Biology subjects

Loosli, F.

Publications and source records attributed to Loosli, F..

9 recordsLinked to original sources

bioRxiv2025

Nanopore duplex sequencing reveals patterns of asymmetric states of 5hmC and 5mC in the medaka brain genome

The nucleotide modification 5-methylcytosine (5mC), has been extensively described in terms of its genomic distribution and function. However, the distribution and functional contributions of its oxidised state, 5-hydroxymethylcytosine (5hmC), and its interplay with 5mC remain to be further characterised. Moreover, the hemi-methylation and hemi-hydroxymethylation of CpG dinucleotides in the genome also remain largely unexplored. Here, we use Nanopore duplex sequencing, i.e, the sequencing of both strands of DNA from a single double stranded molecule, to profile the co-occurrence of 5hmC with 5mC in CpG dinucleotides of medaka (japanese rice paddy fish, Oryzias latipes) brain tissues. We characterised asymmetrical patterns of hemi-modified states of CpGs and show using duplex resolution that in the asymmetrical state 5hmC/5mC, 5hmC is preferentially found in the antisense strand of DNA in intron regions, in particular in splice sites, whereas the exons have an enrichment in 5hmC in the sense strand. Furthermore, we found that in the 5hmC/5mC state, 5hmC preferentially occurs along one strand of the DNA duplex in specific families of transposable elements. Altogether these results shed more light on understanding the function and distribution of 5hmC and its mutual role with 5mC in the genome.

genomics↗

bioRxiv2025

Discovery and characterisation of gene by environment and epistatic genetic effects in a vertebrate model

Phenotypic variation arises from the interplay between genetic and environmental factors. However, disentangling these interactions for complex traits remains challenging in observational cohorts such as human biobanks. Instead, model organisms where genetic (G) and environmental (E) variation can be controlled offer a valuable complement to human studies in the analysis of higher-order genetic effects such as GxE interactions, dominance, and epistasis. Here, we utilized 76 medaka strains of the Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel, to compare heart rate plasticity across temperatures. An F2 segregation analysis identified 16 quantitative trait loci (QTLs), with many exhibiting dominance, GxE, GxG, and GxGxE interactions. We experimentally validated four candidate genes using gene editing, revealing their temperature-sensitive impact on heart function. Finally, we devised simulations to assess how GWAS discovery power is influenced by the choice of statistical models, showing that the apparent additivity in human GWAS is to be expected given study design and sample sizes of current studies. This work demonstrates the value of controlled model organism studies for dissecting the genetics of complex traits and provides guidance on the design of genetic association studies.

genetics↗

bioRxiv2025

Impact of pH and temperature in dairy processing on the infectivity of H5N1 avian influenza viruses

Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 (clade 2.3.4.4b) have crossed the species barrier and caused a mastitis-like infection in dairy cows. The high levels of infectious virus found in the milk raised considerable concerns about the safety of raw milk products. This study examined the effect of temperature and pH on the stability of HPAIV and low-pathogenic avian influenza viruses (LPAIV). We found that H5N1 HPAIV remained infectious in milk at 4{degrees}C for four weeks, with slow decreases at 21{degrees}C, and complete inactivation at 37{degrees}C after four weeks. H5N1 LPAIV was stable at 50{degrees}C for 30 minutes but inactivated at higher temperatures (55{degrees}C for 10 minutes, 60{degrees}C for 1 minute, or 72{degrees}C for 30 seconds). At pH levels between 6 and 10, the virus remained stable but was partially inactivated at pH 5.0 and completely inactivated at pH 4.0. During yogurt production, H5N1 LPAIV was completely inactivated when the pH reached 4.3. In cheese production, the lowest pH reached was between 5.0 and 5.3. When H5N1 LPAIV was incubated with soft and semi-hard cheese for one day at 4 {degrees}C, infectious virus titers decreased by 5.1 and 3.9 log10, respectively. When H5N1 LPAIV was incubated with buffer adjusted to pH 5.0, infectious virus titer dropped by only 3.3 log10, suggesting that, alongside pH, other processes of cheese ripening likely influence virus stability. In conclusion, H5N1 avian influenza viruses are largely inactivated during lactic acid fermentation of raw milk. Future studies will assess the required cheese ripening time for complete inactivation.

