Biology subjects

Wittbrodt, J.

Publications and source records attributed to Wittbrodt, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

2-photon laser printing to mechanically stimulate multicellular systems in 3D

Most biological activities take place in 3D environments, where cells communicate with each other in various directions and are located in a defined, often microstructured, space. To investigate the effect of defined cyclic mechanical forces on a multicellular system, we develop a sub-millimeter sized stretching device for mechanical stimulation of a structurally restricted, soft multicellular microenvironment. For the stretching device, a multimaterial 3D microstructure made of PDMS and gelatine-based hydrogel is printed via 2-photon polymerization (2PP) method. The printed structures are first characterized microscopically and mechanically to study the effect of different printing parameters. With 2PP, organotypic cell cultures are then directly printed into the hydrogel structures to achieve true 3D cell culture systems. These are mechanically stimulated with a cantilever by indenting the stretching device at a defined point. As a most important result, the cells in the 3D organotypic cell culture change morphology and actin orientation when exposed to cyclic mechanical stretch, even within short timescales of just 30 minutes. As a proof of concept, we encapsulated a Medaka retinal organoid in the same structure to demonstrate that even preformed organoids can be stimulated by our method. The results demonstrate the power of 2PP to manufacturing multifunctional soft devices for mechanically controlling multicellular systems at micrometer resolution and thus mimicking mechanical stress situations, as they occur in vivo.

biophysics↗

bioRxiv2023

Genotype imputation in F2 crosses of inbred lines

MotivationCrosses among inbred lines are a fundamental tool for the discovery of genetic loci associated with phenotypes of interest. In organisms for which large reference panels or SNP chips are not available, imputation from low-pass whole-genome sequencing is an effective method for obtaining genotype data from a large number of individuals. To date, a structured analysis of the conditions required for optimal genotype imputation has not been performed. ResultsWe report a systematic exploration of the effect of several design variables on imputation performance in F2 crosses of inbred medaka lines using the imputation software STITCH. We determined that, depending on the number of samples, imputation performance reaches a plateau when increasing the per-sample sequencing coverage. We also systematically explored the trade-offs between cost, imputation accuracy, and sample numbers. We developed a computational pipeline to streamline the process, enabling other researchers to perform a similar cost-benefit analysis on their population of interest. Availability and implementationThe source code for the pipeline is available at https://github.com/birneylab/stitchimpute. While our pipeline has been developed and tested for an F2 population, the software can also be used to analyse populations with a different structure.

bioinformatics↗

bioRxiv2023

Visualisation of gene expression within the context of tissues: an X-ray computed tomography-based multimodal approach

The development of an organism is orchestrated by the spatial and temporal expression of genes. Accurate visualisation of gene expression patterns in the context of the surrounding tissues offers a glimpse into the mechanisms that drive morphogenesis. We developed correlative light-sheet fluorescence microscopy and X-ray computed tomography approach to map gene expression patterns to the whole organisms 3D anatomy at cellular resolution. We show that this multimodal approach is applicable to gene expression visualised by protein-specific antibodies and fluorescence RNA in situ hybridisation, offering a detailed understanding of individual phenotypic variations in model organisms. Furthermore, the approach provides a unique possibility to identify tissues together with their 3D cellular and molecular composition in anatomically less-defined in vitro models, such as organoids. We anticipate that the visual and quantitative insights into the 3D distribution of gene expression within tissue architecture, by the multimodal approach developed here, will be equally valuable for reference atlases of model organisms development, as well as for comprehensive screens and morphogenesis studies of in vitro models.

