Bidirectional crosstalk between Hypoxia-Inducible Factor and glucocorticoid signalling in zebrafish larvae.

In the last decades in vitro studies highlighted the potential for crosstalk between Hypoxia-Inducible Factor-(HIF) and glucocorticoid-(GC) signalling pathways. However, how this interplay precisely occurs in vivo is still debated. Here, we use zebrafish larvae (Danio rerio) to elucidate how and to what degree hypoxic signalling affects the endogenous glucocorticoid pathway and vice versa, in vivo. Firstly, our results demonstrate that in the presence of upregulated HIF signalling, both glucocorticoid receptor (Gr) responsiveness and endogenous cortisol levels are repressed in 5 days post fertilisation larvae. In addition, despite HIF activity being low at normoxia, our data show that it already impedes both glucocorticoid activity and levels. Secondly, we further analysed the in vivo contribution of glucocorticoids to HIF activity. Interestingly, our results show that both glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) play a key role in enhancing it. Finally, we found indications that glucocorticoids promote HIF signalling via multiple routes. Cumulatively, our findings allowed us to suggest a model for how this crosstalk occurs in vivo.

Cholinergic left-right asymmetry in the habenulo-interpeduncular pathway.

The habenulo-interpeduncular pathway, a highly conserved cholinergic system, has emerged as a valuable model to study left-right asymmetry in the brain. In larval zebrafish, the bilaterally paired dorsal habenular nuclei (dHb) exhibit prominent left-right differences in their organization, gene expression, and connectivity, but their cholinergic nature was unclear. Through the discovery of a duplicated cholinergic gene locus, we now show that choline acetyltransferase and vesicular acetylcholine transporter homologs are preferentially expressed in the right dHb of larval zebrafish. Genes encoding the nicotinic acetylcholine receptor subunits α2 and ß4 are transcribed in the target interpeduncular nucleus (IPN), suggesting that the asymmetrical cholinergic pathway is functional. To confirm this, we activated channelrhodopsin-2 specifically in the larval dHb and performed whole-cell patch-clamp recording of IPN neurons. The response to optogenetic or electrical stimulation of the right dHb consisted of an initial fast glutamatergic excitatory postsynaptic current followed by a slow-rising cholinergic current. In adult zebrafish, the dHb are divided into discrete cholinergic and peptidergic subnuclei that differ in size between the left and right sides of the brain. After exposing adults to nicotine, fos expression was activated in subregions of the IPN enriched for specific nicotinic acetylcholine receptor subunits. Our studies of the newly identified cholinergic gene locus resolve the neurotransmitter identity of the zebrafish habenular nuclei and reveal functional asymmetry in a major cholinergic neuromodulatory pathway of the vertebrate brain.

Unveiling the ototoxic effects of paraquat on zebrafish larva.

Paraquat is a widely utilized herbicide in agricultural fields posing a significant impact on human health and the environment due to its potent oxidant properties. Rampant paraquat usage leads to serious health hazards to farmers and the ecosystem, particularly the water bodies. Paraquat exposure can damage dopaminergic neurons causing Parkinson's disease in humans and other animal models. Extensive research has been done regarding the mode of action, pathophysiology and molecular mechanisms of paraquat-induced Parkinson's disease. Meanwhile, the ototoxic effect of paraquat remains poorly understood. Potential ototoxins can cause sensorineural hearing loss, one of the most common sensory disabilities in humans. In this study, we investigated the harmful effects of paraquat on neuromast hair cells in zebrafish larvae, a powerful model organism for auditory research. We treated sub-lethal concentrations (125 µM to 1000 µM) of paraquat to 3 and 4 dpf zebrafish larvae to investigate its ototoxic effects via rheotaxis behavioral assay, neuromast staining and scanning electron microscopy. The behavioral assay findings showed a drastic decline in the rheotaxis behavior in all the concentrations of paraquat-treated larvae. Furthermore, DASPEI neuromast vital staining displayed a dose-dependent reduction in the neuromast hair cells as we increased the paraquat concentration. The scanning electron microscope data revealed the significant shortening of kinociliary length, a decrease in stereociliary density and changes in semilunar peridermal cell morphology signifying the damaging effects of paraquat at the cellular level. Collectively, the behavioral, anatomical and morphological studies highlight the potential ototoxic effects of paraquat on zebrafish neuromast hair cells, further signifying its potential role in causing hearing loss in humans.

