Left–Right Determination: Involvement of Molecular Motor KIF3, Cilia,and Nodal Flow

Mammalian left–right determination is a good example for how multiple cell biological processes coordinate in the formation of a basic body plan. The leftward movement of fluid at the ventral node, called nodal flow, is the central process in symmetry breaking on the left–right axis. Nodal flow is autonomously generated by the rotation of posteriorly tilted cilia that are built by transport via KIF3 motor on cells of the ventral node. How nodal flow is interpreted to create left–right asymmetry has been a matter of debate. Recent evidence suggests that the leftward movement of sheathed lipidic particles, called nodal vesicular parcels (NVPs), may result in the activation of the noncanonical hedgehog signaling pathway, an asymmetric elevation in intracellular Ca²⁺ and changes in gene expression.Although the human body is apparently bilaterally symmetrical on the surface, the visceral organs are arranged asymmetrically in a stereotyped manner. The heart, spleen, and pancreas reside on the left side of the body, whereas the gall bladder and most of the liver are on the right side (Fig. 1A). Because the human body is formed from a spherically symmetrical egg (oocyte), symmetry breakdown is one of the fundamental processes of development.Open in a separate windowFigure 1.(A) Left–right asymmetric arrangements of internal organs in the human body. (Left) Normal arrangement (situs solitus). Most humans (>99%) have the heart on the left side and the liver on the right side. (Right) Mirrored arrangement (situs inversus). Half of patients with Kartagener''s syndrome have this arrangement, whereas the remaining patients are normal. Therefore, the left–right bilateral symmetry is randomly broken in this disease. (B–E) Scanning electron micrographs of wild-type (B, D) and Kif3b^−/− (C, E) mouse embryos. (B, C) Full-length images. Wild-type embryos at this stage have already turned with a right-sided tail (B), whereas Kif3b^−/− embryos remain unturned (C). In panel C, the dilated pericardial sac has been removed, and the heart loop is inverted (arrow). (D, E) Higher-magnification images and schematic representations of the heart loops showing a normal loop in the wild-type embryo (D) and an inverted loop in the mutant embryo (E). (F–I) Scanning electron micrographs of a mouse node. (F) Low-magnification view of a mouse embryo at 7.5 days postcoitum. Reichert''s membrane is removed, and the embryo is observed from the ventral side. The node is indicated by a black rectangle. The orientation is indicated in the panel as anterior (A), posterior (P), left (L), and right (R). Scale bar = 100 µm. (G) Higher-magnification view of the mouse node. The orientation is the same as in panel A. Scale bar = 20 µm. (H) Higher-magnification view of the nodal cilia (arrows) and nodal pit cells. Scale bar = 5 µm. (I) Nodal pit cells of Kif3b^−/− embryos. Nodal cilia are absent in these genetically manipulated embryos. (J) Intraflagellar transport. Protein components in the cilia and flagella are transported by KIF3A/B complexes (light and dark blue) along the doublet microtubules of the axoneme. (Panels A and J were reproduced with permission from JT Biohistory Research Hall/TokyoCinema. B–I were modified from Nonaka et al. 1998, Okada et al. 2005, and Hirokawa et al. 2006, with permission.)In many lower vertebrates and invertebrates, eggs are asymmetrical even before fertilization (as in Drosophila [Gilbert 2003]). In some organisms, such as fish and frog, the dorsoventral (DV) and anteroposterior (AP) axes are determined at fertilization by the distribution of the yolk and the entry position of the sperm (Gilbert 2003). In human, mouse, and other mammals, the embryo is initially cylindrically symmetrical when it implants itself into the wall of the uterus. The DV axis is first to be specified as the proximal–distal axis from the implantation site. Subsequently, the AP axis is arbitrarily determined in the plane perpendicular to the DV axis (Alarcon and Marikawa 2003; Beddington and Robertson 1999). In either case, L/R is thus the last axis to be determined, and needs to be consistent with the preceding DV and AP axes. Because the chirality of the body is predetermined by chiral molecules, such as amino acids and nucleic acids, the laterality or orientation of the L/R axis is established theoretically or potentially once the AP and DV axes are determined. The problem here is how this potentially established laterality is materialized through developmental events. This mechanism is still totally unknown for the invertebrates. However, recent studies of mouse embryo clarified the L/R determination mechanism in mammalian embryos (Hirokawa et al. 2006).Until recently, little was known about the mechanism for breaking L/R symmetry. The initial clue to tackling this question was a human genetic disease called Kartagener''s syndrome. Approximately half of these patients have their organs in the reversed orientation (situs inversus). Thus, the L/R determination is randomized in this syndrome. Patients with Kartagener''s syndrome also suffer from sinusitis and bronchiectasis (Kartagener 1933). A number of male patients with Kartagener''s syndrome