Biology subjects

Wittbrodt, J.

Publications and source records attributed to Wittbrodt, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Temperature scaling of developmental tempo reveals reaction-dominated morphogen patterning

A central question in developmental biology is how embryonic patterning remains robust when developmental tempo changes. In ectotherms, temperature strongly alters developmental speed and biochemical reaction rates, whereas diffusion is expected to change only weakly. How morphogen gradients required for patterning remain correctly scaled under these conditions is unclear. We addressed this problem in medaka embryos by quantifying developmental speed, morphogen clearance, diffusion, and endogenous Nodal dynamics across a broad temperature range. Developmental tempo, morphogen production, clearance, and tissue movement increased approximately two- to threefold per 10{degrees}C, whereas effective morphogen diffusion remained largely unchanged. Despite this mismatch, germ-layer proportions and morphogen patterning remained robust. Our mathematical simulations suggest that this robustness arises because the system operates in a reaction-dominated regime, in which changes in reaction and growth kinetics - rather than diffusion - are sufficient to rescale morphogen gradients. Together, our experiments and simulations provide new insights into how morphogen gradients adjust to maintain accurate tissue patterning across changes in developmental time and tempo.

developmental biology↗

bioRxiv2026

Fast calcium-dependent fluorescent labeling for recording of neuronal activation

Calcium transients encode cellular and neuronal activity across timescales ranging from milliseconds to hours, yet linking these transient signals to downstream molecular states remains a major challenge. We recently introduced Caprola, a calcium-dependent protein labeling tool that converts calcium transients into permanent fluorescent marks for later analysis. In this way, Caprola enables tracking of neuronal activities in animal models as well as retrospective identification of labeled cells for isolation and transcriptomic analysis. However, the relatively slow labeling kinetics of Caprola required high concentrations of fluorophore probe and relatively long labeling times, which limits its sensitivity and applicability, in particular in vivo. To address this limitation, we generated Caprola variants with up to 29-fold faster labeling rates than their predecessor. We demonstrate that our new Caprola variants record calcium transients in cells and in zebrafish larval brains under conditions where previous Caprola variants did not show labeling. We further expand the applicability of Caprola to activity-dependent marking of postsynaptic compartments, opening new avenues for coupling functional activity histories with downstream molecular and transcriptomic analyses.

neuroscience↗

bioRxiv2026

Quantitative mapping of heterochrony to species-specific phenotypes

The genetic program of animal development is conserved, but its rate of execution varies across species. Heterochrony, shifts in the relative timing of developmental events, generates phenotypic variation, but its prevalence and origin are unclear. Here, we compare two vertebrates with a 3-fold difference in developmental rate, zebrafish (Danio rerio) and medaka (Oryzias latipes) and characterize heterochrony with embryo-scale single-cell genomics. We generated an atlas of >1.2M single-cell transcriptomes of medaka from blastula to hatch and developed a new approach to represent medaka development in "zebrafish time", uncovering many cryptic, cell-level timing shifts not predicted by medakas slower development. We confirm a divergent heterochrony in the medaka notochord using in vivo imaging, revealing that coupled acceleration and delay of sister cell types shapes the species-specific morphology of this tissue. Our results point to cell type-specific timing deviations as a reservoir of phenotypic variation and we propose that species-specific developmental rate can emerge from these cell-level differences.

Developmental Biology↗

bioRxiv2026

Cycles upon cycles - Temperature Scaling of Medaka Development

How organisms develop in dynamic environmental conditions is a fundamental question. We asked how day-night temperature cycles impact embryonic axis elongation and segmentation, itself a cyclic process linked to the segmentation clock, using the Japanese rice fish medaka. We developed an unbiased dimensional reduction approach, based on Singular Value Decomposition (SVD), to reliably identify the dynamic modes of segmentation clock oscillations across all temperature conditions. We reveal that the two major dynamic modes show opposite temperature sensitivities: while the temporal oscillation (mode 1) varies strongly with temperature, the spatial phase gradient (mode 2) appears largely temperature invariant. In addition, we found developmental parameters with intermediate, sub-scaled temperature responses, such as axis elongation. We used theoretical modeling to understand how dynamic modes emerge from the underlying local oscillation dynamics and axis elongation. We then exposed embryos to circadian and ultradian temperature cycles to reveal dynamic response patterns of oscillations and axis elongation, and found how these responses are integrated into morphological features. Combined, our theoretical-experimental results support a model in which the dynamic integration of temporal (i.e. segmentation clock related) and spatial (i.e. axis elongation) processes, in particular their sub-scaled temperature response patterns, quantitatively compensate each other to yield a robust, temperature-invariant axis patterning outcome.

