Takahiko Yokoyama

Cloning of inv, a gene that controls left/right asymmetry and kidney development

Most vertebrate internal organs show a distinctive left/right asymmetry. The inv (inversion of embryonic turning) mutation in mice was created previously by random insertional mutagenesis; it produces both a constant reversal of left/right polarity (situs inversus) and cyst formation in the kidneys. Asymmetric expression patterns of the genes nodal and lefty are reversed in the inv mutant, indicating that inv may act early in left/right determination. Here we identify a new gene located at the inv locus. The encoded protein contains 15 consecutive repeats of an Ank/Swi6 motif, at its amino terminus. Expression of the gene is the highest in the kidneys and liver among adult tissues, and is seen in presomite-stage embryos. Analysis of the transgenic genome and the structure of the candidate gene indicate that the candidate gene is the only gene that is disrupted in inv mutants. Transgenic introduction of a minigene encoding the candidate protein restores normal left/right asymmetry and kidney development in the inv mutant, confirming the identity of the candidate gene.

The left-right determinant Inversin is a component of node monocilia and other 9+0 cilia

Inversin (Inv), a protein that contains ankyrin repeats, plays a key role in left-right determination during mammalian embryonic development, but its precise function remains unknown. Transgenic mice expressing an Inv and green fluorescent protein (GFP) fusion construct (Inv::GFP) were established to facilitate characterization of the subcellular localization of Inv. The Inv::GFP transgene rescued the laterality defects and polycystic kidney disease of Inv/Inv mice, indicating that the fusion protein is functional. In transgenic embryos, Inv::GFP protein was detected in the node monocilia. The fusion protein was also present in other 9+0 monocilia, including those of kidney epithelial cells and the pituitary gland, but it was not localized to 9+2 cilia. The N-terminal region of Inv (InvΔC) including the ankyrin repeats also localized to the node cilia and rescued the left-right defects of Inv/Inv mutants. Although no obvious abnormalities were detected in the node monocilia of Inv/Inv embryos, the laterality defects of such embryos were corrected by an artificial leftward flow of fluid in the node, suggesting that nodal flow is impaired by the Inv mutation. These results suggest that the Inv protein contributes to left-right determination as a component of monocilia in the node and is essential for the generation of normal nodal flow.

Reactive Oxygen Species Derived from NOX1/NADPH Oxidase Enhance Inflammatory Pain

The involvement of reactive oxygen species (ROS) in an augmented sensitivity to painful stimuli (hyperalgesia) during inflammation has been suggested, yet how and where ROS affect the pain signaling remain unknown. Here we report a novel role for the superoxide-generating NADPH oxidase in the development of hyperalgesia. In mice lacking Nox1 (Nox1−/Y), a catalytic subunit of NADPH oxidase, thermal and mechanical hyperalgesia was significantly attenuated, whereas no change in nociceptive responses to heat or mechanical stimuli was observed. In dorsal root ganglia (DRG) neurons of Nox1+/Y, pretreatment with chemical mediators bradykinin, serotonin, or phorbol 12-myristate 13-acetate (PMA) augmented the capsaicin-induced calcium increase, whereas this increase was significantly attenuated in DRG neurons of Nox1−/Y. Concomitantly, PMA-induced translocation of PKCε was markedly perturbed in Nox1−/Y or Nox1+/Y DRG neurons treated with ROS-scavenging agents. In cells transfected with tagged PKCε, hydrogen peroxide induced translocation and a reduction in free sulfhydryls of full-length PKCε but not of the deletion mutant lacking the C1A domain. These findings indicate that NOX1/NADPH oxidase accelerates the translocation of PKCε in DRG neurons, thereby enhancing the TRPV1 activity and the sensitivity to painful stimuli.

