Single-cell analysis identifies a key role for Hhip in murine coronal suture development

Abstract

Craniofacial development depends on formation and maintenance of sutures between bones of the skull. In sutures, growth occurs at osteogenic fronts along the edge of each bone, and suture mesenchyme separates adjacent bones. Here, we perform single-cell RNA-seq analysis of the embryonic, wild type murine coronal suture to define its population structure. Seven populations at E16.5 and nine at E18.5 comprise the suture mesenchyme, osteogenic cells, and associated populations. Expression of Hhip, an inhibitor of hedgehog signaling, marks a mesenchymal population distinct from those of other neurocranial sutures. Tracing of the neonatal Hhip-expressing population shows that descendant cells persist in the coronal suture and contribute to calvarial bone growth. In Hhip-/- coronal sutures at E18.5, the osteogenic fronts are closely apposed and the suture mesenchyme is depleted with increased hedgehog signaling compared to those of the wild type. Collectively, these data demonstrate that Hhip is required for normal coronal suture development.

Conflict of interest statement

The authors declare no competing interests.

© 2021. The Author(s).

Figures

Fig. 1. scRNA-seq analysis of the wild-type coronal suture at E16.5.

a Uniform Manifold Approximation and Projection (UMAP) plot of cell clusters detected by unsupervised graph clustering of suture-specific populations (i.e., suture mesenchyme, osteoblasts) from two replicates at E16.5. b Average expression of the top five differentially expressed marker genes (boxed) ranked by fold change (FC) (FDR % 0.01, lnFC R 0.1) for suture-specific populations. Genes selected for smFISH in c are colored according to their population membership. c Localization of populations by smFISH. Each pseudo-colored panel shows an individual section hybridized with probes for the indicated genes. The schematic summarizes the spatial distribution of populations, color-coded as in smFISH. Dashed outlines indicate frontal (f) and parietal (p) bones; white horizontal lines indicate osteoid. Sections are in the transverse plane. smFISH was performed on three independent samples with similar results. Scale bar, 50 μm. d Significant GO BP categories of population-specific expression signatures. Gene Ontology enrichment was performed with ranked query and multiple testing analytical correction (p ≤ 0.05, right of dashed line).

Fig. 2. scRNA-seq analysis of the wild-type coronal suture at E18.5.

a UMAP plot of cell clusters detected by unsupervised graph clustering of suture-specific populations (i.e., suture mesenchyme, osteoblasts) from two replicates at E18.5. b Average expression of the top five differentially expressed marker genes (boxed) ranked by FC (FDR % 0.01, lnFC R 0.1) for suture-specific populations. Genes selected for smFISH in c are colored according to their population membership. For CS8-6, Cxcl12, which is within the top ten markers of this population, was used for its known expression in dura mater. c Localization of populations by smFISH. Each pseudo-colored panel shows an individual section hybridized with probes for the indicated genes. The schematic summarizes the spatial distribution of populations, color-coded as in smFISH. Dashed outlines indicate frontal (f) and parietal (p) bones; white horizontal lines indicate osteoid. Sections are in the transverse plane. smFISH was performed on three independent samples with similar results. Scale bar, 50 μm. d Significant GO BP categories of population-specific expression signatures. Gene Ontology enrichment was performed with ranked query and multiple testing analytical correction (p ≤ 0.05, right of dashed line).

Fig. 3. Enrichment of Hhip expression in SM is specific to the coronal suture.

a Average bulk gene expression changes of the significant marker genes (rows) of all SM populations across the four calvarial sutures. Expression changes are shown as the log2FC between the SM and osteogenic fronts (OF) at E16.5 and E18.5, ranked by their ‘coronal specificity score’ (CS), representing the difference between the average expression across the coronal suture data and the average expression across the remaining sutures (lambdoid, sagittal, and frontal). Frontal OF (FR), parietal OF (PA), interparietal OF (IP). b Average bulk gene expression changes of the combined top ten marker genes of SM populations CS6-4 and CS8-2 across the four calvarial sutures. Expression changes are shown as in a. c Average bulk gene expression changes of key hedgehog pathway genes across the four calvarial sutures. Expression changes are shown as in a.

Fig. 4. Fate mapping of Hhip-CreERT2-expressing suture mesenchyme.

a Graph abstraction of the relationships between CS8-1 to CS8-9. Inset shows coloring by cluster identity. The root state of the inferred trajectory is indicated by the number 1 at the center of the cells with the earliest pseudotime. b TdTomato-expressing (tdTomato + ) cells at postnatal day (P)2 after 1 day of induction. n = 3. c TdTomato+ cells at P3 after two consecutive days of induction. n = 3. d TdTomato+ cells at P6 after five consecutive days of induction. n = 1. e TdTomato+ cells at P30 after 1 day of induction at P1. Dashed box is enlarged at right to show cluster of tdTomato+ osteoblasts within ALPL domain (dashed lines). n = 1. f TdTomato+ cells at P90 after two consecutive days of induction at P1 and P2. n = 3. g TdTomato+ cells at P90 after five consecutive days of induction between P1-P5. n = 2. White and yellow empty/solid arrowheads indicate tdTomato+ osteoblasts/osteocytes in parietal and frontal bones, respectively. f frontal bone, p parietal bone. Sections are in the sagittal plane. Scale bars, 100 μm except inset in e, 50 μm.

