Septins and a formin have distinct functions in anaphase chiral cortical rotation in the Caenorhabditis elegans zygote

Many cells and tissues exhibit chirality that stems from the chirality of proteins and polymers. In the Caenorhabditis elegans zygote, actomyosin contractility drives chiral rotation of the entire cortex circumferentially around the division plane during anaphase. How contractility is translated to cell-scale chirality, and what dictates handedness, are unknown. Septins are candidate contributors to cell-scale chirality because they anchor and cross-link the actomyosin cytoskeleton. We report that septins are required for anaphase cortical rotation. In contrast, the formin CYK-1, which we found to be enriched in the posterior in early anaphase, is not required for cortical rotation but contributes to its chirality. Simultaneous loss of septin and CYK-1 function led to abnormal and often reversed cortical rotation. Our results suggest that anaphase contractility leads to chiral rotation by releasing torsional stress generated during formin-based polymerization, which is polarized along the cell anterior–posterior axis and which accumulates due to actomyosin network connectivity. Our findings shed light on the molecular and physical bases for cellular chirality in the C. elegans zygote. We also identify conditions in which chiral rotation fails but animals are developmentally viable, opening avenues for future work on the relationship between early embryonic cellular chirality and animal body plan.     

Genetic and Phenotypic Characterization of Roller Mutants of CAENORHABDITIS ELEGANS

Eighty-eight mutants of C. elegans that display a roller phenotype (a helically twisted body) have been isolated and characterized genetically and phenotypically. The mutations are located in 14 different genes. Most genes contain a number of alleles. Their distribution among the chromosomes appears nonrandom, with seven of the genes being located on linkage group II, some very closely linked. The phenotypes of the mutants suggest that there are five different classes of genes, each class representing a set of similar phenotypic effects: Left Roller (four genes), Right Roller (one gene), Left Squat (one gene), Right Squat (two genes) and Left Dumpy Roller (six genes). The classes of mutants differ with respect to a number of characteristics that include the developmental stages affected and the types of aberrations observed in cuticle structure. A variety of gene interactions were found, arguing that these genes are involved in a common developmental process. The presence of alterations in cuticle morphology strongly suggests that these genes are active in the formation of the nematode cuticle.     

Maternal effect of low temperature on handedness determination in C. elegans embryos

C. elegans embryos, larvae, and adults exhibit several left-right asymmetries with an invariant dextral handedness, which first becomes evident in the embryo at the 6-cell stage. Reversed (sinistral) handedness was not observed among > 10,000 N2 adults reared at 16°C or 20°C under standard conditions. However, among the progeny of adults reproducing at 10°C, the frequency of animals with sinistral handedness was increased to ∼0.5%. Cold pulse experiments indicated that the critical period for this increase was in early oogenesis, several hours before the first appearance of left-right asymmetry in the embryo. Hermaphrodites reared at 10°C and mated with males reared at 20°C produced sinistral outcross as well as sinistral self-progeny, indicating that the low temperature effect on oocytes was sufficient to cause reversals. Increased frequency of reversal was also observed among animals developed from embryos lacking the egg shell. Possible mechanisms for the control of embryonic handedness are discussed in the context of these results, including the hypothesis that handedness could be dictated by the chirality of a gametic component. © 1996 Wiley-Liss, Inc.     

Development and evolution of a variable left-right asymmetry in nematodes: the handedness of P11/P12 migration

In Caenorhabditis elegans, two lateral blast cells called P11/12L and P11/12R are symmetric left-right homologs at hatching, migrate subsequently in opposite anteroposterior directions during the first larval stage, and adopt two different fates, thus breaking the symmetry between them. Our results show that, unlike most other cell fate decisions in C. elegans, the orientation of P11/12L/R migration is highly biased, but not fixed. The handedness of their migration is linked to whole body handedness and is randomized in lin-12/Notch mutants and by ablation of the Y cell. Migration handedness is independent of P11 and P12 fate determination, previously shown to require the LIN-44/Wnt and the LIN-3/EGF pathways (L. I. Jiang and P. W. Sternberg, 1998, Development 125, 2337-2347). We further show that several changes in P11/12L/R asymmetry have occurred during nematode evolution: loss of asymmetry or reversals in orientation of migration. Strikingly, for most species studied, handedness of migration is highly biased but not fixed. Thus, whereas the final cell fate pattern of P11/12 is invariant, the developmental route leading to it is subject both to developmental indeterminacy and to evolutionary variations.     