Pkd1l1 complexes with Pkd2 on motile cilia and functions to establish the left-right axis

The internal organs of vertebrates show distinctive left-right asymmetry. Leftward extracellular fluid flow at the node (nodal flow), which is generated by the rotational movement of node cilia, is essential for left-right patterning in the mouse and other vertebrates. However, the identity of the pathways by which nodal flow is interpreted remains controversial as the molecular sensors of this process are unknown. In the current study, we show that the medaka left-right mutant abecobe (abc) is defective for left-right asymmetric expression of southpaw, lefty and charon, but not for nodal flow. We identify the abc gene as pkd1l1, the expression of which is confined to Kupffers vesicle (KV, an organ equivalent to the node). Pkd1l1 can interact and interdependently colocalize with Pkd2 at the cilia in KV. We further demonstrate that all KV cilia contain Pkd1l1 and Pkd2 and left-right dynein, and that they are motile. These results suggest that Pkd1l1 and Pkd2 form a complex that functions as the nodal flow sensor in the motile cilia of the medaka KV. We propose a new model for the role of cilia in left-right patterning in which the KV cilia have a dual function: to generate nodal flow and to interpret it through Pkd1l1-Pkd2 complexes.

Localization of Inv in a distinctive intraciliary compartment requires the C-terminal ninein-homolog-containing region

The primary cilium is an antenna-like structure extending from the surface of most vertebrate cells. Loss or mutation of ciliary proteins can lead to polycystic kidney disease and other developmental abnormalities. inv mutant mice develop multiple renal cysts and are a model for human nephronophthisis type 2. The mouse Inv gene encodes a 1062-amino-acid protein that is localized in primary cilia. In this study, we show that the Inv protein (also known as inversin) is localized at a distinctive proximal segment of the primary cilium, using GFP-tagged Inv constructs and anti-Inv antibody. We named this segment the Inv compartment of the cilium. Further investigation of the Inv protein showed that 60 amino acids at its C-terminal, which contains ninein homologous sequences, are crucial for its localization to the Inv compartment. Fluorescence recovery after photobleaching analysis revealed that the Inv protein was dynamic within this compartment. These results suggest that localization of the Inv protein to the Inv compartment is actively regulated. The present study revealed that the primary cilium has a distinct molecular compartment in the body of the primary cilium with a specific confining and trafficking machinery that has not been detected previously by morphological examination.

Inv acts as a molecular anchor for Nphp3 and Nek8 in the proximal segment of primary cilia.

A primary cilium is an antenna‐like structure extending from the surface of most vertebrate cells. It is structurally divided along its vertical axis into sub‐compartments that include the ciliary tip, the shaft, the ciliary necklace segment, the transitional zone and the basal body. We recently discovered that the shaft of the primary cilia has a distinct molecular compartment, termed the “Inv compartment”, which is characterized by the accumulation of Inv at the base of primary cilia. Inv was discovered as a causative gene in inv mutant mice. It was later found to be responsible for the infantile type of nephronophthisis (NPHP2). Nephronophthisis (NPHP) is an autosomal recessive kidney disease. Nine causative genes have been identified, with all examined products thought to function in cilia, basal body and/or centrioles. However, their exact intra‐ciliary localization and relationship have not been clear. Here, we report that products of Nphp3 and Nek8 (the mouse orthologs of the causative genes for NPHP3 and NPHP9, respectively) localize to the Inv compartment. We also show that Inv is essential for the compartmental localization of Nphp3 and Nek8, whereas localization of Inv does not require Nphp3 or Nek8. Nphp1 and Nphp4 also localize at the proximal region of the cilium, but not in Inv compartment. Our results indicate that Inv acts as an anchor for Nphp3 and Nek8 in the Inv compartment, and suggest that Inv compartment is a candidate site for intra‐ciliary interaction of Inv, Nphp3 and Nek8.

Calmodulin binds to inv protein: Implication for the regulation of inv function

Establishment of the left–right asymmetry of internal organs is essential for the normal development of vertebrates. The inv mutant in mice shows a constant reversal of left–right asymmetry and although the inv gene has been cloned, its biochemical and cell biological functions have not been defined. Here, we show that calmodulin binds to mouse inv protein at two sites (IQ1 and IQ2). The binding of calmodulin to the IQ2 site occurs in the absence of Ca2+ and is not observed in the presence of Ca2+. Injection of mouse inv mRNA into the right blastomere of Xenopus embryos at the two‐cell stage randomized the left–right asymmetry of the embryo and altered the patterns of Xnr‐1 and Pitx2 expression. Importantly, inv mRNA that lacked the region encoding the IQ2 site was unable to randomize left–right asymmetry in Xenopus embryos, implying that the IQ2 site is essential for inv to randomize left–right asymmetry in Xenopus. These results suggest that calmodulin binding may regulate inv function. Based on our findings, we propose a model for the regulation of inv function by calcium–calmodulin and discuss its implications.

