Biology subjects

Scholpp, S.

Publications and source records attributed to Scholpp, S..

13 recordsLinked to original sources

bioRxiv2026

Lost and found: beer belly is a novel, ancient regulator of animal development

Single-cell RNA sequencing (scRNA-seq) can provide new insights into the regulation of developmental processes by revealing unknown or overlooked genes. We identified a novel gene called beer belly (bebe) as a marker of a segment addition zone (SAZ) associated cell cluster in a single-cell atlas of the spider Parasteatoda tepidariorum. Here we show that bebe is an ancient toolkit gene encoding a previously uncharacterised protein predicted to be secreted, which likely plays an important role in the development of most animal phyla. However, we could not identify a bebe ortholog in any mammal or holometabolous insect, suggesting it has been lost in these lineages and explaining why it has been overlooked to date. Knock-down of bebe expression in P. tepidariorum demonstrates that it regulates SAZ size and consequently segment size by interacting with Wnt8. Our findings also suggest that bebe may regulate segmentation in other invertebrates and play additional developmental roles in bilaterian and non-bilaterian animals, likely ancestrally, through modulation of Wnt signalling. This study emphasises the promise of applying single-cell sequencing across a diverse range of species to better understand the regulation of development beyond candidate genes from traditional models, allowing us to better characterise the animal developmental toolkit and its evolution.

developmental biology↗

bioRxiv2026

Calcium signals at cytoneme contacts amplify Wnt/β-catenin signalling

Wnt signalling controls morphogenesis, patterning and homeostasis across tissues. Because Wnt ligands are lipid-modified, their movement between cells cannot rely on simple diffusion and instead requires specialised transport mechanisms. Signalling filopodia, also known as cytonemes, have emerged as a key mediator of contact-dependent morphogen delivery. The mechanisms that stabilise signalling interfaces between Wnt ligand-bearing and receptor-bearing membranes at the cytoneme contact sites, a prerequisite for efficient Wnt ligand-receptor complex assembly and downstream pathway activation, remain poorly understood. Here, we show that HeLa cell cytonemes carry canonical Wnt components and form contact sites marked by focal Ca2+ transients. At these contacts, local Wnt and Ca2+ signals act in a positive feedback loop to promote longer-lasting interactions between neighbouring cells. Mechanistically, we find that E-cadherin/Cadherin 1 (CDH1) co-accumulates at these cytoneme contact sites in a Ca2+ - and Wnt-dependent manner, suggesting that contact stabilisation can be mediated by local adhesion. Consistent with this, presentation of Wnt ligands and CDH1 at cytoneme tips, together with Ca2+ availability, enhances Wnt signalosome formation at contact sites and, consequently, nuclear beta-catenin accumulation in the target cells. Our data support a model in which local Ca2+ transients stabilise cytoneme contacts and facilitate efficient Wnt ligand transfer to the receiving membrane, thereby enhancing canonical Wnt/beta-catenin signalling. We propose that focal Ca2+ signalling can serve as a contact-authentication step for morphogen signalling, converting transient cytoneme encounters into effective activators of the signal transduction cascade.

cell biology↗

bioRxiv2026

Rab8a-positive vesicles transport Wnt8a along cytonemes in zebrafish embryogenesis.

Wnt signalling is a conserved pathway that orchestrates key developmental processes by regulating cell fate, proliferation, and tissue organisation. While the production and secretion of Wnt ligands are well characterised, less is known about how lipid-modified Wnts are delivered for long-range communication. Recently, cytonemes - actin-based signalling filopodia - have been identified as transporters of Wnts over distances to target specific cells in embryogenesis. Here, we characterise Rab8a-dependent vesicular trafficking as a crucial step in this process. Using human cell lines and zebrafish embryos, we show that Wnt ligands, such as Wnt8a, are transported with their carrier protein Wntless (Wls) in Rab8a-positive vesicles along cytonemes, where they fuse at the tips to enable hand-over and signal activation in neighbouring cells. Disruption of Rab8a function results in the intracellular retention of Wnt8a, reduces Wnt spreading and paracrine signalling, and alters embryonic patterning, consistent with reduced Wnt/{beta}-catenin function. Conversely, Rab8a activation enhances Wnt8a dissemination, leading to increased long-range signalling and, consequently, patterning defects in embryogenesis. Our findings uncover a dedicated intracellular trafficking route for Wnt delivery to and along cytonemes, offering new insights into how the spatial precision of Wnt spreading in vertebrate tissues is achieved.