Identification of key genes and signalling pathways in clear cell renal cell carcinoma: An integrated bioinformatics approach.

BACKGROUND: Clear cell Renal Cell Carcinoma (ccRCC) is one of the most prevalent types of kidney cancer. Unravelling the genes responsible for driving cellular changes and the transformation of cells in ccRCC pathogenesis is a complex process. OBJECTIVE: In this study, twelve microarray ccRCC datasets were chosen from the gene expression omnibus (GEO) database and subjected to integrated analysis. METHODS: Through GEO2R analysis, 179 common differentially expressed genes (DEGs) were identified among the datasets. The common DEGs were subjected to functional enrichment analysis using ToppFun followed by construction of protein-protein interaction network (PPIN) using Cytoscape. Clusters within the DEGs PPIN were identified using the Molecular Complex Detection (MCODE) Cytoscape plugin. To identify the hub genes, the centrality parameters degree, betweenness, and closeness scores were calculated for each DEGs in the PPIN. Additionally, Gene Expression Profiling Interactive Analysis (GEPIA) was utilized to validate the relative expression levels of hub genes in the normal and ccRCC tissues. RESULTS: The common DEGs were highly enriched in Hypoxia-inducible factor (HIF) signalling and metabolic reprogramming pathways. VEGFA, CAV1, LOX, CCND1, PLG, EGF, SLC2A1, and ENO2 were identified as hub genes. CONCLUSION: Among 8 hub genes, only the expression levels of VEGFA, LOX, CCND1, and EGF showed a unique expression pattern exclusively in ccRCC on compared to other type of cancers.

Angiogenesis Assay for Live and Fixed Zebrafish Embryos/Larvae.

Angiogenesis is the process of new blood vessel formation from preexisting vasculature. It is an integral component in normal embryonic development and tissue repair. Dysregulation of angiogenesis might lead to tissue ischemia (resulting from reduced blood vessel formation) or major diseases such as cancer (abnormal vascular growth). This makes angiogenesis an excellent area of research for cancer therapeutics, and various animal models including zebrafish are used to study blood vessel development. As most of the techniques used to study angiogenesis are complex and cumbersome, in this chapter, we provide two simple assays to study angiogenesis with live and fixed zebrafish embryos/larvae.

Induction of Paraquat-Mediated Parkinsonian Phenotype in Zebrafish.

Paraquat (PQ) is a well-known neurotoxin closely associated with neurodegenerative Parkinson's disease (PD). Zebrafish are utilized as a model for PD research because of their well-defined neuropathology and locomotor behavior. Here, we highlight protocols for inducing PD using PQ and analyzing locomotor activity in adult zebrafish. Basic Protocol 1 details the treatment of adult male zebrafish with 60 mg/kg PQ via intraperitoneal injection to induce a PD-like phenotype, followed by the steps to perform a locomotor assay. Basic Protocol 2 provides step-by-step guidance for processing the acquired videos in ToxTrac software to understand the locomotor parameters of 0.9% saline- and 60 mg/kg PQ-injected adult zebrafish. The simplicity of the treatment strategy, low-cost video acquisition setup, and free video processing make these protocols accessible without prior experience. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Development of Parkinson's disease features in adult zebrafish Basic Protocol 2: ToxTrac analysis for locomotor assay.

Insights on the Role of α- and ß-Tubulin Isotypes in Early Brain Development.

Tubulins are the highly conserved subunit of microtubules which involve in various fundamental functions including brain development. Microtubules help in neuronal proliferation, migration, differentiation, cargo transport along the axons, synapse formation, and many more. Tubulin gene family consisting of multiple isotypes, their differential expression and varied post translational modifications create a whole new level of complexity and diversity in accomplishing manifold neuronal functions. The studies on the relation between tubulin genes and brain development opened a new avenue to understand the role of each tubulin isotype in neurodevelopment. Mutations in tubulin genes are reported to cause brain development defects especially cortical malformations, referred as tubulinopathies. There is an increased need to understand the molecular correlation between various tubulin mutations and the associated brain pathology. Recently, mutations in tubulin isotypes (TUBA1A, TUBB, TUBB1, TUBB2A, TUBB2B, TUBB3, and TUBG1) have been linked to cause various neurodevelopmental defects like lissencephaly, microcephaly, cortical dysplasia, polymicrogyria, schizencephaly, subcortical band heterotopia, periventricular heterotopia, corpus callosum agenesis, and cerebellar hypoplasia. This review summarizes on the microtubule dynamics, their role in neurodevelopment, tubulin isotypes, post translational modifications, and the role of tubulin mutations in causing specific neurodevelopmental defects. A comprehensive list containing all the reported tubulin pathogenic variants associated with brain developmental defects has been prepared to give a bird's eye view on the broad range of tubulin functions.