developmental biology↗

bioRxiv2023

Natural genetic variation quantitatively regulates heart-rate and -dimension

The polygenic contribution to heart development and function along the health-disease continuum remains unresolved. To gain insight into the genetic basis of quantitative cardiac phenotypes, we utilize highly inbred Japanese rice fish models, Oryzias latipes, and Oryzias sakaizumii. Employing automated quantification of embryonic heart rates as core metric, we profiled phenotype variability across five inbred strains. We observed maximal phenotypic contrast between individuals of the HO5 and the HdrR strain. HO5 showed elevated heart rates associated with embryonic ventricular hypoplasia and impaired adult cardiac function. This contrast served as the basis for genome-wide mapping. In a segregation population of 1192 HO5 x HdrR F2 embryos, we mapped 59 loci (173 genes) associated with heart rate. Experimental validation of the top 12 candidate genes in loss-of-function models revealed their causal and distinct impact on heart rate, development, ventricle size, and arrhythmia. Our study uncovers new diagnostic and therapeutic targets for developmental and electrophysiological cardiac diseases and provides a novel scalable approach to investigate the intricate genetic architecture of the vertebrate heart. One-Sentence SummaryKey loci for vertebrate heart function mapped and validated, highlighting diagnostic and potential therapeutic targets for cardiac disorders.

genetics↗

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

The inceptionist's guide to base editing - de novo PAM generation to reach initially inaccessible target-sites

Base editing by CRISPR crucially depends on the presence of a PAM in proper distance to the editing-site. Here we present and validate an efficient one-shot approach termed "inception", relaxing these constraints. This is achieved by sequential, combinatorial base editing: de novo generated synonymous, non-synonymous or intronic PAM-sites facilitate subsequent base editing at nucleotide positions inaccessible before, increasing the targeting range of highly precise editing approaches.

genetics↗

bioRxiv2021

Efficient single copy integration via homology-directed repair (scHDR) by 5'modification of large DNA donor fragments in mice

CRISPR/Cas approaches have largely replaced conventional gene targeting strategies. However, homology-directed repair (HDR) in the mouse genome is not very efficient, and precisely inserting longer sequences using HDR remains challenging, given that donor constructs preferentially integrate as concatemers. Here, we show that injecting 5biotinylated donor DNA in mouse embryos at the two-cell stage leads to efficient single-copy HDR (scHDR) alleles. Our dedicated genotyping strategy showed that these alleles occurred with a frequency of 19%, 20%, and 26%, respectively, in three independent gene loci, indicating that scHDR is dramatically boosted by 5biotinylation. Thus, we suggest that a combination of a 5biotinylated donor and diligent analysis of concatemer integration are prerequisites for efficiently and reliably generating conditional alleles or other large fragment knock-ins into the mouse genome.

molecular biology↗

bioRxiv2021

Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction

Single nucleotide variants (SNVs) are prevalent genetic factors shaping individual trait profiles and disease susceptibility. The recent development and optimizations of base editors, rubber and pencil genome editing tools now promise to enable direct functional assessment of SNVs in model organisms. However, the lack of bioinformatic tools aiding target prediction limits the application of base editing in vivo. Here, we provide a framework for adenine and cytosine base editing in medaka (Oryzias latipes) and zebrafish (Danio rerio), ideal for scalable validation studies. We developed an online base editing tool ACEofBASEs (a careful evaluation of base-edits), to facilitate decision-making by streamlining sgRNA design and performing off-target evaluation. We used state-of-the-art adenine (ABE) and cytosine base editors (CBE) in medaka and zebrafish to edit eye pigmentation genes and transgenic GFP function with high efficiencies. Base editing in the genes encoding troponin T and the potassium channel ERG faithfully recreated known cardiac phenotypes. We finally validated missense mutations in novel candidate genes of congenital heart disease (CHD) dapk3, ube2b, usp44, and ptpn11 and present the resulting heart specific phenotypes. This base editing framework applies to a wide range of SNV-susceptible traits accessible in fish, facilitating straight-forward candidate validation and prioritization for detailed mechanistic downstream studies.

