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Notch signaling is crucial for fate specification and maturation of thymus-seeding progenitors along the T-cell lineage. Recent studies have extended the role of Notch signaling to thymic epithelial cells (TECs), showing that Notch regulates TEC progenitor maintenance and emergence of medullary TECs (mTECs) in fetal thymopoiesis. Based on immunohistochemistry studies of spatiotemporal regulation of Notch activation in the postnatal thymus, we show that in vivo Notch activation is not confined to fetal TECs. Rather, Notch signaling, likely mediated through the Notch1 receptor, is induced in postnatal cortical and medullary TECs, and increases significantly with age in the latter, in both humans and mice, suggesting a conserved role for Notch signaling in TEC homeostasis during thymus aging. To investigate the functional impact of Notch activation in postnatal TEC biology, we used a mouse model in which RPBJκ, the transcriptional effector of canonical Notch signaling, is deleted in epithelial cells, including TECs, under the control of the transcription factor Foxn1. Immunohistochemistry and flow cytometry analyses revealed no significant differences in TEC composition in mutant (RPBJκ-KO TEC ) and wild-type (WT) littermate mice at early postnatal ages. However, a significant reduction of the medullary region was observed in mutant compared to WT older thymi, which was accompanied by an accelerated decrease of postnatal mTEC numbers. Also, we found that organization and integrity of the postnatal thymic medulla critically depends on activation of the canonical Notch signaling pathway, as abrogation of Notch signaling in TECs led to the disruption of the medullary thymic microenvironment and to an accelerated thymus atrophy. These features paralleled a significant increase in the proportion of intrathymic non-T lineage cells, mostly B cells, and a slight decrease of DP thymocyte numbers compatible with a compromised thymic function in mutant mice. Therefore, impaired Notch signaling induced in embryonic development impacts postnatal TECs and leads to an accelerated mTEC degeneration and a premature thymus involution. Collectively, our data have uncovered a new role for Notch1 signaling in the control of adult mTEC homeostasis, and point toward Notch signaling manipulation as a novel strategy for thymus regeneration and functional recovery from immunosenescence., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 García-León, Mosquera, Cela, Alcain, Zuklys, Holländer and Toribio.)

Cancer metastasis is a multistep process during which tumor cells leave the primary tumor mass and form distant secondary colonies that are lethal. Circulating tumor cells (CTCs) are transported by body fluids to reach distant organs, where they will extravasate and either remain dormant or form new tumor foci. Development of methods to study the behavior of CTCs at the late stages of the intravascular journey is thus required to dissect the molecular mechanisms at play. Using recently developed microfluidics approaches, we have demonstrated that CTCs arrest intravascularly, through a two-step process: (a) CTCs stop using low energy and rapidly activated adhesion receptors to form transient metastable adhesions and (b) CTCs stabilize their adhesions to the endothelial layer with high energy and slowly activated adhesion receptors. In this methods chapter, we describe these easy-to-implement quantitative methods using commercially available microfluidic channels. We detail the use of fast live imaging combined to fine-tuned perfusion to measure the adhesion potential of CTC depending on flow velocities. We document how rapidly engaged early metastable adhesion can be discriminated from slower activated stable adhesion using microfluidics. Finally, CTC extravasation potential can be assessed within this setup using long-term cell culture under flow. Altogether, this experimental pipeline can be adapted to probe the adhesion (to the endothelial layer) and extravasation potential of any circulating cell.

Macrophage activation by Toll receptors is an essential event in the development of the response against pathogens. NOTCH signaling pathway is involved in the control of macrophage activation and the inflammatory processes. In this work, we have characterized NOTCH signaling in macrophages activated by Toll-like receptor (TLR) triggering and determined that DLL1 and DLL4 are the main ligands responsible for NOTCH signaling. We have identified ADAM10 as the main protease implicated in NOTCH processing and activation. We have also observed that furin, which processes NOTCH receptors, is induced by TLR signaling in a NOTCH-dependent manner. NOTCH3 is the only NOTCH receptor expressed in resting macrophages. Its expression increased rapidly in the first hours after TLR4 activation, followed by a gradual decrease, which was coincident with an elevation of the expression of the other NOTCH receptors. All NOTCH1, 2 and 3 contribute to the increased NOTCH signaling detected in activated macrophages. We also observed a crosstalk between NOTCH3 and NOTCH1 during macrophage activation. Finally, our results highlight the relevance of NOTCH3 in the activation of NF-κB, increasing p65 phosphorylation by p38 MAP kinase. Our data identify, for the first time, NOTCH3 as a relevant player in the control of inflammation.

