Biology subjects

Tolwinski, N. S.

Publications and source records attributed to Tolwinski, N. S..

10 recordsLinked to original sources

bioRxiv2026

A Cross-Species Systems Genetics Framework Identifies Causal Genes in Diabetic Nephropathy

Diabetic nephropathy (DN) is the leading cause of kidney failure in the developed world, but the genetic architecture of DN susceptibility is not well characterised. Here we apply a systems genetics approach in a mouse model of DN to discover novel QTLs for clinically relevant phenotypes including albuminuria, glomerulosclerosis, and macrophage infiltration. For context and prioritisation, we combined single-cell-transcriptomics-guided pQTL and eQTL mapping with cell-type-specific co-expression networks, identifying 192 candidate pGenes for albuminuria. While many were novel, 27% had prior genetic associations, and 40% were validated in a human diabetic cohort. Twelve genes belong to a podocyte network enriched for human GWAS signals. Among those, functional significance of the E3 ubiquitin ligases DCAF6 and ZNRF2 was confirmed by knockdown in Drosophila nephrocytes. Our systems genetics approach identified DN susceptibility genes previously validated in human GWAS while uncovering potential new genes and pathways that could be exploited for risk stratification and therapeutics development.

systems biology↗

bioRxiv2026

Junctional β-Catenin Stabilization Links Wnt Signaling and Force Generation

{beta}-catenin plays two fundamental roles in animal tissues: it acts as a transcriptional effector of canonical Wnt signaling and as a core structural component of adherens junctions that mediate cell-cell adhesion. In canonical Wnt signaling, the post-transcriptional regulation of {beta}-catenin abundance, primarily through regulated phosphorylation, ubiquitination, and proteasomal degradation, determines whether the pathway is "off" or "on." Despite the central importance of {beta}-catenin stabilization, in vivo measurements of {beta}-catenin protein lifetime and stabilization dynamics during development remain limited. Here, we measure the stability of endogenous {beta}-catenin in vivo using tandem fluorescent protein timers (tFPs; "Timers") inserted as minimally disruptive cassettes within the endogenous locus. Timers allow simultaneous visualization of a newly synthesized, rapidly accumulating pool (fast-maturing GFP) and a long-lived, stabilized pool (slow-maturing RFP). Surprisingly, the strongest stabilization does not occur in canonical Wnt patterning stripes; instead, we observe marked stabilization of junctional {beta}-catenin at the leading edge during dorsal closure, a force-generating morphogenetic process. This stabilization is not explained by canonical Wnt ligand input and seems to reflect a stability program linked to {beta}-catenins adhesive function in adherens junctions. We suggest that a stable junctional pool of {beta}-catenin is vital for dorsal closure mechanics and provide evidence that this stabilization is regulated by Dishevelled and JNK, thus connecting Wnt pathway components to mechanotransduction.

developmental biology↗

bioRxiv2021

Wnt signaling rescues amyloid beta induced stem cell loss

Previously, we established an optogenetic model to induce Amyloid-{beta} intracellular oligomerization to model distinct disease etiologies (LO_SCPLOWIMC_SCPLOW et al. 2020). Here we examine the effect of Wnt signaling on Amyloid in this model. We observe that Wnt activation rescues the detrimental effects of Amyloid expression and oligomerization. We analyze the gene expression changes downstream of Wnt that contribute to this rescue and find changes in aging related genes, protein misfolding, metabolism and inflammation. We propose that Wnt expression reduces inflammation through repression of Toll activating factors and confirm that chronic Toll activation reduces lifespan. We propose that the protective effect observed for Lithium treatment functions at least in part through Wnt activation and inhibition of inflammation.

cell biology↗

bioRxiv2021

A Non-Canonical Raf Function Is Required for Dorsal-Ventral Patterning During Drosophila Embryogenesis

