Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease

Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or ‘primary’ cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic

Escapement of Fishes from Modified Fyke Nets with Differing Throat Configurations

We performed a field experiment in five eastern South Dakota lakes to investigate fish escapement differences between modified fyke nets with two common throat configurations (restricted and unrestricted). Nets with restricted and unrestricted throats were deployed in pairs for 24 h on similar habitat. Captured fish were measured for TL and were given day-specific marks. The paired nets were redeployed, and marked fish were randomly assigned to be stocked into the restricted or unrestricted net for 24 h; stocking densities (stratified into low, medium, and high) were species specific. Marked fish that were retained after 24 h were used to quantify escapement, whereas newly captured fish were used to estimate differences in mean CPUE and size structure. Mean CPUE of Black Bullheads Ameiurus melas, Black Crappies Pomoxis nigromaculatus, and BluegillsLepomis macrochirus approximately doubled when restricted nets were used. Mean TL of Black Crappies was 31 mm greater (95% confidence interval [CI] = 6–57 mm greater) and mean TL of Bluegills was 21 mm greater (95% CI = 8–35 mm greater) in restricted nets than in unrestricted nets. Escapement from restricted nets was 4.4% for Black Crappies and 10.3% for Bluegills, whereas escapement from unrestricted nets was 71.7% for Black Crappies and 58.4% for Bluegills. We urge researchers to consider the influence of varying fyke-net throat configurations on calculated population metrics, and we recommend inclusion of the restricted throat feature in gear specifications for North American standard modified fyke nets

A391V is a potential pathogenic allele.

<p>(A) Sanger Sequence confirmation of the 1172 C>T point mutation in Gas8^AV mice reproducing the A391V missense mutation found in the human patient. (B) Ciliary beat frequency analysis on tracheal cilia of Gas8^GT/WT and Gas8^GT/AV mice shows no difference between controls and compound heterozygotes (n = 86 points from 3 trachea for Gas8^GT/WT (13.04 Hz), n = 76 points from 3 tracheas for Gas8^GT/AV (13.34 Hz)). (C) Tracking of red fluorescent latex beads added to lateral ventricles shows a trending but not significant decrease in ability of Gas8^GT/AV cilia to move fluid. (n = 3 for Gas8^GT/WT (163.7μm/sec), n = 2 for Gas8^GT/AV (135.9μm/sec)). (D) Nissl stained brains of 10 week old Gas8^GT/WT, Gas8^WT/AV, and Gas8^GT/AV mice. Mild to moderate hydrocephalus is present in the Gas8^GT/AV brains. Scale is 1mm (Arrowhead indicates mild, arrow indicates moderate) (n = 4). (E) Swim speed quantification of rescue of DRC4-D198K construct in <i>pf2</i> deficient <i>Chlamydomonas</i>. “<i>pf2</i>” denotes <i>pf2</i> deficient <i>Chlamydomonas</i>, “DK-GFP” denotes <i>pf2</i> deficient <i>Chlamydomonas</i> expressing the DRC4-D198K-GFP construct, “DRC4-GFP” denotes <i>pf2</i> deficient <i>Chlamydomonas</i> expressing the DRC4-GFP wild-type construct. * = significant difference from WT (p<0.05), # = significant difference from <i>pf2</i> (p<0.05), § = significant difference between DK-GFP and DRC4-GFP (p<0.05) (n = 390 for WT (123.9μm/sec), n = 271 for <i>pf2</i> (40.1μm/sec), n = 180 for DK-GFP (108.3μm/sec), n = 299 for DRC4-GFP (120.9μm/sec)).</p