Identification of gene involved in late stage zebrafish craniofacial development

描述（由申请人提供）：骨形成过程是所有脊椎动物共有的过程，通常在人类骨骼发育不良中受到影响，导致颅面骨骼发生的显著缺陷。颅骨由软骨内骨和膜内骨形成，软骨内骨通过软骨的矿化而发育，膜内骨直接由浓缩的间充质细胞发育。软骨形成和/或骨形成过程中的缺陷导致颅面缺陷和骨骼发育不良，并且许多负责这些骨骼畸形的基因仍然未知。虽然鱼类和人类的头骨明显不同，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提供了一个理想的系统来研究人类颅面骨骼发生和骨骼发育不良。我们的实验室已经确定了一个自发突变，称为koliber（kol），它显示了迟发型骨骼表型。骨和软骨染色显示由过度骨化的骨组成的缺陷，以及颅面骨骼生长板中的软骨损失和骨融合。利用定位克隆，我们已经缩小了kol关键区域的1号染色体，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共享基因组同线性;在这一关键区域内的基因以前没有涉及斑马鱼骨骼发育，这表明kol突变可能为颅面骨骼发生提供新的见解。该提案旨在表征kol突变体，确认kol突变并确定受kol突变影响的信号通路。涉及使用骨和软骨染色，基因组编辑和转基因报告系的技术将用于比较正常和突变鱼的头骨形成过程。以前在斑马鱼和小鼠中发现的大多数突变都会影响对早期发育至关重要的基因。 在颅面骨骼发育的各个阶段，基因的表达水平都很高，而参与后期阶段的基因在很大程度上尚未研究。发现幼年脊椎动物骨形成所必需的新基因或途径将为儿童颅面骨骼发育问题提供重要的见解。kol突变体可能为骨骼发育这一相当未研究的领域提供新的线索。

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.