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 Kupffer's 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.

Ktu/PF13 is required for cytoplasmic pre-assembly of axonemal dyneins.

Cilia and flagella are highly conserved organelles that have diverse roles in cell motility and sensing extracellular signals. Motility defects in cilia and flagella often result in primary ciliary dyskinesia. However, the mechanisms underlying cilia formation and function, and in particular the cytoplasmic assembly of dyneins that power ciliary motility, are only poorly understood. Here we report a new gene, kintoun (ktu), involved in this cytoplasmic process. This gene was first identified in a medaka mutant, and found to be mutated in primary ciliary dyskinesia patients from two affected families as well as in the pf13 mutant of Chlamydomonas. In the absence of Ktu/PF13, both outer and inner dynein arms are missing or defective in the axoneme, leading to a loss of motility. Biochemical and immunohistochemical studies show that Ktu/PF13 is one of the long-sought proteins involved in pre-assembly of dynein arm complexes in the cytoplasm before intraflagellar transport loads them for the ciliary compartment.

Deficiency of mastl, a mitotic regulator, results in cell detachment from developing tissues of zebrafish embryos.

To form tissues with unique functions and structures, it is important that the cells that comprise them maintain physical contact. On the other hand, with each mitosis, drastic changes in cell shapes, cell adhesion, and cytoskeletal architecture may cause such contacts to be temporarily weakened, risking improper development and maintenance of tissues. Despite such risks, tissues form properly during normal development. However, it is not well understood whether mitotic abnormalities affect tissue formation. Here, analysis of zebrafish embryos with aberrant mitosis shows that proper progression of mitosis is important to maintain cell contact in developing tissues. By screening mutants with abnormal trunk and tail development, we obtained a mutant with perturbed expression of some tissue-specific genes in embryonic caudal regions. The responsible gene is mastl/gwl, which is involved in progression of mitosis. Analysis focusing on the chordo-neural hinge (CNH), the primordium of axial tissues, shows that cell detachment from the CNH is increased in mastl mutant embryos. Time-lapse imaging reveals that this cell detachment occurs during mitosis. These results suggest that cells are unable to maintain contact due to abnormalities in progression of mitosis in mastl mutants.

The establishment of rotational polarity in the airway and ependymal cilia: analysis with a novel cilium motility mutant mouse.

The airway is covered by multicilia that beat in a metachronous manner toward the mouth to eliminate debris and infectious particles. Coordinated one-directional beating is an essential feature of multicilia in the airway to guarantee proper mucociliary clearance. Defects in ciliary motility lead to primary ciliary dyskinesia (PCD), with major symptoms including bronchitis and other chronic respiratory diseases. Recent work suggested that ciliary motility and planar polarity are required in the process of ciliary alignment that produces coordinated beating. However, the extent to which cilia motility is involved in this process in mammals has not yet been fully clarified. Here, to address the role of ciliary motility in the process of coordinated ciliary alignment, we analyzed Kintoun mice mutants (Ktu(-/-)). Ktu(-/-) exhibited typical phenotypes of PCD with complete loss of ciliary motility in trachea and another ciliated tissue, the brain ependyma. Immunohistochemistry using antibodies against axonemal dynein confirmed the loss of multiple axonemal dynein components in mutant cilia. Observation of cilia orientation based on basal foot directions revealed that ciliary motility was not required in the alignment of airway cilia, whereas a strong requirement was observed in brain ependymal cells. Thus we conclude that the involvement of ciliary motility in the establishment of coordinated ciliary alignment varies among tissues.

Dynein axonemal intermediate chain 2 is required for formation of the left-right body axis and kidney in medaka.

Ciliary defects lead to various diseases, such as primary ciliary dyskinesia (PCD) and polycystic kidney disease (PKD). We isolated a medaka mutant mii, which exhibits defects in the left-right (LR) polarity of organs, and found that mii encodes dynein axonemal intermediate chain 2a (dnai2a). Ortholog mutations were recently reported to cause PCD in humans. mii mutant embryos exhibited loss of nodal flow in Kupffer's Vesicle (KV), which is equivalent to the mammalian node, and abnormal expression of the left-specific gene. KV cilia in the mii mutant were defective in their outer dynein arms (ODAs), indicating that Dnai2a is required for ODA formation in KV cilia. While the mii mutant retained motility of the renal cilia and failed to show PKD, the loss of dnai2a and another dnai2 ortholog dnai2b led to PKD. These findings demonstrate that Dnai2 proteins control LR polarity and kidney formation through regulation of ciliary motility.