are sterile. Affected individuals have immotile sperm and defective cilia in their airway (Afzelius 1976). Airway cilia and sperm from these patients have abnormal ultrastructures. Specifically, the axonemes, the interior supramolecular complexes that produce the movement of cilia and flagella, lack dynein arms, the molecular motors required for cilia and flagella motility (Afzelius 1976). However, the relationships between these phenotypes and the causes of situs inversus remained unknown for more than 20 years.Approximately 10 years ago, molecular biological studies identified several genes that are asymmetrically expressed in the L/R orientation before L/R asymmetric morphogenesis of the embryo. Morphological L/R asymmetry first becomes apparent with the orientation of the heart-tube loop (Fig. 1B,D) (Kaufman 1992), but initial L/R asymmetric gene expression precedes the morphological changes. In early embryos (7.5 days for mice) at the stage of somatogenesis, genes such as Lefty-2 (Ebaf), Nodal, and Pitx2 are expressed in the left lateral plate mesoderm, a structure located on the side of the embryos (Capdevila et al. 2000; Hamada 2002; Harvey 1998; Levin 2005; Yost 1999). However, the upstream phenomena that cause asymmetrical expression of these genes remain enigmatic.Many studies have suggested that the so-called node, a concave triangular region transiently formed during gastrulation at the ventral midline surface of early embryos, is important for L/R determination (Harvey 1998). When viewed from above the ventral side, the node of mouse embryos appears as a roughly triangular depression with the apex pointed toward the anterior (Fig. 1F,G), and it is 50–100 µm in width and 10–20 µm in depth. This nodal pit is covered by Reichert''s membrane, and the cavity is filled with extraembryonic fluid. The ventral embryonic surface of the nodal pit consists of an epithelial sheet of a few hundred monociliated cells (nodal pit cells).Nodal pit cells have one or sometimes two cilia that appear as rodlike protrusions approximately 5 µm in length and 0.3 µm in diameter (Fig. 1G,H). Because Kartagener''s syndrome suggests a potential link between cilia motility and L/R determination, the cilia in the node have been postulated to be motile and responsible for L/R determination.However, the ultrastructure of these nodal cilia is similar to that of immotile primary cilia. In motile cilia and flagella, nine pairs of doublet microtubules are arranged longitudinally along the axes (Fig. 2A) (Satir and Christensen 2007). Adjacent pairs of doublet microtubules are connected by dynein arms, which generate the motility of cilia and flagella. In addition, there are two microtubules in the center of cilia and flagella, referred to as the central pair, which define the direction of the beating plane. This essential central pair of microtubules is missing in primary cilia. Similar to other immotile primary cilia, the monocilia of nodal pit cells lack the central pair of microtubules and thus have a 9 + 0 microtubule arrangement. Therefore, based on their ultrastructure and initial video-microscopic observations, nodal monocilia were originally considered immotile (Bellomo et al. 1996).Open in a separate windowFigure 2.(A) Ultrastructures of normal cilia and primary cilia. (Left) Normal cilia and flagella have nine pairs of doublet microtubules (yellow) and two central microtubules (yellow). Adjacent doublet microtubules are connected with dynein motors (blue and green). The orientation of the central pair of microtubules is considered to determine the beating plane (purple). (Right) The central pair of microtubules is missing in immotile primary cilia and nodal cilia. In nodal cilia, the dynein motors remain in a chiral arrangement and produce a rotation-like movement (purple). (B–E) Rotation of nodal cilia and leftward nodal flow. The images are views from the ventral side. The orientation is indicated in the panels as anterior (A), posterior (P), left (L), and right (R). (B) Trajectory of a fluorescent bead attached to the tip of a nodal cilium (Movies 1 and 2). Three consecutive video frames with 33-ms exposures at 16-ms intervals (interlaced scan) are shown. The moving bead produces arc-shaped images (traced by green arrows). The beads rotate clockwise when viewed above the node. (C) Trajectories of the tips of nodal cilia traced from a high-speed video sequence (500 frames/s) (Movie 3). The red circles show the positions of the ends of the cilia at 10-ms time intervals, and the yellow circles show the positions of the roots of the cilia. The white ellipses show the trajectories of the tips. Scale bar = 5 µm. (D) The positions of beads that entered the node from the right edge traced for 4 seconds at 0.33-second intervals. Different symbols indicate different beads. Most beads go straight to the left edge of the node. Scale bar = 20 µm. (E) The trajectories of four beads selected to illustrate the streamline of the nodal flow. The flow is mostly laminar and straight in the middle of the node, but often makes small vortices near the left edge (arrowheads). (Panel A was reproduced with permission from JT Biohistory Research Hall/TokyoCinema. B–E were modified from Okada et al. [1999, 2005] with permission.)