microbiology↗

bioRxiv2024

Measurement and classification of bold-shy behaviours in medaka fish

MotivationBoldness-shyness is considered a fundamental axis of behavioural variation in humans and other species, with obvious adaptive causes and evolutionary implications. Besides an individuals own genetics, this phenotype is also affected by the genetic makeup of peers in the individuals social environment. To identify genetic determinants of variation along the bold-shy behavioural axis, a reliable experimental and analytical setup able to highlight direct and indirect genetic effects is needed. ResultsWe describe a custom assay designed to detect bold-shy behaviours in medaka fish, combining an open-field and novel-object component. We use this assay to explore direct and social genetic effects on the behaviours of 307 pairs of fish from five inbred medaka strains. Applying a Hidden Markov Model (HMM) to classify behavioural modes, we find that direct genetic effects influence the proportions of time the five strains spent in slow-moving states, explaining up to 29.7% of the variance in time spent in those states. We also found that an individuals behaviour is influenced by the genetics of its tank partner, explaining up to 8.64% of the variance in the time spent in slow-moving states. Our behavioural assay in combination with the HMM analysis is applicable to follow-up genetic linkage studies of genetic variants involved in direct behavioural effects and indirect social genetic effects. A suitable genetic resource for such studies, the Medaka Inbred Kiyosu-Karlsruhe panel (MIKK) has recently been established.

bioinformatics↗

bioRxiv2023

Modular control of time and space during vertebrate axis segmentation

How temporal and spatial control of developmental processes are linked remains a fundamental question. Do underlying mechanisms form a single functional unit or are these dissociable modules? We address this question by studying the periodic process of embryonic axis segmentation, using genetic crosses of inbred medaka fish strains representing two species, Oryzias sakaizumii and latipes. Our analysis revealed correlated interspecies differences with regard to the timing of segmentation, the size of segments and of the presomitic mesoderm (PSM), from which segments are periodically formed. We then did interspecies crosses and real-time imaging quantifications, which revealed extensive phenotypic variation in [~]600 F2 embryos. Importantly, while the F2 analysis showed correlated changes of PSM and segment size, these spatial measures were not correlated to the timing of segmentation. This shows that the control of time and space of axis segmentation can, in principle, be decoupled. In line with this finding, we identified, using developmental quantitative trait loci (devQTL) mapping, distinct chromosomal regions linked to either the control of segmentation timing or PSM size. We were able to validate the devQTL findings using a CRISPR/Cas9 loss-of-function approach on several candidate genes in vivo. Combined, this study reveals that a developmental constraint mechanism underlies spatial scaling of axis segmentation, while its spatial and temporal control are dissociable modules. Our findings emphasise the need to reveal the selective constraints linking these modules in the natural environment.

developmental biology↗

bioRxiv2022

A Yap-dependent transcriptional program directs cell migration for embryo axis assembly

The condensation of the embryo primary axis is a fundamental landmark in the establishment of the vertebrate body plan. Although the complex morphogenetic movements directing cell convergence towards the midline have been described extensively, little is known on how gastrulating cells interpret mechanical cues. Yap proteins are among the best characterized transcriptional mechanotransducers, yet their role in gastrulation has remained elusive. Here we show that the double knockout of yap and its paralog yap1b in medaka results in an axis assembly failure. Quantitative live imaging reveals that mutant cells display reduced displacement and migratory persistence. By characterizing the Yap-dependent transcriptional program, we identified genes involved in cytoskeletal organization and cell-ECM adhesion, rather than in germ layer specification, as direct Yap targets. Dynamic analysis of Tead sensors and downstream targets reveals Yap is acting in migratory cells, and not as a midline beacon, to direct gastrulating precursors trajectories by promoting cortical actin recruitment and focal adhesions assembly. We propose that Yap is engaged in a mechano-regulatory loop that is essential to maintain the directed cell migration sustaining embryo axis formation.