Chemically induced models of Parkinson's disease.

Environmental toxins are harmful substances detrimental to humans. Constant exposure to these fatal neurotoxins can cause various neurodegenerative disorders. Although poisonous, specific neurotoxins at optimal concentrations mimic the clinical features of neurodegenerative diseases in several animal models. Such chemically-induced model systems are beneficial in deciphering the molecular mechanisms of neurodegeneration and drug screening for these disorders. One such neurotoxin is 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a widely used chemical that recapitulates Parkinsonian features in various animal models. Apart from MPTP, other neurotoxins like 6-hydroxydopamine (6-OHDA), paraquat, rotenone also induce specific clinical features of Parkinson's disease in animal models. These chemically-induced Parkinson's disease models are playing a crucial role in understanding Parkinson's disease onset, pathology, and novel therapeutics. In this review, we provide a concise overview of various neurotoxins that can recapitulate Parkinsonian features in different in vivo and in vitro model systems specifically focusing on the different treatment methodologies of neurotoxins.

Novel insights on acetylcholinesterase inhibition by Convolvulus pluricaulis, scopolamine and their combination in zebrafish.

Acetylcholinesterase (AChE) inhibitors increase the retention of acetylcholine (ACh) in synapses. Although they alleviate cognitive deficits in Alzheimer's disease, their limited benefits warrant investigations of plant extracts with similar properties. We studied the anti-AChE activity of Convolvulus pluricaulis (CP) in a zebrafish model of cognitive impairment induced by scopolamine (SCOP). CP is a perennial herb with anti-amnesiac and anxiolytic properties. It contains alkaloid, anthocyanin, coumarin, flavonoid, phytosterol and triterpenoid components. Isoxazole (ISOX) was used as a positive control for AChE inhibition. CP-treated 168 hpf larvae showed a similar pattern of AChE inhibition (in the myelencephalon and somites) as that of ISOX-treated larvae. CP was superior to ISOX as evidenced by the retention of avoidance response behavior in adult zebrafish. Molecular docking studies indicated that ISOX binds Ser203 of the catalytic triad on the human AChE. The active components of CP-scopoletin and kaempferol-were bound by His447 of the catalytic triad, the anionic subsite of the catalytic center, and the peripheral anionic site. This suggested the ability of CP to mediate both competitive and non-competitive modes of inhibition. Surprisingly, SCOP showed AChE inhibition in larvae, possibly mediated via the choline-binding sites. CP + SCOP induced a concentration-dependent increase in AChE inhibition and ACh depletion. Abnormal motor responses were observed with ISOX, CP, ISOX + SCOP, and CP + SCOP, indicative of undesirable effects on the peripheral cholinergic system. Our study proposes the examination of CP, SCOP, and CP + SCOP as potential AChE inhibitors for their ability to modulate cognitive deficits.

A Simple Blood Vessel Imaging Protocol for Live Zebrafish Larva.

Zebrafish larva is a simple model system to study blood vessel development because of its excellent optical properties. Current methods to study blood vessels in zebrafish involve utilizing either blood vessel-specific transgenic lines or specialized techniques such as microangiography and video capillaroscopy. In this study, we have developed a simple protocol using ImageJ to observe the blood vessels of live zebrafish larva at different developmental stages, namely 1- to 4-day postfertilization (dpf). To validate this protocol, we treated 1 dpf zebrafish embryos with 100 µM quercetin, and analyzed the impaired development of subintestinal vein in the quercetin-treated 3 dpf larvae.

Simple and High-Throughput Rheotaxis Behavioral Assay for Zebrafish Larva.