Single-cell analysis of somatotopic map formation in the zebrafish lateral line system.

The zebrafish lateral line is a simple sensory system comprising a small number of neurons in addition to their sensory organs, the neuromasts. We have adopted this system as a model for single-cell level analyses of topographic map formation and examined when and how the lateral line topographic map is established. Single-neuron labeling demonstrated that somatotopic organization of the ganglion emerges by 54 hr postfertilization, but also that this initial map is not as accurate as that observed at 6 days postfertilization. During this initial stage, individual neurons exhibit extensively diverse behavior and morphologies. We identified leader neurons, the axons of which are the first to reach the tail, and later-appearing axons that contribute to the initial map. Our data suggest that lateral line neurons are heterogeneous from the beginning of lateral line development, and that some of them are intrinsically fate determined to contribute to the somatotopic map.

Integrinalpha5-dependent fibronectin accumulation for maintenance of somite boundaries in zebrafish embryos.

Boundary formation and epithelialization are crucial processes in the morphological segmentation of vertebrate somites. By a genetic screening procedure with zebrafish, we identified two genes, integrinalpha5 (itga5) and fibronectin (fn), required for these processes. Fibronectin proteins accumulate at somite boundaries in accordance with epithelialization of the somites. Both Fibronectin accumulation and the epithelialization are dependent on itga5, which is expressed in the most medial part of somites. Although somite boundaries are initially formed, but not maintained, in the anterior trunk of the mutant embryos deficient in either gene, their maintenance is defective at all axial levels of embryos deficient for both of these genes. Therefore, Integrinalpha5-directed assembly of Fibronectin appears critical for epithelialization and boundary maintenance of somites. Furthermore, with an additional deficiency in ephrin-B2a, the segmental defect in itga5 or fn mutant embryos is expanded posteriorly, indicating that both Integrin-Fibronectin and Eph-Ephrin systems function cooperatively in maintaining somite boundaries.

Groucho-associated transcriptional repressor ripply1 is required for proper transition from the presomitic mesoderm to somites.

Concomitant with the transition from the presomitic mesoderm (PSM) to somites, the periodical gene expression characteristic of the PSM is drastically changed and translated into the segmental structure. However, the molecular mechanism underlying this transition has remained obscure. Here, we show that ripply1, encoding a nuclear protein associated with the transcriptional corepressor Groucho, is required for this transition. Zebrafish ripply1 is expressed in the anterior PSM and in several newly formed somites. Ripply1 represses mesp-b expression in the PSM through a Groucho-interacting motif. In ripply1-deficient embryos, somite boundaries do not form, the characteristic gene expression in the PSM is not properly terminated, and the initially established rostrocaudal polarity in the segmental unit is not maintained, whereas paraxial mesoderm cells become differentiated. Thus, ripply1 plays dual roles in the transition from the PSM to somites: termination of the segmentation program in the PSM and maintenance of the rostrocaudal polarity.

Zebrafish maternal-effect mutations causing cytokinesis defect without affecting mitosis or equatorial vasa deposition.

Maternal-effect genes play essential roles in early embryogenesis particularly before activation of the zygotic genes. A genetic screen for mutations affecting such maternal-effect genes was carried out employing an F3 screen strategy, identifying six recessive mutations out of 60 mutagenized genomes. Three of the mutations (acytokinesis mutations: ackkt5, ackkt62 and ackkt119) caused absence of cell cleavage in the embryos derived from homozygous females regardless of the paternal genotype, without affecting nuclear divisions. These embryos are defective in generating contractile rings, ackkt62 mutation abolishing reactions to organize cortical F-actin, while other mutations causing abortive contractile ring-like structures at ectopic sites. Defect of contractile ring formation in the affected embryos leads to the absence of microtubule arrays at the prospective cleavage plane. Thus, these mutations reveal the sequence of events associated with cytokinesis, in particular, the cortical actin dynamics. It is remarkable that in all acytokinetic embryos, daughter nuclei after mitosis are arranged in spatially normal positions, and maternal vasa mRNAs accumulate in the prospective planes of the first and second cell cleavages in the total absence of cytokinesis. This indicates that the basic cell architectures of early embryos are largely established by the autonomous activities of the mitotic apparatus, without much dependence on the cell cleavage machinery.

