Smad6 deficient zebrafish as a model for multifactorial craniosynostosis

颅缝闭塞（CS）是最常见的颅面出生缺陷之一，影响近1/2000

Craniosynostosis (CS) is one of the most common craniofacial birth defects, affecting nearly 1/2000 infants. About 20% of cases are caused by mutations in single genes; most common are activating mutations in the genes encoding FGF receptors, with additional mutations identified in other genes, both transmitted and de novo. Existing mouse models for single gene CS have yielded significant insights into the molecular and developmental basis of CS and serves as a powerful illustration of the value of accurate animal models. However, the majority of CS cases are nonsyndromic, and the underlying genetic risk is more complex and probably also involves interactions with environmental factors. Recent human genetics studies have implicated several specific genes in some of these complex cases. Genome wide association studies (GWASs) of CS cases identified risk loci near the BMP2 and BMP7 genes. Subsequent whole exome sequencing (WES) of individual CS patients found a high incidence of mutations inactivating one copy of the gene encoding Smad6, a downstream inhibitor of BMP signaling. Individuals carrying the risk locus near BMP2 and one SMAD6 mutant allele reportedly had a greatly increased incidence of CS, suggesting a straightforward hypothesis that the combination of increased levels of BMP2 and reduced inhibitory Smad6 causes CS. Although this hypothesis has been challenged by more recent studies, there is also evidence that mutations in other genes can interact with SMAD6 to increase CS risk. To directly test these hypotheses, we propose to develop a zebrafish model to study the complex genetic risk factors underlying CS. We have generated inactivating mutations in the two zebrafish smad6 genes, and in Aim 1 we will characterize the phenotypes of fish lacking one or both copies of smad6a and smad6b. In Aim 2, we will take a two– pronged approach to assess genetic CS risk in zebrafish. First, we will cross the smad6 mutants with existing mutants for inhibitors of the BMP pathway, and with mutants for other genes implicated in human patients. We will assay the resulting fish for CS and other craniofacial and skeletal abnormalities, using histological staining and live confocal imaging. Second, we will use overexpression of human SMAD6 variants in zebrafish embryos as an efficient assay, to test the functional consequences of sequence variants identified in human patients. Through successful completion of our aims, we will establish an accurate model system for the complex genetics underlying the majority of CS cases. Zebrafish are amenable to in vivo imaging and direct manipulations during all stages of skull and suture formation, providing insight into the pathophysiology of CS. Also importantly, the model can be used to test other genes that may interact with smad6 mutations to increase CS risk, and will provide a sensitized genetic background to assess potentially contributing environmental factors.