Bone elongation in the embryo occurs without column formation in the growth plate

Chondrocyte columns, which are a hallmark of growth plate architecture, play a central role in bone elongation. Columns are formed by clonal expansion following rotation of the division plane, resulting in a stack of cells oriented parallel to the growth direction. However, despite decades of research, column structure has thus far been studied only in two dimensions. To fill this knowledge gap, we analyzed hundreds of Confetti multicolor clones in growth plates of mouse embryos using a pipeline comprising 3D imaging and algorithms for morphometric analysis. Surprisingly, analysis of the elevation angles between neighboring pairs of cells revealed that most cells did not display the typical stacking pattern associated with column formation, implying incomplete rotation of the division plane. Morphological analysis revealed that although embryonic clones were elongated, they formed clusters oriented perpendicular to the growth direction. Analysis of growth plates of postnatal mice revealed both complex columns, composed of both ordered and disordered cell stacks, and small, disorganized clusters located in the outer edges. Our finding that embryonic growth plates function without forming columns suggests that longitudinal bone growth is regulated by different cellular mechanisms during pre- and postnatal development. Moreover, the observed complex columnar and cluster arrangements may serve other, yet unknown morphogenetic functions. More broadly, our findings provide a new understanding of the cellular mechanisms underlying growth plate activity and bone elongation during development.