Biology subjects

Varga, M.

Publications and source records attributed to Varga, M..

16 recordsLinked to original sources

bioRxiv2026

SCAMP - an open-source tool for the quantification of calcification in fish larvae

Quantifying skeletal mineralization phenotypes in larval fish is complicated by the natural curvature of the notochord and by sample-to-sample variability in orientation, staining and imaging. Consequently, many studies rely on summary measures such as vertebral counts or total stain intensity. Here we present SCAMP (Spinal Calcification & Mineralization Profiler), an open-source, GUI-based Python tool that computationally straightens the curved notochord of Alizarin Red S-stained fish larvae and generates standardized mineralization profiles along the spinal axis. This approach reduces positional and shape variability, allowing direct, quantitative comparison of calcification patterns within and between experimental cohorts, without requiring programming expertise. We validate SCAMP using a zebrafish model of Pseudoxanthoma elasticum (abcc6aelu15/elu15), recovering genotype-specific differences in the intensity, extent and spatial distribution of ectopic calcification. Using SCAMP, we further show that inorganic pyrophosphate (PPi) supplementation of the medium suppresses ectopic notochord calcification, alters the anterior-posterior distribution of mineralized regions in homozygous mutants, and promotes mineralization at physiological vertebral sites. We also show that methylene blue, a routine antifungal additive in fish medium, reduces baseline calcification, with the most pronounced effects observed in heterozygous controls. SCAMP is freely available and has the potential to be adapted to other fish species used in skeletal and mineralization research.

developmental biology↗

bioRxiv2026

Optic nerve innervation promotes Wnt/b-catenin pathway activity and progenitor cell proliferation in the zebrafish optic tectum

In the zebrafish visual system, accurate retinotopic mapping occurs throughout life as new neurons are generated and integrated into existing circuitry in both the retina and optic tectum (OT). To explore how OT development changes relative to innervation from the retina, we examined cell death and proliferation in genetic and surgical models that disrupt retinal innervation of the OT. Specifically, we compared lakritz (lak) mutants, which have no optic nerves due to a lesion in the atoh7 gene, with either wild-type or one-eyed fish generated through surgical eye removal. We observed elevated cell death, fewer proliferating progenitors, and fewer sox2+ OT neuroepithelial stem cells in lak mutant and denervated OT lobes. To examine whether light-mediated vision contributes to proliferation and survival in the optic tectum, we reared fish in constant darkness and then compared survival and proliferation of OT cells in innervated and non-innervated tecta. We found that OT cells were still more likely to survive and proliferate in the presence of optic nerve innervation even when fish were reared in the dark. To identify molecular pathways that could regulate OT growth, we examined the expression of known mitogens in the zebrafish optic tectum and found evidence that Wnt/{beta}-catenin pathway activity could promote innervation-dependent proliferation in lak mutant tecta. Expression of both wnt3a and the Wnt/{beta}-catenin target gene axin2, as detected by in situ hybridization and RT-qPCR, is decreased in non-innervated tectal lobes. Further supporting an innervation-dependent role for Wnt/{beta}-catenin pathway activation in the zebrafish OT, we found that lak mutants treated with a Wnt-pathway agonist, BIO, exhibited levels of OT cell proliferation that were indistinguishable from wild-type. Together these findings suggest that progenitor cells in the optic tectum produce Wnt3a in response to innervation by the optic nerve, providing new insight into how a vertebrate visual system coordinates growth across its sensory and recipient tissues.

Developmental Biology↗

bioRxiv2025

Genome-wide association study reveals KCNQ3 gene as a risk factor for fear-related behavior in dogs and zebrafish