Proper embryonic development requires directional axes to pattern cells into embryonic structures. In Drosophila, spatially discrete expression of transcription factors determines the anterior to posterior organization of the early embryo, while the Toll and TGF{beta} signalling pathways determine the early dorsal to ventral pattern. Embryonic MAPK/ERK signaling contributes to both anterior to posterior patterning in the terminal regions and to dorsal to ventral patterning during oogenesis and embryonic stages. Here we describe a novel loss of function mutation in the Raf kinase gene, which leads to loss of ventral cell fates as seen through the loss of the ventral furrow, the absence of Dorsal/NF{kappa}B nuclear localization, the absence of mesoderm determinants Twist and Snail, and the expansion of TGF{beta}. Gene expression analysis showed cells adopting ectodermal fates much like loss of Toll signaling. Our results combine novel mutants, live imaging, optogenetics and transcriptomics to establish a novel role for Raf, that appears to be independent of the MAPK cascade, in embryonic patterning.

developmental biology↗

bioRxiv2021

Mutated in Colorectal Cancer (MCC): a centrosomal protein that relocalizes to the ncMTOC during intestinal cell differentiation

Mutated in Colorectal Cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests, in the pathogenesis of hereditary human colon cancer. To date, however, the contributions of MCC to intestinal homeostasis remain unclear. Here, we examine the subcellular localization of MCC, both at the mRNA and protein levels, in the adult intestinal epithelium. Our findings reveal that Mcc transcripts are restricted to proliferating crypt cells, including Lgr5+ stem cells, and that Mcc protein is distinctly associated with the centrosome in these cells. Upon intestinal cellular differentiation, Mcc is redeployed to the non-centrosomal microtubule organizing center (ncMTOC) at the apical domain of villus cells. Using intestinal organoids, we show that the shuttling of the Mcc protein depends on phosphorylation by Casein Kinases 1{delta}/{varepsilon}, which are critical modulators of WNT signaling. Together, our findings support a putative role for MCC in establishing and maintaining the cellular architecture of the intestinal epithelium as a component of both the centrosome and ncMTOC.

cell biology↗

bioRxiv2019

Application of Optogenetic Amyloid-β Distinguishes Between Metabolic and Physical Damage in Neurodegeneration

The brains of Alzheimers Disease patients show a decrease in brain mass and a preponderance of extracellular Amyloid-{beta} plaques. These plaques are formed by aggregation of polypeptides that are derived from Amyloid Precursor Protein (APP). Amyloid-{beta} plaques are thought to play either a direct or an indirect role in disease progression, however the exact role of aggregation and plaque formation in the ethology of Alzheimers Disease is subject to debate, not least because the biological effects of soluble and aggregated Amyloid-{beta} peptides are difficult to separate in vivo. To investigate the consequences of formation of Amyloid-{beta} oligomers in living tissues, we developed a fluorescently tagged, optogenetic Amyloid-{beta} peptide that oligomerizes rapidly in the presence of blue light. We applied this system to the crucial question of how intracellular Amyloid-{beta} oligomers underlie the pathologies of Alzheimers Disease. We show that, although both expression and induced oligomerization of Amyloid-{beta} were detrimental to lifespan and healthspan, we were able to separate the metabolic and physical damage caused by light-induced Amyloid-{beta} oligomerization from Amyloid-{beta} expression alone. The physical damage caused by Amyloid-{beta} oligomers also recapitulated the catastrophic tissue loss that is a hallmark of late AD. We show that the lifespan deficit induced by Amyloid-{beta} oligomers was reduced with Li+ treatment. Our results present the first model to separate different aspects of disease progression.

