Mammalian DRC, Ciliary, Motility and Left‐Right Body Asymmetry

Abstract

Many advances made in our understanding of the molecular basis of ciliopathies in humans have come through the identification and analysis of candidate ciliary proteins in lower model systems. For example, Growth Arrest Specific 8 (Gas8) has been implicated in cilia motility based on its Chlamydomonas homolog pf2 showing it is a component of the Nexin-dynein regulatory complex (NDRC). However, the study of Gas8 in mammalian systems is very limited, and unexpectedly, recent data indicate that Gas8 localizes to the base of primary non-motile cilia and knockdown studies indicate that Gas8 has a role as a positive regulator of Smoothened activity and its ciliary localization. Here we investigate the in vivo role of Gas8 by generating a mutant mouse line. Gas8 mutant mice did not have any overt phenotypes expected if Hedgehog signaling is affected. Gas8 mutants did present with situs inversus implicating the NDRC in mammalian left-right body axis specification and in the normal function of node cilia. In addition, to the LR defects, Gas8 mutants invariably develop hydrocephalus and were sterile. Our analyses of cilia motility in the ventricles of the brain and trachea show abnormalities in cilia generated fluid movement associated with changes in beat frequency, waveform, and structure of the cilium. We show Gas8 is required for proper assembly of the inner dynein arms (IDA) and the Nexin-Dynein Regulatory Complex (NDRC). Additionally, a screen for GAS8 mutations in human patients with situs defects identified two independent homozygous missense mutations. The two independent mutations at c.595G>A and c.1172C>T interrupt the Gas8 Microtubule Association Domain (GMAD) and the putative Smoothened binding domain, respectively. We hypothesize that both of these mutations will result in disruption of NDRC assembly and cause dyskinetic motile cilia. The pathogenicity of these mutations and their effect on NDRC formation is being analyzed in Chlamydomonas, zebrafish, and mouse lines that express the human disease alleles.