Zebrafish (Danio rerio) is used as a model for studying sensorineural hearing loss. The damage to the hair cells can be assessed by scoring rheotaxis behavior in zebrafish. In this study, we newly designed a rheotaxis behavioral assay protocol capable of quantifying rheotaxis behavior in zebrafish larvae. We chemically induced ototoxicity in the larvae using copper sulfate, a well-known ototoxin, and determined rheotaxis at different flow velocities. The simple, cost-effective, and high-throughput rheotaxis assay system can provide great insights into drug development and other behavioral studies.

Identification of zebrafish fumarate hydratase active site by molecular docking and simulation studies.

Fumarate hydratase (FH), one of the members of TCA cycle, acts as a catalyte for the synthesis of malate from fumarate. FH has been proposed to play as a tumour suppressor leading to the pathogenicity of leiomyomas, renal cell carcinoma and paraganglioma. Mutations in the active site of FH lead to alteration in the protein structure. Similarly, binding of several chemical inhibitors to the active site also leads to the disruption of protein structural integrity thereby leading to protein dysfunction. Therefore, in order to address this mechanism leading to cancer, the binding efficiency of potential human FH inhibitor citrate to zebrafish fh has been extensively analysed in this study by molecular docking and simulation experiments followed by quantification of fumarate hydratase enzyme activity to validate and confirm the findings. Molecular docking revealed stronger interaction of zebrafish fh protein with inhibitor citrate when compared to natural substrate fumarate. Study on the dynamics of docked structures further confirmed that citrate was found to possess more binding affinity than fumarate. In vitro biochemical analysis also revealed concentration dependent potential inhibitory effect of citrate on zebrafish fh, thus confirming the findings of the in-silico experiments.Communicated by Ramaswamy H. Sarma.

Investigation of the role of VHL-HIF signaling in DNA repair and apoptosis in zebrafish.

pVHL is a tumor suppressor. The lack of its function leads to various tumors, among which ccRCC (clear cell renal cell carcinoma) has the most serious outcome due to its resistance to chemotherapies and radiotherapies. Although HIF promotes the progression of ccRCC, the precise mechanism by which the loss of VHL leads to tumor initiation remains unclear. We exploited two zebrafish vhl mutants, vhl and vll, and Tg (phd3:: EGFP)i144 fish to identify crucial functions of Vhl in tumor initiation. Through the mutant analysis, we found that the role of pVHL in DNA repair is conserved in zebrafish Vll. Interestingly, we also discovered that Hif activation strongly suppressed genotoxic stress induced DNA repair defects and apoptosis in vll and brca2 mutants and in embryos lacking ATM activity. These results suggest the potential of HIF as a clinical modulator that can protect cells from accumulating DNA damage and apoptosis which can lead to cancers and neurodegenerative disorders.

A simple method to study motor and non-motor behaviors in adult zebrafish.

BACKGROUND: Motor and non-motor behavior analyses are increasingly utilizied in drug discovery and screening, detection of neurobehavioral disorders and chemical toxicology. The emergence of computational approaches has helped to develop different tools to analyse complex behaviors. Analysis of locomotor behavior helps in understanding the motor neuron disorders like Parkinson's Disease. Although many animal models are available to study the locomotion, adult zebrafish has emerged as a simple and efficient model to study this behavior. An inexpensive and easily customizable tool is required to replace the licensed and expensive set-up to analyse the locomotor behavior. NEW METHOD: In this study we have optimized the ImageJ plugin wrMTrck to analyse motor and non-motor behaviors in adult zebrafish. We have generated a macro to simplify the preprocessing and tracking. Subsequently, we have developed a data analysis sheet to analyse various behavioral end points. RESULTS: We have successfully developed an inexpensive video acquisition set-up and optimized wrMTrck for adult zebrafish. In order to demonstrate the efficacy of this method, adult zebrafish were injected with MPTP and motor and non-motor behaviors were analysed. Expectedly, MPTP injected fish showed decrease in dopamine level and dat expression level, which subsequently led to locomotor behavioral defects as well as anxiety, a non-motor symptom of PD. COMPARISON WITH EXISTING METHOD(S): Further, the obtained results were validated by another ImageJ macro developed by Pelkowski et al. (2011) and we observed identical trajectories. CONCLUSIONS: The usefulness of popular ImageJ plugin wrMTrck and this extended protocol will be helpful to quantify motor and non-motor behavioral parameters.

