Understanding the role of osteoclasts in wild-type craniofacial development and genetic mapping of skeletal mutants in zebrafish

Date
2023
DOI
Authors
Miao, Kelly Z.
Version
OA Version
Citation
Abstract
Detailed understanding of skeletal development, especially in the craniofacial space, requires close observation of growth patterns along with understanding of the genetics involved. Prior work on craniofacial development has focused on imaging data taken at specific timepoints on fixed specimens. The zebrafish offers advantages as a model organism to address the extant challenges in this field of research as many aspects of skull formation and general skeletal development are conserved from zebrafish to mammals. Zebrafish remain optically clear well into the juvenile stage, encompassing the core period of bone development in the skull. Taking advantage of many fluorescent transgenic lines marking cells that contribute to skeletal development, our lab has developed protocols to perform confocal live imaging multiple times on individual fish. I have used those imaging protocols for two main objectives: 1) to establish atlases of tissue patterning during cranial development; and 2) to characterize craniofacial and general skeletal development in mutants identified through forward genetic screens. Using serial low-magnification confocal imaging, fish carrying transgenic markers for osteoblasts (sp7:mCherry) and chondrocytes (col1a1:eGFP) were tracked for approximately 30 days, focusing on bones of the skull vault. We defined three stages of growth: initiation by osteoblasts on a cartilage scaffold; a period of rapid planar growth by the frontal and parietal bones; and a plateauing of growth and maturation of the sutures. We captured confocal Z-stacks which were converted into 3D models. To further understand the complex cell interactions of craniofacial development, we used a transgene marking osteoclasts, bone-resorbing cells which represent the innate immune component within the bone. Osteoclasts are critical in homeostasis of adult skeletal structures, but there is limited information on the role of osteoclasts in craniofacial development. We used confocal microscopy of live transgenic fish to determine the location of osteoclasts in the developing skull daily over several weeks, from 5.18 mm to 9.6 mm standard length (approximately 15 to 34 days post fertilization). The overall distribution of osteoclasts is consistent among individuals, but they are sparse in most areas of the skull and the exact locations vary among fish and across developmental time points. We observed osteoclasts concentrating at areas of remodeling associated with neuromasts and their associated nerves, the hyomandibular foramina and around the supraorbital lateral line. This suggests that they play a special role in bone remodeling around neuromasts and nerves during craniofacial development. In csfr1amh5 mutants lacking functional osteoclasts, the morphology of the cranial bone was disrupted in both areas. The hyomandibular foramen is present in the initial cartilage template, but after ossification has begun, the diameter of the canal is significantly smaller in the absence of osteoclasts. The diameter of the supraorbital lateral line canals was also reduced in the mutants, but the more significant difference was the paucity of small foramina in and around the canals, which allow passage of efferent nerves through the bone. These findings define important and previously unappreciated roles for osteoclast activity during craniofacial development. With this data on baseline developmental benchmarks and tissue patterning in wild-type individuals we then focused on two skeletal mutants, toth and jinx, identified in a genetic screen. toth was originally identified due to severely lagging growth and structural skull abnormalities, including numerous pits in the frontal and parietal bones. Mutants for toth have a dramatic accumulation of osteoclasts on the frontal and parietal bones corresponding to the pits in the bone. jinx mutants have craniofacial irregularities but have additional skeletal abnormalities, including sporadic fusion of vertebral bodies. Using the sp7:mcherry transgenic line and fluorescent vital staining of mineralized bone, I have documented uneven mineralization and vertebral fusion events in live jinx mutants during development. We have localized the genes for toth and jinx based on filtering bulked sequencing for single nucleotide polymorphisms. I have further refined the mapping of jinx using microsatellite markers to implicate the candidate gene entpd5a, which has previously been implicated in bone mineralization in zebrafish. I am performing additional experiments to verify the gene identity and mechanisms underlying the defects. My thesis presents a body of work demonstrating the power of live imaging in zebrafish to reveal developmental processes. We created an atlas of bone and cartilage morphology during normal skull development and building on that foundation demonstrated previously unknown requirements for osteoclasts in sculpting skull morphology. We have applied the same imaging tools to characterize developmental processes in two zebrafish mutants with craniofacial and skeletal defects, and in future experiments will be able to correlate those defects with specific genetic lesions.
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