Expression of the FGF receptor 2 gene (fgfr2) during embryogenesis in the zebrafish Danio rerio.

We isolated a full-length cDNA clone for the zebrafish homologue of fibroblast growth factor receptor (FGFR) 2. The deduced protein sequence is typical of vertebrate FGFRs in that it has three Ig-like domains in the extracellular region. The expression of fgfr2 is initiated during epiboly in the paraxial mesoderm. During early somitogenesis, fgfr2 expression was noted in the anterior neural plate as well as in newly formed somites. Whereas fgfr2 expression in somites is transient, it increases in the central nervous system (CNS), i.e. in the ventral telencephalon, anterior diencephalon, midbrain, and respective rhombomeres of the hindbrain, from the mid-somitogenesis stage. The dorsal telencephalon and the region around the midbrain-hindbrain boundary are devoid of fgfr2 expression. Essentially the same expression pattern is observed until 48 h post-fertilization in the CNS, although rhombomeric expression in the hindbrain is progressively confined to narrower stripes. After somitogenesis, fgfr2 expression was also observed in the lens, hypochord, endoderm, and fin mesenchyme. We compared the expression of the four fgfr genes (fgfr1-4) in the CNS of zebrafish embryos and show that fgfr1 is the only fgfr gene that is expressed in the dorsal telencephalon and isthmic region from which expression of fgfr2-4 is absent.

Expression of the FGF receptor 2 gene (fgfr2) during embryogenesis in the zebrafish Danio rerio.

We isolated a full-length cDNA clone for the zebrafish homologue of fibroblast growth factor receptor (FGFR) 2. The deduced protein sequence is typical of vertebrate FGFRs in that it has three Ig-like domains in the extracellular region. The expression of fgfr2 is initiated during epiboly in the paraxial mesoderm. During early somitogenesis, fgfr2 expression was noted in the anterior neural plate as well as in newly formed somites. Whereas fgfr2 expression in somites is transient, it increases in the central nervous system (CNS), i.e. in the ventral telencephalon, anterior diencephalon, midbrain, and respective rhombomeres of the hindbrain, from the mid-somitogenesis stage. The dorsal telencephalon and the region around the midbrain-hindbrain boundary are devoid of fgfr2 expression. Essentially the same expression pattern is observed until 48 h post-fertilization in the CNS, although rhombomeric expression in the hindbrain is progressively confined to narrower stripes. After somitogenesis, fgfr2 expression was also observed in the lens, hypochord, endoderm, and fin mesenchyme. We compared the expression of the four fgfr genes (fgfr1-4) in the CNS of zebrafish embryos and show that fgfr1 is the only fgfr gene that is expressed in the dorsal telencephalon and isthmic region from which expression of fgfr2-4 is absent.

Activator-to-repressor conversion of T-box transcription factors by the Ripply family of Groucho/TLE-associated mediators.

The T-box family of transcription factors, defined by a conserved DNA binding domain called the T-box, regulate various aspects of embryogenesis by activating and/or repressing downstream genes. In spite of the biological significance of the T-box proteins, how they regulate transcription remains to be elucidated. Here we show that the Groucho/TLE-associated protein Ripply converts T-box proteins from activators to repressors. In cultured cells, zebrafish Ripply1, an essential component in somite segmentation, and its structural relatives, Ripply2 and -3, suppress the transcriptional activation mediated by the T-box protein Tbx24, which is coexpressed with ripply1 during segmentation. Ripply1 associates with Tbx24 and converts it to a repressor. Ripply1 also antagonizes the transcriptional activation of another T-box protein, No tail (Ntl), the zebrafish ortholog of Brachyury. Furthermore, injection of a high dosage of ripply1 mRNA into zebrafish eggs causes defective development of the posterior trunk, similar to the phenotype observed in homozygous mutants of ntl. A mutant form of Ripply1 defective in association with Tbx24 also lacks activity in zebrafish embryos. These results indicate that the intrinsic transcriptional property of T-box proteins is controlled by Ripply family proteins, which act as specific adaptors that recruit the global corepressor Groucho/TLE to T-box proteins.