Freezing behavior, characterised by attentive immobility as a reaction to a perceived threat, is widely studied in the context of fear, anxiety and stress. To uncover the genetic factors underlying this behavior, we conducted a genome-wide association study (GWAS) in kennel-housed beagle dogs. Our analysis identified a single-nucleotide polymorphism (SNP) in intron 5 of the KCNQ3 gene on chromosome 13 associated with freezing behavior in response to unfamiliar environments and people. To validate this finding, we used a zebrafish model, where CRISPR/Cas9-induced kcnq3 deficiencies led to heightened fear and arousal in two behavioral tests. KCNQ3 is implicated in several neurodevelopmental and psychiatric disorders, and our results highlight its evolutionarily conserved role in modulating fear responses. In dogs, an enriched environment can mitigate the adverse effects of KCNQ3 deficiency by reducing threat perception, highlighting the role of gene-environment interactions in shaping behavioral responses. TeaserKCNQ3 influences fear responses across species, with gene-environment interactions shaping freezing behavior in dogs.

animal behavior and cognition↗

bioRxiv2025

Transposon insertion causes ctnnb2 transcript instability that results in the maternal effect zebrafish ichabod (ich) mutation

The maternal-effect mutation ichabod (ich) results in ventralized zebrafish embryos due to impaired induction of the dorsal canonical Wnt-signaling pathway. While previous studies linked the phenotype to reduced ctnnb2 transcript levels, the causative mutation remained unidentified. Using long-read sequencing, we discovered that the ich phenotype stems from the insertion of a non-autonomous CMC-Enhancer/Suppressor-mutator (CMC-EnSpm) transposon in the 3UTR of the gene. Through reporter assays, we demonstrate that while wild type ctnnb2 mRNAs exhibit remarkably high stability throughout the early stages of development, the insertion of the transposon dramatically reduces transcript stability. Genome-wide mapping of the CMC-EnSpm transposons across multiple zebrafish strains also indicated ongoing transposition activity in the zebrafish genome. Our findings not only resolve the molecular basis of the ich mutation but also highlight the continuing mutagenic potential of endogenous transposons and reveal unexpected aspects of maternal transcript regulation during early zebrafish development.

developmental biology↗

bioRxiv2024

Paradise fish (Macropodus opercularis) as novel translational model for emotional and cognitive function

Zebrafish have revolutionised physiological screening in vertebrates, but the interpretation of individual-based behavioural assays is burdened by the strong sociality of this species. We propose the use of a solitaire fish species, the paradise fish to keep the advantages and compensate for the limitations of the zebrafish model. We compared juvenile paradise fish and zebrafish in social and non-social exploratory tasks, anxiety tests and in a working memory assay to assess their performance in these individual-based models. We found that in contrast to zebrafish, paradise fish did not show social approach in the U-shape sociability test, their novelty exploration was not biased by the presence of a conspecific in the slalom test, and was not impaired by social isolation in the showjump or in the swimming plus-maze (SPM) anxiety tests. While social circumstances did not affect the anxiety of paradise fish, it was sensitive to the anxiolytic drug buspirone. Intra- and interest repeatability measures of the anxiety tests revealed that paradise fish express more consistent exploratory and defensive behaviours regarding time and context compared to zebrafish. Behavioural consistency in paradise fish was also supported by arm alternation as a predominant choice of exploration in the y-maze task. In summary, our results indicate that the behaviour of paradise fish is less biased by social cues and is more effective and repeatable in individual-based assays than zebrafish behaviour. We hypothesise that the two phenomena are connected and novelty exploration, anxiety and working memory can be more reliably measured and are translatable in a solitaire species.

animal behavior and cognition↗

bioRxiv2024

The zebrafish (Danio rerio) snoRNAome

Small nucleolar RNAs (snoRNAs) are one of the most abundant and evolutionary ancient group of functional non-coding RNAs. They were originally described as guides of post-transcriptional rRNA modifications, but emerging evidence suggests that snoRNAs fulfil an impressive variety of cellular functions, some of which are only being discovered. To reveal the true complexity of snoRNA-dependent functions, we need to catalogue first the complete repertoire of snoRNAs in a given cellular context. While the systematic mapping and characterization of "snoRNAomes" for some species have been described recently, this has not been done hitherto for the zebrafish (Danio rerio). Using size-fractionated RNA sequencing data from adult zebrafish tissues, we created an interactive "snoRNAome" database for this species. Our custom-designed analysis pipeline allowed us to identify with high-confidence 68 previously unannotated snoRNAs in the zebrafish genome, resulting in the most complete set of snoRNAs to date in this species. Reanalyzing multiple previously published datasets, we also provide evidence for the dynamic expression of some snoRNAs during the early stages of zebrafish development and tissue-specific expression patterns for others in adults. To facilitate further investigations into the functions of snoRNAs in zebrafish, we created a novel interactive database, snoDanio, which can be used to explore small RNA expression from transcriptomic data.

