Right isomerism of the brain in inversus viscerum mutant mice

Left-right (L-R) asymmetry is a fundamental feature of higher-order neural function. However, the molecular basis of brain asymmetry remains unclear. We recently reported L-R asymmetry of hippocampal circuitry caused by differential allocation of N-methyl-D-aspartate receptor (NMDAR) subunit GluRepsilon2 (NR2B) in hippocampal synapses. Using electrophysiology and immunocytochemistry, here we analyzed the hippocampal circuitry of the inversus viscerum (iv) mouse that has a randomized laterality of internal organs. The iv mouse hippocampus lacks L-R asymmetry, it exhibits right isomerism in the synaptic distribution of the epsilon2 subunit, irrespective of the laterality of visceral organs. This independent right isomerism of the hippocampus is the first evidence that a distinct mechanism downstream of the iv mutation generates brain asymmetry.     

Left-right axis development: examples of similar and divergent strategies to generate asymmetric morphogenesis in chick and mouse embryos

Left-right asymmetry of internal organs is widely distributed in the animal kingdom. The chick and mouse embryos have served as important model organisms to analyze the mechanisms underlying the establishment of the left-right axis. In the chick embryo many genes have been found to be asymmetrically expressed in and around the node, while the same genes in the mouse show symmetric expression patterns. In the mouse there is strong evidence for an establishment of left-right asymmetry through nodal cilia. In contrast, in the chick and in many other organisms left-right asymmetry is probably generated by an early-acting event involving membrane depolarization. In both birds and mammals a conserved Nodal-Lefty-Pitx2 module exists that controls many aspects of asymmetric morphogenesis. This review also gives examples of divergent mechanisms of establishing asymmetric organ formation. Thus there is ample evidence for conserved and non-conserved strategies to generate asymmetry in birds and mammals.     

Functional motor asymmetry in three species of mouse rodents from natural populations

Motor asymmetry of paws was studied in three species of mouse rodents (Apodemus agrarius, Clethrionomys glareolus, Clethrionomys rutilus) from natural populations. The prevalence of right-hand asymmetry was revealed in both males and females of all studied species. The spatial and temporal variability of asymmetry structures was shown. The increased number of left-handed rodents was observed in unfavorable habitats. A possible adaptive role of the functional motor asymmetry exemplified by small rodents is discussed.     

Wernicke's area homologue in chimpanzees (Pan troglodytes) and its relation to the appearance of modern human language

Human language is distinctive compared with the communication systems of other species. Yet, several questions concerning its emergence and evolution remain unresolved. As a means of evaluating the neuroanatomical changes relevant to language that accompanied divergence from the last common ancestor of chimpanzees, bonobos and humans, we defined the cytoarchitectonic boundaries of area Tpt, a component of Wernicke''s area, in 12 common chimpanzee brains and used design-based stereologic methods to estimate regional volumes, total neuron number and neuron density. In addition, we created a probabilistic map of the location of area Tpt in a template chimpanzee brain coordinate space. Our results show that chimpanzees display significant population-level leftward asymmetry of area Tpt in terms of neuron number, with volume asymmetry approaching significance. Furthermore, asymmetry in the number of neurons in area Tpt was positively correlated with asymmetry of neuron numbers in Brodmann''s area 45, a component of Broca''s frontal language region. Our findings support the conclusion that leftward asymmetry of Wernicke''s area originated prior to the appearance of modern human language and before our divergence from the last common ancestor. Moreover, this study provides the first evidence of covariance between asymmetry of anterior and posterior cortical regions that in humans are important to language and other higher order cognitive functions.     

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 Ca(2+) and is not observed in the presence of Ca(2+). 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.     

Cilia are at the heart of vertebrate left-right asymmetry

Handed asymmetry of the shape and position of the internal organs is found in all vertebrates, and is essential for normal cardiac development. Recent genetic and embryological experiments in mouse embryos have demonstrated that left-right asymmetry is established by directional flow of extraembryonic fluid surrounding the node, which is driven by motile monocilia.     

On the origin of lateral asymmetry

Lateral asymmetry refers to unequal fluorescent intensity between adjacent regions of sister chromatids. It has been observed in the centromeric regions of mitotic chromosomes of mouse or human origin when cells are grown in 5-bromo-2-deoxyuridine (BrdU) for a single round of DNA synthesis. The chromosome-orientation fluorescence in situ hybridization (CO-FISH) technique was used with pseudodiploid mouse cells to show that the regions of asymmetrical brightness coincide with major satellite repetitive DNA, and that the more heavily BrdU-substituted chromatid is the one that fluoresces less brightly. These observations support a 20 year old hypothesis on the origin of lateral asymmetry. Other observations suggest that differential loss of DNA from the heavily substituted chromatid also contributes to lateral asymmetry.     