Evolution of mirror images by sexually asymmetric mating behavior in hermaphroditic snails

Abstract Directionally asymmetric animals generally exhibit no variation in handedness of whole-body architecture. In contrast, reversed chirality in both coil and entire anatomy has frequently evolved in snails. We demonstrate a nonrandom pattern and deterministic process of chiral evolution, as predicted by the following hypothesis. Bimodal shell shapes are associated with discrete mating behaviors in hermaphroditic pulmonates. Flat-shelled species mate reciprocally, face-to-face. This sexual symmetry prevents interchiral mating because genitalia exposed by a sinistral on its left side cannot be joined with those exposed by a dextral on its right. Thus, selection against the chiral minority, resulting from mating disadvantage, stabilizes chiral monomorphism. Tall-shelled species mate nonreciprocally: the 'male' copulates by mounting the 'female's' shell, mutually aligned in the same direction. This sexual asymmetry permits interchiral copulation with small behavioral adjustments. Therefore, the positive frequency-dependent selection is relaxed, and reversal alleles persist longer in populations of tall-shelled species. We verified both the assumption and the prediction of this hypothesis: significantly lower interchiral mating success in a low-spired species and higher chiral evolution rate in high-spired taxa. Sexual asymmetry is the key to understanding the accelerated chiral evolution in high-spired pulmonates.     

Construction of a backcross progeny collection of dextral and sinistral individuals of a freshwater gastropod,<Emphasis Type="Italic"> Lymnaea stagnalis</Emphasis>

The handedness of gastropods is genetically determined, but the molecular nature of the gene responsible remains unknown. In order to identify the gene by a forward genetic approach, we carried out backcross breeding between dextral and sinistral inbred strains of a freshwater gastropod, Lymnaea stagnalis, cultivated in the laboratory. We used the dextral animals as donor "fathers" and the sinistral animals as recurrent "mothers". Each of the backcross progeny obtained was typed for the chirality of the next generation offspring oviposited by it, because the genotype for the handedness locus emerges as filial chirality. We constructed a collection of DNA and RNA specimens that included about 200 of the backcross F(2) progeny. Success in breeding was confirmed by the expected inheritance behavior of strain-specific DNA markers in the progeny.     

The enigma of reversed asymmetry in lithodid crabs: absence of evidence for heritability or induction of morphological handedness in Lopholithodes foraminatus

SUMMARY Mutations or environmental factors that result in reversal of conspicuous left–right asymmetries provide an opportunity to study developmental mechanisms. They may also provide insight into evolutionary changes in asymmetry states within and between species. King crabs (family Lithodidae) have a larger right claw and females typically exhibit a dextrally offset abdomen. Nevertheless, I observed a high incidence of left handedness in laboratory reared box crabs ( Lopholithodes foraminatus ) and captured the first known egg-bearing female lithodid to exhibit reversed asymmetry. This provided a unique opportunity to characterize the reversed phenotype and to compare the incidence of reversed asymmetry in the offspring of normal and reversed females. Asymmetry of the chelae became apparent in the first postzoeal stage (glaucothoe) and handedness was maintained through subsequent instars. Females with larger left claws developed reversed abdominal asymmetry by the fourth crab stage. No reversed asymmetry was observed in the mandibles of zoea larvae or juveniles of either handedness. The incidence of reversed asymmetry in glaucothoe reared from one reversed and three normal females was high (between 20% and 30%), and independent of maternity ( P =0.67). Removal of the right cheliped of fourth stage zoeae, and the major cheliped of glaucothoe, did not reverse the direction of asymmetry. Elevated larval rearing temperature also did not affect the frequency of reversed individuals. This lack of evidence for either heritability or induction of handedness is enigmatic. Further investigation of reversed asymmetry in lithodid crabs may provide valuable insights into the development and evolution of bilateral asymmetries.     

Isolation and characterization of a Japanese flounder clonal line, reversed, which exhibits reversal of metamorphic left-right asymmetry.