Effect of Fadrozole Exposure on Socioreproductive Behaviors and Neurochemical Parameters in Betta splendens.

Induction of all-male population in Siamese fighting fish, Betta splendens, has potential application in ornamental fish trade. In addition, the sexually dimorphic nature of aggressive behavior exhibited by this species has made it into an emerging model for behavioral studies. Fadrozole, an aromatase inhibitor, which has been used widely in masculinization, has captivated us to use it in this study. Twenty one days postfertilization (dpf), B. splendens fry were subjected to discrete immersion treatment with various concentrations of fadrozole, and eventually, analyses of various socioreproductive behaviors and analyses of stress markers such as dopamine in brain samples, sex hormones, cortisol, and glucose in plasma samples were performed. We observed that 91% of 50 µg/L fadrozole treated fish developed as males. Interestingly, reproductive analyses of these males gave rise to two subgroups (A and B). Subsequent sociobehavioral analyses demonstrated a timid and subdued behavior in subgroup B males. Furthermore, estimation of stress markers such as dopamine levels in the brain tissue, cortisol, and glucose levels in blood plasma and sex hormone levels in blood plasma exhibited an endocrine disruption-mediated stress leading to altered behavior in these males. These findings will help in understanding the fadrozole-mediated masculinization and behavioral alterations following endocrine disruption.

Transition Metal Ion (Mn2+, Fe2+, Co2+, and Ni2+)-Doped Carbon Dots Synthesized via Microwave-Assisted Pyrolysis: A Potential Nanoprobe for Magneto-fluorescent Dual-Modality Bioimaging.

Heteroatom-doped carbon dots (C-dots) have captured widespread research interest owing to high fluorescence and biocompatibility for multimodal bioimaging applications. Here, we exemplify a rapid, facile synthesis of ethylenediamine (EDA)-functionalized transition metal ion (Mn2+, Fe2+, Co2+, and Ni2+)-doped C-dots via one-pot microwave (MW)-assisted pyrolysis at 800 W within 6 min using Citrus limon (lemon) extract as a carbon source. During MW pyrolysis, the precursor extract undergoes simultaneous carbonization and doping of metal ions onto C-dot surfaces in the presence of EDA. The EDA-functionalized transition metal ion-doped C-dots (i.e., Mn/C, Fe/C, Co/C, and Ni/C-dots) are collectively termed as TMCDs. The water-soluble TMCDs exhibited a size of 3.2 ± 0.485 nm and were enriched with amino and oxo functionalities and corresponding metal-oxide traces on the surfaces, as revealed from Fourier transfer infrared and X-ray photoelectron spectroscopy analyses. Interestingly, TMCDs demonstrated excitation-wavelength-dependent emission with brighter photoluminescence (PL) at 460 nm. Compared to pristine C-dots with a PL quantum yield (QY) of 48.31% and a fluorescence lifetime of 3.6 ns, the synthesized Mn/C, Fe/C, Co/C, and Ni/C-dots exhibited PL QY values of 35.71, 41.72, 75.07, and 50.84% as well as enhanced fluorescence lifetimes (τav) of 9.4, 8.6, 9.2, and 8.9 ns, respectively. The TMCDs significantly exhibited enhanced biocompatibility in human colon cancer cells (SW480) for fluorescence bioimaging and showed ferromagnetic and superparamagnetic behavior with vibrant T1-contrast ability. Interestingly, the maximum longitudinal (r1) relaxivity of 0.341 mM-1 s-1 was observed for Mn/C-dots in comparison to that of 3.1-3.5 mM-1 s-1 of clinically used Gd-DTPA magnetic resonance (MR)-contrast agent in vitro (1.5 T). Similarly, the maximum longitudinal relaxivity (r1) of 0.356 mM-1 s-1 was observed for Ni/C-dots (1.5 T) with respect to 4.16 ± 0.02 mM-1 s-1 attained for Gd-DTPA in vivo (8.45 T). Thus, the rapid, energy-efficient MW-assisted pyrolysis presents lemon extract derived, EDA-functionalized TMCDs with enhanced PL and efficient T1 contrast as potential magneto-fluorescent nanoprobes for dual-modality bioimaging applications.