genomics↗

bioRxiv2024

Silicon-rhodamine functionalized evocalcet probes (EvoSiR) potently and selectively label calcium sensing receptors (CaSR) in vitro, in vivo and ex vivo

The calcium sensing receptor (CaSR) is a ubiquitously expressed G-protein coupled receptor (GPCR) that regulates extracellular calcium signals via the parathyroid glands. CaSR has recently also been implicated in non-calcitropic pathophysiologies like asthma, gut inflammation and cancer. To date, molecular tools that enable the bioimaging of CaSR in tissues are lacking. Based on in silico analyses of available structure-activity relationship data on CaSR ligands, we designed and prepared silicon-rhodamine (SiR) conjugates of the clinically approved drug evocalcet. The new probes EvoSiR4 and EvoSiR6, with differing linker lengths at the evocalcet carboxyl end, both showed a 6-fold and 3-fold increase in potency towards CaSR (EC50<45 nM) compared to evocalcet and the evocalcet-linker conjugate, respectively, in a FLIPR(R)-based cellular functional assay. The specificity of the EvoSiR probes towards CaSR binding and the impact of albumin was evaluated in live cell experiments. Both probes showed strong albumin binding, which facilitated the clearance of nonspecific binding interactions. Accordingly, in zebrafish embryos, EvoSiR4 specifically labelled the high CaSR expressing neuromasts of the lateral line in vivo. EvoSiR4 was also assessed in human parathyroid tissues ex vivo, showing a specific absolute CaSR associated fluorescence compared to parathyroid autofluorescence. In summary, functionalization of evocalcet by SiR led to the preparation of potent and specific fluorescent CaSR probes. EvoSiR4 is a versatile small molecular probe that can be employed in CaSR-related biomedical analyses where antibodies are not applicable.

pharmacology and toxicology↗

bioRxiv2023

The reference genome of the paradise fish (Macropodus opercularis)

Over the decades, a small number of model species, each representative of a larger taxa, have dominated the field of biological research. Amongst fishes, zebrafish (Danio rerio) has gained popularity over most other species and while their value as a model is well documented, their usefulness is limited in certain fields of research such as behavior. By embracing other, less conventional experimental organisms, opportunities arise to gain broader insights into evolution and development, as well as studying behavioral aspects not available in current popular model systems. The anabantoid paradise fish (Macropodus opercularis), an "air-breather" species from Southeast Asia, has a highly complex behavioral repertoire and has been the subject of many ethological investigations, but lacks genomic resources. Here we report the reference genome assembly of Macropodus opercularis using long-read sequences at 150-fold coverage. The final assembly consisted of {approx}483 Mb on 152 contigs. Within the assembled genome we identified and annotated 20,157 protein coding genes and assigned {approx}90% of them to orthogroups. Completeness analysis showed that 98.5% of the Actinopterygii core gene set (ODB10) was present as a complete ortholog in our reference genome with a further 1.2 % being present in a fragmented form. Additionally, we cloned multiple genes important during early development and using newly developed in situ hybridization protocols, we showed that they have conserved expression patterns.

genomics↗

bioRxiv2021

Nicotinate degradation in a microbial eukaryote: a novel, complete pathway extant in Aspergillus nidulans