Movie 1

Download video file.^{(3.2M, mov)}Leftward nodal flow. Nodal flow was visualized by adding fluorescent beads to the medium surrounding the ventral node of a mouse embryo at the early somite stage. 4× time lapse.

Movie 2

Open in a separate windowClick here to view.^{(104K, gif)}Rotatory movement of nodal cilia.

Movie 3

Download video file.^{(39M, mov)}Posteriorly-tilted rotation of nodal cilia. Nodal cilia were observed by high-speed video microscopy (500 frames/s). The focus was adjusted to approximately 3 µm above the surface of the ventral node. The images of the cilia thus blur when they are near the floor and become clear when they come up to the focal plane. Note that the particle (highlighted by a red circle) does not go down to the surface but eventually goes toward the left side of the node due to the movement of the cilia. Reproduced from Okada et al. (2005) with permission.Through studies of the flow of materials within cells, we serendipitously found that nodal cilia are actually motile and vigorously rotating. This rotation generates the leftward flow of extraembryonic fluid in the nodal pit. The directionality of this flow, termed nodal flow, determines laterality. Thus, quite unexpectedly, a physical process, fluid flow, was identified as the initial L/R symmetry-breaking event. In this review, we first summarize the discovery of nodal flow and then discuss how this leftward linear flow is generated in a fluid dynamic manner by the rotational movement of cilia. We further discuss the mechanisms by which L/R asymmetry is determined by this nodal flow.     

Ordering process of self-organizing maps improved by asymmetric neighborhood function

The Self-organizing map (SOM) is an unsupervised learning method based on the neural computation, which has found wide applications. However, the learning process sometime takes multi-stable states, within which the map is trapped to an undesirable disordered state including topological defects on the map. These topological defects critically aggravate the performance of the SOM. In order to overcome this problem, we propose to introduce an asymmetric neighborhood function for the SOM algorithm. Compared with the conventional symmetric one, the asymmetric neighborhood function accelerates the ordering process even in the presence of the defect. However, this asymmetry tends to generate a distorted map. This can be suppressed by an improved method of the asymmetric neighborhood function. In the case of one-dimensional SOM, it is found that the required steps for perfect ordering is numerically shown to be reduced from O(N ³) to O(N ²). We also discuss the ordering process of a twisted state in two-dimensional SOM, which can not be rectified by the ordinary symmetric neighborhood function.     