Fig. 5. Coronal suture dysgenesis in Hhip−/− mice at E16.5.

a Staining for alkaline phosphatase activity (ALPL, red) in preosteoblasts and osteoblasts for wild type (WT) and mutant (Hhip−/−) coronal sutures. f frontal bone; p parietal bone, sm suture mesenchyme. b Immunohistochemistry for RUNX2 (green). Sections are the same as shown in a. c Immunohistochemistry for SP7 (green). d Proliferation detected by EdU incorporation (green). Sections are counterstained for ALPL activity (red) and DAPI (blue). e Quantification of EdU incorporation in osteogenic fronts (OF) and suture mesenchyme (SM). WT and Hhip−/− are indicated in black and red, respectively. n = 3 WT and three Hhip−/−. f Quantification of cell numbers per section in OFs and SM. n = 3 WT and three Hhip−/−. g TUNEL assay for apoptotic cells. Inset, example of an apoptotic cell in non-sutural tissue on the same slide. White dashed outlines, frontal and parietal bones. Data in e and f are presented as dot plots showing mean ± standard deviation. Results presented in a, b, c, d, and g are representative of n = 3 WT and three Hhip−/− independent samples. Sections are in the transverse plane. Scale bar, 50 μm. Source data are provided as a Source Data file.

Fig. 6. Coronal suture dysgenesis in Hhip−/− mice at E18.5.

a Staining for alkaline phosphatase activity (ALPL, red) in preosteoblasts and osteoblasts for WT and Hhip−/− coronal sutures. f, frontal bone; p, parietal bone; sm, suture mesenchyme. b Immunohistochemistry for RUNX2 (green). Sections are the same as shown in a. c Immunohistochemistry for SP7 (green). d Proliferation detected by EdU incorporation (green). Sections are counterstained for ALPL activity (red) and DAPI (blue). e Quantification of EdU incorporation in osteogenic fronts (OF) and suture mesenchyme (SM). WT and Hhip−/− are indicated in black and red, respectively. Asterisk indicates a significant difference using the two-tailed Student’s t-test (P = 0.030). n = 6 WT and six Hhip−/−. f Quantification of cell numbers per section in OFs and SM. Asterisk indicates a significant difference using the two-tailed Student’s t-test (P = 0.018, 0.008, and 0.00004, respectively). n = 6 WT and six Hhip−/−. g TUNEL assay for apoptotic cells. Inset, example of an apoptotic cell in non-sutural tissue on the same slide. White dashed outlines, frontal and parietal bones. Data in e and f are presented as dot plots showing mean ± standard deviation. Results presented in a, b, c, and g are representative of n = 3 WT and three Hhip−/− independent samples. Sections are in the transverse plane. Scale bar, 100 μm. Source data are provided as a Source Data file.

Fig. 7. Results of principal components analyses (PCA) of data from microcomputed tomography (microCT) imaging of Hhip−/− heads at E18.5.

Scatter plots of results of PC1 and PC2 scores based on PCA analysis of unique linear distances among 3D landmarks for a form and b shape (adjusting for the effects of allometry) of WT (n = 4) and Hhip−/− (n = 7) skulls. c 3D reconstruction of microCT images showing dorsal views of the frontal (f) and parietal (p) bones of WT and Hhip−/− calvaria. Insets show lateral views of the whole skull. Images are representative n = 4 WT and seven Hhip−/− skulls. Scale bars, 1 mm.

Fig. 8. Localization of Hhip−/− coronal suture populations by smFISH.

Each pseudo-colored panel shows an individual section hybridized with probes for the indicated genes at each age. Expression of the E18.5 CS8-2 marker, Cd34, can be compared to WT in Supplementary Fig. 3a. Schematics summarize the spatial distribution of populations, color-coded as in smFISH. White arrowheads indicate novel Mmp13 expression. Dashed outlines indicate frontal (f) and parietal (p) bones; white horizontal lines indicate osteoid. Sections are in the transverse plane. smFISH was performed on three independent samples with similar results. Scale bars, 50 μm.

Fig. 9. Altered HH signaling in the Hhip−/− coronal suture.

a LacZ staining (blue) in WT, Hhip+/−, and Hhip−/− coronal sutures at E18.5, counterstained with nuclear fast red. b–e smFISH for b, dPtch1, and c, eGli1 in WT and Hhip−/− coronal sutures at b, c E16.5 and d, e E18.5. Dashed outlines indicate frontal (f) and parietal (p) bones; sm, suture mesenchyme. Sections are in the transverse plane. Results presented in a are representative of n = 2 WT, two Hhip+/−, and two Hhip−/− independent samples. Results presented in b–e are representative of n = 3 WT and three Hhip−/− independent samples. Scale bars, 50 μm.