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Requirement Of Wnt Modulator R-spondin3 In Craniofacial Morphogenesis, Dental Development And Bone Homeostasis
Nora Alhazmi, BDS, MS1, Shannon H. Carroll, PhD2, Katherine Woronowicz, PhD3, Claudio Macias Trevino, BS4, Shawn Hallet, BA5, Edward Li, BA4, Francesca Gori, PhD1, Matthew Harris, PhD3, Eric Chien-Wei Liao, MD, PhD6.
1Harvard School of Dental Medicine, Boston, MA, USA, 2Shriners Hospital for Children, Boston, MA, USA, 3Childernís Hospital Boston, Boston, MA, USA, 4Harvard Medical School, Boston, MA, USA, 5Michigan School of Dental Medicine, Boston, MA, USA, 6Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Boston, MA, USA.

PURPOSE:
Investigating the expression and function of Wnt modulator rspo3 in craniofacial development will improve our molecular understanding of craniofacial development.
METHODS:
Using ChIPseq and RNAseq, we identified rspo3 as an irf6 target gene. In zebrafish, rescue of irf6 mutant was sufficient to rescue rspo3 gene expression, suggesting that rspo3 acts downstream of irf6. Single-cell gene detection technology (RNAscope) was used to delineate rspo3 expression in the craniofacial region during development. CRISPR gene editing was used to generate rspo3 mutant and detailed phenotype analysis was carried out to analyze ossification and bone density of the mutants.
RESULTS:
Using RT-qPCR, rspo3 mRNA was reduced in irf6 mutant. However, injection of irf6 mRNA was able to rescue the expression of rspo3. RNAscope showed co-expression of irf6 and rspo3 mRNA in the pharyngeal arches indicating its importance in zebrafish craniofacial development. RNAscope technique identified a unique expression of rspo3 in the perichondrium of the trabecula as well as in Meckelís cartilage. CRISPR targeted mutagenesis successfully generated rspo3 mutants. The embryonic palate and Meckelís cartilage morphology were very modestly disrupted in the rspo3 mutant. In contrast, as the cartilage elements underwent ossification and the animals matured, the mutant craniofacial skeleton exhibited dramatic defects in bone density, dysmorphology of maxillary and mandibular elements, disorganized relationship between bones, and pathogenic fractures. Formation, maturation and morphology of the dentition were also disrupted.
CONCLUSIONS:
We discovered that rspo3 is a target gene of irf6, and is expressed in perichondrial cells that are juxtaposed to the oral epithelium and cartilage structures. We show that rspo3 function is required in a non-cell autonomous way to regulate osteogenesis of the chondrogenic cells, and that rspo3 is also required for bone homeostasis. In contrast to the many genes known to affect embryonic craniofacial development, this study highlights the key function of rspo3 and Wnt signaling in bone and tooth homeostasis. We are now interrogating the genetic interaction between rspo3 with Wnt genes such as wls, wnt9a and gpcs. Taken together, this work uncovered the transcriptional activation of rspo3 by irf6 in craniofacial morphogenesis and integrates rspo3 function and Wnt signaling during palatogenesis.


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