Several strikingly different aerobic and anaerobic pathways of nicotinate utilization had been described in bacteria. No similar work is extant in any eukaryote. Here we elucidate a complete eukaryotic nicotinate utilization pathway, by constructing single or multiple gene deleted strains and identifying metabolic intermediates by ultra-high performance liquid chromatography - high-resolution mass spectrometry. Enzymes catalyzing each step and all intermediate metabolites were identified. We previously established that the cognate eleven genes organized in three clusters constitute a regulon, strictly dependent on HxnR, a pathway-specific transcription factor. The first step, hydroxylation of nicotinic acid to 6-hydroxynicotinic acid is analogous to that occurring in bacterial pathways and is catalyzed by an independently evolved molybdenum-containing hydroxylase. The following enzymatic steps have no prokaryotic equivalents: 6-hydroxynicotinic acid is converted to 2,3,6-trihydroxypyridine through 2,5-dihydroxypiridine and the trihydroxylated pyridine ring is then saturated to 5,6-dihydroxypiperidine-2-one followed by the oxidation of the C6 hydroxyl group resulting in 3-hydroxypiperidine-2,6-dione. The latter two heterocyclic compounds are newly identified cellular metabolites, while 5,6-dihydroxypiperidine-2-one is a completely new chemical compound. Ring opening between C and N results in -hydroxyglutaramate, an unprecedented compound in prokaryotic nicotinate catabolic routes. The pathway extant in A. nidulans, and in many other ascomycetes, is different from any other previously analyzed in bacteria. Our earlier phylogenetic analysis of Hxn proteins together with the complete novel biochemical pathway we now describe further illustrates the convergent evolution of catabolic pathways between fungi and bacteria. Significance StatementThis eukaryotic nicotinate catabolic pathway illustrates the convergent evolution of prokaryotic and microbial eukaryotic metabolism. It brings to light newly identified metabolites and step processing enzymes. The identification of hitherto undescribed metabolites - which could serve as precursor biosynthetic molecules - is potentially relevant to both pharmaceutical and agrochemical industries.

microbiology↗

bioRxiv2021

Bloom syndrome helicase contributes to germ line development and longevity in zebrafish

RecQ helicases - also known as the guardians of the genome - play crucial roles in genome integrity maintenance through their involvement in various DNA metabolic pathways. Aside from being conserved from bacteria to vertebrates, their importance is also reflected in the fact that in humans impaired function of multiple RecQ helicase orthologs are known to cause severe sets of problems, including Bloom, Werner or Rothmund-Thomson syndromes. Our aim was to create and characterize a zebrafish (Danio rerio) disease model for Bloom syndrome, a recessive autosomal disorder. In humans, this syndrome is characterized by short stature, skin rashes, reduced fertility, increased risk of carcinogenesis and shortened life expectancy brought on by genomic instability. We show that zebrafish blm mutants recapitulate major hallmarks of the human disease, such as shortened lifespan and reduced fertility. Moreover, similarly to other factors involved in DNA repair, some functions of zebrafish Blm bear additional importance in germ line development, and consequently in sex differentiation. Unlike fanc genes and rad51, however, blm appears to effect its function independent of tp53. Therefore, our model will be a valuable tool for further understanding the developmental and molecular attributes of this rare disease, along with providing novel insights into the role of genome maintenance proteins in somatic DNA repair and fertility.

genetics↗

bioRxiv2020

The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins

SARS-CoV-2 emerged in late 2019, leading to the COVID-19 pandemic that continues to cause significant global mortality in human populations. Given its sequence similarity to SARS-CoV, as well as related coronaviruses circulating in bats, SARS-CoV-2 is thought to have originated in Chiroptera species in China. However, whether the virus spread directly to humans or through an intermediate host is currently unclear, as is the potential for this virus to infect companion animals, livestock and wildlife that could act as viral reservoirs. Using a combination of surrogate entry assays and live virus we demonstrate that, in addition to human ACE2, the Spike glycoprotein of SARS-CoV-2 has a broad host tropism for mammalian ACE2 receptors, despite divergence in the amino acids at the Spike receptor binding site on these proteins. Of the twenty-two different hosts we investigated, ACE2 proteins from dog, cat and rabbit were the most permissive to SARS-CoV-2, while bat and bird ACE2 proteins were the least efficiently used receptors. The absence of a significant tropism for any of the three genetically distinct bat ACE2 proteins we examined indicates that SARS-CoV-2 receptor usage likely shifted during zoonotic transmission from bats into people, possibly in an intermediate reservoir. Interestingly, while SARS-CoV-2 pseudoparticle entry was inefficient in cells bearing the ACE2 receptor from bats or birds the live virus was still able to enter these cells, albeit with markedly lower efficiency. The apparently broad tropism of SARS-CoV-2 at the point of viral entry confirms the potential risk of infection to a wide range of companion animals, livestock and wildlife.