The canonical Wnt signaling pathway is not involved in renal cyst development in the kidneys of inv mutant mice

Recent studies have identified several genes whose defects cause hereditary renal cystic diseases with most of the gene products located in the primary cilia. It has been suggested that primary cilia are involved in signaling pathways, defects of which result in abnormal cell proliferation and randomization of oriented cell division in the kidney leading to cyst formation. Mice with a mutation in the inv gene are a model for human nephronophthisis type 2 and develop multiple renal cysts. Inv protein (also called inversin) is located in the base of primary cilia and acts as a switch from canonical to non-canonical Wnt signaling. Here, we studied the orientation of cell division and proliferation in the kidneys of inv mutant mice, as its loss is thought to maintain activation of the canonical Wnt signaling. To establish if canonical signaling was involved in this process, we mated inv mutant with BATlacZ mice to measure canonical Wnt activity. Based on these reporter mice, nuclear localization and phosphorylation of β-catenin, and responsiveness to Wnt ligands in inv mutant cells, we found that random oriented cell division is an initial event for renal tubule expansion and precedes cell proliferation. Thus, our results do not support the hypothesis that canonical Wnt signaling causes renal cyst development in these mice.

Photoreceptor-specific activity of the human interphotoreceptor retinoid-binding protein (IRBP) promoter in transgenic mice.

In order to define the cellular specificity of the interphotoreceptor retinoid-binding protein (IRBP) promoter in the retina, we linked the human IRBP promoter to the beta-galactosidase (lacZ) gene and made five lines of transgenic mice. In three of the five transgenic mouse lines, retinas showed positive staining upon incubation with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). Mice from one line (OVE278B) showed positive X-gal staining throughout the retina except for the most peripheral regions. Interestingly, the staining was heterogeneous throughout the retina. Heavily stained regions were interspersed with lightly stained areas. Mice in two other lines showed highly mosaic X-gal staining patterns. Histological examination demonstrated that staining was confined to photoreceptor cells in all three expressing families. Furthermore, electron microscopy showed that the promoter is active in both rod and cone cells. Our results demonstrate that the human IRBP promoter can be used to obtain photoreceptor-specific gene expression in transgenic mice.

Reversal of left-right asymmetry induced by aberrant Nodal signaling in the node of mouse embryos

The node at the anterior tip of the primitive streak serves as an initial generator of the left-right (L-R) axis in mammalian embryos. We now show that a small disturbance in molecular signaling at the node is responsible for the L-R reversal of visceral organs in the inv mutant mouse. In the node of wild-type embryos, the expression of Nodal and Cerl2 (Dand5), which encodes an inhibitor of Nodal, is asymmetric, with the level of Nodal expression being higher on the left side and that of Cerl2 expression higher on the right. In inv/inv embryos, however, a localized reduction in the level of Cerl2 expression results in upregulation of the Nodal signal and a consequent induction of Lefty expression in the node. The ectopic expression of Lefty1 delays the onset of Nodal expression in the lateral plate mesoderm. L-R asymmetry of Cerl2 expression in the node also becomes reversed in a manner dependent on the Nodal signal. Nodal expression in the lateral plate mesoderm then appears on the right side, probably reflecting the balance between Nodal and Cerl2 in the node. The inhibition of Cerl2 expression by the Nodal signal suggests a mechanism for amplification of the cue for L-R asymmetry provided by nodal flow and for stabilization of asymmetric gene expression around the node. In inv/inv embryos, this system may function in reverse as a result of ectopic production of Lefty, which inhibits the Nodal signal on the left side in a manner dependent on leftward nodal flow.