developmental biology↗

bioRxiv2026

A Scalable, High-Throughput Optomotor Response Pipeline for Quantitative Analysis of Vision Using Infinity-Pools

The optomotor response (OMR) provides a robust readout assay for evaluating visually guided locomotion and has been widely applied to various animal models. We present a scalable, high-throughput OMR platform with tuneable and moving stripe patterns simultaneously projected to the side of 50 petri dishes. The setup is optimized for hatchling-stage medaka (Oryzias latipes and Oryzias sakaizumii) and larval stage zebrafish (Danio rerio) and integrates custom software for automated generation of visual stimuli and specimen detection, enabling precise and automated quantification of optomotor behaviour across thousands of individuals. Using this platform, we systematically assess the visual performance of zebrafish and five medaka strains. By varying stripe width, direction, speed, contrast, and colour, we address spatial resolution, contrast, and colour sensitivity. Albino mutant medaka exhibited the highest sensitivity, responding at lower stripe widths and across a broader range of contrasts and chromatic conditions. Interestingly, some Cab and HO5 hatchlings displayed uni-directional response, revealing strain-specific visuomotor differences. Our platform enables reliable detection of OMR behaviour already at hatching and supports robust analysis of visuomotor function. The modular design, automated detection, analysis pipeline, and capacity for large sample numbers make it a powerful tool for comparative vision research, high-throughput genetic screening, and systematic behavioural profiling.

animal behavior and cognition↗

bioRxiv2025

Extrinsic polarity cues control lamination versus cluster-based organisation in vertebrate retinal development

1.Photosensitive organs are essential for most animals to perceive and respond to their environment. While the gene regulatory networks establishing retinal identity are deeply conserved across metazoans (reviewed in Gehring, 2012; Vopalensky & Kozmik, 2009; Hahn et al., 2023), the retinal architecture varies widely--from invertebrate compound eyes to vertebrate camera-type eyes (Lamb et al., 2007; Schwab, 2017; Arendt & Wittbrodt, 2001). Despite this morphological diversity, early eye anlagen in both, invertebrates and vertebrates, share an initial pseudo-stratified epithelial organization (Weasner & Kumar, 2022; Randlett et al., 2010; Das et al., 2003; Kitambi & Malicki, 2008), which is maintained and elaborated into multi-layered retinae in vertebrates. In contrast, the invertebrate neuroepithelium is re-organized as ommatidia develop. Laminar organisation of the vertebrate retina appears to be a consequence of initial polarisation of the retinal neuroepithelium. This is, however, challenging to test in the organismal context. To address the plasticity of retinal architecture and the impact of epithelial polarity on the structuring of retinal tissue, we take advantage of retinal organoids derived from medaka (Oryzias latipes) (Zilova et al., 2021) that allow to modulate polarity cues and test their impact on the level of epithelialization and structural organisation of the forming retina. We show that under specific culture conditions, medaka retinal organoids undergo a striking morphological switch depending on the level of apico-basal polarity imposed. When polarity cues are continuously provided, a laminated retinal epithelium is established in the organoid. The absence of polarity cues results in the formation of horizontal cellular clusters containing the retinal cell types, which form the vertical retinal column in the developing embryo. We demonstrate that the emergence of this alternative retinal architecture is associated with a loss of epithelial polarity, notably the absence of extracellular matrix (ECM) components, such as laminin, which efficiently rescues lamination. Our findings indicate that tissue-level polarization and lamination in vertebrate retinae require specific extrinsic cues, and that in their absence, differentiating retinal cell types self-organize into structurally distinct, retinal units. This reveals an unexpected plasticity in vertebrate retinal development and indicates a potential for alternative modes of retinal patterning. O_FIG O_LINKSMALLFIG WIDTH=155 HEIGHT=200 SRC="FIGDIR/small/688026v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@100bb1eorg.highwire.dtl.DTLVardef@fceb9borg.highwire.dtl.DTLVardef@d036a7org.highwire.dtl.DTLVardef@1b41483_HPS_FORMAT_FIGEXP M_FIG C_FIG Retinal cells in medaka organoids adopt either a continuous layered epithelium when supported by laminin or a unit-based, ommatidia-like organization when epithelial continuity is lost. This dual outcome suggests that epithelial integrity represents a branching point between vertebrate and invertebrate strategies of retinal patterning, providing an experimental system to replay alternative evolutionary trajectories of eye design.