We have isolated a clonal line reversed (rev) of homozygous Japanese flounder through gynogenesis. The homozygous offspring gynogenetically produced from rev exhibited reversal of organization of the metamorphic L/R asymmetry such as the direction of eye-migration at a high frequency (20-30%). The molecular analysis using a left-specific marker pitx2 revealed that the embryonic L/R axis was ambiguously established: in more than half of rev embryos, pitx2 was expressed bilaterally in the lateral plate mesoderm (LPM). Previous studies in other animals demonstrated that ectopic pitx2 expression in the LPM could cause laterality defects of the visceral organs. Likewise, our results using rev imply that bilateral pitx2 expression could lead to randomization of the visceral organs. Coincidence of ectopic pitx2 expression and reversal of the direction of eye-migration in the population of rev offspring suggests that the rev locus is critical in specification of both the metamorphic and the visceral L/R asymmetries. However, reversal of the sidedness of the orientation of the visceral organs was not always accompanied by reversal of the direction of metamorphic eye-migration, suggesting that different mechanisms should be involved downstream of the rev locus in directing these two phases of asymmetric morphogenesis in the Japanese flounder.     

Left–right asymmetry in Drosophila melanogaster gut development

While left-right (LR) asymmetric morphogenesis is common to various animal species, there have been no systematic studies of the LR asymmetry of body structures of Drosophila melanogaster. In the present paper the LR asymmetric development of the Drosophila gut is described, in which three major parts, the foregut, midgut and hindgut, show almost invariant LR asymmetry. The asymmetry is generated by a twist of each part in particular orientations, resulting in a left-handed (sinistral) convolution as a whole. The frequency of spontaneous reversal of LR orientations is very low (< 0.6%) and reversal of each part of the gut occurs independently. The bicoid mutation causes duplication of the posterior half of the gut, essentially keeping the left-handed twist, suggesting that the LR asymmetry may depend on some intrinsic nature of the cells or tissues rather than a graded distribution of morphogens in the egg. The handedness of particular gut parts was randomized or became symmetric in mutants of brachyenteron, huckebein and patched, suggesting that different gene pathways can interfere in determining LR asymmetry of the gut. It is noteworthy that all of these genes are expressed LR symmetrically.     

Action anticipation in human infants reveals assumptions about anteroposterior body-structure and action

Animal actions are almost universally constrained by the bilateral body-plan. For example, the direction of travel tends to be constrained by the orientation of the animal''s anteroposterior axis. Hence, an animal''s behaviour can reliably guide the identification of its front and back, and its orientation can reliably guide action prediction. We examine the hypothesis that the evolutionarily ancient relation between anteroposterior body-structure and behaviour guides our cognitive processing of agents and their actions. In a series of studies, we demonstrate that, after limited exposure, human infants as young as six months of age spontaneously encode a novel agent as having a certain axial direction with respect to its actions and rely on it when anticipating the agent''s further behaviour. We found that such encoding is restricted to objects exhibiting cues of agency and does not depend on generalization from features of familiar animals. Our research offers a new tool for investigating the perception of animate agency and supports the proposal that the underlying cognitive mechanisms have been shaped by basic biological adaptations in humans.     

Magnetic compass sense in the large yellow underwing moth, Noctua pronuba L.

Many animals are now known to have a magnetic sense which they use when moving from one place to another. Among insects, this sense has only been studied in any detail in the honey bee. A role for a magnetic compass sense in cross-country migration has not so far been demonstrated for any insect. On clear nights the large yellow underwing moth, Noctua pronuba, has been shown to orientate by both the moon and the stars. However, radar studies have shown moths to be well-oriented on overcast nights as well as clear nights. We report here that when large yellow underwings are placed in an orientation cage on overcast nights and the Earth's normal magnetic field is reversed, there is a corresponding reversal in the orientation of the moth. We conclude that this species makes use of the Earth's magnetic field in maintaining compass orientation on overcast nights. We also show that the preferred compass orientation to the Earth's magnetic field is the same as the compass direction that results from orientation to the moon and stars.     

