IADR Abstract Archives
Unravelling the Molecular Heterogeneity of Synchondrosis Development Using Spatial Transcriptomics
Objectives: Our preliminary data suggests a significant role for the master osteoblast regulatory gene, RUNX2, in the development of the postnatal cranial base spheno-occipital synchondrosis (SOS). In this study, we used spatial transcriptomic analysis to examine the molecular heterogeneity of synchondrosis cells in a spatiotemporal manner during physiologic development and in response to Runx2 ablation.
Methods: We utilized a tamoxifen-inducible Fgfr3-creER line and Runx2-floxed alleles to ablate Runx2 in Fgfr3+ synchondrosis chondrocytes. Control (Fgfr3-creER; Runx2fl/+) and Runx2cKO (Fgfr3-creER; Runx2fl/fl) mice were pulsed with tamoxifen at postnatal day 3 (P3) and chased to P21. Highly multiplex targeted single cell spatial profiling analyses (Xenium in situ, 10x Genomics, Inc.) of murine SOS’s at P21 was performed using a custom designed panel of 350 unique genes specific to musculoskeletal development. Sub-regional analyses (chondrocyte layers, primary spongiosa, periosteum) were performed between control and Runx2cKO SOS tissue sections. A novel computational pipeline was designed, validated, and implemented for this spatially resolved transcriptomic analysis.
Results: In the absence of Runx2, Runx2cKO synchondrosis chondrocytes presented significant downregulation of major signaling pathways, including Bmp (Bmp2, Bmpr2, Grem1), extracellular matrix formation (Col1a1, Postn), cell proliferation (Pcna), Notch (Notch1, Jag1) and osteoblast-related pathways (Dlx6, Erg, Rln1). Cells within the Runx2cKO synchondrosis primary spongiosa displayed upregulation of Amelx, Foxj1 and Sftpd and downregulation of Thbs1 and Vwf. Lastly, periosteal cells were significantly upregulated for Col2a1, Cdk1 and Tac1 but were downregulated for Col23a1, Angptl1, Mmp15 and Ephb2.
Conclusions: Collectively, our data suggest distinct molecular properties of spatially resolved zones within the postnatal SOS and point to Runx2 being a possible novel regulator of key Bmp, extracellular matrix and cell proliferation-related pathways amongst others. These data provide a robust transcriptomic toolkit for discerning the molecular regulation of the postnatal murine cranial base synchondrosis.
Methods: We utilized a tamoxifen-inducible Fgfr3-creER line and Runx2-floxed alleles to ablate Runx2 in Fgfr3+ synchondrosis chondrocytes. Control (Fgfr3-creER; Runx2fl/+) and Runx2cKO (Fgfr3-creER; Runx2fl/fl) mice were pulsed with tamoxifen at postnatal day 3 (P3) and chased to P21. Highly multiplex targeted single cell spatial profiling analyses (Xenium in situ, 10x Genomics, Inc.) of murine SOS’s at P21 was performed using a custom designed panel of 350 unique genes specific to musculoskeletal development. Sub-regional analyses (chondrocyte layers, primary spongiosa, periosteum) were performed between control and Runx2cKO SOS tissue sections. A novel computational pipeline was designed, validated, and implemented for this spatially resolved transcriptomic analysis.
Results: In the absence of Runx2, Runx2cKO synchondrosis chondrocytes presented significant downregulation of major signaling pathways, including Bmp (Bmp2, Bmpr2, Grem1), extracellular matrix formation (Col1a1, Postn), cell proliferation (Pcna), Notch (Notch1, Jag1) and osteoblast-related pathways (Dlx6, Erg, Rln1). Cells within the Runx2cKO synchondrosis primary spongiosa displayed upregulation of Amelx, Foxj1 and Sftpd and downregulation of Thbs1 and Vwf. Lastly, periosteal cells were significantly upregulated for Col2a1, Cdk1 and Tac1 but were downregulated for Col23a1, Angptl1, Mmp15 and Ephb2.
Conclusions: Collectively, our data suggest distinct molecular properties of spatially resolved zones within the postnatal SOS and point to Runx2 being a possible novel regulator of key Bmp, extracellular matrix and cell proliferation-related pathways amongst others. These data provide a robust transcriptomic toolkit for discerning the molecular regulation of the postnatal murine cranial base synchondrosis.