PROTEOGLYCAN MODULATION OF BMP SIGNALLING IN ENDOCHONDRAL AND INTRAMEMBRANOUS OSSIFICATION: INSIGHTS FROM ZEBRAFISH CRANIOFACIAL AND FIN REGENERATION

During the early stages of embryo development, the formation of the skeletal system is carefully controlled by specialized growth factors that are secreted locally. One such group of growth factors is known as bone morphogenetic proteins (BMPs), which serve multiple functions and play crucial roles in the maturation of cartilage and the differentiation of osteoblasts. To transmit the BMP signals from the cell membrane receptors to the nucleus, two pathways are involved: the canonical Smad pathway and the noncanonical p38 mitogen-activated protein kinase (MAPK) pathway. The extracellular regulation of BMP signalling occurs through interactions with proteoglycans (PGs) present in the extracellular matrix (ECM). These PGs generally modulate the efficiency of binding between the ligands and their receptors. During the process of endochondral ossification, which involves bone formation through cartilage, chondrocytes (cartilage cells) produce an ECM abundant in PGs. Following the formation of cartilage, bone formation occurs in the surrounding perichondrium as certain chondrocytes express Indian hedgehog (Ihh), a factor that induces osteoblasts, while undergoing a precisely regulated maturation process. Interestingly, the PG-rich ECM somehow inhibits the maturation of cartilage, including cellular hypertrophy and the expression of Ihh and Col10a1 genes. Given that BMPs are known to promote cartilage maturation, I hypothesize that PGs normally act as inhibitors of BMP signalling. To investigate this hypothesis, using various experimental manipulations we evaluated BMP signalling in chondrocytes of both wild-type zebrafish and zebrafish with a mutation in the PG gene fam20b-/-, which encodes a kinase responsible for phosphorylating xylose in the GAG side chain. The results showed that the levels of phosphorylated Smad1/5/9 (p-Smad1/5/9) were increased only when mutant fam20b chondrocytes secreted PGs, while the phosphorylation of p38 (p-p38) remained unaffected. As anticipated, the levels of p-Smad1/5/9 decreased in chondrocytes treated with DMH1, an inhibitor of the BMP receptor kinase domain, and in chondrocytes of zebrafish with a dominant-negative BMP receptor (dnBMPR). Only treatment with DMH1, and not the dnBMPR condition, reduced p-p38 levels in the chondrocytes. However, in both DMH1-treated and dnBMPR zebrafish the expression of two markers of chondrocyte maturation, ihha and col10a1, was decreased, and formation of perichondral bone was diminished. Next, to confirm the regulatory\nrole of PGs in BMP-dependent cartilage maturation and in the timing of endochondral ossification, we were able to rescue the early expression of ihha and col10a1 genes as well as the formation of perichondral bone in fam20b mutant zebrafish by treating them with either DMH1 or dnBMPR. These rescue findings supported the hypothesis that PGs normally inhibit canonical BMP-dependent cartilage maturation, thereby influencing the rate and onset time of endochondral ossification. Additionally, we hypothesized that PGs typically inhibit BMP signalling during the process of intramembranous (or dermal) ossification, where bone tissue develops directly from mesenchymal cells without the presence of a cartilage precursor. We investigated the development of dermal bones in the craniofacial region of zebrafish to further test our hypothesis. Using a Tg(5xBMPRE-Xla.Id3:GFP)ir1189 transgenic zebrafish line that exhibits green fluorescent protein (GFP) expression in response to BMP signalling, the involvement of BMP signalling in the development of ossified dermal elements was confirmed. However, when embryos were treated with DMH1, the development of craniofacial dermal bone remained unchanged. While most craniofacial dermal bones of fam20b-/- zebrafish showed no discernible differences in their formation during embryonic development, we did observe an early initiation of dermal bone formation derived from the perichondrium, specifically in the dentary and quadrate bones. To test the hypothesis further, the process of fin regeneration was used as an additional model for investigating intramembranous ossification. Through the analysis of GFP expression in two BMP-responsive reporter lines, namely Tg(5xBMPRE-Xla.Id3:GFP)ir1189 and Tg(BMPRE:EGFP)pt510, a relationship was observed, linking the GFP signal as an indicator to the development of bony rays during fin regeneration. Furthermore, regeneration of caudal fin dermal bony rays in fam20b-/- zebrafish was not impaired. In adult zebrafish, the repercussions of DMH1 treatment were clear as it significantly hindered the regenerative capacity of adult fins, supporting the involvement of BMP signalling in this process. Overall, this study demonstrates the inhibitory role of PGs in BMP signalling during cartilage maturation and the influence of PGs on the rate and onset time of endochondral ossification. Furthermore, this study unveils a novel observation, the early formation of dermal bones originating from the perichondrium in fam20b-/- zebrafish mutants.

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