Defining the Progenitor Population in Adult Zebrafish Jaw Bone Regeneration

 描述（由申请人提供）：每年发生约800万例骨折，其中10%为延迟性骨折和骨不连骨折，尽管进行了治疗干预，但仍无法愈合。目前的治疗策略受到自体骨供应有限和人工材料机械性能差的严重阻碍。斑马鱼是再生的大师，我发现成年斑马鱼在切除后可以迅速再生一半的下颌骨。颅面骨发育在斑马鱼和人类之间是高度保守的，因此了解斑马鱼的大规模骨再生可能会导致患者的新治疗。斑马鱼颌骨再生的一个独特特征是涉及一种不寻常的骨生成软骨细胞群。在本申请中，我的目标是确定在大规模骨再生过程中产生这些骨化软骨细胞的祖细胞群，以及巨噬细胞在刺激这一祖细胞群中的作用。利用Cre-based的转基因谱系追踪策略，我将测试在骨膜中的前破骨细胞的Runx 2 +/Sp 7-群体在再生过程中产生软骨愈伤组织。使用基因消融策略，然后我将测试巨噬细胞是骨修复中最早期事件所必需的，即骨膜细胞从稳态期间制造骨到修复期间制造软骨的转变。最后，我建议利用RUNX 2：GFP转基因分离这些骨膜细胞，对表达的mRNA进行深度测序，这将有助于识别损伤后骨膜细胞中激活的巨噬细胞依赖性基因。总之，积极的发现将阐明免疫系统在骨修复过程中将骨膜细胞的命运从骨转移到软骨中的作用，斑马鱼模型中获得的知识将在未来用于开发新的患者骨修复策略。

 DESCRIPTION (provided by applicant): Approximately 8 million bone fractures occur annually and 10% of these are delayed and nonunion fractures which fail to heal despite therapeutic intervention. Current therapeutic strategies are severely hindered by the limited supplies of autograft bone and the poor mechanical properties of artificial materials. Zebrafish are masters of regeneration, and I have found that adult zebrafish can rapidly regenerate up to half of its lower jawbone after resection. Craniofacial bone development is highly conserved between zebrafish and humans, and hence understanding large-scale bone regeneration in zebrafish may lead to novel treatments in patients. A unique feature of zebrafish jawbone regeneration is the involvement of an unusual bone-producing chondrocyte population. In this application, I aim to identify the progenitor population that generates these ossifying chondrocytes during large-scale bone regeneration, as well as the role of macrophages in stimulating this progenitor population. Utilizing Cre-based transgenic lineage tracing strategies, I will test that a Runx2+/Sp7- population of pre-osteolasts in the periosteum generate the cartilage callus during regeneration. Using a genetic ablation strategy, I will then test that macrophages are required for the earliest events in bone repair, namely the shift of periosteal cells from making bone during homeostasis to making cartilage during repair. Lastly, I propose to utilize a RUNX2:GFP transgene to isolate these periosteal cells for deep sequencing of expressed mRNAs, which will help identify macrophage-dependent genes activated in periosteal cells after injury. Together, positive findings will illuminate the role of the immune system in shifting the fate of periosteal cells from bone to cartilage during bone repair, with knowledge gained in the zebrafish model being used in the future to develop new bone repair strategies in patients.