Left-right asymmetry defect in the hippocampal circuitry impairs spatial learning and working memory in iv mice

Although left-right (L-R) asymmetry is a fundamental feature of higher-order brain function, little is known about how asymmetry defects of the brain affect animal behavior. Previously, we identified structural and functional asymmetries in the circuitry of the mouse hippocampus resulting from the asymmetrical distribution of NMDA receptor GluR ε2 (NR2B) subunits. We further examined the ε2 asymmetry in the inversus viscerum (iv) mouse, which has randomized laterality of internal organs, and found that the iv mouse hippocampus exhibits right isomerism (bilateral right-sidedness) in the synaptic distribution of the ε2 subunit, irrespective of the laterality of visceral organs. To investigate the effects of hippocampal laterality defects on higher-order brain functions, we examined the capacity of reference and working memories of iv mice using a dry maze and a delayed nonmatching-to-position (DNMTP) task, respectively. The iv mice improved dry maze performance more slowly than control mice during acquisition, whereas the asymptotic level of performance was similar between the two groups. In the DNMTP task, the iv mice showed poorer accuracy than control mice as the retention interval became longer. These results suggest that the L-R asymmetry of hippocampal circuitry is critical for the acquisition of reference memory and the retention of working memory.     

Beta-catenin asymmetry is regulated by PLA1 and retrograde traffic in C. elegans stem cell divisions

Asymmetric division is an important property of stem cells. In Caenorhabditis elegans, the Wnt/beta-catenin asymmetry pathway determines the polarity of most asymmetric divisions. The Wnt signalling components such as beta-catenin localize asymmetrically to the cortex of mother cells to produce two distinct daughter cells. However, the molecular mechanism to polarize them remains to be elucidated. Here, we demonstrate that intracellular phospholipase A(1) (PLA(1)), a poorly characterized lipid-metabolizing enzyme, controls the subcellular localizations of beta-catenin in the terminal asymmetric divisions of epithelial stem cells (seam cells). In mutants of ipla-1, a single C. elegans PLA(1) gene, cortical beta-catenin is delocalized and the asymmetry of cell-fate specification is disrupted in the asymmetric divisions. ipla-1 mutant phenotypes are rescued by expression of ipla-1 in seam cells in a catalytic activity-dependent manner. Furthermore, our genetic screen utilizing ipla-1 mutants reveals that reduction of endosome-to-Golgi retrograde transport in seam cells restores normal subcellular localization of beta-catenin to ipla-1 mutants. We propose that membrane trafficking regulated by ipla-1 provides a mechanism to control the cortical asymmetry of beta-catenin.     

Non‐visual cues and indirect strategies that enable discrimination of asymmetric mates

The postulates of developmental instability–sexual selection hypothesis is intensely debated among evolutionary biologists, wherein despite a large amount of empirical data, evidence for or against it has been largely inconclusive. A key assumption of this hypothesis is that animals assess symmetry in potential mates as an indicator of genetic quality (developmental stability), and consequently use this information to discriminate against those with higher asymmetries while choosing mates. However, the perceptional basis that must underlie such discriminatory behavior (is symmetry a signal or is symmetry signaled) is not clearly defined. It is also argued that since asymmetry levels in natural populations are very low, the low signal‐to‐noise ratio would make accurate assessment of symmetry both difficult and costly. Rather than attempting to validate this hypothesis or even as to whether animals assess mate symmetry, this review simply aims to examine the plausibility that animals perceive symmetry (directly or indirectly) and consequently discriminate against asymmetric mates in response to perceived irregularities during courtship. For this, we review mate choice and courtship literature to identify potential sensory cues that might advertise asymmetry or lead to discrimination of asymmetric individuals. Although signaling associated with mate choice is commonly multimodal, previous studies on asymmetry have mainly focused on visual perception. In the light of a recent study (Vijendravarma et al., 2022, Proceedings of the National Academy of Sciences of the United States of America, 119, e2116136119), this review attempts to balance this bias by emphasizing on non‐visual perception of asymmetry. In conclusion, we discuss the methodological challenges associated with testing the role of multimodal cues in detecting mate asymmetry, and highlight the importance of considering ecological, behavioral, and evolutionary aspects of animals while interpreting empirical data that test such hypothesis.     

