Thread by @paul_sandeep

Hi Everyone,
Very excited to present our recent work @eLife. We wanted to see whether conversion of the cartilage template to bone is conserved from zebrafish to mammals.
elifesciences.org/articles/42736

It is reported that chondrocytes in mammals undergo programmed cell death while invading blood vessels bring in osteoblasts that form trabecular bone.
cell.com/developmental-…

Other studies suggest many chondrocytes escape cell death and give rise to osteoblasts/osteocytes adipocytes.
pnas.org/content/early/…

Adult fish chondral bones are fat-filled yet do not support hematopoiesis.
doi.org/10.1111/j.1469…

Here we address the long term fate of growth plate chondrocytes and their plasticity in zebrafish using the bone Ceratohyal (CH) that exhibits properties similar to mammalian long bones.
dev.biologists.org/content/143/12…

We performed histology from juvenile to adult. We found evidence of bone collar remodeling along with cartilage matrix degradation from 12-16mm Standard Length (SL) with first adipocytes visible by 16mm SL.

This breakdown coincides with vascularization of the CH.

To address the long term fate of chondrocytes we generate inducible Cre lines expressed in chondrocytes, Sox10:CreERT2, Col2:CreERT2 and crossed them with reporter line bactin2:loxP-tagBFP-stop-loxP-DsRed (Blue to Red conversion: B>R)

Both lines showed the contribution of Sox10 and Col2 lineages to osteoblasts and adipocytes. The Col2 line was made using an “R2” enhancer that is highly specific to chondrocytes

doi.org/10.1016/j.devc…

The chondrocyte lineage osteoblasts co-express osteoblast markers sp7:GFP and ocn:GFP and get embedded in the mineralized matrix.

A limitation of Cre base lineage tracing is ruling out the contribution of cells other than chondrocytes, viz periosteal or perichondral due to low-level expression of chondrocyte promoters.

To rule this out we generated Col2a1a:Histone2A-mCherry-T2A-GFP-CAAX that expressed long-lived Histone-mCherry fusion protein and short-lived GFPcaax that serve as a “timer” for promoter activity.

We observed histone-mCherry+ nuclei in matrix-embedded osteocytes and osteoblasts marked by co-expression of RUNX2:GFP, sp7:GFP and ocn:GFP, early to late osteoblast makers.

Furthermore, we found that hypertrophic chondrocytes re-enter cell cycle (BrdU+ve) and start expressing Leptin Receptor (lepr) while mmp9 is expressed in chondrocytes during remodeling.

mmp9-/- we generated resulted in an increase in growth plate consistent with loss of remodeling and a decrease in adipocyte numbers.

To test whether MMP9 is required in hematopoietic lineage vs chondrocytes we performed, we performed transplanted WT neural crest (chondrocyte precursors) into mmp9-/- and rescued growth plate size and adipocyte numbers phenotype.

The key takeaway point is that zebrafish chondral bones are very similar to those of mammals and can serve as a great model where (live) imaging or drug screening is desired.

Thanks for reading till the end. Would love to discuss more. Also a shout out to my former PI @CrumpLab and collaborators @D_F_GIOVANNONE and @simonejohanna81 and lastly thanks @Josh_Currie1 for inspiring me to write this summary with the one he wrote for his paper.

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