A major goal in human health is to improve the ability of large fractures and skeletal wounds to heal. In contrast to mammals, many amphibia and lizards do have a remarkable ability to reform entire limb and/or tail skeletons, yet the relative lack of genetic tools in these species have limited progress towards the underlying cellular and molecular mechanisms. Here, we present a new model of skeletal regeneration in the genetically tractable zebrafish. In a matter of just a few weeks, adult zebrafish can regenerate nearly two-thirds of their lower jawbone, and they appear to do so through an unusual chondrocyte population that directly produces woven bone. As potentially similar cells have been observed during mammalian fracture repair, a better understanding of these cells during skeletal repair, as well as how they contribute to more extensive regeneration in lower vertebrates, will aid in developing novel therapies for improving bone repair in patients. In the first aim, purification and expression profiling of regenerating chondrocytes, which express markers of both chondrocytes and osteoblasts, will determine the extent to which these cells are hybrid chondrocytes/ osteoblasts. Genes specifically upregulated in early regenerating chondrocytes will also indicate potential pathways that induce these cells in response to injury. Next, we use newly developed Cre/Lox transgenic lines to test the origins and long-term fate of regenerating chondrocytes. In particular, we test that the periosteum is a major source of regenerating chondrocytes, with these directly converting into the osteoblasts that produce woven bone. Using a novel intersectional transgenic strategy to specifically ablate regenerating chondrocytes, we then test that these cells are required for the large-scale regeneration of bone in the zebrafish jaw. During the development of endochondral bone, the majority of chondrocytes undergo hypertrophy and apoptosis, with bony matrix being produced by invading osteoblasts. Quite differently during regeneration, our preliminary data suggest that many chondrocytes directly differentiate into osteoblasts. Using an adult viable ihha mutant and a transgenic strategy to inhibit Hh signaling only in regenerating chondrocytes, we test in the second aim that persistently high Ihh signaling is essential for regenerating chondrocytes to differentiate into osteoblasts. The completion of these Aims will test a model that the ability of regenerating chondrocytes to directly make bone allows a rapid restoration of rigidity in a damaged body part, with the initial woven bone later being remodeled into mature bone. In the long-term, we plan to use lessons taken from this new zebrafish model to devise strategies to augment the inherent ability of the skeleton to repair critical size defects. !

Public Health Relevance

A major challenge in the clinic is the inability of large skeletal wounds to adequately repair. In contrast, lower vertebrates such as zebrafish have a much higher capacity to regenerate their skeletons after injury. The study of the genes and cell behaviors required for such robust skeletal regeneration will therefore provide novel insights into how to augment skeletal repair in patients.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21DE023899-01
Application #
8620576
Study Section
Skeletal Biology Development and Disease Study Section (SBDD)
Program Officer
Lumelsky, Nadya L
Project Start
2014-09-01
Project End
2016-08-30
Budget Start
2014-09-01
Budget End
2015-08-30
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Southern California
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Paul, Sandeep; Schindler, Simone; Giovannone, Dion et al. (2016) Ihha induces hybrid cartilage-bone cells during zebrafish jawbone regeneration. Development 143:2066-76
Paul, Sandeep; Crump, J Gage (2016) Lessons on skeletal cell plasticity from studying jawbone regeneration in zebrafish. Bonekey Rep 5:853