Identifying Osteoporosis Genes by Whole Genome Sequencing and Functional Validation in Zebrafish

Large-scale biological approaches such as forward screens and systems biology-based investigations are recognized to be vital for the advancement of biomedical research. Zebrafish (a small tropical freshwater fish that has gained prominence for its use in developmental biology) are ideal for such approaches due to their unique attributes such as optical transparency, ease of genetic manipulation, small size, and low cost. We are currently developing new bone phenotyping technologies in zebrafish for rapid mapping of gene-to-phenotype relationships. An example can be seen in the above schematic, which demonstrates the generation of a musculoskeletal “bar­code” containing 576 different descriptors of axial muscle and bone morphology for a single zebrafish. The barcode was computed using a rapid MicroCT-based phenotyping platform which we developed. Such barcodes can be used to identify emergent or coordinated behaviors between many different muscles and bones in normal and pathological conditions.

We are currently using these phenotyping technologies to understand the genetic archiecture of skeletal diseases. One such disease is osteoporosis, which is a major, worldwide public health burden. For NIH/NIAMS grant AR072199, we are using human genetic studies in conjunction with rapid-throughput screening in zebrafish to identify new druggable targets to overcome the current treatment crisis in osteoporosis. In collaboration with Dr. Yi-Hsiang Hsu (Harvard), we are utilizing whole genome sequencing in large well-phenotyped populations, as well as deep phenotyping in CRISPR-edited zebrafish mutants, to identify potential causal variants and targeted genes influencing skeletal integrity.

Neuroskeletal Systems Biology in the Zebrafish SkeletonMontage

The use of simple vertebrate models to identify the molecular and cellular basis of neuromuscular regulation of bone represents a powerful yet unexplored strategy to uncover homologous pathways in higher organisms. In this context, in vivo screens and rapid gene knockdown strategies hold unique potential to identify extraskeletal pathways regulating osteogenesis, however such strategies are largely inaccessible in traditional in vivo models of bone anabolism. Toward overcoming this hurdle, in this project we will develop the regenerating zebrafish tail fin, a model of intramembranous ossification that recapitulates the major phases of mammalian bone formation, as a rapid genetic platform for neuroskeletal pathway discovery.

Following fin amputation, osteoblasts at the stump de-differentiate to form a proliferative mass of cells called the blastema, and then re-differentiate to undergo bone formation. The rate of bone growth during this process is remarkable, as new bone segments are readily observed within 3-5 days following amputation (with the majority of lost bone, joints, nerves, skin and blood vessels restored within a few weeks).

For NIH/NIAMS grant AR066061, we are currently integrating novel bone phenotyping technologies and exploiting the amenability of zebrafish to genetic manipulation and high-throughput approaches to pursue large-scale, systems-based investigations of bone formation and mineralization in the regenerating fin. In particular, our central objective is to integrate quantitative bone imaging in the regenerating fin with zebrafish knockdown and screening strategies to identify novel neural regulators of bone outgrowth, patterning, and mineralization. If successful, these studies will establish powerful in vivo assays for neuroskeletal pathway assessment in the regenerating fin, develop the technological toolbox for measuring bone growth and mineralization in this process, and identify valuable chemical entry points for neuroskeletal discovery. In doing so, this project will advance the regenerating zebrafish fin as a novel in vivo model for the emerging field of neuroskeletal systems biology, and catalyze broader pathway and therapeutic screening efforts in the zebrafish skeleton.