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Abstract:
Bones are essential vertebrate structures that arise during embryonic development from mesenchymal condensations. In the skull vault, bones arise from condensations above the eye, which later expand to cover the brain. Contrarily to most bones in the axial skeleton, which develop through an intermediate cartilage template, the skull vault develops from the direct differentiation of mesenchymal progenitors into bone cells, or osteoblasts. The molecular details of this process have however remained elusive, since systems allowing the dynamical observation of cells while they undergobone differentiation have been lacking. Recent data in the Tabler lab, acquired by imaging skull caps ex vivo, has uncovered that differentiation takes place during expansion stages, and that a progressive wave of differentiation underlies the presence of intermediate states between progenitors and osteoblasts. In this thesis, we focused on the cranial mesenchyme to study the differentiation trajectory of skull bone progenitors at single cell resolution. By harnessing single cell RNA-Sequencing, we elucidated the heterogeneity of cells in the cranial mesenchyme, describing in molecular terms meningeal, dermal, osteoblastic and progenitor populations. By modelling dynamics from single cell RNA-Seq data, we inferred that a population expressing intermediate levels of cartilage specific genes, such as Col2a1, underlies differentiation towards dermal, meningeal and bone fate. Using single cell resolution in situ RNA hybridisation, we mapped the anatomical location of progenitors in a layer between the meninges and the bone. To better understand the relationship between the phenotype of these progenitors and differentiated cartilage, we examined the presence of cartilage-like ECM in the tissue. Finally, we asked whether similar progenitors may be present in other intramembranous bones outside the skull by re-applying these tools on the clavicle. Taken together, our data lead us to hypothesise a mechanism for differentiation of the cranial mesenchyme, which can explain previous phenotypes reported in the literature and supports a role for cartilage-like cells in intramembranous ossification.
