Biology subjects

Dale, J. K.

Publications and source records attributed to Dale, J. K..

6 recordsLinked to original sources

bioRxiv2026

A high throughput system reveals distinct segmentation clock phase responses in hiPSC-derived organoids

During somitogenesis, the vertebrate body axis segments into transient periodic structures known as somites. Somite formation is regulated by a multicellular molecular oscillator known as the segmentation clock. Recent advances in human induced pluripotent stem cell (hiPSC) culture have shown that hiPSC-derived somitogenesis organoids (somitoids) exhibit segmentation clock oscillations and can be produced at scale, making them an excellent model system for high throughput investigation of the mechanisms underpinning the segmentation clock. However, somitoids interact both biochemically and mechanically, and accurate high-throughput sampling of segmentation clock phases is required to exploit this system effectively. Here we address these challenges using an image-based, high-content screening workflow. Individual hiPSC-derived somitoids carrying a segmentation clock reporter are cultured in 384-well plates, and a programmable feeding schedule is used to initiate oscillations that are monitored using fluorescence microscopy. We develop an automated pipeline for image segmentation and data analysis, represent oscillations using a compact set of parameters, and construct predictive mathematical models to interpret data. We find that: (i) a staggered feeding schedule that sequentially initiates oscillations yields large numbers of somitoids at defined stages of the segmentation clock cycle; (ii) media exchange in established oscillations induces a Type 0-like phase response, resetting the segmentation clock to a state characterised by low NOTCH pathway transcription; and (iii) control wells in media exchange experiments exhibit a Type 1 phase response in which the segmentation clock is delayed non-uniformly across the cycle. Using a mathematical model of segmentation clock dynamics along the anterior-posterior axis, we show that periodic activation of a Type 1 phase response could segment a continuous phase gradient -- an insight with potential implications for the determination of somite boundaries in vivo. IMPORTANTO_LIManuscripts submitted to Review Commons are peer reviewed in a journal-agnostic way. C_LIO_LIUpon transfer of the peer reviewed preprint to a journal, the referee reports will be available in full to the handling editor. C_LIO_LIThe identity of the referees will NOT be communicated to the authors unless the reviewers choose to sign their report. C_LIO_LIThe identity of the referee will be confidentially disclosed to any affiliate journals to which the manuscript is transferred. C_LI GUIDELINESO_LIFor reviewers: https://www.reviewcommons.org/reviewers C_LIO_LIFor authors: https://www.reviewcommons.org/authors C_LI CONTACTThe Review Commons office can be contacted directly at: office@reviewcommons.org

developmental biology↗

bioRxiv2024

NOTCH1 S2513 is critical for the regulation of NICD levels impacting the segmentation clock in hiPSC-derived PSM cells and somitoids

The segmentation clock is a molecular oscillator that regulates the timing of somite formation in the developing vertebrate embryo. NOTCH signalling is one of the key pathways required for proper functioning of the segmentation clock. Aberrant NOTCH signalling results in developmental abnormalities such as congenital scoliosis as well as diseases such as T-cell acute lymphoblastic lymphoma (T-ALL). In this study we analyse the effects of a mutation detected in T-ALL patients on somitogenesis using human iPS derived PSM cells and somitoids. Mutation of NOTCH1 Serine 2513 into Alanine compromises the interaction of Notch intracellular domain (NICD) with the F-box protein FBXW7 and consequently increases NICD stability and NICD levels in PSM cells. Moreover, the mutation impairs several aspects of clock gene oscillations and restricts the ability of somitoids to polarise, elongate and form paired somites. The data suggest a mechanism by which post-translational modification of a key segmentation clock component plays a crucial role in vertebrate axis segmentation.

developmental biology↗

bioRxiv2024

A toolkit for mapping cell identities in relation to neighbours reveals Notch-dependent heterogeneity within neuromesodermal progenitor populations

