Biology subjects

Tsuchida-Straeten, N.

Publications and source records attributed to Tsuchida-Straeten, N..

3 recordsLinked to original sources

bioRxiv2025

Quantitative high-resolution imaging of mouse nephron formation to study Wnt signaling dynamics

IntroductionNephron formation is initiated when Wnt9b from the ureteric bud acts on nephron progenitor cells in the metanephric mesenchyme. For mouse embryonic kidneys, this process can be studied in real time using ex vivo organ cultures. Previous imaging methods relied on Transwell filters with a long distance between objective and sample as well as low signal-to-noise ratio due to the filter membrane. Moreover, Wnt signaling was previously visualized with the expression reporters TCF/Lef:H2B-GFP. MethodsWe developed an ex vivo culture system based on low-volume media and fibronectin coating for real time imaging. We further made use of endogenously tagged Cherry-Lef1 and Ctnnb1-Venus reporter mouse lines as reporters for Wnt signaling during kidney development. Furthermore, we established an adaptive feedback microscopy pipeline to track the signal with high resolution. ResultsUsing these approaches, we found that Cherry-Lef1 proteins are only expressed in the nascent nephron and derivatives. Expression is graded along distal-proximal nephron axis suggesting that the ureteric bud-derived Wnt9b is controlling its expression. Interestingly, we observed that Cherry-Lef1 gradient shifts from distal to proximal axis during nephron patterning. {beta}-catenin-Venus, on the other hand, first became visible in the ureteric bud, then in the nascent nephron after Lef1. ConclusionsUsing a novel imaging approach we demonstrate a dynamic regulation of Wnt signaling that correlates with nephrogenesis in mouse kidney development. Combined with our two new Wnt signaling reporters, nephron formation can be studied in real-time with high resolution.

developmental biology↗

bioRxiv2021

Glycolytic flux-signaling controls mouse embryo mesoderm development

How cellular metabolic state impacts cellular programs is a fundamental, unresolved question. Here we investigated how glycolytic flux impacts embryonic development, using presomitic mesoderm (PSM) patterning as the experimental model. First, we identified fructose 1,6-bisphosphate (FBP) as an in vivo sentinel metabolite that mirrors glycolytic flux within PSM cells of post-implantation mouse embryos. We found that medium-supplementation with FBP, but not with other glycolytic metabolites, such as fructose 6-phosphate and 3-phosphoglycerate, impaired mesoderm segmentation. To genetically manipulate glycolytic flux and FBP levels, we generated a mouse model enabling the conditional overexpression of dominant active, cytoplasmic Pfkfb3 (cytoPfkfb3). Overexpression of cytoPfkfb3 indeed led to increased glycolytic flux/FBP levels and caused an impairment of mesoderm segmentation, paralleled by the downregulation of Wnt-signaling, reminiscent of the effects seen upon FBP-supplementation. To probe for mechanisms underlying glycolytic flux-signaling, we performed subcellular proteome analysis and revealed that cytoPfkfb3 overexpression altered subcellular localization of certain proteins, including glycolytic enzymes, in PSM cells. Specifically, we revealed that FBP supplementation caused depletion of Pfkl and Aldoa from the nuclear-soluble fraction. Combined, we propose that FBP functions as a flux-signaling metabolite connecting glycolysis and PSM patterning, potentially through modulating subcellular protein localization.

developmental biology↗

bioRxiv2021

Arnold tongue entrainment reveals dynamicalprinciples of the embryonic segmentation clock

Living systems exhibit an unmatched complexity, due to countless, entangled interactions across scales. Here we aim to understand a complex system, i.e. segmentation timing in mouse embryos, without a reference to these detailed interactions. To this end, we develop a coarse-grained approach, in which theory guides the experimental identification of the segmentation clock entrainment responses. We demonstrate period- and phase-locking of the segmentation clock across a wide range of entrainment parameters, including higher-order coupling. These quantifications allow to derive the phase response curve (PRC) and Arnold tongues of the segmentation clock, revealing its essential dynamical properties. Our results indicate that the somite segmentation clock has characteristics reminiscent of a highly non-linear oscillator close to an infinite period bifurcation and suggests the presence of long-term feedbacks. Combined, this coarse-grained theoretical-experimental approach reveals how we can derive simple, essential features of a highly complex dynamical system, providing precise experimental control over the pace and rhythm of the somite segmentation clock.

developmental biology↗