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Osteoporotic Phenotype of Newly Identified Zebrafish Mutant
Sophia A. Bidny, BA1,2, Joanna K. Ledwon, PhD1, Jacek Topczewski, PhD3, Arun K. Gosain, MD4, Jolanta Topczewska, PhD1.
1Northwestern University Feinberg School of Medicine, Department of Surgery Plastic Division, Stanley Manne Children's Research Institute, Chicago, IL, USA, 2University of Illinois at Chicago, Chicago, IL, USA, 3Northwestern University Feinberg School of Medicine, Department of Pediatrics, Chicago, IL, USA, 4Division of Pediatric Plastic Surgery, Ann and Robert H. Lurie Children's Hospital of Chicago and Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

PURPOSE: A novel mutation was discovered in our laboratory with a striking feature of a misshapen, smaller skull, prompting the “microcephaly” name. By genetic crossing of the mutant with wild type zebrafish it was determined that the mutation has Mendelian and recessive inheritance. We evaluated the membranous ossification, focusing on the flat bones of the cranium: frontal and parietal. These results revealed that membranous bones become porous in adult mutants as compared to normal siblings. We developed a working hypothesis that the porosity of the flat bones is caused by too much bone-resorbing activity provided by osteoclasts.
METHODS: The characterization of the mutant was undertaken through several different methods. Cranial vault development was evaluated in order to establish a timeline of bone defects by using alizarin red and alcian blue staining. Paraffin embedded cross sections of the skull were analyzed histologically and by TRAP stain to estimate osteoclasts' activity. In order to evaluate development of osteoblasts and osteoclasts, specific genetic markers were examined through RNAscope in situ hybridization on paraffin sections. Search of the genetic identity of the mutation is in progress. The genomic DNA isolated from 50 mutant and 50 normal siblings was sequenced and comparatively analyzed for loss of heterozygosity. This mapped the mutation on chromosome 15. Using various primers specific to this region and for PCR method we narrowed down this region to a few candidate genes.
RESULTS: The skeletal staining revealed smaller and irregular frontal and parietal bones in the adult mutant, as opposed to normal siblings. In some places the bone was so thin as to produce holes in the cranium. These malformations can be seen in Figure 1 - the normal head skeleton of the adult zebrafish is shown in (A), the mutant in (B), dissected flat bones of the wild type in (C) and porous bones of the mutant in (D).
Figure 1:

To verify the osteoclast hypothesis we performed TRAP staining. This revealed much stronger activity of the osteoclasts in the mutant (Figure 2, B) as compared to the wild type (Figure 2, A). The RANKL expression presented in Figure 2 (C,D) shows that it is also increased in the mutant (D), supporting our initial hypothesis that increased osteoclast activity is causing the porous effect.
Figure 2:

CONCLUSION: The phenotype of the porous cranium suggests two possibilities: overactive osteoclasts, or more osteoclasts developed in this mutant, causing this osteoporotic defect. Once the verification of these results would be completed, and the mutation would be identified, the microcephaly mutant could provide a model for osteoporosis study. Zebrafish are especially suitable for pharmacological treatment as drugs can be provided directly to the fish water, allowing to simultaneously evaluate the benefits and toxicity of drugs.

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