microbiology↗

bioRxiv2020

The GINS complex is required for the survival of rapidly proliferating retinal and tectal progenitor cells during zebrafish development

Efficient and accurate DNA replication is particularly critical in stem and progenitor cells for successful proliferation and survival. The replisome, an amalgam of protein complexes, is responsible for binding potential origins of replication, unwinding the double helix, and then synthesizing complimentary strands of DNA. According to current models, the initial steps of DNA unwinding and opening are facilitated by the CMG complex, which is composed of a GINS heterotetramer that connects Cdc45 with the mini-chromosome maintenance (Mcm) helicase. In this work, we provide evidence that in the absence of GINS function DNA replication is cell autonomously impaired, and we also show that gins1 and gins2 mutants exhibit elevated levels of apoptosis restricted to actively proliferating regions of the central nervous system (CNS). Intriguingly, our results also suggest that the rapid cell cycles during early embryonic development in zebrafish may not require the function of the GINS complex as neither Gins1 nor Gins2 seem to be present during these stages.

genetics↗

bioRxiv2019

Conserved serotonergic background of experience-dependent challenge-responding in zebrafish (Danio rerio)

Forming effective responses to threatening stimuli requires the adequate and coordinated emergence of stress-related internal states. Such ability depends on early-life experiences and, in connection, the adequate formation of neuromodulatory systems, particularly serotonergic signaling. Here, we assess the serotonergic background of experience-dependent behavioral responsiveness employing a zebrafish (Danio rerio) model. For the first time, we have characterized a period during the behavioral metamorphosis in which zebrafish are highly reactive to their environment. Absence of social stimuli during this phase established by isolated rearing fundamentally altered the behavioral phenotype of post-metamorphic zebrafish in a challenge-specific manner, partially due to a decline in responsiveness and an inability to develop stress-associated arousal state. In line with this, isolation differently affected whole-brain 5-HT signaling in resting and stress-induced conditions, an effect that was present at the level of the dorsal pallium and was negatively associated with responsiveness. Administration of the 5HT1AR partial agonist buspirone prevented the isolation-induced serotonin response to novelty in the forebrain and rescued stress-induced arousal along with challenge-induced behaviors, which altogether indicates a functional connection between these changes. In summary, there is a consistent negative association between behavioral responsiveness and serotonergic signaling in zebrafish, which is well recognizable through the modifying effects of developmental perturbation and pharmacological manipulations as well. Our results imply a conserved serotonergic mechanism that context-dependently modulates environmental reactivity and is highly sensitive to experiences acquired during a specific early-life time-window, a phenomenon that was previously only suggested in mammals.\n\nSignificance statementThe ability to respond to challenges is a fundamental factor in survival. We show that zebrafish that lack appropriate social stimuli in a sensitive developmental period show exacerbated alertness in non-stressful conditions while failing to react adequately to stressors. This shift is reflected inversely by central serotonergic signaling, a system that is implicated in numerous mental disorders in humans. Serotonergic changes in brain regions modulating responsivity and behavioral impairment were both prevented by the pharmacological blockade of serotonergic function. These results imply a serotonergic mechanism in zebrafish that transmits early-life experiences to the later phenotype by shaping stress-dependent behavioral reactivity, a phenomenon that was previously only suggested in mammals. Zebrafish provide new insights into early-life-dependent neuromodulation of behavioral stress-responses.

neuroscience↗

bioRxiv2019

Loss of cell cycle control renders cells nonresponsive to local extrinsic differentiation cues