Smad5 is essential for left-right asymmetry in mice

Left-right (L-R) asymmetry of the vertebrate body plan is established from an originally morphologically symmetric embryo. Recent studies have implicated several TGF-beta family signaling proteins (i.e., nodal, lefty-1, lefty-2, activin receptor type IIB, and Smad2) in L-R axis determination in the mouse. However, the genetic pathways underlying L-R patterning are still unclear. Smad5 is a downstream component in the TGF-beta family signaling cascade, and lack of Smad5 results in embryonic lethality between E9.5 and E11.5. In this report, we demonstrate that Smad5 mutant embryos have defects in heart looping and embryonic turning which are the first signs of L-R asymmetry in mice. To gain more insights into the molecular basis of the laterality defects in the Smad5-deficient embryos, we examined the expression of lefty-1, lefty-2, nodal, and Pitx2 since the asymmetric expression of these genes always closely correlates with the direction of heart looping and embryonic turning. In the absence of Smad5, lefty-1 was expressed at very low or undetectable levels, while nodal, lefty-2, and Pitx2 were expressed bilaterally. These data suggest that Smad5 is upstream of lefty-1, nodal, and lefty-2, and as a consequence also of Pitx2, and Smad5 is essential for L-R axis determination.     

De novo formation of left-right asymmetry by posterior tilt of nodal cilia

In the developing mouse embryo, leftward fluid flow on the ventral side of the node determines left–right (L-R) asymmetry. However, the mechanism by which the rotational movement of node cilia can generate a unidirectional flow remains hypothetical. Here we have addressed this question by motion and morphological analyses of the node cilia and by fluid dynamic model experiments. We found that the cilia stand, not perpendicular to the node surface, but tilted posteriorly. We further confirmed that such posterior tilt can produce leftward flow in model experiments. These results strongly suggest that L-R asymmetry is not the descendant of pre-existing L-R asymmetry within each cell but is generated de novo by combining three sources of spatial information: antero-posterior and dorso-ventral axes, and the chirality of ciliary movement.     

De Novo Formation of Left–Right Asymmetry by Posterior Tilt of Nodal Cilia

In the developing mouse embryo, leftward fluid flow on the ventral side of the node determines left–right (L-R) asymmetry. However, the mechanism by which the rotational movement of node cilia can generate a unidirectional flow remains hypothetical. Here we have addressed this question by motion and morphological analyses of the node cilia and by fluid dynamic model experiments. We found that the cilia stand, not perpendicular to the node surface, but tilted posteriorly. We further confirmed that such posterior tilt can produce leftward flow in model experiments. These results strongly suggest that L-R asymmetry is not the descendant of pre-existing L-R asymmetry within each cell but is generated de novo by combining three sources of spatial information: antero-posterior and dorso-ventral axes, and the chirality of ciliary movement.     

Genetic control of cuticle formation during embryonic development of Drosophila melanogaster

The embryonic cuticle of Drosophila melanogaster is deposited by the epidermal epithelium during stage 16 of development. This tough, waterproof layer is essential for maintaining the structural integrity of the larval body. We have characterized mutations in a set of genes required for proper deposition and/or morphogenesis of the cuticle. Zygotic disruption of any one of these genes results in embryonic lethality. Mutant embryos are hyperactive within the eggshell, resulting in a high proportion reversed within the eggshell (the "retroactive" phenotype), and all show poor cuticle integrity when embryos are mechanically devitellinized. This last property results in embryonic cuticle preparations that appear grossly inflated compared to wild-type cuticles (the "blimp" phenotype). We find that one of these genes, krotzkopf verkehrt (kkv), encodes the Drosophila chitin synthase enzyme and that a closely linked gene, knickkopf (knk), encodes a novel protein that shows genetic interaction with the Drosophila E-cadherin, shotgun. We also demonstrate that two other known mutants, grainy head (grh) and retroactive (rtv), show the blimp phenotype when devitellinized, and we describe a new mutation, called zeppelin (zep), that shows the blimp phenotype but does not produce defects in the head cuticle as the other mutations do.     

Orientation by magnetic field in leaf-cutter ants, Atta colombica (Hymenoptera: Formicidae)

Leaf‐cutter ants (Atta colombica) use trail following to travel between foraging sites and the home nest. However, this combination of pheromone and visual cues is likely to be complemented by a directional reference system such as a compass, used not only when foraging but also during colony formation, where foraging trails degrade or where ants become displaced. One candidate system is the magnetic polarity compass. We tested the orientation of leaf‐cutter ants under a magnetic field of reversed‐polarity, with the prediction that the ants would show 180° deflection compared with control ants in an unchanged geomagnetic field. When the sun's disc was unobstructed by clouds, orientation was the same as that of control ants, implying that magnetic cues were not used to orient. However, when the sky was overcast, ants in the experimental treatment significantly shifted their mean orientation both in comparison with controls and reversed‐polarity ants under the sun. Although a total reversal in orientation was not induced, the results demonstrate that Atta respond to magnetic reversal in the absence of sunlight cues, and suggest a role for magnetic cues in determining direction during orientation.     

