The role of Hedgehog signaling in bone formation Tyler Huycke

Bone formation occurs via two pathways Endochondral Intramembranous Are these mechanisms genetically similar? Question: What is the functional role of ihh (indian hedgehog) in bone formation? n/Hum12wk/images/headbone.jpg

Bone formation occurs via two pathways Osteoblasts intramembranous chondrocytes perichondrium Osteoblasts endochondral

The role of Hedgehog signaling in endochondral ossification Hedgehog pathway mediates signaling between cartilage and perichondrium during endochondral ossification Maturing chondrocytes express ihh (indian hedgehog) Ihh is secreted to the surrounding tissue, the perichondrium Signal is hypothesized to induce the differentiation of perichondral cells to osteoblasts Ihh perichondrium chondrocytes

Ihh -/- mice lack osteoblast development in endochondral bones In addition, Ihh -/- show reduced chondrocyte proliferation and skeletal dwarfism wt Ihh -/- St Jacques et al., 1999 wt Ihh -/- wtIhh -/-

ihh is expressed during zebrafish development Zebrafish express both ihha and ihhb ihha and ihhb expression detected during skeletal development in craniofacial elements Avaron et al., 2005

ihha mutant zebrafish likely contain a null allele 3 exons Mutant allele in zebrafish has a nonsense mutation in exon 1 Y44  stop codon Results in truncated N-terminal peptide which is essential to proper signaling mutation

Assaying for a mutant phenotype Used Alizarin Red live staining to assess amount of chondral bone wt ihha -/- 6dpf 10dpf op bsr op bsr ch hm bsr op Dermal Bone Chondral bone

ihha mutants show reduced chondral bone Assayed for the presence of chondral bone at 6 and 10dpf Separated fish based on elements ossified 70% of fish with reduced ossification at both 6 and 10 days (n=20) were mutants 0% with heavy ossification at both day 6 and 10 were mutant (n=19) wt ihha -/- 6dpf 10dpf ch hm

Ossification of chondral bone is recovered later in development Little phenotypic difference in chondral bone observed at 17 and 21dpf 17dpf21dpf wt ihha -/- ch hm Bone recovered by ihhb?

Bone formation occurs via two pathways Osteoblasts intramembranous chondrocytes perichondrium Osteoblasts endochondral

ihha mutants display patterning defects in the opercular series Opercular series and Branchiostegal rays arise through intramembranous ossification op sop iop bsr op sop iop bsr wt ihha -/-

Ihha mutants display bone fusion and ectopic ossification ihha -/- wt 10dpf14dpf17dpf21dpf

Conclusions ihha -/- fish display less chondral bone than wild type the factor appears to be inducing osteoblast differentiation during endochondral ossification ihha also appears to be involved with intramembranous ossification, but mechanism is unclear

Future directions Determine the mechanism of ihh in intramembranous ossification – Transgenic background – Molecular markers Is it acting similarly or different than in endochondral ossification? Analyze other mutants in the hedgehog pathway such as ptc ptc2 -/- wt

Thank you Kimmel Lab SPUR Charles Kimmel Peter O’Day Brian Eames Chelsie Fish Mark Sasaki SPUR participants April DeLaurier Jamie Nichols Jared Talbot Bonnie Ullmann John Dowd Vishesh Khanna