Biology subjects

Malaguti, M.

Publications and source records attributed to Malaguti, M..

6 recordsLinked to original sources

bioRxiv2026

KELPE: knock-in exchangeable dual landing pad embryonic stem cells enable efficient screening of synthetic gene circuits

The establishment of genetic circuits in pluripotent stem cells (PSCs) allows to model and manipulate developmental events. However, prototyping complex circuitry remains challenging, due to limitations in screening circuit components and transgene silencing. Here, we introduce KELPE: PSCs with two silencing-resistant insulated genomic landing pads targeted to genomic safe harbour sites. KELPE cells enable the stable integration of multiple transgenes into the same genomic region, facilitating fair comparisons of genetic circuit components. We demonstrate this by fine-tuning "synthetic neighbour-labelling" technologies. We first generate optimised PUFFFIN PSCs, which report on cell-cell interactions by fluorescently labelling wild-type neighbours. We then generate new synNotch "receiver" PSCs, which can trigger expression of any transgene following interaction with a synthetic ligand presented by "sender" cells of interest. We describe an optimised circuit syntax that abolishes ligand-independent transgene induction in receiver PSCs, and showcase this by synthetically programming cell death in receiver cells engineered to express a toxin following interaction with sender cells. In summary, we describe a new cell line that facilitates silencing-resistant transgene expression and prototyping of synthetic biology tools in a developmentally-relevant model.

developmental biology↗

bioRxiv2023

PUFFFIN: A novel, ultra-bright, customisable, singleplasmid system for labelling cell neighbourhoods

Cell communication orchestrates development, homeostasis, and disease. Understanding these processes depends on effective methods to study how cells influence their neighbours. Here, we present Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), a novel cell neighbour-labelling system based upon secretion and uptake of a positively supercharged fluorescent protein s36GFP. To achieve sensitive labelling, we amplified the fluorescent signal by fusing s36GFP to the ultra-bright protein mNeonGreen. Secretor cells transfer s36GFP-mNeonGreen to neighbours while retaining a nuclear mCherry, making it straightforward to unambiguously identify, isolate, and profile neighbours of secretor cells. Transfer of the s36GFP-mNeonGreen fusion occurs within minutes, enabling even relatively transient interactions to be captured or tracked over time with live-imaging or flow cytometry. To enhance flexibility of use, we incorporated HaloTag technology to facilitate colour-of-choice labelling. To further increase the range of applications, we engineered a customisable single-plasmid construct composed of interchangeable components with option to incorporate any additional transgene. This allows users to manipulate the properties of a cell, while at the same time applying a fluorescent label to the surrounding cells. PUFFFIN offers a simple, sensitive, customisable approach to profile non-cell-autonomous responses to natural or induced changes in cell identity or behaviour in mammalian cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/556381v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1ea5bcaorg.highwire.dtl.DTLVardef@1303810org.highwire.dtl.DTLVardef@b0d863org.highwire.dtl.DTLVardef@4e7b1f_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2021

SyNPL: Synthetic Notch pluripotent cell lines to monitor and manipulate cell interactions in vitro and in vivo.

Cell-cell interactions govern differentiation and cell competition in pluripotent cells during early development, but the investigation of such processes is hindered by a lack of efficient analysis tools. Here we introduce SyNPL: clonal pluripotent stem cell lines which employ optimised Synthetic Notch (SynNotch) technology to report cell-cell interactions between engineered "sender" and "receiver" cells in cultured pluripotent cells and chimaeric mouse embryos. A modular design makes it straightforward to adapt the system for programming differentiation decisions non-cell-autonomously in receiver cells in response to direct contact with sender cells. We demonstrate the utility of this system by enforcing neuronal differentiation at the boundary between two cell populations. In summary, we provide a new tool which could be used to identify cell interactions and to profile changes in gene or protein expression that result from direct cell-cell contact with defined cell populations in culture and in early embryos, and which can be adapted to generate synthetic patterning of cell fate decisions. SUMMARY STATEMENTOptimised Synthetic Notch circuitry in mouse pluripotent stem cells provides a modular tool to monitor cell-cell interactions and program synthetic patterning of cell fates in culture and in embryos.

developmental biology↗

bioRxiv2019

Competence to epithelialise coincides with competence to differentiate in pluripotent cells

Pluripotent cells reorganise themselves into an epithelium before they initiate differentiation, but it is not clear how these two events are mechanistically linked. Here we use quantitative imaging approaches to measure cellular rearrangements that accompany exit from naive pluripotency. We show that competence to epithelialise, like competence to differentiate, is a regulated process. The pro-differentiation transcription factor Tcf15 prospectively identifies cells that are competent to epithelialise. We identify early upregulation of the laminin receptor integrin alpha3 prior to differentiation and show that Tcf15 helps to regulate this change. Finally, we show that Tcf15 identifies and is required for efficient differentiation of a primed subpopulation of pluripotent cells. We conclude that competence to epithelialise is actively regulated and linked to differentiation-competence through the transcription factor Tcf15.

developmental biology↗

bioRxiv2019

The transcription factor E2A drives neural differentiation in pluripotent cells

The intrinsic mechanisms that link extracellular signalling to the onset of neural differentiation are not well understood. In pluripotent mouse cells, BMP blocks entry into the neural lineage via transcriptional upregulation of Inhibitor of Differentiation (Id) factors. We have previously identified that the major binding partner of Id proteins in pluripotent cells is the basic helix-loop-helix (bHLH) transcription factor (TF), E2A. Id1 can prevent E2A from forming heterodimers with bHLH TFs or from forming homodimers. Here, we show that overexpression of a forced E2A homodimer is sufficient to drive robust neural commitment in pluripotent cells, even under non-permissive conditions. Conversely, we find that E2A null cells display a defect in their neural differentiation capacity. E2A acts as an upstream activator of neural lineage genes, including Sox1 and Foxd4, and as a repressor of Nodal signalling. Our results suggest a crucial role for E2A in establishing neural lineage commitment in pluripotent cells.

developmental biology↗

bioRxiv2019

N-cadherin stabilises neural identity by dampening anti-neural signals

A switch from E-to N-cadherin regulates the transition from pluripotency to neural identity but the mechanism by which cadherins regulate differentiation was previously unknown. Here we show that the acquisition of N-cadherin stabilises neural identity by dampening anti-neural signals. We use quantitative image-analysis to identify an effect of N-cadherin to promote neural differentiation independently of effects on cell cohesiveness. We reveal that cadherin switching diminishes the level of nuclear {beta}-catenin, and that N-cadherin also dampens FGF activity and consequently stabilises neural fate. Finally, we compare the timing of cadherin switching and differentiation in vivo and in vitro, and find that this process becomes dysregulated during in vitro differentiation. We propose that N-cadherin helps to propagate a stable neural identity throughout the emerging neuroepithelium, and that dysregulation of this process contributes to asynchronous differentiation in culture.

developmental biology↗