developmental biology↗

bioRxiv2025

Optimised genome editing for precise DNA insertion and substitution using Prime Editors in zebrafish

CRISPR/Cas9-mediated genome editing has rapidly become a popular tool for studying gene functions and generating genetically modified organisms. However, using this system, stochastic integration of random insertions and deletions restricts precise genome manipulation. Advanced CRISPR/Cas9 technologies using Prime Editors (PEs), Cas9 proteins fused with reverse transcriptase, enable programmed integration of short DNA modifications into the genome. However, its application in precise genome editing in animal models is challenging. Here, we utilise a nickase-and a nuclease-based PE to perform programmed short DNA substitution and insertion in various loci in the zebrafish genome. Whereas nickase-based PE2 mediated a higher ratio of precise prime edits to the total edits, nuclease-based PEn was more efficient for short DNA modifications, achieving up to 27.3% precise insertion. To further evaluate our approach, we inserted a nuclear localisation signal into a reporter transgene to incorporate longer fragments by prime editing. These gene modifications were transmitted to the next generation. We show that PE-mediated prime editing can efficiently manipulate genome information in zebrafish without using exogenous donor DNA. Prime editing, CRISPR/Cas9, zebrafish, PE2, PEn

molecular biology↗

bioRxiv2024

Amyloid-β can activate JNK signalling via WNT-5A/ROR2 to reduce synapse formation in Alzheimer's disease.

Wnt signalling is an essential signalling system in neurogenesis, and recent studies have highlighted the critical role of this signalling network in regulating synaptic plasticity, neuronal survival, and neurogenesis, processes that are disrupted in Alzheimers disease (AD). From the Wnt network, the Wnt/{beta}-catenin pathway has been studied for its neuroprotective role, and this is suppressed in AD. However, the involvement of the non-canonical pathway, which operates independently of {beta}-catenin and involves the planar cell polarity (PCP), remains to be determined in AD. In this work, we analyse the function of the orphan receptor tyrosine kinase ROR2, an essential co-receptor of the Wnt/PCP signalling pathway. We find that activation of WNT-5A/ROR2 signalling activates JNK signalling, reducing pre- and postsynaptic clusters on neurites in mature SH-SY5Y neurons. This observation is similar to SH-SY5Y neurons treated with the Amyloid-{beta} peptide A{beta}1-42 or DKK1, which are both increased in AD. Surprisingly, the effect of A{beta}1-42 and DKK1 signalling on synaptogenesis can be mitigated by blocking ROR2 and JNK signalling, suggesting that A{beta} and DKK1 signalling depends on ROR2/JNK signalling. Finally, we find an increase of WNT-5A/ROR2 clusters on neurites of iPSC-derived cortical neurons carrying the PSEN1 A75V mutation, known to enhance the pathological A{beta}42/40 ratio. Simultaneously, the number of pre- and post-synaptic clusters decreased in the mutant line. Inhibition of ROR2/JNK signalling in PSEN1A75V cortical neurons partially rescues the reduction in synaptogenesis, suggesting that ROR2 signalling may act in a positive feedback loop with A{beta}1-42 and DKK1 signalling to augment JNK signalling as seen in AD.

cell biology↗

bioRxiv2023

Wnt-7a-positive dendritic cytonemes induce synaptogenesis in cortical neurons.

SummaryNeuronal circuits evolve as a precisely patterned network. In this context, a growing neuron must locate the appropriate target area on a neurite of a neighbouring cell with which to connect. Controlled target selection involves dendritic filopodial contacts and requires the exact apposition of synaptic components. Calcium signalling has been postulated to trigger the transformation from dendritic filopodia into functional synapses. However, calcium is a rather unspecific signalling system, and it needs to be clarified how the exact development of synaptic connections is controlled. Similarly, Wnt/{beta}-catenin signalling promotes synapse formation; however, how secreted Wnts induce and maintain synapses on neuronal dendrites is not well understood. Here, we show that Wnt-7a is tethered to the tips of dynamic dendritic filopodia during spine formation in human cortical neurons. These filopodia can activate Wnt signalling precisely at the contact sites on the dendrites of an adjacent neuron. Subsequently, local calcium transients can be observed at these Wnt-positive contact sites. Depleting either the filopodial-loaded Wnt or the extracellular calcium pool blocks the clustering of pre- and post-synaptic markers, hence the establishment of stable connections. Therefore, we postulate that local Wnt-7a signalling from the tip of the dendritic filopodia, verified by simultaneous calcium signalling, provides an elegant mechanism for orchestrating focal synapse maturation.