Left-right patterning in the mouse requires Epb4.1l5-dependent morphogenesis of the node and midline

The mouse node is a transient early embryonic structure that is required for left-right asymmetry and for generation of the axial midline, which patterns neural and mesodermal tissues. The node is a shallow teardrop-shaped pit that sits at the distal tip of the early headfold (e7.75) embryo. The shape of the node is believed to be important for generation of the coherent leftward fluid flow required for initiation of left-right asymmetry, but little is known about the morphogenesis of the node. Here we show that the FERM domain protein Lulu/Epb4.1l5 is required for left-right asymmetry in the early mouse embryo. Unlike other genes previously shown to be required for left-right asymmetry in the mouse, lulu is not required for specification of node cell identity, for Nodal signaling in the node or for ciliogenesis. Instead, lulu is required for proper morphogenesis of the node and midline. The precursors of the wild-type node undergo a series of rapid morphological transitions. First, node precursors arise from an epithelial-to-mesenchymal transition at the anterior primitive streak. While in the mesenchymal layer, the node precursors form several ciliated rosette-like clusters; they then rapidly undergo a mesenchymal-to-epithelial transition to insert into the outer, endodermal layer of the embryo. In lulu mutants, node precursor cells are specified and form clusters, but those clusters fail to coalesce to make a single continuous node epithelium. The data suggest that the assembly of the contiguous node epithelium from mesenchymal clusters requires a rapid reorganization of apical-basal polarity that depends on Lulu/Epb4.1l5.     

Random determination of a developmental process: reversal of normal visceral asymmetry in the mouse.

Situs inversus viscerum in the mouse has been shown to be inherited as an autosomal recessive trait (gene symbol iv) with reduced penetrance. It is hypothesized that the normal allele at the iv locus exhibits complete dominance and controls normal visceral asymmetry. Absence of this control allows the situs of visceral asymmetry to be determined in a random fashion. This hypothesis also appears to apply to the inheritance of situs inversus in man and to the experimental production of situs inversus.     

A DNA-binding fraction of mouse kidney 3-ketosteroid reductase: Comparison with androgen receptors

Since approximately 1% of 3-ketosteroid reductase (which metabolizes dihydrotestosterone [17β-hydroxy-5α-androstan-3-one] to 5α-androstane-3α,17β-diol or 5α-androstane-3α,17β-diol) from mouse kidney cytosol adheres to DNA under conditions that allow virtually complete androgen receptor binding, these two DNA-binding activities were compared in cytosol extracts of mouse kidney and hypothalamus-preoptic area. This DNA-binding fraction of 3-ketosteroid reductase was distinguished from androgen receptor in several ways: (1) its pattern of elution from DNA-cellulose with steps of increasing NaC1 concentration differed from that for receptors from wild-type kidney; (2) it was influenced differently by the mutation Tfm, both in level and in DNA-cellulose elution pattern; (3) in mouse kidney cytosol it was relatively stable at moderate (25°C) temperatures which rapidly inactivated ligand-free androgen receptors in the same cytosols; (4) the DNA-binding was not proportional to androgen receptor levels between two wild-type tissues, the hypothalamus-preoptic area and kidney. By these criteria, a simple relationship of androgen receptors and a DNA-binding fraction of 3-ketosteroid reductase activity is unlikely.     

The bounce of the body in hopping,running and trotting: different machines with the same motor

The bouncing mechanism of human running is characterized by a shorter duration of the brake after ‘landing’ compared with a longer duration of the push before ‘takeoff’. This landing–takeoff asymmetry has been thought to be a consequence of the force–velocity relation of the muscle, resulting in a greater force exerted during stretching after landing and a lower force developed during shortening before takeoff. However, the asymmetric lever system of the human foot during stance may also be the cause. Here, we measure the landing–takeoff asymmetry in bouncing steps of running, hopping and trotting animals using diverse lever systems. We find that the duration of the push exceeds that of the brake in all the animals, indicating that the different lever systems comply with the basic property of muscle to resist stretching with a force greater than that developed during shortening. In addition, results show both the landing–takeoff asymmetry and the mass-specific vertical stiffness to be greater in small animals than in large animals. We suggest that the landing–takeoff asymmetry is an index of a lack of elasticity, which increases with increasing the role of muscle relative to that of tendon within muscle–tendon units.     