genetics↗

bioRxiv2021

Zic1 advances epaxial myotome morphogenesis to cover the neural tube via Wnt11r

The dorsal axial muscles, or epaxial muscles, are a fundamental structure covering the spinal cord and vertebrae, as well as mobilizing the vertebrate trunk. To date, mechanisms underlying the morphogenetic process shaping the epaxial myotome are largely unknown. To address this, we used the medaka zic1/zic4-enhancer mutant Double anal fin (Da), which exhibits ventralized dorsal trunk structures resulting in impaired epaxial myotome morphology and incomplete coverage over the neural tube. In wild type, dorsal dermomyotome (DM) cells, progenitors of myotomal cells, reduce their proliferative activity after somitogenesis and subsequently form unique large protrusions extending dorsally, potentially guiding the epaxial myotome dorsally. In Da, by contrast, DM cells maintain the high proliferative activity and form mainly small protrusions. By combining RNA- and ChIP-sequencing analyses, we revealed direct targets of Zic1 which are specifically expressed in dorsal somites and involved in various aspects of development, such as cell migration, extracellular matrix organization and cell-cell communication. Among these, we identified wnt11r as a crucial factor regulating both cell proliferation and protrusive activity of DM cells. We propose that the dorsal movement of the epaxial myotome is guided by DM cells and that Zic1 empowers this activity via Wnt11r to achieve the neural tube coverage.

developmental biology↗

bioRxiv2021

hei-tag: a highly efficient tag to boost targeted genome editing

Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems1. While active in all species tested so far, editing efficiencies still leave room for improvement. To reach its target, the bacterial Cas9 needs to be efficiently shuttled into the nucleus as attempted by fusion of nuclear localization signals (NLSs) to the Cas9 protein2. Additional domains such as FLAG- or myc-tags are added for immediate detection or straight-forward purification3. To avoid steric hinderance impacting on activity, amino acid linkers are employed connecting Cas9 and additional domains. We present the hei-tag (high efficiency-tag), boosting the activity of the wide variety of CRISPR/Cas genome editing tools. The addition of the hei-tag to Cas9 or a C-to-T base editor dramatically enhances the respective targeting efficiency in model systems ranging from fish to mammals, including tissue culture applications. This allows to instantly upgrade existing and potentially highly adapted systems as well as establish novel highly efficient tools.

genetics↗

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↗

bioRxiv2021

The human FLT1 regulatory element directs vascular expression and modulates angiogenesis pathways in vitro and in vivo

There is growing evidence that mutations in non-coding cis-regulatory elements (CREs) disrupt proper development. However, little is known about human CREs that are crucial for cardiovascular development. To address this, we bioinformatically identified cardiovascular CREs based on the occupancy of the CRE by the homeodomain protein NKX2-5 and cardiac chromatin histone modifications. This search defined a highly conserved CRE within the FLT1 locus termed enFLT1. We show that the human enFLT1 is an enhancer capable of driving reporter transgene expression in vivo throughout the developing cardiovascular system of medaka. Deletion of the human enFLT1 enhancer ({Delta}enFLT1) triggered molecular perturbations in extracellular matrix organisation and blood vessel morphogenesis in vitro in endothelial cells derived from human embryonic stem cells and vascular defects in vivo in medaka. These findings highlight the crucial role of the human FLT1 enhancer and its function as a regulator and buffer of transcriptional regulation in cardiovascular development.

genomics↗

bioRxiv2021

In vivo identification and validation of novel potential predictors for human cardiovascular diseases

Genetics crucially contributes to cardiovascular diseases (CVDs), the global leading cause of death. Since the majority of CVDs can be prevented by early intervention there is a high demand for predictive markers. While genome wide association studies (GWAS) correlate genes and CVDs after diagnosis and provide a valuable resource for such markers, preferentially those with previously known or suspected function are addressed further. To tackle the unaddressed blind spot of understudied genes, we particularly focused on the validation of heart GWAS candidates with little or no apparent connection to cardiac function. Building on the high conservation of basic heart function and underlying genetics from fish to human we combined CRISPR/Cas9 genome editing of the orthologs of human GWAS candidates in isogenic medaka with automated high-throughput heart rate analysis. Our functional analyses of understudied human candidates uncovered a prominent fraction of heart rate associated genes from adult human patients displaying a heart rate effect in embryonic medaka already in the injected generation. Following this pipeline, we identified 16 GWAS candidates with potential diagnostic and predictive power for human CVDs.

