A diverse family of cytoskeletal dynein motors powers various cellular transport systems, including axonemal dyneins generating the force for ciliary and flagellar beating essential to movement of ...extracellular fluids and of cells through fluid. Multisubunit outer dynein arm (ODA) motor complexes, produced and preassembled in the cytosol, are transported to the ciliary or flagellar compartment and anchored into the axonemal microtubular scaffold via the ODA docking complex (ODA-DC) system. In humans, defects in ODA assembly are the major cause of primary ciliary dyskinesia (PCD), an inherited disorder of ciliary and flagellar dysmotility characterized by chronic upper and lower respiratory infections and defects in laterality. Here, by combined high-throughput mapping and sequencing, we identified CCDC151 loss-of-function mutations in five affected individuals from three independent families whose cilia showed a complete loss of ODAs and severely impaired ciliary beating. Consistent with the laterality defects observed in these individuals, we found Ccdc151 expressed in vertebrate left-right organizers. Homozygous zebrafish ccdc151ts272a and mouse Ccdc151Snbl mutants display a spectrum of situs defects associated with complex heart defects. We demonstrate that CCDC151 encodes an axonemal coiled coil protein, mutations in which abolish assembly of CCDC151 into respiratory cilia and cause a failure in axonemal assembly of the ODA component DNAH5 and the ODA-DC-associated components CCDC114 and ARMC4. CCDC151-deficient zebrafish, planaria, and mice also display ciliary dysmotility accompanied by ODA loss. Furthermore, CCDC151 coimmunoprecipitates CCDC114 and thus appears to be a highly evolutionarily conserved ODA-DC-related protein involved in mediating assembly of both ODAs and their axonemal docking machinery onto ciliary microtubules.
Heterotaxy results from a failure to establish normal left-right asymmetry early in embryonic development. By whole-exome sequencing, whole-genome sequencing and high-throughput cohort resequencing, ...we identified recessive mutations in MMP21 (encoding matrix metallopeptidase 21) in nine index cases with heterotaxy. In addition, Mmp21-mutant mice and mmp21-morphant zebrafish displayed heterotaxy and abnormal cardiac looping, respectively, suggesting a new role for extracellular matrix remodeling in the establishment of laterality in vertebrates.
A central role for cilia in congenital heart disease (CHD) was recently identified in a large-scale mouse mutagenesis screen. Although the screen was phenotype-driven, the majority of genes recovered ...were cilia-related, suggesting that cilia play a central role in CHD pathogenesis. This partly reflects the role of cilia as a hub for cell signaling pathways regulating cardiovascular development. Consistent with this, many cilia-transduced cell signaling genes were also recovered, and genes regulating vesicular trafficking, a pathway essential for ciliogenesis and cell signaling. Interestingly, among CHD-cilia genes recovered, some regulate left-right patterning, indicating cardiac left-right asymmetry disturbance may play significant roles in CHD pathogenesis. Clinically, CHD patients show a high prevalence of ciliary dysfunction and show enrichment for de novo mutations in cilia-related pathways. Combined with the mouse findings, this would suggest CHD may be a new class of ciliopathy.
Congenital heart disease (CHD) is the most prevalent birth defect, affecting nearly 1% of live births; the incidence of CHD is up to tenfold higher in human fetuses. A genetic contribution is ...strongly suggested by the association of CHD with chromosome abnormalities and high recurrence risk. Here we report findings from a recessive forward genetic screen in fetal mice, showing that cilia and cilia-transduced cell signalling have important roles in the pathogenesis of CHD. The cilium is an evolutionarily conserved organelle projecting from the cell surface with essential roles in diverse cellular processes. Using echocardiography, we ultrasound scanned 87,355 chemically mutagenized C57BL/6J fetal mice and recovered 218 CHD mouse models. Whole-exome sequencing identified 91 recessive CHD mutations in 61 genes. This included 34 cilia-related genes, 16 genes involved in cilia-transduced cell signalling, and 10 genes regulating vesicular trafficking, a pathway important for ciliogenesis and cell signalling. Surprisingly, many CHD genes encoded interacting proteins, suggesting that an interactome protein network may provide a larger genomic context for CHD pathogenesis. These findings provide novel insights into the potential Mendelian genetic contribution to CHD in the fetal population, a segment of the human population not well studied. We note that the pathways identified show overlap with CHD candidate genes recovered in CHD patients, suggesting that they may have relevance to the more complex genetics of CHD overall. These CHD mouse models and >8,000 incidental mutations have been sperm archived, creating a rich public resource for human disease modelling.