neuroscience↗

bioRxiv2022

Cytoneme-mediated transport of active Wnt5b/Ror2 complexes in zebrafish gastrulation

Chemical signaling is the primary means by which cells communicate in the embryo. The underlying principle refers to a group of ligand-producing cells and a group of cells that respond to this signal because they express the appropriate receptors. In the zebrafish embryo, Wnt5b binds to the receptor Ror2 to trigger the Wnt/Planar Cell Polarity (Wnt/PCP) signaling pathway to regulate tissue polarity and cell migration. However, it is still unclear how this lipophilic ligand is transported from the source cells through the aqueous extracellular space to the target tissue. Here we show that Wnt5b, together with Ror2, is loaded on long protrusions. The active Wnt5b/Ror2 complexes are handed over from these cytonemes to the receiving cell to trigger Wnt/PCP signaling, regardless of whether the cell expresses functional receptors. On the tissue level, we show that cytoneme-dependent spreading of active Wnt5b/Ror2 affects convergence and extension in the zebrafish gastrula.

developmental biology↗

bioRxiv2022

Cancer-associated fibroblast-derived ROR2 induces WNT/PCP activation and polarized migration in receiving gastric cancer cells.

Bone marrow-derived mesenchymal stem cells and cancer-associated fibroblasts in the tumor-stromal environment have been linked to cancer progression in many studies. These fibroblasts provide signaling factors to the tumor cells that promote proliferation, survival, invasion, and metastasis. One signaling pathway influencing tumor cell behavior is the WNT/Planar Cell Polarity (PCP) signaling in gastric cancer. Here, we show that the gastric tumor cell line, AGS, can respond to the PCP ligand WNT5A, however, express a very low level of the bona-fide WNT/PCP receptor, ROR2. At the same time, we find that CAF display long filopodia and had significantly higher levels of ROR2 than normal gastric fibroblasts. By high-resolution imaging, we observe a direct, cytoneme-mediated transfer of a complex containing ROR2 and WNT5A from CAF to the gastric cancer cells. The amount of ROR2 transferred correlated with JNK signaling in receiving cells, showing a direct requirement for receptor transfer. Co-culture of AGS with CAF expressing a dominant-negative form of ROR2 exhibited reduced actin polarization and migration compared to wild-type CAF. Furthermore, induction of migration via paracrine ROR2 transfer was observed in a zebrafish in vivo model. These unexpected findings demonstrate a fresh role in the direct transfer of a Wnt receptor from a signal-producing cell to a receiving cell and explain the mechanism by which gastric cancer cells expressing low levels of ROR2 can respond to a WNT5A-high tumor microenvironment.

cancer biology↗

bioRxiv2022

The scaffolding protein Flot2 regulates cytoneme-based transport of Wnt3 in gastric cancer

The Wnt/{beta}-catenin signalling pathway regulates multiple cellular processes during development and many diseases, including cell proliferation, migration, and differentiation. Despite their hydrophobic nature, Wnt proteins exert their function over long distances to induce paracrine signalling. Recent studies have identified several factors involved in Wnt secretion, however, our understanding of how Wnt ligands are transported between cells to interact with their cognate receptors is still debated. Here, we demonstrate that gastric cancer cells utilise cytonemes to transport Wnt3 intercellularly to promote proliferation. Furthermore, we identify the membrane-bound scaffolding protein Flotillin-2 (Flot2), frequently overexpressed in gastric cancer, as a regulator of these cytonemes. Together with the Wnt co-receptor and cytoneme initiator Ror2, Flot2 determines the number and length of Wnt3 cytonemes in gastric cancer. Finally, we show that Flot2 is necessary for Wnt8a cytonemes during zebrafish embryogenesis, suggesting a conserved mechanism for Flot2-mediated Wnt transport on cytonemes in development and disease.

cancer biology↗

bioRxiv2021

Human rare variant and zebrafish CRISPR/Cas9-mediated mutant analyses reveal novel functions for API5, HSPB7, and LMO2 in heart failure