developmental biology↗

bioRxiv2017

Slowing ageing using drug synergy in C. elegans

Pharmacological interventions that target human ageing would extend individual healthspan and result in dramatic economic benefits to rapidly ageing societies worldwide. For such interventions to be contemplated they need to comprise drugs that are efficacious when given to adults and for which extensive human safety data are available. Here we show that dramatic lifespan extension can be achieved in C.elegans by targeting multiple, evolutionarily conserved ageing pathways using drugs that are already in human use. By targeting multiple synergistic ageing pathways, we are able to slow ageing rate, double lifespan and improves healthspan while minimize developmental and fitness trade-offs. Moreover, we established that there is no synergistic benefit in a daf-2 or daf-7 background, implying the involvement of the TGF{beta} and IGF pathways in this synergy. Employing lipidomics and transcriptomics analysis we found lipid metabolism to be affected resulting in increased monounsaturated fatty acids (MUFA) and decrease membrane peroxidation index. Our best drug combination showed a conserved lifespan extension in fruit flies. To the best of our knowledge, this is the largest lifespan effect ever reported for any adult-onset drug treatment in C. elegans. This drug-repurposing approach, using drugs already approved for humans to target multiple conserved aging pathways simultaneously, could lead to interventions that prevent age-related diseases and overall frailty in a rapidly ageing population.

biochemistry↗

bioRxiv2017

An Embryonic System To Assess Wnt Transcriptional Targets

During animal development, complex signals determine and organize a vast number of tissues using a very small number of signal transduction pathways. These developmental signaling pathways determine cell fates through a coordinated transcriptional response that remains poorly understood. The Wnt pathway is involved in a variety of these cellular functions, and its signals are transmitted in part through a {beta}-catenin/TCF transcriptional complex. Here we report an in vivo Drosophila assay that can be used to distinguish between activation, de-repression and repression of transcriptional responses, separating upstream and downstream pathway activation and canonical/non-canonical Wnt signals in embryos. We find specific sets of genes downstream of both {beta}-catenin and TCF with an additional group of genes regulated by Wnt, while the non-canonical Wnt4 regulates a separate cohort of genes. We correlate transcriptional changes with phenotypic outcomes of cell differentiation and embryo size, showing our model can be used to characterize developmental signaling compartmentalization in vivo.

developmental biology↗

bioRxiv2017

Coupling optogenetics and light-sheet microscopy to study signal transduction in vivo

Optogenetics allows precise, fast and reversible intervention in biological processes. Light-sheet microscopy allows observation of the full course of embryonic development from egg to larva. Bringing the two approaches together allows unparalleled precision into the temporal regulation of signaling pathways and cellular processes in vivo. To develop this method, we investigated the regulation of canonical Wnt signaling during anterior-posterior patterning of the Drosophila embryonic epidermis. Cryptochrome 2 (CRY2) from Arabidopsis Thaliana was fused to mCherry fluorescent protein and Drosophila {beta}-catenin to form an easy to visualize optogenetic switch. Blue light illumination caused oligomerization of the fusion protein and inhibited downstream Wnt signaling in vitro and in vivo. Temporal inactivation of {beta}-catenin confirmed that Wnt signaling is required not only for Drosophila pattern formation, but also for maintenance later in development. We anticipate that this method will be easily extendable to other developmental signaling pathways and many other experimental systems.

developmental biology↗

bioRxiv2017

Decoding temporal interpretation of the morphogen Bicoid in the early Drosophila embryo

Morphogen gradients provide essential spatial information during development. Not only the local concentration but also duration of morphogen exposure is critical for correct cell fate decisions. Yet, how and when cells temporally integrate signals from a morphogen remains unclear. Here, we use optogenetic manipulation to switch off Bicoid-dependent transcription in the early Drosophila embryo with high temporal resolution, allowing time-specific and reversible manipulation of morphogen signalling. We find that Bicoid transcriptional activity is dispensable for embryonic viability in the first hour after fertilization, but persistently required throughout the rest of the blastoderm stage. Short interruptions of Bicoid activity alter the most anterior cell fate decisions, while prolonged inactivation expands patterning defects from anterior to posterior. Such anterior susceptibility correlates with high reliance of anterior gap gene expression on Bicoid. Therefore, cell fates exposed to higher Bicoid concentration require input for longer duration, demonstrating a previously unknown aspect of morphogen decoding.

developmental biology↗