Objective and approachesAberrantly proliferating cells are linked to a number of diseases including cancers and developmental defects.To determine the extent to which local extrinsic signals contribute to or ameliorate mutant cell behaviors, we examined survival and differentiation of mutant cells in wild-type retinal environments by generating chimeric zebrafish embryos comprised of unlabeled host cells and GFP-labeled neural progenitor donor cells. In addition, we examined the fate of retinal progenitor cells when cdkn1c, a cyclin dependent kinase inhibitor, was induced in clones within wild-type and hdac1 mutant retinae.\n\nResultsWe found that seven of the ten mutants examined exhibited apoptosis when grafted into wild-type tissue, with cells from two slowly cycling mutants, elys and emi1, noticeably differentiating in a wild-type environment. Observations of the one hyperproliferative mutant, hdac1, revealed that these mutant cells did not appear to die or differentiate but instead survived and formed tumor-like rosettes in a wild-type environment. Ectopic expression of cdkn1c was unable to force cell cycle exit and differentiation of the majority of hdac1 mutant cells.\n\nConclusionsTogether, these results suggest that although a wild-type environment rarely encourages cell cycle exit and differentiation of neural progenitors with cell cycle defects, wild-type survival signals may enable hyperproliferative progenitor cells to persist instead of die.

developmental biology↗

bioRxiv2019

Family aggregation analysis reveals a heritable background of equine grass sickness (dysautonomia)

Equine grass sickness (also known as dysautonomia) is a life-threatening polyneuropathic disease affecting horses with approx. 80% mortality. Since its first description over a hundred years ago, several factors including phenotypic, environmental, management, climate, and intestinal microbiome) have been associated with increased risk of dysautonomia. But despite the extensive research on dysautonomia, its causative factors have yet been identified. A retrospective pedigree and phenotype based genetic epidemiological study was performed to analyze the associations of disease occurrence and the kinship in a Hungarian large scale stud. The pedigree data set containing 1233 horses with 49 affected animals was used in the analysis. The first finding was that among the descendants of some stallions the proportion of affected animals are unexpectedly high, with a maximum of 25% of a stallions descendants affected. Animals with affected siblings have higher odds to be a case (OR: 1.27, 95% CI: 1.01-1.57, p=0.033). Among males in the affected population the odds of dysautonomia is higher than in females (OR: 1.76, 95% CI: 0.95-3.29, p=0.057). Significant familial clustering was observed among the affected animals (GIF p=0.001). Further subgroups were identified with significant (p<0.001) aggregation among close relatives using kinship-based methods. Our analysis of the data and the observed higher disease frequency in males suggests that dysautonomia may have X-linked recessive inheritance as a causal factor. This is the first study providing ancestry data and suggesting a genetic contribution to the likely multifactorial causes of the disease.

genetics↗

bioRxiv2018

The swimming plus-maze test: a novel high-throughput model for assessment of anxiety-related behaviour in larval zebrafish. (Danio rerio)

Larval zebrafish (Danio rerio) has the potential to supplement rodent models due to the availability of resource efficient methods implying high-throughput screening and high-resolution imaging techniques. Although behavioural models are available in larvae, only a few, insensitive approaches can be employed to assess anxiety. Here we present the swimming plus-maze (SPM) test paradigm to assess anxiety-related states in young zebrafish. The \"+\" shaped apparatus consists of arms of different depth representing differentially aversive context. The paradigm was validated i.) in larval and juvenile zebrafish, ii.) after administration of compounds affecting human anxiety and iii.) in differentially aversive experimental conditions. Furthermore, we compared the SPM with conventional \"anxiety tests\" of larvae such as the open tank and light/dark tank tests to identify their shared characteristics. We clarified that the preference towards deeper water is conserved trough the ontogenesis and can be abolished by anxiolytic or enhanced by anxiogenic agents, respectively. The behavioural read-out is insensitive to the aversiveness of the platform and unrelated to behaviours assessed by conventional tests utilizing larval fish. Taken together, we developed a sensitive high-throughput test measuring anxiety-related responses of larval zebrafish, which likely reflect bottom-dwelling behaviour of adults, potentially supporting larva-based integrative approaches.

animal behavior and cognition↗