Analyzing Locomotor Activity in Zebrafish Larvae Using wrMTrck.

The zebrafish has emerged as a new model system in behavioral neurobiology research. There are numerous assays available to analyze locomotor behavior, but most of these assays require sophisticated equipment and proprietary software. A freely available plugin, wrMTrck, originally developed to analyze locomotor response in Caenorhabditis elegans, has been used in this study to measure locomotion in zebrafish.

A zebrafish model to study and therapeutically manipulate hypoxia signaling in tumorigenesis.

Hypoxic signaling is a central modulator of cellular physiology in cancer. Core members of oxygen-sensing pathway including the von Hippel-Lindau tumor suppressor protein (pVHL) and the hypoxia inducible factor (HIF) transcription factors have been intensively studied, but improved organismal models might speed advances for both pathobiologic understanding and therapeutic modulation. To study HIF signaling during tumorigenesis and development in zebrafish, we developed a unique in vivo reporter for hypoxia, expressing EGFP driven by prolyl hydroxylase 3 (phd3) promoter/regulatory elements. Modulation of HIF pathway in Tg(phd3::EGFP) embryos showed a specific role for hypoxic signaling in the transgene activation. Zebrafish vhl mutants display a systemic hypoxia response, reflected by strong and ubiquitous transgene expression. In contrast to human VHL patients, heterozygous Vhl mice and vhl zebrafish are not predisposed to cancer. However, upon exposure to dimethylbenzanthracene (DMBA), the vhl heterozygous fish showed an increase in the occurrence of hepatic and intestinal tumors, a subset of which exhibited strong transgene expression, suggesting loss of Vhl function in these tumor cells. Compared with control fish, DMBA-treated vhl heterozygous fish also showed an increase in proliferating cell nuclear antigen-positive renal tubules. Taken together, our findings establish Vhl as a genuine tumor suppressor in zebrafish and offer this model as a tool to noninvasively study VHL and HIF signaling during tumorigenesis and development.

A zebrafish model for VHL and hypoxia signaling.

The von Hippel-Lindau (VHL) tumor suppressor gene encodes an adaptor protein that regulates an array of transcription-dependent and -independent cellular and physiological processes. Mutations in this gene cause VHL disease, congenital polycythemia, and several sporadic tumor types. The last 15 years of fundamental and clinical research have helped define the phenotypic spectrum of VHL-associated diseases and have introduced new cellular functions for pVHL. Here, we review the current knowledge of VHL function, and the different animal models for VHL disease, with a particular focus on the zebrafish. Zebrafish vhl mutants develop key aspects of the human disease condition, including activation of the hypoxia-inducible factor (HIF) signaling pathway, polycythemia, excessive neovascularization, macular edema, and pronephric abnormalities. The zebrafish vhl model offers a platform for the identification of genetic pathways, modifiers, and interactors involved in the development of VHL-associated neoplasms. Vhl mutants represent a unique and clinically relevant in vivo model for studying genotype-phenotype correlations and the identification of prognostic biomarkers. The amenability of zebrafish for chemical genetic screens will not only be helpful to identify novel therapeutic agents but may also reveal novel processes that require regulation by VHL.

Tbx2b is required for the development of the parapineal organ.

Structural differences between the left and right sides of the brain exist throughout the vertebrate lineage. By studying the zebrafish pineal complex, which exhibits notable asymmetries, both the genes and the cell movements that result in left-right differences can be characterized. The pineal complex consists of the midline pineal organ and the left-sided parapineal organ. The parapineal is responsible for instructing the asymmetric architecture of the bilateral habenulae, the brain nuclei that flank the pineal complex. Using in vivo time-lapse confocal microscopy, we find that the cells that form the parapineal organ migrate as a cluster of cells from the pineal complex anlage to the left side of the brain. In a screen for mutations that disrupted brain laterality, we identified a nonsense mutation in the T-box2b (tbx2b) gene, which encodes a transcription factor expressed in the pineal complex anlage. The tbx2b mutant makes fewer parapineal cells, and they remain as individuals near the midline rather than migrating leftward as a group. The reduced number and incorrect placement of parapineal cells result in symmetric development of the adjacent habenular nuclei. We conclude that tbx2b functions to specify the correct number of parapineal cells and to regulate their asymmetric migration.