Structure-activity relationships of rosiglitazone for peroxisome proliferator-activated receptor gamma transrepression

Anti-inflammatory effects of peroxisome proliferator-activated receptor gamma (PPRAγ) ligands are thought to be largely due to PPARγ-mediated transrepression. Thus, transrepression-selective PPARγ ligands without agonistic activity or with only partial agonistic activity should exhibit anti-inflammatory properties with reduced side effects. Here, we investigated the structure-activity relationships (SARs) of PPARγ agonist rosiglitazone, focusing on transrepression activity. Alkenic analogs showed slightly more potent transrepression with reduced efficacy of transactivating agonistic activity. Removal of the alkyl group on the nitrogen atom improved selectivity for transrepression over transactivation. Among the synthesized compounds, 3l exhibited stronger transrepressional activity (IC₅₀: 14 μM) and weaker agonistic efficacy (11%) than rosiglitazone or pioglitazone.     

Rules for connectivity of secondary structure elements in protein: Two–layer αβ sandwiches

In protein structures, the fold is described according to the spatial arrangement of secondary structure elements (SSEs: α‐helices and β‐strands) and their connectivity. The connectivity or the pattern of links among SSEs is one of the most important factors for understanding the variety of protein folds. In this study, we introduced the connectivity strings that encode the connectivities by using the types, positions, and connections of SSEs, and computationally enumerated all the connectivities of two‐layer αβ sandwiches. The calculated connectivities were compared with those in natural proteins determined using MICAN, a nonsequential structure comparison method. For 2α‐4β, among 23,000 of all connectivities, only 48 were free from irregular connectivities such as loop crossing. Of these, only 20 were found in natural proteins and the superfamilies were biased toward certain types of connectivities. A similar disproportional distribution was confirmed for most of other spatial arrangements of SSEs in the two‐layer αβ sandwiches. We found two connectivity rules that explain the bias well: the abundances of interlayer connecting loops that bridge SSEs in the distinct layers; and nonlocal β‐strand pairs, two spatially adjacent β‐strands located at discontinuous positions in the amino acid sequence. A two‐dimensional plot of these two properties indicated that the two connectivity rules are not independent, which may be interpreted as a rule for the cooperativity of proteins.     

Expression of a fungal <Emphasis Type="Italic">laccase</Emphasis> fused with a bacterial cellulose-binding module improves the enzymatic saccharification efficiency of lignocellulose biomass in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Delignification is effective for improving the saccharification efficiency of lignocellulosic biomass materials. We previously identified that the expression of a fungal laccase (Lac) fused with a bacterial cellulose-binding module domain (CBD) improved the enzymatic saccharification efficiency of rice plants. In this work, to evaluate the ability of the Lac-CBD fused chimeric enzyme to improve saccharification efficiency in a dicot plant, we introduced the chimeric gene into a dicot model plant, Arabidopsis thaliana. Transgenic plants expressing the Lac-CBD chimeric gene showed normal morphology and growth, and showed a significant increase of enzymatic saccharification efficiency compared to control plants. The transgenic plants with the largest improvement of enzymatic saccharification efficiency also showed an increase of crystalline cellulose in their cell wall fractions. These results indicated that expression of the Lac-CBD chimeric protein in dicotyledonous plants improved the enzymatic saccharification of plant biomass by increasing the crystallinity of cellulose in the cell wall.     

Adaptive plasticity in the life history strategy of a canopy tree species, <Emphasis Type="Italic">Pterocarya rhoifolia</Emphasis>, along a gradient of maximum snow depth

The Japanese wingnut Pterocarya rhoifolia is a riparian canopy tree species that grows under a variety of climate conditions, including heavy snowfall, despite the difficulties posed to canopy trees in such environments. This suggests that P. rhoifolia might adapt its life history strategy to different snowfall conditions. This study compared several life history traits of this tree species in a cool temperate mountainous area in central Japan along a gradient of maximum snow depth. The following trends were observed with increasing maximum snow depth: (1) diameter at breast height decreased, maximum stem length and tree height shortened, and trees tended toward a ‘dwarf shrub’ form; (2) the number of sprout stems increased significantly, and these sprouts contributed to maintaining the population; and (3) seed production decreased. Our results suggest trade-offs between clonal growth (sprouting) and sexual reproduction (seed production), and between sprouting and height growth. We concluded that the life history strategy P. rhoifolia demonstrated adaptive plasticity in response to a gradient of maximum snow depth.     