Parental age influences developmental stability of the progeny in Drosophila

The stochastic nature of biochemical processes is a source of variability that influences developmental stability. Developmental instability (DI) is often estimated through fluctuating asymmetry (FA), a parameter that deals with within-individual variation in bilateral structures. A relevant goal is to shed light on how environment, physiology and genotype relate to DI, thus providing a more comprehensive view of organismal development. Using Drosophila melanogaster isogenic lines, we investigated the effect of parental age, parental diet and offspring heterozygosity on DI. In this work, we have uncovered a clear relationship between parental age and offspring asymmetry. We show that asymmetry of the progeny increases concomitantly with parental age. Moreover, we demonstrate that enriching the diet of parents mitigates the effect of age on offspring symmetry. We show as well that increasing the heterozygosity of the progeny eliminates the effect of parental age on offspring symmetry. Taken together, our results suggest that diet, genotype and age of the parents interact to determine offspring DI in wild populations. These findings provide us with an avenue to understand the mechanisms underlying DI.     

Nonspecific activation of murine spleen cells in vitro by a synthetic immunoadjuvant (N-acetyl-muramyl-L-alanyl-D-isoglutamine)

We reported previously that synthetic N-acetyl-muramyl-l-alanyl-d-isoglutamine (MDP) displayed marked adjuvant activity but was devoid of mitogenicity in vitro. The data reported here establish that, under different cultural conditions, thymidine uptake and blast cells can be increased by MDP in spleen cells of DBA/2 and Balb/c mouse strains. Optimal responses were obtained on culture in a serum-free medium supplemented with 2-mercaptoethanol for 4 or 5 days. This effect was also obtained with spleen cells of Balb/c nude mice. When the synthetic MDP was compared to a natural water-soluble adjuvant (neo-WSA), extracted from Mycobacterium smegmatis cells, both were found to stimulate [³H] thymidine incorporation by mouse spleen cells. However, with the neo-WSA, the effect peaked on Day 2 and was weak or absent on Days 4 and 5. When the cells were cultured in a medium containing fetal calf serum, neo-WSA activation was completely abolished, while MDP-mediated stimulation was decreased.     

Paw-Dragging: a Novel,Sensitive Analysis of the Mouse Cylinder Test

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore by rearing and touching the walls of the cylinder with their forelimb paws for postural support. Following ischemic injury to the forelimb sensorimotor cortex, rats rely more heavily on their unaffected forelimb paw for postural support resulting in fewer touches with their affected paw which is termed forelimb asymmetry. In contrast, focal ischemic damage in the mouse brain fails to result in comparable consistent deficits in forelimb asymmetry. While forelimb asymmetry deficits are infrequently observed, mice do demonstrate a novel behaviour post stroke termed “paw-dragging”. Paw-dragging is the tendency for a mouse to drag its affected paw along the cylinder wall rather than directly push off from the wall when dismounting from a rear to a four-legged stance. We have previously demonstrated that paw-dragging behaviour is highly sensitive to small cortical ischemic injuries to the forelimb motor cortex. Here we provide a detailed protocol for paw-dragging analysis. We define what a paw-drag is and demonstrate how to quantify paw-dragging behaviour. The cylinder test is a simple and inexpensive test to administer and does not require pre-training or food deprivation strategies. In using paw-dragging analysis with the cylinder test, it fills a niche for predicting cortical ischemic injuries such as photothrombosis and Endothelin-1 (ET-1)-induced ischemia – two models that are ever-increasing in popularity and produce smaller focal injuries than middle cerebral artery occlusion. Finally, measuring paw-dragging behaviour in the cylinder test will allow studies of functional recovery after cortical injury using a wide cohort of transgenic mouse strains where previous forelimb asymmetry analysis has failed to detect consistent deficits.     

Left-right patterning: conserved and divergent mechanisms

The left-right (LR) asymmetry of visceral organs is fundamental to their function and position within the body. Over the past decade or so, the molecular mechanisms underlying the establishment of such LR asymmetry have been revealed in many vertebrate and invertebrate model organisms. These studies have identified a gene network that contributes to this process and is highly conserved from sea urchin to mouse. By contrast, some specific steps of the process, such as the symmetry-breaking event and situs-specific organogenesis, appear to have diverged during evolution. Here, we summarize the common and divergent mechanisms by which LR asymmetry is established in vertebrates.