TTC25 Deficiency Results in Defects of the Outer Dynein Arm Docking Machinery and Primary Ciliary Dyskinesia with Left-Right Body Asymmetry Randomization.

Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice.

Pih1d3 is required for cytoplasmic preassembly of axonemal dynein in mouse sperm.

Axonemal dynein complexes are preassembled in the cytoplasm before their transport to cilia, but the mechanism of this process remains unclear. We now show that mice lacking Pih1d3, a PIH1 domain-containing protein, develop normally but manifest male sterility. Pih1d3(-/-) sperm were immotile and fragile, with the axoneme of the flagellum lacking outer dynein arms (ODAs) and inner dynein arms (IDAs) and showing a disturbed 9+2 microtubule organization. Pih1d3 was expressed specifically in spermatogenic cells, with the mRNA being most abundant in pachytene spermatocytes. Pih1d3 localized to the cytoplasm of spermatogenic cells but was not detected in spermatids or mature sperm. The levels of ODA and IDA proteins were reduced in the mutant testis and sperm, and Pih1d3 was found to interact with an intermediate chain of ODA as well as with Hsp70 and Hsp90. Our results suggest that Pih1d3 contributes to cytoplasmic preassembly of dynein complexes in spermatogenic cells by stabilizing and promoting complex formation by ODA and IDA proteins.

Maternal bisphenol A oral dosing relates to the acceleration of neurogenesis in the developing neocortex of mouse fetuses.

Bisphenol A (BPA), an endocrine-disruptor, is widely used in the production of plastics and resins. Human perinatal exposure to this chemical has been proposed to be a potential risk to public health. Animal studies indicate that postnatal exposure to BPA may affect neocortex development in embryos by accelerated neurogenesis and causing neuronal migration defects. The detailed phenotypes and pathogenetic mechanisms, especially with regard to the proliferation and differentiation of neural stem/progenitor cells, however, have not been clarified. C57BL/6J pregnant mice were orally administered BPA at 200µg/kg from embryonic day (E) 8.5 to 13.5, and the fetuses were observed histologically at E14.5. To clarify the histological changes, especially in terms of neurogenesis, proliferation and cell cycle, we performed histological analysis using specific markers of neurons/neural stem cells and cell cycle-specific labeling experiments using thymidine-analog substances. Cortical plate was hyperplastic and the number of neural stem/progenitor cells was decreased after the exposure to BPA. In particular, the maternal BPA oral dosing related to the effects on intermediate progenitor cells (IPCs, neural progenitor cells) in the subventricular zone (SVZ) of dorsal telencephalon. Exposure to BPA associated the promotion of the cell cycle exit in radial glial cells (RGCs, neural stem cells) and IPCs, and decreased the proliferation resulting from the prolong cell cycle length of IPCs in the SVZ. Our data show that maternal oral exposure to BPA related to the disruption of the cell cycle in IPCs and the effects of neurogenesis in the developing neocortex.

Fluid flow and interlinked feedback loops establish left-right asymmetric decay of Cerl2 mRNA.

Breaking of left-right symmetry in mouse embryos requires fluid flow at the node, but the precise action of the flow has remained unknown. Here we show that the left-right asymmetry of Cerl2 expression around the node, a target of the flow, is determined post-transcriptionally by decay of Cerl2 mRNA in a manner dependent on its 3' untranslated region. Cerl2 mRNA is absent specifically from the apical region of crown cells on the left side of the node. Preferential decay of Cerl2 mRNA on the left is initiated by the leftward flow and further enhanced by the operation of Wnt-Cerl2 interlinked feedback loops, in which Wnt3 upregulates Wnt3 expression and promotes Cerl2 mRNA decay, whereas Cerl2 promotes Wnt degradation. Mathematical modelling and experimental data suggest that these feedback loops behave as a bistable switch that can amplify in a noise-resistant manner a small bias conferred by fluid flow.

Cilia at the node of mouse embryos sense fluid flow for left-right determination via Pkd2.

Unidirectional fluid flow plays an essential role in the breaking of left-right (L-R) symmetry in mouse embryos, but it has remained unclear how the flow is sensed by the embryo. We report that the Ca(2+) channel Polycystin-2 (Pkd2) is required specifically in the perinodal crown cells for sensing the nodal flow. Examination of mutant forms of Pkd2 shows that the ciliary localization of Pkd2 is essential for correct L-R patterning. Whereas Kif3a mutant embryos, which lack all cilia, failed to respond to an artificial flow, restoration of primary cilia in crown cells rescued the response to the flow. Our results thus suggest that nodal flow is sensed in a manner dependent on Pkd2 by the cilia of crown cells located at the edge of the node.