Congenital heart disease (CHD) affects up to 1% of live births. Although a genetic etiology is indicated by an increased recurrence risk, sporadic occurrence suggests that CHD genetics is complex. ...Here, we show that hypoplastic left heart syndrome (HLHS), a severe CHD, is multigenic and genetically heterogeneous. Using mouse forward genetics, we report what is, to our knowledge, the first isolation of HLHS mutant mice and identification of genes causing HLHS. Mutations from seven HLHS mouse lines showed multigenic enrichment in ten human chromosome regions linked to HLHS. Mutations in Sap130 and Pcdha9, genes not previously associated with CHD, were validated by CRISPR-Cas9 genome editing in mice as being digenic causes of HLHS. We also identified one subject with HLHS with SAP130 and PCDHA13 mutations. Mouse and zebrafish modeling showed that Sap130 mediates left ventricular hypoplasia, whereas Pcdha9 increases penetrance of aortic valve abnormalities, both signature HLHS defects. These findings show that HLHS can arise genetically in a combinatorial fashion, thus providing a new paradigm for the complex genetics of CHD.
The ciliary kinase NEK8 plays a critical role in situs determination and cystic kidney disease, yet its exact function remains unknown. In this study, we identify ANKS6 as a target and activator of ...NEK8. ANKS6 requires NEK8 for localizing to the ciliary inversin compartment (IC) and activates NEK8 by binding to its kinase domain. Here we demonstrate the functional importance of this interaction through the analysis of two novel mouse mutations, Anks6(Streaker) and Nek8(Roc). Both display heterotaxy, cardiopulmonary malformations and cystic kidneys, a syndrome also characteristic of mutations in Invs and Nphp3, the other known components of the IC. The Anks6(Strkr) mutation decreases ANKS6 interaction with NEK8, precluding NEK8 activation. The Nek8(Roc) mutation inactivates NEK8 kinase function while preserving ANKS6 localization to the IC. Together, these data reveal the crucial role of NEK8 kinase activation within the IC, promoting proper left-right patterning, cardiopulmonary development and renal morphogenesis.
Cyclic dimeric GMP (c-di-GMP) regulates numerous processes in Gram-negative bacteria, yet little is known about its role in Gram-positive bacteria. Here we characterize two c-di-GMP ...phosphodiesterases from the filamentous high-GC Gram-positive actinobacterium Streptomyces coelicolor, involved in controlling colony morphology and development. A transposon mutation in one of the two phosphodiesterase genes, SCO0928, hereby designated rmdA (regulator of morphology and development A), resulted in decreased levels of spore-specific gray pigment and a delay in spore formation. The RmdA protein contains GGDEF-EAL domains arranged in tandem and possesses c-di-GMP phosphodiesterase activity, as is evident from in vitro enzymatic assays using the purified protein. RmdA contains a PAS9 domain and is a hemoprotein. Inactivation of another GGDEF-EAL-encoding gene, SCO5495, designated rmdB, resulted in a phenotype identical to that of the rmdA mutant. Purified soluble fragment of RmdB devoid of transmembrane domains also possesses c-di-GMP phosphodiesterase activity. The rmdA rmdB double mutant has a bald phenotype and is impaired in aerial mycelium formation. This suggests that RmdA and RmdB functions are additive and at least partially overlapping. The rmdA and rmdB mutations likely result in increased local pools of intracellular c-di-GMP, because intracellular c-di-GMP levels in the single mutants did not differ significantly from those of the wild type, whereas in the double rmdA rmdB mutant, c-di-GMP levels were 3-fold higher than those in the wild type. This study highlights the importance of c-di-GMP-dependent signaling in actinomycete colony morphology and development and identifies two c-di-GMP phosphodiesterases controlling these processes.
Cilia are ubiquitous microtubule-based eukaryotic organelles that project from the cell to generate motility or function in cellular signaling. Motile cilia or flagella contain axonemal dynein motors ...and other complexes to achieve beating. Primary cilia are immotile and act as signaling hubs, with receptors shuttling between the cytoplasm and ciliary compartment. In both cilia types, an intraflagellar transport (IFT) system powered by unique kinesin and dynein motors functions to deliver the molecules required to build cilia and maintain their functions. Cryo-electron tomography has helped to reveal the organization of protein complex arrangement along the axoneme and the structure of anterograde IFT trains as well as the structure of primary cilia. Only recently, single-particle analysis (SPA) cryo-electron microscopy has provided molecular details of the protein organization of ciliary components, helping us to understand how they bind to microtubule doublets and how mechanical force propagated by dynein conformational changes is converted into ciliary beating. Here we highlight recent structural advances that are leading to greater knowledge of ciliary function.
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