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The rarity and inaccessibility of the earliest primordial germ cells (PGCs) in the mouse embryo thwart efforts to investigate molecular mechanisms of germ-cell specification. stella (also called Dppa3) marks the rare founder population of the germ lineage (1,2). Here we differentiate mouse embryonic stem cells carrying a stella-transgenic reporter into putative PGCs in vitro. The [Stella.sup.+] cells possess a transcriptional profile similar to embryo-derived PGCs, and like their counterparts in vivo, lose imprints in a time-dependent manner. Using inhibitory RNAs to screen candidate genes for effects on the development of [Stella.sup.+] cells in vitro, we discovered that Lin28, a negative regulator of let-7 microRNA processing (3-6), is essential for proper PGC development. Furthermore, we show that Blimp1 (also called Prdm1), a let-7 target and a master regulator of PGC specification (7-9), can rescue the effect of Lin28 deficiency during PGC development, thereby establishing a mechanism of action for Lin28 during PGC specification. Overexpression of Lin28 promotes formation of [Stella.sup.+] cells in vitro and PGCs in chimaeric embryos, and is associated with human germ-cell tumours. The differentiation of putative PGCs from embryonic stem cells in vitro recapitulates the early stages of gamete development in vivo, and provides an accessible system for discovering novel genes involved in germ-cell development and malignancy., The transcriptional program responsible for specifying PGCs has been investigated through molecular analysis of cDNA created from single cells (10,11), but because transgenic or knockout methods required to verify roles [...]

The canonical NF-κB transcription factor RELA is a master regulator of immune and stress responses and is upregulated in pancreatic ductal adenocardinoma (PDAC) tumours. In this study, we characterised previously unexplored endogenous RELA-GFP dynamics in PDAC cell lines through live single-cell imaging. Our observations revealed that TNFα stimulation induces rapid, sustained, and non-oscillatory nuclear translocation of RELA. Through Bayesian analysis of single-cell datasets with variation in nuclear RELA, we predicted that RELA heterogeneity in PDAC cell lines is dependent on F-actin dynamics. RNA-seq analysis identified distinct clusters of RELA-regulated gene expression in PDAC cells, including TNFα-induced RELA upregulation of the actin regulators NUAK2 and ARHGAP31. Further, siRNA-mediated depletion of ARHGAP31 and NUAK2 altered TNFα-stimulated nuclear RELA dynamics in PDAC cells, establishing a novel negative feedback loop that regulates RELA activation by TNFα. Additionally, we characterised the NF-κB pathway in PDAC cells, identifying how NF-κB/IκB proteins genetically and physically interact with RELA in the absence or presence of TNFα. Taken together, we provide computational and experimental support for interdependence between the F-actin network and the NF-κB pathway with RELA translocation dynamics in PDAC., Competing Interests: FB, JS, LD, CB No competing interests declared, (© 2024, Butera et al.)

Mechanical forces that capillary endothelial cells generate in their cytoskeleton and exert on their extracellular matrix adhesions feed back to modulate cell sensitivity to soluble angiogenic factors, and thereby control vascular development. Here we describe various genetic, biochemical, and engineering methods that can be used to study, manipulate, and probe this physical mechanism of developmental control. These techniques are useful as in vitro angiogenesis models and for analyzing the molecular and biophysical basis of vascular control.