Targeting Notch signaling has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL), particularly in NOTCH1-mutated patients. We provide first evidence that the Notch ligand DLL4 is a potent stimulator of Notch signaling in NOTCH1-mutated CLL cells while increases cell proliferation. Importantly, DLL4 is expressed in histiocytes from the lymph node, both in NOTCH1-mutated and -unmutated cases. We also show that the DLL4-induced activation of the Notch signaling pathway can be efficiently blocked with the specific anti-Notch1 antibody OMP-52M51. Accordingly, OMP-52M51 also reverses Notch-induced MYC, CCND1, and NPM1 gene expression as well as cell proliferation in NOTCH1-mutated CLL cells. In addition, DLL4 stimulation triggers the expression of protumor target genes, such as CXCR4, NRARP, and VEGFA, together with an increase in cell migration and angiogenesis. All these events can be antagonized by OMP-52M51. Collectively, our results emphasize the role of DLL4 stimulation in NOTCH1-mutated CLL and confirm the specific therapeutic targeting of Notch1 as a promising approach for this group of poor prognosis CLL patients.

Cancer metastasis is a process whereby a primary tumor spreads to distant organs. We have demonstrated previously that blood flow controls the intravascular arrest of circulating tumor cells (CTCs) through stable adhesion to endothelial cells. We now aim to define the contribution of cell adhesion potential and identify adhesion receptors at play. Early arrest is mediated by the formation of weak adhesion, depending on CD44 and integrin αvβ3. Stabilization of this arrest uses integrin α5β1-dependent adhesions with higher adhesion strength, which allows CTCs to stop in vascular regions with lower shear forces. Moreover, blood flow favors luminal deposition of fibronectin on endothelial cells, an integrin α5β1 ligand. Finally, we show that only receptors involved in stable adhesion are required for subsequent extravasation and metastasis. In conclusion, we identified the molecular partners that are sequentially exploited by CTCs to arrest and extravasate in vascular regions with permissive flow regimes., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

T-cell development is a complex dynamic process that relies on ordered stromal signals delivered to thymus-seeding progenitors that migrate throughout different thymus microenvironments (TMEs). Particularly, Notch signaling provided by thymic epithelial cells (TECs) is crucial for T-cell fate specification and generation of mature T cells. Four canonical Notch ligands (Dll1, Dll4, Jag1 and Jag2) are expressed in the thymus, but their spatial distribution in functional TMEs is largely unknown, especially in humans, and their impact on Notch1 activation during T-lymphopoiesis remains undefined. Based on immunohistochemistry and quantitative confocal microscopy of fetal, postnatal and adult human and mouse thymus samples, we show that spatial regulation of Notch ligand expression defines discrete Notch signaling niches and dynamic species-specific TMEs. We further show that Notch ligand expression, particularly DLL4, is tightly regulated in cortical TECs during human thymus ontogeny and involution. Also, we provide the first evidence that NOTCH1 activation is induced in vivo in CD34 + progenitors and developing thymocytes at particular cortical niches of the human fetal and postnatal thymus. Collectively, our results show that human thymopoiesis involves complex spatiotemporal regulation of Notch ligand expression, which ensures the coordinated delivery of niche-specific NOTCH1 signals required for dynamic T-cell development., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

NOTCH1 is a prevalent signaling pathway in T cell acute lymphoblastic leukemia (T-ALL), but crucial NOTCH1 downstream signals and target genes contributing to T-ALL pathogenesis cannot be retrospectively analyzed in patients and thus remain ill defined. This information is clinically relevant, as initiating lesions that lead to cell transformation and leukemia-initiating cell (LIC) activity are promising therapeutic targets against the major hurdle of T-ALL relapse. Here, we describe the generation in vivo of a human T cell leukemia that recapitulates T-ALL in patients, which arises de novo in immunodeficient mice reconstituted with human hematopoietic progenitors ectopically expressing active NOTCH1. This T-ALL model allowed us to identify CD44 as a direct NOTCH1 transcriptional target and to recognize CD44 overexpression as an early hallmark of preleukemic cells that engraft the BM and finally develop a clonal transplantable T-ALL that infiltrates lymphoid organs and brain. Notably, CD44 is shown to support crucial BM niche interactions necessary for LIC activity of human T-ALL xenografts and disease progression, highlighting the importance of the NOTCH1/CD44 axis in T-ALL pathogenesis. The observed therapeutic benefit of anti-CD44 antibody administration in xenotransplanted mice holds great promise for therapeutic purposes against T-ALL relapse.

A key unsolved question regarding the developmental origin of conventional and plasmacytoid dendritic cells (cDCs and pDCs, respectively) resident in the steady-state thymus is whether early thymic progenitors (ETPs) could escape T cell fate constraints imposed normally by a Notch-inductive microenvironment and undergo DC development. By modeling DC generation in bulk and clonal cultures, we show here that Jagged1 (JAG1)-mediated Notch signaling allows human ETPs to undertake a myeloid transcriptional program, resulting in GATA2-dependent generation of CD34 + CD123 + progenitors with restricted pDC, cDC, and monocyte potential, whereas Delta-like1 signaling down-regulates GATA2 and impairs myeloid development. Progressive commitment to the DC lineage also occurs intrathymically, as myeloid-primed CD123 + monocyte/DC and common DC progenitors, equivalent to those previously identified in the bone marrow, are resident in the normal human thymus. The identification of a discrete JAG1 + thymic medullary niche enriched for DC-lineage cells expressing Notch receptors further validates the human thymus as a DC-poietic organ, which provides selective microenvironments permissive for DC development., (© 2017 Martín-Gayo et al.)