Paf1 complex homologues are required for Notch-regulated transcription during somite segmentation.

Members of the yeast polymerase-associated factor 1 (Paf1) complex, which is composed of at least five components (Paf1, Rtf1, Cdc73, Leo1 and Ctr9), are conserved from yeast to humans. Although these proteins have been implicated in RNA polymerase II-mediated transcription, their roles in vertebrate development have not been explained. Here, we show that a zebrafish mutant with a somite segmentation defect is deficient in rtf1. In addition, embryos deficient in rtf1 or ctr9 show abnormal development of the heart, ears and neural crest cells. rtf1 is required for correct RNA levels of the Notch-regulated genes her1, her7 and deltaC, and also for Notch-induced her1 expression in the presomitic mesoderm. Furthermore, the phenotype observed in rtf1-deficient mutants is enhanced by an additional deficiency in mind bomb, which encodes an effector of Notch signalling. Therefore, zebrafish homologues of the yeast Paf1 complex seem to preferentially affect a subset of genes, including Notch-regulated genes, during embryogenesis.

Zebrafish hairy/enhancer of split protein links FGF signaling to cyclic gene expression in the periodic segmentation of somites.

Notch and fibroblast growth factor (FGF) signaling pathways have been implicated in the establishment of proper periodicity of vertebrate somites. Here, we show evidence that a Hes6-related hairy/Enhancer of split-related gene, her13.2, links FGF signaling to the Notch-regulated oscillation machinery in zebrafish. Expression of her13.2 is induced by FGF-soaked beads and decreased by an FGF signaling inhibitor. her13.2 is required for periodic repression of the Notch-regulated genes her1 and her7, and for proper somite segmentation. Furthermore, Her13.2 augments autorepression of her1 in association with Her1 protein. Therefore, FGF signaling appears to maintain the oscillation machinery by supplying a binding partner, Her13.2, for Her1.

Inhibition of BMP activity by the FGF signal promotes posterior neural development in zebrafish.

The expression patterns of region-specific neuroectodermal genes and fate-map analyses in zebrafish gastrulae suggest that posterior neural development is initiated by nonaxial signals, distinct from organizer-derived secreted bone morphogenetic protein (BMP) antagonists. This notion is further supported by the misexpression of a constitutively active form of zebrafish BMP type IA receptor (CA-BRIA) in the zebrafish embryos. It effectively suppressed the anterior neural marker, otx2, but not the posterior marker, hoxb1b. Furthermore, we demonstrated that the cells in the presumptive posterior neural region lose their neural fate only when CA-BRIA and Xenopus dominant-negative fibroblast growth factor (FGF) receptors (XFD) are coexpressed. The indications are that FGF signaling is involved in the formation of the posterior neural region, counteracting the BMP signaling pathway within the target cells. We then examined the functions of Fgf3 in posterior neural development. Zebrafish fgf3 is expressed in the correct place (dorsolateral margin) and at the correct time (late blastula to early gastrula stages), the same point that the most precocious posterior neural marker, hoxb1b, is first activated. Unlike other members of the FGF family, Fgf3 had little mesoderm-inducing activity. When ectopically expressed, Fgf3 expands the neural region with suppression of anterior neural fate. However, this effect was mediated by Chordino (zebrafish Chordin), because Fgf3 induces chordino expression in the epiblast and Fgf3-induced neural expansion was substantially suppressed in dino mutants with mutated chordino genes. The results obtained in the present study reveal multiple actions of the FGF signal on neural development: it antagonizes BMP signaling within posterior neural cells, induces the expression of secreted BMP antagonists, and suppresses anterior neural fate.