developmental biology↗

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

Establishing an auxin-inducible GFP nanobody-based acute protein knockdown system to mimic hypomorphic mutations during early medaka embryogenesis

Creating hypomorphic mutations are crucial to study gene function in vivo, especially when null mutations result in (embryonic) lethality. This is especially the case for enzymes involved in glycosylation that, when mutated in human patients, are causing the disease congenital disorders of glycosylation (CDG). To resemble the patient conditions, it would be ideal to acutely modulate the proteins in question to directly interfere with protein levels of such essential enzymes. These methods offer to establish pathogenic enzyme levels resembling net enzyme activity reported from patients suffering from CDG, with Phosphomannomutase 2 - CDG (PMM2-CDG) as the most common form. We established an auxin-inducible acute protein knockdown system for the use in the teleost fish medaka (Oryzias latipes) by combining an improved degron (AID2) technology with a mAID-nanobody targeting endogenously GFP-tagged Pmm2 protein. We generated a fishline expressing a functional Pmm2-GFP fusion protein, by single copy integration of GFP into the pmm2 locus. Upon induction, the degron system efficiently reduced Pmm2-GFP levels and enzyme activity, recapitulating the activity level of the hypomorphic mutations associated with PMM2-CDG in patients. This broadly applicable approach enables the investigation of CDG disease mechanisms during early embryonic development through reduction of protein abundance mimicking hypomorphic mutations and thus substantially expands the range of the genetic toolbox. Summary StatementThe combination of TIR1F74G and mAID-GFP-nanobody enables efficient acute knockdown of endogenously GFP-tagged proteins in medaka. This approach successfully reduced Pmm2 enzyme activity to pathological levels as seen in PMM2-CDG patients.

developmental biology↗

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

Inverted Assembly of the Lens Within Ocular Organoids Reveals Alternate Paths to Ocular Morphogenesis

The eye is a complex organ composed of two main structures - the retina and the lens. It forms by the invagination of the lens forming head surface ectoderm embedding into the forming optic cup. This "outside-in" mode of morphogenesis ensures that the light focusing lens is positioned centrally inside of the eye in the highly constrained environment of the developing embryo. Advances in stem cell biology in the last decade introduced organoids as model to study organogenesis under normal and diseased conditions. However, even though strikingly similar at some points, it remained elusive to which extend the generation of individual structural features in organoids recapitulates in vivo organogenesis. Here we describe the generation of fish ocular organoids composed of both, lens and retina, using pluripotent embryonic cells from medaka (Oryzias latipes). Formation of the organoid lens followed the key molecular features of the process in vivo, including the establishment of lens progenitor cells and their subsequent differentiation into lens fiber cells. In a process dependent on the coordinated activity of BMP and FGF signaling, lens formation in ocular organoids was marked by the expression of key genes implicated in organismal lens development. Despite adhering to the basic molecular machinery of lens formation in vivo, the morphogenesis into a spherical lens followed an "inside-out" mode. Lens progenitor cells were initially established and differentiated into a spherical lens directly inside of the retina. Subsequent displacement of the lens from the center of the organoid towards its surface ultimately led to the formation of a cup-like shaped retina with a centrally positioned lens. Our study highlights that the self-organization of the organoid can favor routes that were not selected for in the developing embryo. Those routes can lead to an alternative, though highly similar outcome with the respect to achieving specific structural features in an unconstrained, embryo-free environment.