developmental biology↗

bioRxiv2021

The Medaka Inbred Kiyosu-Karlsruhe (MIKK) Panel

Unraveling the relationship between genetic variation and phenotypic traits remains a fundamental challenge in biology. Mapping variants underlying complex traits while controlling for confounding environmental factors is often problematic. To address this, we have established a vertebrate genetic resource specifically to allow for robust genotype-to-phenotype investigations. The teleost medaka (Oryzias latipes) is an established genetic model system with a long history of genetic research and a high tolerance to inbreeding from the wild. Here we present the Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel: the first near-isogenic panel of 80 inbred lines in a vertebrate model derived from a wild founder population. Inbred lines provide fixed genomes that are a prerequisite for the replication of studies, studies which vary both the genetics and environment in a controlled manner and functional testing. The MIKK panel will therefore enable phenotype-to-genotype association studies of complex genetic traits while allowing for careful control of interacting factors, with numerous applications in genetic research, human health, and drug development and fundamental biology. Here we present a detailed characterisation of the genetic variation across the MIKK panel, which provides a rich and unique genetic resource to the community by enabling large-scale experiments for mapping complex traits.

genomics↗

bioRxiv2021

Genomic variations and epigenomic landscape of the Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel

The teleost medaka (Oryzias latipes) is a well-established vertebrate model system, with a long history of genetic research, and multiple high-quality reference genomes available for several inbred strains (HdrR, HNI and HSOK). Medaka has a high tolerance to inbreeding from the wild, thus allowing one to establish inbred lines from wild founder individuals. We have exploited this feature to create an inbred panel resource: the Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel. This panel of 80 near-isogenic inbred lines contains a large amount of genetic variation inherited from the original wild population. We used Oxford Nanopore Technologies (ONT) long read data to further investigate the genomic and epigenomic landscapes of a subset of the MIKK panel. Nanopore sequencing allowed us to identify a much greater variety of high-quality structural variants compared with Illumina sequencing. We also present results and methods using a pan-genome graph representation of 12 individual medaka lines from the MIKK panel. This graph-based reference MIKK panel genome revealed novel differences between the MIKK panel lines compared to standard linear reference genomes. We found additional MIKK panel-specific genomic content that would be missing from linear reference alignment approaches. We were also able to identify and quantify the presence of repeat elements in each of the lines. Finally, we investigated line-specific CpG methylation and performed differential DNA methylation analysis across the 12 lines. We thus present a detailed analysis of the MIKK panel genomes using long and short read sequence technologies, creating a MIKK panel specific pan genome reference dataset allowing for the investigation of novel variation types that would be elusive using standard approaches.

genomics↗

bioRxiv2019

Automated high-throughput heart rate measurement in medaka and zebrafish embryos under physiological conditions

RationaleAccurate and efficient quantification of heartbeats in small fish models is an important readout to study cardiovascular biology, disease states and pharmacology at large scale. However, dependence on anesthesia, laborious sample orientation or requirement for fluorescent reporters have hampered the establishment of high-throughput heartbeat analysis. ObjectiveTo overcome these limitations, we aimed to develop a high-throughput assay with automated heart rate scoring in medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos under physiological conditions designed for genetic screens and drug discovery and validation. Methods and ResultsWe established an efficient screening assay employing automated label-free heart rate determination of randomly oriented, non-anesthetized specimen in microtiter plates. Automatically acquired bright-field data feeds into an easy-to-use HeartBeat software, a MATLAB algorithm with graphical user interface developed for automated quantification of heart rate and rhythm. Sensitivity of the assay and algorithm was demonstrated by profiling heart rates during entire embryonic development. Our analysis pipeline revealed acute temperature changes triggering rapid adaption of heart rates, which has implications for standardization of experimental layout. The approach is scalable and allows scoring of multiple embryos per well resulting in a throughput of >500 embryos per 96-well plate. In a proof of principle screen for compound testing, our assay captured concentration-dependent effects of nifedipine and terfenadine over time. ConclusionA novel workflow and HeartBeat software provide efficient means for reliable and direct quantification of heart rate and rhythm of small fish in a physiological environment. Importantly, confounding factors such as anesthetics or laborious mounting are eliminated. We provide detailed profiles of embryonic heart rate dynamics in medaka and zebrafish as reference for future assay development. Ease of sample handling, automated imaging, physiological conditions and software-assisted analysis now facilitate various large-scale applications ranging from phenotypic screening, interrogation of gene functions to cardiovascular drug development pipelines.

developmental biology↗