Patterning of cell fates is central to embryonic development, tissue homeostasis, and disease. Quantitative analysis of patterning reveals the logic by which cell-cell interactions orchestrate changes in cell fate. However, it is challenging to quantify patterning when graded changes in identity occur over complex 4D trajectories, or where different cell states are intermingled. Furthermore, comparing patterns across multiple individual embryos, tissues, or organoids is difficult because these often vary in shape and size. Here we present a toolkit of computational approaches to tackle these problems. These strategies are based on measuring properties of each cell in relation to the properties of its neighbours to quantify patterning, and on using embryonic landmarks in order to compare these patterns between embryos. We use this toolkit to characterise patterning of cell identities within the caudal lateral epiblast of E8.5 embryos, revealing local patterning in emergence of early mesoderm cells that is sensitive to inhibition of Notch activity.

developmental biology↗

bioRxiv2023

Notch signalling influences cell fate decisions and HOX gene induction in axial progenitors

The generation of the post-cranial embryonic body relies on the coordinated production of spinal cord neurectoderm and presomitic mesoderm cells from neuromesodermal progenitors (NMPs). This process is orchestrated by pro-neural and pro-mesodermal transcription factors that are co-expressed in NMPs together with Hox genes, which are critical for axial allocation of NMP derivatives. NMPs reside in a posterior growth region, which is marked by the expression of Wnt, FGF and Notch signalling components. While the importance of Wnt and FGF in influencing the induction and differentiation of NMPs is well established, the precise role of Notch remains unclear. Here, we show that the Wnt/FGF-driven induction of NMPs from human embryonic stem cells (hESCs) relies on Notch signalling. Using hESC-derived NMPs and chick embryo grafting, we demonstrate that Notch directs a pro-mesodermal character at the expense of neural fate. We show that Notch also contributes to activation of HOX gene expression in human NMPs, partly in a non cell-autonomous manner. Finally, we provide evidence that Notch exerts its effects via the establishment of a negative feedback loop with FGF signalling.

developmental biology↗

bioRxiv2018

CDK1 and CDK2 regulate phosphorylation-dependent NICD1 turnover and the periodicity of the segmentation clock

All vertebrates share a segmented body axis. Segments form periodically from the rostral end of the presomitic mesoderm (PSM) and this periodicity is regulated by the segmentation clock, a molecular oscillator that drives dynamic clock gene expression across the PSM with a periodicity that matches somite formation. Notch signalling is crucial to this process. Altering Notch intracellular domain (NICD) stability affects both the clock period and somite size. However, the mechanistic details of how NICD stability is regulated are unclear.\n\nWe identified a highly conserved site crucial for NICD recognition by the SCF E3 ligase, which targets NICD for degradation. We demonstrate both CDK1 and CDK2 can phosphorylate NICD in the domain where this crucial residue lies and that NICD levels vary in a cell cycle-dependent manner. Inhibiting CDK1 or CDK2 activity increases NICD levels both in vitro and in vivo, leading to a delay of clock gene oscillations.

developmental biology↗

bioRxiv2017

MYC activity is required for maintenance of the Neuromesodermal Progenitor signalling network and for correct timing of segmentation clock gene oscillations

The Myc transcriptional regulators are implicated in a range of cellular functions, including proliferation, cell cycle progression, metabolism and pluripotency maintenance. Here, we investigated the expression, regulation and function of Myc during mouse embryonic axis elongation and segmentation. Expression of both cMyc and MycN in the domains where neuromesodermal progenitors (NMPs) and underlying caudal pre-somitic mesoderm (cPSM) cells reside is coincident WNT and FGF signals; factors known to maintain progenitors in an undifferentiated state. Pharmacological inhibition of MYC activity, downregulates expression of WNT/FGF components. In turn, we find that cMyc expression is WNT, FGF and NOTCH regulated, placing it centrally in the signalling circuit that operates in the tail end that both sustains progenitors and drives maturation of the PSM into somites. Interfering with MYC function in the PSM, where it displays oscillatory expression, delays the timing of segmentation clock oscillations and thus of somite formation. In summary, we identify Myc as a component that links NMP maintenance and PSM maturation during the body axis elongation stages of mouse embryogenesis.\n\nSummary StatementMYC operates in a positive feedback loop with WNT/FGF signals to maintain the progenitors which facilitate body axis elongation while its activity is crucial for timing of the segmentation clock.

developmental biology↗