developmental biology↗

bioRxiv2025

Immune surveillance and pruning of neuronal stem cells in the medaka retina

Stem cell populations in tissues require precise regulation of their number and quality to maintain proper organ growth. Amongst various regulatory mechanisms, immune cells are emerging to directly regulate stem cell populations. The medaka retinal stem cell (RSC) niche, a model for lifelong neurogenic growth, provides a system to study immune-stem cell interactions. We investigate how microglia, resident macrophages of the central nervous system, regulate the RSC niche. We identify that bona fide RSCs express the chemokine Ccl25b while its cognate receptor, Ccr9a, is expressed in microglia. These microglia form a surveillance ring adjacent to the RSC niche and actively phagocytose RSCs. Interference with microglia by deletion of spi1b reveals that microglia absence leads to increased numbers of ccl25b-positive RSCs and results in morphological defects of the retina. Targeted mutation of ccl25b specifically affects microglia mobility under injury conditions, however, we did not observe any morphological defects indicating that Ccl25b-Ccr9a signaling is not essential for stem cell maintenance. Overall, our data show that under homeostatic conditions the individual RSCs, essential for proper eye development, are actively phagocytosed by immune surveillance.

developmental biology↗

bioRxiv2025

Deep learning predicts tissue outcomes in retinal organoids

Retinal organoids have become important models for studying development and disease, yet stochastic heterogeneity in the formation of cell types, tissues, and phenotypes remains a major challenge. This limits our ability to precisely experimentally address the early developmental trajectories towards these outcomes. Here, we utilize deep learning to predict the differentiation path and resulting tissues in retinal organoids well before they become visually discernible. Our approach effectively bypasses the challenge of organoid-related heterogeneity in tissue formation. For this, we acquired a high-resolution time-lapse imaging dataset comprising about 1,000 organoids and over 100,000 images enabling precise temporal tracking of organoid development. By combining expert annotations with advanced image analysis of organoid morphology, we characterized the heterogeneity of the retinal pigmented epithelium (RPE) and lens tissues, as well as global organoid morphologies over time. Using this training set, our deep learning approach accurately predicts the emergence and size of RPE and lens tissue formation on an organoid-by-organoid basis at early developmental stages, refining our understanding of when early lineage decisions are made. This approach advances knowledge of tissue and phenotype decision-making in organoid development and can inform the design of similar predictive platforms for other organoid systems, paving the way for more standardized and reproducible organoid research. Finally, it provides a direct focus on early developmental time points for in-depth molecular analyses, alleviated from confounding effects of heterogeneity.

developmental biology↗

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↗

bioRxiv2024

FEHAT: Efficient, Large scale and Automated Heartbeat Detection in Medaka Fish Embryos.

High resolution imaging of model organisms allows the quantification of important physiological measurements. In the case of fish with transparent embryos, these videos can visualise key physiological processes, such as heartbeat. High throughput systems can provide enough measurements for the robust investigation of developmental processes as well as the impact of system perturbations on physiological state. However, few analytical schemes have been designed to handle thousands of high-resolution videos without the need for some level of human intervention. We developed a software package, named FEHAT, to provide a fully automated solution for the analytics of large numbers of heart rate imaging datasets obtained from developing Medaka fish embryos in 96 well plate format imaged on an Acquifer machine. FEHAT uses image segmentation to define regions of the embryo showing changes in pixel intensity over time, followed by the classification of the most likely position of the heart and Fourier Transformations to estimate the heart rate. Here we describe some important features of the FEHAT software, showcasing its performance across a large set of medaka fish embryos and compare its performance to established, less automated solutions. FEHAT provides reliable heart rate estimates across a range of temperature-based perturbations and can be applied to tens of thousands of embryos without the need for any human intervention.

bioinformatics↗

bioRxiv2024

Characterizing medaka visual features using a high-throughput optomotor response assay