Effects of temperature on body color change in the grasshopper Atractomorpha lata (Orthoptera: Pyrgomorphidae) with reference to sex differences in color morph frequencies

The effects of five environmental factors, background color, density, humidity, photoperiod and temperature, on the body color of a pyrgomorphid green/brown polymorphic grasshopper, Atractomorpha lata, were examined in rearing experiments, in which the differences in color morph frequencies between males and females were also examined. Field censuses were conducted to determine whether the sex difference in body‐color polymorphism occurs under field conditions. Among the environmental factors examined, temperature was the only factor that significantly affected body color: the frequency of brown morphs tended to be higher at higher temperatures. The frequency of brown morphs in the female was higher than in the male, a consistent finding in both the rearing experiments and the field censuses. The threshold temperature at which body color changes may be lower in the female than in the male.     

Natural variation in immune responses to neonatal Mycobacterium bovis Bacillus Calmette-Guerin (BCG) Vaccination in a Cohort of Gambian infants

Background

There is a need for new vaccines for tuberculosis (TB) that protect against adult pulmonary disease in regions where BCG is not effective. However, BCG could remain integral to TB control programmes because neonatal BCG protects against disseminated forms of childhood TB and many new vaccines rely on BCG to prime immunity or are recombinant strains of BCG. Interferon-gamma (IFN-γ) is required for immunity to mycobacteria and used as a marker of immunity when new vaccines are tested. Although BCG is widely given to neonates IFN-γ responses to BCG in this age group are poorly described. Characterisation of IFN-γ responses to BCG is required for interpretation of vaccine immunogenicity study data where BCG is part of the vaccination strategy.

Methodology/Principal Findings

236 healthy Gambian babies were vaccinated with M. bovis BCG at birth. IFN-γ, interleukin (IL)-5 and IL-13 responses to purified protein derivative (PPD), killed Mycobacterium tuberculosis (KMTB), M. tuberculosis short term culture filtrate (STCF) and M. bovis BCG antigen 85 complex (Ag85) were measured in a whole blood assay two months after vaccination. Cytokine responses varied up to 10 log-fold within this population. The majority of infants (89–98% depending on the antigen) made IFN-γ responses and there was significant correlation between IFN-γ responses to the different mycobacterial antigens (Spearman''s coefficient ranged from 0.340 to 0.675, p = 10⁻⁶–10⁻²²). IL-13 and IL-5 responses were generally low and there were more non-responders (33–75%) for these cytokines. Nonetheless, significant correlations were observed for IL-13 and IL-5 responses to different mycobacterial antigens

Conclusions/Significance

Cytokine responses to mycobacterial antigens in BCG-vaccinated infants are heterogeneous and there is significant inter-individual variation. Further studies in large populations of infants are required to identify the factors that determine variation in IFN-γ responses.     

Lrp4 Modulates Extracellular Integration of Cell Signaling Pathways in Development

The extent to which cell signaling is integrated outside the cell is not currently appreciated. We show that a member of the low-density receptor-related protein family, Lrp4 modulates and integrates Bmp and canonical Wnt signalling during tooth morphogenesis by binding the secreted Bmp antagonist protein Wise. Mouse mutants of Lrp4 and Wise exhibit identical tooth phenotypes that include supernumerary incisors and molars, and fused molars. We propose that the Lrp4/Wise interaction acts as an extracellular integrator of epithelial-mesenchymal cell signaling. Wise, secreted from mesenchyme cells binds to BMP''s and also to Lrp4 that is expressed on epithelial cells. This binding then results in the modulation of Wnt activity in the epithelial cells. Thus in this context Wise acts as an extracellular signaling molecule linking two signaling pathways. We further show that a downstream mediator of this integration is the Shh signaling pathway.