Plastic Surgery Research Council

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Osteoclast Activity Required For Cranial Suture Patency
Jolanta M. Topczewska, Ph.D.1,2, Joanna K. Ledwon, Ph.D.1,2, Cecil S. Qiu, B.S.1, Andrew S. Bi, B.S.1, Jacek Topczewski, Ph.D.1,2, Arun K. Gosain, M.D.1,2.
1Northwestern Feinberg School of Medicine, Chicagp, IL, USA, 2Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA.

Purpose:
Zebrafish continually produce new bone material, reshape it, and repair it as needed to sustain lifetime growth and skeletal integrity. This is accomplished by synchronized efforts of osteogenic cells such as osteoblasts, osteocytes, and osteoclasts. The elastic joints formed between flat bones of the cranium called sutures provide adaptation, which can be compromised by suture obliteration and bone fusion associated with disorders such as craniosynostosis. Here, we describe the genetic and histological study of the zebrafish koliber mutant, which presents with sporadically fused frontal bones.
The koliber mutation has a regulatory character with recessive, Mendelian inheritance. During embryonic development, heterozygotes and homozygotes are undistinguished phenotypically from the wild type siblings. Morphological changes, such as the shortening of body length and a noticeably rounded head, become visible at the larval stage. The mutant abnormalities advance with age significantly, though variations of the phenotype are typical for this mutation. Simultaneous staining for bone and cartilage revealed progressive loss of cartilage, severe compression of vertebral column, fusions of vertebrae, and sporadically fused frontal bones. We hypothesize that the koliber phenotype results from a functional imbalance between osteogenic cells.
Methods:
RNAscope in situ hybridization on paraffin sections was used to analyze genes' expression pattern. Histological and TRAP staining for osteoclast activity was also performed on paraffin sections. The qRT-PCR was used to verify genes expression level. To analyze the cellular organization of the sutural tissue, we implemented TEM. The osteogenic cells were statistically evaluated based on their position within the sutural tissue. All results were concluded based on a minimum of three independent experiments. Lurie Children's IACUC committee approved zebrafish husbandry and experimental methods.
Results:
Statistical analysis applied to histologically stained specimens revealed significantly more cells attached to the frontal bone of the mutant than wild type (72.7%) and 64.5% respectively (p = 0.02). RNAscope in situ identified these cells as terminally differentiated osteoblasts positive for bglap, suggesting intensive bone matrix synthesis. These results were supported by TEM evaluation, which additionally exposed more complex, collagen-rich ECM in the mutant. Concurrently, we observed a prominent reduction (by 50%) of acp5a transcription, the gene encoding TRAP activity, indicating lower activity of osteoclasts in the mutant. This was further confirmed by histological staining for TRAP enzyme. The qRT-PCR analysis for genes regulating osteogenic cells function revealed a significant deficiency in the transcription of genes required for osteoclasts' maturation but not for the development of their precursors. In addition, we observed increased expression of osteocyte markers such as phex and spp1.
Conclusion:
Our results suggest an inadequate balance between osteogenic cells, with more osteoid synthesis and a simultaneous deficiency in bone remodeling in the mutant. We propose that the excessive/irregular frontal bone growth observed sporadically in the wild type is normally corrected by recruited osteoclasts. In contrast, in the koliber mutant reduced osteoclast activity interferes with this functionality, resulting in bone fusion.


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