Tissue patterning in any developmental system requires well-coordinated signaling events that result in the formation of distinct cell types in the appropriate locations. While many of these signaling mechanisms are known, how they are initially established during development is less understood. Endochondral ossification involves cartilage replacement by bone in sites within the cartilage where chondrocytes become hypertrophic. In long bones, this occurs first in the mid-region of the cartilage, then on the distal ends. This process results in the formation of growth plates (GP), segments of cartilage in between these ossification zones that drive bone elongation via a combination of chondrocyte proliferation and endochondral ossification of GP cartilage. Two signals, Indian Hedgehog (Ihh), which promotes the process of ossification by inducing hypertrophic differentiation of chondrocytes near the mid-region ossification zone, and Parathyroid Hormone-Like Hormone (Pthlh), which represses hypertrophic differentiation of chondrocytes via a gradient that originates near the distal ossification zones, control the rate and direction of bone growth. In addition to these, mechanical force plays important roles in tissue maintenance, as illustrated by the effects that zero gravity has on the bones and muscles of astronauts in orbit at the International Space Station. While Ihh is essential for the formation of hypertrophic zones (HZ) in embryonic cartilages, it is not expressed at the initial stages of HZ formation, suggesting that alternative signaling components are needed to begin the ossification process. Similarly, whether or not Pthlh plays a role in initial patterning of HZs and ossification remains unclear. In my thesis, I study the role of Pthlh signaling in the early patterning of HZs and explore the role of mechanical force in the process of endochondral ossification using the zebrafish jaw cartilages as a model.

Using a transgenic reporter line for the entpd5a gene, I show that HZs develop earlier than previously reported in zebrafish cartilages. Using this result as a base, I look at the expression pattern of pthlha and find expression domains of pthlha that inversely correlate with HZ patterns. Knockout of pthlha leads to an expanded HZ and increased ossification rates, results congruent with the known role of Pthlh in GPs. Mosaic misexpression of pthlha in chondrocytes results in disruption of HZs and bone formation as a function of the number and proximity of pthlha-expressing cells to the site of bone formation. In addition, mosaic misexpression of pthlha in chondrocytes leads to the mispatterning of the HZ such that it forms ectopically. These results suggest that Pthlh signaling is necessary and sufficient to pattern the initial HZs in embryonic cartilages and suggest that Pthlh signaling also helps establish the Ihh/Pthlh negative feedback loop that regulates GP homeostasis. In addition to these findings, I show that mechanical force is specifically required for chondrocyte hypertrophic differentiation in developing HZs and for chondrocyte proliferation in zones of the cartilage that do not initially become hypertrophic. This result suggests that an additional signal may modulate the effects of mechanical force on chondrocytes, and we speculate that this signal is Pthlh. Therefore, this work establishes Pthlh as the early patterning signal of endochondral ossification and reveals a role for mechanical force in initiating the process.