Caleb is a rising senior at Kansas State University studying bioengineering. He is currently working in Dr. Joel Boerkel’s lab studying the mechanobiological properties of embryonic bone development. Caleb plans to advance to a PhD program in biomedical engineering after graduation.
Osteoprogenitor Lineage Progression is Spatiotemporally Determined in Embryonic Bone Morphogenesis
Long bone morphogenesis requires the spatiotemporal coordination of osteoprogenitor invasion and lineage progression. During endochondral ossification, osteoprogenitors mobilize into the hypertrophic cartilage anlage and differentiate into osteoblasts that highly express collagen 1. Osteoblast lineage progression is coordinated by a series of morphogenic and mechanical cues in vitro, however, the spatial regulation of osteoblast maturity in utero is unclear. Here, we tested the hypothesis that immature osteoprogenitors present preferentially near the line of remodeling cartilage within the primary spongiosa, relative to other bone regions. We used a dual transgenic fluorescent reporter mouse model that co-expresses cyan fluorescent protein (CFP) and green fluorescent protein (GFP) under the control of the 3.6kb (Col3.6) and 2.3kb (Col2.3) fragments of the collagen 1 promoter, respectively. The Col3.6-CFP reporter marks immature osteoblasts/precursors, while the Col2.3-GFP reporter marks mature, further differentiated osteoblasts. Using cryohistology and fluorescent microscopy, E17.5 femurs were evaluated in ImageJ. Reporter-positive areas were evaluated by region of interest analysis in the primary ossification center (POC) and the bone collar. The bone collar exhibited a higher proportion of Col2.3(+) and Col3.6(+) cells per area than the POC, with peak expression occurring at the distal and proximal ends. Immature osteoblasts were found at a higher rate in the POC and are primarily present at the leading edge of the primary spongiosa. Comparatively, the immature osteoblasts in the bone collar were more uniformly distributed axially through the tissue. Together, these data reveal that lineage progression of osteoblasts is spatially regulated along the primary axis of these regions. This enhanced understanding of spatiotemporal lineage progression will aid in further characterization of microenvironmental factors that facilitate proper bone formation.