Medaka fish (Oryzias latipes) is a powerful model to study genetics underlying the developmental and functional traits of the vertebrate visual system. We established a simple and high-throughput optomotor response (OMR) assay utilizing medaka larvae to study visual functions including visual acuity and contrast sensitivity. Our assay presents multiple adjustable stripes in motion to individual fish in a linear arena. For that the OMR assay employs a tablet display and the Fish Stripes software to adjust speed, width, color, and contrast of the stripes. Our results demonstrated that optomotor responses were robustly induced by black and white stripes presented from below in the linear-pool-arena. We detected robust strain specific differences in the OMR when comparing long established medaka inbred strains. We observed an interesting training effect upon the initial exposure of larvae to thick stripes, which allowed them to better respond to narrower stripes. The OMR setup and protocol presented here provide an efficient tool for quantitative phenotype mapping, addressing visual acuity, trainability of cortical neurons, color sensitivity, locomotor response, retinal regeneration and others. Our open-source setup presented here provides a crucial prerequisite for ultimately addressing the genetic basis of those processes.

animal behavior and cognition↗

bioRxiv2024

Multi-species community platform for comparative neuroscience in teleost fish

Studying neural mechanisms in complementary model organisms from different ecological niches in the same animal class can leverage the comparative brain analysis at the cellular level. To advance such a direction, we developed a unified brain atlas platform and specialized tools that allowed us to quantitatively compare neural structures in two teleost larvae, medaka (Oryzias latipes) and zebrafish (Danio rerio). Leveraging this quantitative approach we found that most brain regions are similar but some subpopulations are unique in each species. Specifically, we confirmed the existence of a clear dorsal pallial region in the telencephalon in medaka lacking in zebrafish. Further, our approach allows for extraction of differentially expressed genes in both species, and for quantitative comparison of neural activity at cellular resolution. The web-based and interactive nature of this atlas platform will facilitate the teleost communitys research and its easy extensibility will encourage contributions to its continuous expansion.

neuroscience↗

bioRxiv2024

Minimal-invasive 3D laser printing of microimplants in organismo

Multi-photon 3D laser printing has gathered much attention in recent years as a means of manufacturing biocompatible scaffolds that can modify and guide cellular behavior in vitro. However, in vivo tissue engineering efforts have been limited so far to the implantation of beforehand 3D printed biocompatible scaffolds and in vivo bioprinting of tissue constructs from bioinks containing cells, biomolecules, and printable hydrogel formulations. Thus, a comprehensive 3D laser printing platform for in vivo and in situ manufacturing of microimplants raised from synthetic polymer-based inks is currently missing. Here we present a platform for minimal-invasive manufacturing of microimplants directly in the organism by one-photon photopolymerization and multi-photon 3D laser printing. Employing a commercially available elastomeric ink giving rise to biocompatible synthetic polymer-based microimplants, we demonstrate first applicational examples of biological responses to in situ printed microimplants in the teleost fish Oryzias latipes and in embryos of the fruit fly Drosophila melanogaster. This provides a framework for future studies addressing the suitability of inks for in vivo 3D manufacturing. Our platform bears great potential for the direct engineering of the intricate microarchitectures in a variety of tissues in model organisms and beyond.

bioengineering↗

bioRxiv2024

DNA microbeads for spatio-temporally controlled morphogen release within organoids

Organoids have proven to be powerful in vitro model systems that mimic features of the corresponding tissue in vivo. However, across tissue types and species, organoids still often fail to reach full maturity and function, because biochemical cues cannot be provided from within the organoid to guide their development. The establishment of such tools has been identified as a major goal of the field. Here, we introduce DNA microbeads as a novel tool for implementing spatio-temporally controlled morphogen gradients inside of organoids at any point in their life cycle. The DNA microbeads are formed in a simple one-pot process, they can be stored for a year and their viscoelastic behavior and surface modification is tunable to mimic the corresponding tissue. Employing medaka retinal organoids and early embryos, we show that DNA microbeads can be integrated into embryos and organoids by microinjection and erased in a non-invasive manner with light. Coupling a recombinant surrogate Wnt to the DNA microbeads we demonstrate the spatio-temporally controlled release of the morphogen from the microinjection site, which leads to the formation of retinal pigmented epithelium while maintaining neuroretinal ganglion cells. We were thus able to bioengineer retinal organoids to more closely mirror the cell type diversity of in vivo retinas. The DNA microbead technology can easily be adapted to other organoid applications for improved tissue mimicry.

bioengineering↗