Evidence links aryl hydrocarbon receptor (AHR) activation to rheumatoid arthritis (RA) pathogenesis, although results are inconsistent. AHR agonists inhibit pro-inflammatory cytokine expression in macrophages, pivotal cells in RA aetiopathogenesis, which hints at specific circuits that regulate the AHR pathway in RA macrophages. We compared microRNA (miR) expression in CD14(+) cells from patients with active RA or with osteoarthritis (OA). Seven miR were downregulated and one (miR-223) upregulated in RA compared to OA cells. miR-223 upregulation correlated with reduced Notch3 and Notch effector expression in RA patients. Overexpression of the Notch-induced repressor HEY-1 and co-culture of healthy donor monocytes with Notch ligand-expressing cells showed direct Notch-mediated downregulation of miR-223. Bioinformatics predicted the AHR regulator ARNT (AHR nuclear translocator) as a miR-223 target. Pre-miR-223 overexpression silenced ARNT 3'UTR-driven reporter expression, reduced ARNT (but not AHR) protein levels and prevented AHR/ARNT-mediated inhibition of pro-inflammatory cytokine expression. miR-223 counteracted AHR/ARNT-induced Notch3 upregulation in monocytes. Levels of ARNT and of CYP1B1, an AHR/ARNT signalling effector, were reduced in RA compared to OA synovial tissue, which correlated with miR-223 levels. Our results associate Notch signalling to miR-223 downregulation in RA macrophages, and identify miR-223 as a negative regulator of the AHR/ARNT pathway through ARNT targeting.

Notch signaling is essential for definitive hematopoiesis, but its role in human embryonic hematopoiesis is largely unknown. We show that in hESCs the expression of the Notch ligand DLL4 is induced during hematopoietic differentiation. We found that DLL4 is only expressed in a sub-population of bipotent hematoendothelial progenitors (HEPs) and segregates their hematopoietic versus endothelial potential. We demonstrate at the clonal level and through transcriptome analyses that DLL4(high) HEPs are enriched in endothelial potential, whereas DLL4(low/-) HEPs are committed to the hematopoietic lineage, albeit both populations still contain bipotent cells. Moreover, DLL4 stimulation enhances hematopoietic differentiation of HEPs and increases the amount of clonogenic hematopoietic progenitors. Confocal microscopy analysis of whole differentiating embryoid bodies revealed that DLL4(high) HEPs are located close to DLL4(low/-) HEPs, and at the base of clusters of CD45+ cells, resembling intra-aortic hematopoietic clusters found in mouse embryos. We propose a model for human embryonic hematopoiesis in which DLL4(low/-) cells within hemogenic endothelium receive Notch-activating signals from DLL4(high) cells, resulting in an endothelial-to-hematopoietic transition and their differentiation into CD45+ hematopoietic cells.

Epithelial organs develop through tightly coordinated events of cell proliferation and differentiation in which endocytosis plays a major role. Despite recent advances, how endocytosis regulates the development of vertebrate organs is still unknown. Here we describe a mechanism that facilitates the apical availability of endosomal SNARE receptors for epithelial morphogenesis through the developmental upregulation of plasmolipin (pllp) in a highly endocytic segment of the zebrafish posterior midgut. The protein PLLP (Pllp in fish) recruits the clathrin adaptor EpsinR to sort the SNARE machinery of the endolysosomal pathway into the subapical compartment, which is a switch for polarized endocytosis. Furthermore, PLLP expression induces apical Crumbs internalization and the activation of the Notch signalling pathway, both crucial steps in the acquisition of cell polarity and differentiation of epithelial cells. We thus postulate that differential apical endosomal SNARE sorting is a mechanism that regulates epithelial patterning.

Thymocyte differentiation is a complex process involving well-defined sequential developmental stages that ultimately result in the generation of mature T-cells. In this study, we analyzed DNA methylation and gene expression profiles at successive human thymus developmental stages. Gain and loss of methylation occurred during thymocyte differentiation, but DNA demethylation was much more frequent than de novo methylation and more strongly correlated with gene expression. These changes took place in CpG-poor regions and were closely associated with T-cell differentiation and TCR function. Up to 88 genes that encode transcriptional regulators, some of whose functions in T-cell development are as yet unknown, were differentially methylated during differentiation. Interestingly, no reversion of accumulated DNA methylation changes was observed as differentiation progressed, except in a very small subset of key genes (RAG1, RAG2, CD8A, PTCRA, etc.), indicating that methylation changes are mostly unique and irreversible events. Our study explores the contribution of DNA methylation to T-cell lymphopoiesis and provides a fine-scale map of differentially methylated regions associated with gene expression changes. These can lay the molecular foundations for a better interpretation of the regulatory networks driving human thymopoiesis., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)