Identification of gene involved in late stage zebrafish craniofacial development

描述(申请人提供)：所有脊椎动物共有的骨形成过程，在人类骨骼发育不良中经常受到影响，导致颅面部骨骼发育严重缺陷。颅骨由软骨内骨和膜内骨组成，软骨内骨通过软骨矿化发育，膜内骨直接由浓缩的间充质细胞发育而来。软骨形成和/或成骨过程中的缺陷会导致头面部缺陷和骨骼发育不良，许多导致这些骨骼畸形的基因仍不清楚。尽管鱼和人类的头骨截然不同，但许多骨骼元素是同源的，形成这些结构所需的基因是保守的。因此，显示骨骼畸形的斑马鱼突变体为研究人类颅面部骨骼发生和骨骼发育不良提供了一个理想的系统。我们的实验室已经发现了一种自发突变，称为Koliber(Kol)，它表现出晚发的骨骼表型。骨和软骨染色显示颅面部骨骼生长板有骨质疏松症、软骨丢失和骨融合。利用位置克隆，我们已经缩小了Kol关键区到1号染色体的范围，并确定了一个候选基因。有趣的是，斑马鱼基因组的这一区域与哺乳动物具有基因组同步性；这一关键区域内的基因以前没有与斑马鱼的骨骼发育有关，这表明Kol突变可能为研究头面部骨骼发育提供了新的见解。本研究旨在鉴定Kol突变体，确认Kol突变，并确定受Kol突变影响的信号转导途径。涉及骨骼和软骨染色、基因组编辑和转基因报告线的技术将被用来比较正常和突变鱼类的头骨形成过程。以前在斑马鱼和小鼠中发现的大多数突变都会影响早期至关重要的基因 在头面部骨骼发育的各个阶段，而涉及后期阶段的基因在很大程度上还没有被研究。幼年脊椎动物骨形成所必需的新基因或途径的发现将为儿童颅面部骨骼发育问题提供重要的见解。Kol突变体可能会为这一相当未被研究的骨骼发育领域提供新的线索。

DESCRIPTION (provided by applicant): The processes of bone formation, shared by all vertebrates, are often affected in human skeletal dysplasias, resulting in significant defects to craniofacial skeletogenesis. The skull is formed from endochondral bones, which develop through mineralization of cartilage, and intramembranous bones, which develop directly from condensed mesenchymal cells. Defects in the processes of chondrogenesis and/or osteogenesis lead to craniofacial defects and skeletal dysplasias, and many of the genes responsible for these skeletal malformations remain unknown. Although the skulls of fish and humans are distinctly different, many of the skeletal elements are homologous and the genes required to form these structures are conserved. Therefore, zebrafish mutants displaying skeletal dysmorphologies provide an ideal system to study human craniofacial skeletogenesis and skeletal dysplasias. Our laboratory has identified a spontaneous mutant, referred to as koliber (kol), which displays a late onset skeletal phenotype. Bone and cartilage staining show defects consisting of hyperossified bone, as well as cartilage loss and bone fusion in the growth plates of the craniofacial skeleton. Using positional cloning, we have narrowed down the kol critical region to Chromosome 1 and identified a candidate gene. Interestingly, this region of the zebrafish genome shares genomic synteny with mammals; no gene within this critical region has previously been implicated in zebrafish bone development, suggesting the kol mutation may provide novel insight into craniofacial skeletogenesis. This proposal aims to characterize the kol mutant, confirm the kol mutation and determine the signaling pathways affected by the kol mutation. Techniques involving the use of bone and cartilage staining, genome editing and transgenic reporter lines will be used to compare the processes of skull formation in normal and mutant fish. Most mutations previously identified in zebrafish and mice affect genes vital to early stages of craniofacial skeletal development, while genes involved in later stages have largely been unstudied. Discovery of a novel gene or pathway necessary to juvenile vertebrate bone formation will provide significant insight into problems of pediatric craniofacial skeletal development. The kol mutant may shed new light on this considerably unstudied area of bone development.