genetics↗

bioRxiv2021

Fish primary embryonic stem cells self-assemble into retinal tissue mirroring in vivo early eye development

Organoids derived from pluripotent stem cells promise the solution to current challenges in basic and biomedical research. Further progress and widespread applications are however limited by long developmental time, variable success, and lack of direct comparison to an in vivo reference. To overcome those limitations, we derived organoids from rapidly developing teleosts. We demonstrate how primary embryonic stem cells from zebrafish and medaka efficiently self-organize into anterior neural structures, particularly retina. Within four days, blastula-stage cell aggregates reproducibly execute key steps of eye development: retinal specification, morphogenesis and differentiation. The number of aggregated cells as well as genetic factors crucially impacted upon the concomitant morphological changes that were intriguingly reflecting the in vivo situation. High reproducibility and rapid development of fish-derived organoids in combination with advanced genome editing techniques immediately allow addressing aspects of development and disease, and systematically probing the impact of the physical environment on morphogenesis and differentiation.

developmental biology↗

bioRxiv2020

The medaka alg2 mutant is a model for hypo-N-glycosylation-associated retinitis pigmentosa

Patients suffering from Congenital Disorders of Glycosylation (CDG) carry mutations in components of the evolutionarily highly conserved protein-glycosylation-machinery. Employing targeted genome editing, we modeled alleles in medaka fish based on a mutation described in an ALG2-index patient. The multisystemic phenotypes in our alg2 model closely resembled the patients syndromes. Molecularly, the mutation results in a reduction of the abundance of N-glycans without altering the profile of glycan structures in fish as well as in patient fibroblasts. This hypo-N-glycosylation impacted on protein abundance in two directions. We discovered a putative compensatory upregulation of the basic glycosylation and glycoprotein processing machinery highlighting the regulatory topology of the network. Conversely, proteins of the retinal phototransduction machinery were massively downregulated in the alg2 model. Those relate to the specific loss of rod photoreceptors that fail to be maintained in the alg2 mutants, a condition known as retinitis pigmentosa. Transient supply of human or medaka alg2 mRNA efficiently rescued the phenotypic spectrum and restored viability demonstrating that our alg2 model delivers key traits for the potential treatment of the disorder.

developmental biology↗

bioRxiv2020

Deep learning-enhanced light-field imaging with continuous validation

Light-field microscopy (LFM) has emerged as a powerful tool for fast volumetric image acquisition in biology, but its effective throughput and widespread use has been hampered by a computationally demanding and artefact-prone image reconstruction process. Here, we present a novel framework consisting of a hybrid light-field light-sheet microscope and deep learning-based volume reconstruction, where single light-sheet acquisitions continuously serve as training data and validation for the convolutional neural network reconstructing the LFM volume. Our network delivers high-quality reconstructions at video-rate throughput and we demonstrate the capabilities of our approach by imaging medaka heart dynamics and zebrafish neural activity.

bioengineering↗

bioRxiv2020

Igf signalling uncouples retina growth from body size by modulating progenitor cell division

Balancing the relative growth of body and organs is of key importance for coordinating size and function. This is of particular relevance in post-embryonically growing organisms, facing this challenge life-long. We addressed this question in the neuroretina of medaka fish (Oryzias latipes), where growth and size regulation are crucial for functional homeostasis of the visual system. We find that a central growth regulator, Igf1 receptor, is necessary and sufficient for proliferation control in the postembryonic retinal stem cell niche, the ciliary marginal zone (CMZ). Targeted activation of Igf1r signalling in the CMZ uncouples neuroretina growth from body size control, increasing layer thickness while preserving the structural integrity of the retina. The retinal expansion is driven exclusively by enhanced proliferation of progenitor cells while stem cells do not respond to Igf1r modulation. Our findings position Igf signalling as key module controlling retinal size and structure with far reaching evolutionary implications.

developmental biology↗