The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that in vivo models are vital to understand the links between genetic perturbations and functional impacts. Traditional approaches (e.g. genetically-modified mice) are optimal for assessing small numbers of proposed target genes, but less practical when multiple targets are identified. The zebrafish, in contrast, offers great potential for higher throughput in vivo gene functional assessment to aid target prioritisation and support definitive studies undertaken in mice. Here we used whole-exome sequencing and bioinformatics on human patient data to identify 3 genes (API5, HSPB7, and LMO2) suggestively associated with heart failure that were also predicted to play a broader role in disease aetiology. The role of these genes in cardiovascular system development and function was then further investigated using in vivo CRISPR/Cas9-mediated gene mutation analysis in zebrafish. We observed multiple impacts in F0 knockout zebrafish embryos (crispants) following effective somatic mutation, including reductions in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2, there was also a significant impact on cardiovascular function as well as an expected reduction in erythropoiesis. The data generated from both the human in silico and zebrafish in vivo assessments undertaken supports roles for API5, HSPB7, and LMO2 in human cardiovascular disease and identifies them as potential drug targets for further investigation. The data presented also supports the use of human in silico genetic variant analysis, in combination with zebrafish crispant phenotyping, as a powerful approach for assessing gene function as part of an integrated multi-level drug target validation strategy.

molecular biology↗

bioRxiv2020

Vangl2 regulates the dynamics of Wnt cytonemes in vertebrates

The Wnt signalling network regulates cell proliferation and cell differentiation as well as migration and polarity in development of multicellular organisms. However, it is still unclear how distribution of Wnt ligands is precisely controlled to fulfil all of these different functions. Here, we show that the four-pass transmembrane protein Vangl2 occupies a central role in determining the distribution of Wnt by cytonemes in vertebrate tissue. In zebrafish epiblast cells, mouse intestinal telocytes and human gastric cancer cells, activation of Vangl2 leads to the generation of fewer but extremely long cytonemes, which start to branch and deliver Wnt protein to multiple cells. The Vangl2-activated cytonemes increase Wnt/{beta}-catenin signalling in the surrounding cells. Concordantly, inhibition of Vangl2 function leads to the formation of shorter cytonemes and reduced paracrine Wnt/{beta}-catenin signal activation. A mathematical model simulating the observed Vangl2 functions on cytonemes in zebrafish gastrulation predicts an anterior shift of the morphogenetic signalling gradient, altered tissue patterning, and a loss of the sharpness of tissue domains. We confirmed these predictions during anteroposterior patterning in the zebrafish neural plate. In summary, we show that Vangl2 - a core member of the PCP signalling component - is fundamental to paracrine Wnt/{beta}-catenin signalling by controlling cytoneme behaviour in vertebrate development and tissue homeostasis.

developmental biology↗

bioRxiv2018

Pcdh18a-positive tip cells instruct notochord formation in zebrafish

The notochord defines the axial structure of all vertebrates during development. Notogenesis is a result of major cell reorganization in the mesoderm, the convergence and the extension of the axial cells. However, it is currently not known how these processes act together in a coordinated way during notochord formation. Analysing the tissue flow, we determined the displacement of the axial mesoderm and identified, relative to the ectoderm, an actively-migrating notochord tip cell population and a group of trailing notochordal plate cells. Molecularly, these tip cells express Protocadherin18a, a member of the cadherin superfamily. We show that Pcdh18a-mediated recycling of E-cadherin adhesion complexes transforms these tip cells into a cohesive and fast migrating cell group. In turn, these tip cells subsequently instruct the trailing mesoderm. We simulated cell migration during early mesoderm formation using a lattice-based mathematical framework, and predicted that the requirement for an anterior, local motile cell cluster could guide the intercalation of the posterior, axial cells. Indeed, grafting experiments validated the predictions and induced ectopic notochord-like rods. Our findings indicate that the tip cells influence the trailing mesodermal cell sheet by inducing the formation of the notochord.

developmental biology↗

bioRxiv2018

Observing the Cell in Its Native State: Imaging Subcellular Dynamics in Multicellular Organisms

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution without inducing undue stress on either. We combined lattice light sheet microscopy with two-channel adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages, and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.\n\nOne Sentence SummaryCombining lattice light sheet microscopy with adaptive optics enables high speed, high resolution in vivo 3D imaging of dynamic processes inside cells under physiological conditions within their parent organisms.

cell biology↗