Spatiotemporal single-cell atlas of suture stem cell dynamics in craniosynostosis

Abstract

Background: Craniosynostosis is a congenital disorder characterized by premature suture fusion and aberrant skull morphogenesis. The cellular dynamics and regulatory mechanisms of suture mesenchymal stem cells (SuSCs) in this disease remain poorly defined. Methods: We integrated single-cell RNA sequencing and 2-μm-resolution Visium HD spatial transcriptomics to build a spatiotemporal atlas of coronal suture cells in Fgfr2C342Y/+ mice, a murine model recapitulating human Crouzon syndrome, alongside wild-type controls across three key developmental stages (E14.5, E18.5, and P3). To obtain near single-cell spatial resolution, we created SpatialCell, which combines morphology-based segmentation and machine-learning classification using a reference trained on our single-cell datasets. Results: The atlas reveals stage-specific remodeling of SuSC niches and a shift of SuSC spatial associations toward osteogenic mesenchyme in craniosynostosis. Along the SuSC-to-osteoblast trajectory, pre-osteoblasts were depleted earlier than upstream SuSCs, and SuSCs displayed premature acquisition of osteogenic programs near the suture midline. Temporal Gene Ontology patterns indicated early extracellular-matrix disruption, mid-gestation chondrogenic activation, and postnatal mineralization. Network analysis nominated Foxa3 as a candidate regulator in SuSC subsets; siRNA knockdown of Foxa3 reduced ex vivo mineralization in the craniosynostosis background. Spatial communication analyses implicated signals from suture meningeal fibroblasts and immune cells that converge on SuSC fate. Conclusions: Our results support a model where craniosynostosis may involve disrupted temporal coordination of developmental programs, not merely accelerated bone formation. The atlas and analytic framework pinpoint when and where SuSC fate diverges, propose Foxa3 as an intervention target, and provide a high-resolution resource for mechanistic and therapeutic exploration.

1. Introduction

Craniosynostosis is a group of craniofacial disorders involving premature fusion of one or more calvarial sutures in infancy, leading to abnormal skull growth, elevated intracranial pressure, and potential neurodevelopmental impairment [1, 2]. Current treatment relies predominantly on surgical intervention, which is frequently followed by resynostosis and the need for reoperation [3]. These limitations underscore the urgent need for effective therapeutic strategies; however, progress remains hindered by limited understanding of the pathogenesis and progression of craniosynostosis.

Previous studies on craniosynostosis have predominantly focused on mature osteoblasts and osteoprogenitors, whereas the role of suture mesenchymal stem cells (SuSCs)—which reside in the suture midline and give rise to osteogenic precursors during normal cranial development—has received comparatively less attention. Emerging evidence indicates that SuSCs are critical for maintaining suture patency and regulating osteogenic homeostasis [4–6]. Notably, SuSCs have been shown to reverse cranial and neurocognitive abnormalities and promote suture regeneration [7]. However, the mechanisms underlying SuSC fate determination, cellular heterogeneity, and niche regulation in disease contexts remain poorly understood.