAP E13.5 dermal cells by condition. (B) UMAP of E14.5 dermal cells by condition (left) or by Lef1 and Sox2 (right). (C) Diffusion maps by Wnt and DC components. (D) Differentially-abundant (DA) clusters of control and K14Cre;βcat^fl//fl dermal cells (P < 0.01). (E) Volcano plot of control and mutant DA1 DEGs with genes (navy) that dually correlate with Wnt and DC components. (F) Violin plots of Lef1 and Ptch1 by DA clusters. (G) Pseudo-order showing covarying Ptch1 and Lef1 levels. (H) Quantification of Ptch1 levels across space (n=4). (I) Cartoon depicting SHH and Wnt signal levels by components. Data as mean± SEM; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA; ns, not significant; scale bars, 50 µm. Related to Figure S3.

Figure 4:. SHH is essential for progression onto the DC component and genetically defines a critical transition stage of DC genesis

(A) Whole mount of E14.5 control and SHH cKO skin. (B) UMAP of control and SHH cKO dermal cells by condition or by DA clusters (left); Sox2 on UMAP (right). (C) Diffusion maps of SHH cKO and control showing Sox2 and Lef1. (D,E) Diffusion maps (left) and pseudo-order (right) of control and K14Cre;βcat^fl//fl or control and SHH cKO color-coded by DA clusters. Dashed lines delineating DA populations corresponding to Regions 1 and 2. (F) Cartoon of Regions 1 and 2 and transition zone (bracket). Scale bars, 50 µm. Related to Figure S4.

Figure 5:. Dermal SHH activation is required for the rapid transition to quiescence and mature DC differentiation.

(A) Lef1 levels in Bmp4+ DCs at E14.5 and E15.5. (B) FISH showing decreased Lef1 expression in Bmp4+ SHH cKO pseudo-DCs. (C) Proliferation rate of Dkk1+ peri-DC and upper dermal cells at E14.5. (D) Pseudo-order of control and SHH cKO dermal cells at E14.5. (E) FISH showing proliferative Bmp4+ pseudo-DCs in SHH cKO at E14.5 and E15.5. (F) Number of Bmp4+ cells in SHH cKO and control over time. (G) %EdU+ of Bmp4+ population over time in control and SHH cKO. (H) Diffusion maps of E14.5 and E15.5 control and SHH cKO dermal cells showing that SHH cKO pseudo-DCs progress only on the Wnt component by E15.5. (I) Cartoon showing aberrant proliferation of SHH cKO Dkk1+ peri-DC cells and slow transition to quiescence. Data as mean± SEM; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA. Scale bars, 50 µm. Related to Figure S5.

Figure 6:. High dermal SHH activation in early Wnt-active cells deterministically reproduces events of DC genesis over more intermediates

(A) E14.5 control and SmoM2YFP whole mount showing proliferative mutant Sox2+ clusters. (B) Number of Sox2+ cells per dermal cluster in control and SmoM2YFP at E14.5 and E15.5. (C) Diffusion maps of E14.5 control and SmoM2YFP cells. (D) Pseudo-order with Regions 1 and 2 demarcated. (E) FISH of E14.5 control and mutant showing virtually all Sox2+ cells co-express Ptch1 and Lef1. (F) Pseudo-order of indicated genes in mutant and control. (G) E14.5 FISH showing EdU, Sox2, and Dkk1. (H) FISH at E15.5 after EdU pulse in control and SmoM2YFP skin. (I) FISH 24 hours after EdU chase. (J) %EdU+ cells by population in E14.5 mutant and control. (K) %EdU+ of Sox2+ cells at E15.5 after EdU pulse or 24-hour chase. (L) E14.5 SmoM2;βcat^fl/EX3 FISH showing Sox2+ clusters in upper and lower dermis that co-express Ptch1 and Lef1. (M) Sox2+ clusters with %EdU+ of indicated populations by condition at E14.5. (N) Depiction of transition rate and number of intermediates affected by modulating SHH and Wnt signaling. Data as mean ± SEM, one-way ANOVA. Scale bars, 50 µm. Related to Figure S6 and S7.

Figure 7:. The spatial patterning of SHH and Wnt signaling gradients regulates the number of transitioning intermediates

(A) E13.5 whole mounts across indicated conditions. (B) FISH of E13.5 embryos by condition showing increased Lef1 levels in SmoM2 and SmoM2YFP;βcat^fl/EX3 embryos. (C) Number of Sox2+ cells per cluster by condition. (D) Average Lef1 levels by condition parsed by Ptch1 mutant status. (E) % EdU+ cells in upper dermis by condition and Ptch1 status. (F) Model plots illustrating that the spatiotemporal distance between threshold levels of Wnt and SHH signaling determines the number of transitioning intermediates and the length of transition to DC status. Bottom, depiction of the transition pseudo-order across conditions dependent on levels of SHH and Wnt signaling (orange, DC markers; blue, transitioning state; dashed lines, SHH and Wnt thresholds). Data as mean ± SEM, one-way ANOVA; scale bars, 50 µm. Related to Figure S7.