Microfiber reinforcement of an arthropod cuticle

Summary 1. According to its fine structure arthropod cuticle is a laminated composite material. The exoskeleton of a spider is surveyed in an electron microscopical study as to the characteristics of its fiber reinforcement.2. Five types of fiber arrangement are distinguished, ranging from unidirectional to continuously rotating fiber orientation and with three types combining elements of both of these extreme cases. The unidirectional type is rare and its occurrence confined to the innermost part of the walking leg endocuticle and to parts of articular membranes. Quite contrary a continuous and regular rotation of fiber direction is common. In hard cuticle (exocuticle) this type of reinforcement is found exclusively.3. Lamellar width, indicating a 180° rotation of the fiber direction, varies within wide limits. Variation is due to varying numbers of fiber layers and the amount of directional change between them (ca. 0.6–24°, type E).4. The diameter of the chitin/protein microfiber amounts to ca. 35 Å. Its length measured up to 1,5 m in the preparations. A periodicity of ca. 80 Å is noted. The precision of fiber alignment within the cuticle is high.5. In the cuticle of the opisthosoma and the articular membranes endocuticular lamellae are taking a wavy course, which is interpreted to be in relation to the cuticle's softness.6. The mechanical implications of both an increasing number of fiber directions in a composite material and of the different types of fiber reinforcement found in the spider cuticle are demonstrated by a calculation of the directional properties of the moduli of elasticity in tension and shear. The predictable strong influence of both the fiber content and the binder material's E-modulus on the mechanical properties of cuticle is illustrated.7. Instead of a trajectorial design a continuous rotation of the fiber orientation is found in all exocuticles. Thus the spider does not make use of the special advantage (high strength/weight ratio) of unidirectional fiber arrangement in places where it seemed most likely.Many thanks are due to Ms. H. Tscharntke for expert assistance with electron microscopy and photographic work. Dipl.-Ing. F. Och and Dr. G. Haberl (Messerschmitt-Bölkow-Blohm GmbH, Ottobrunn) provided the calculations for Figs. 14 and 15. Their generous help is gratefully acknowledged. Dr. K. Frahm (Universität München, Fakultät f. Tiermedizin) kindly shared his densitometer. Ms. F. Althaus carefully carried out the drawings.     

C3-symmetrical self-assembled structures investigated by vibrational circular dichroism

We demonstrate by using vibrational circular dichroism (VCD) spectroscopy that it is possible to investigate the chirality of a supramolecular polymeric system in relatively dilute solutions. Chiral C(3)-symmetrical discotic molecules, based on a trialkylbenzene-1,3,5-carboxamide, form supramolecular columnar stacks with a right-handed helical structure in solution due to intermolecular hydrogen bonds. The handedness of the supramolecular chirality is determined using electronic spectroscopy measurements. Under dilute conditions (at 10(-3) M concentrations), it was also possible to probe the hydrogen bonding moieties with IR and VCD spectroscopy on these self-assembled structures. In combination with density functional theory (DFT) calculations, we could verify the preference for a right-handed chirality in the helical stacks and the nonplanar orientation of the carbonyl groups present in the molecule. This chiral arrangement is in agreement with the structure determined for a related benzene-1,3,5-tricarboxamide by X-ray diffraction. Chirality, 2008. (c) 2008 Wiley-Liss, Inc.     

文昌鱼左右体轴建立机制的研究进展

两侧对称动物左右体轴建立机制研究是发育生物学领域重要的基础科学问题之一.文昌鱼(amphioxus)由于其特殊的进化地位以及与脊椎动物相似的胚胎发育模式和身体构筑方式,是研究动物左右体轴建立机制的理想模式物种.近年来随着文昌鱼室内全人工繁育技术、高效显微注射技术和基因敲除技术的建立,国内外学者在左右体轴建立机制研究上取...