Biology subjects

Benedito, R.

Publications and source records attributed to Benedito, R..

13 recordsLinked to original sources

bioRxiv2025

Mitochondrial genetics defines anti-tumour immunity through mitochondrial ROS and PD-1 signalling

As central players in cell metabolism, mitochondria influence numerous aspects of health and disease, including the initiation and progression of cancer. Although mitochondrial DNA (mtDNA) mutations have been extensively documented in human cancers for decades, the functional impact of mitochondrial haplogroups on tumour biology remains largely unexplored. Here, we investigate the role of mitochondrial variability in tumour biology using conplastic mouse strains, which are animal models with identical nuclear genomes but different mtDNA haplotypes. We showed that the physiologically relevant variation in mitochondrial ROS (mROS) generation, associated with specific clusters of mtDNA single nucleotide polymorphisms (SNPs), modulated immune responses within the tumour microenvironment and altered tumour growth. We observed strain-dependent differences in the abundance of multiple immune subsets and in PD-1 expression in tumour-infiltrating lymphocytes (TILs). In addition, mtDNA haplotypes influenced cancer progression by modulating tumour angiogenesis through an mROS-independent mechanism. These findings connect nucleo-mitochondrial genetic variability to tumour progression, de novo vessel formation and anti-tumour immunity. Tumour immunotherapies should incorporate the spatial and temporal dynamics of cancer evolution and consider mitochondrial genetics as a targetable layer influencing treatment efficacy.

cancer biology↗

bioRxiv2025

Disrupted astrocyte-endothelial crosstalk drives hemangioblastoma lesions in VHL disease

Hemangioblastomas (HBs) are highly vascularized central nervous system (CNS) tumours that can become life-threatening, especially in the context of Von Hippel-Lindau (VHL) disease, caused by the loss of VHL function. The limited pharmacological options targeting VHL-HBs stem from an incomplete understanding of their cellular origin, development, and molecular pathogenesis. Here we use advanced mouse genetics to show that mosaic deletion of Vhl in Apln+ cells leads to the formation of precursor tumour-like lesions, composed by clusters of Vhl-knockout (VhlKO) astrocytes and surrounding Vhl-wild-type (VhlWT) vessels that become malformed, resembling early-stage HBs linked to VHL disease. VhlKO astrocytes morphologically and transcriptomically resembled the reactive astrocytes characteristic of ischemic CNS injury. They exhibited metabolic rewirement towards glycolysis and upregulation of cell growth pathways. They also expressed several secreted proangiogenic molecules that activate and prevent the normal maturation of neighbouring vessels, leading to VHL-HBs. Temporal conditional genetic analysis revealed that Vhl loss need to happen during postnatal development for HBs to form, and that lesions become quiescent in early adulthood. HIF-2 deletion, or MTORC1 inhibition with rapamycin, efficiently inhibited VHL-HBs growth and the associated vascular malformations. Our work shows that the loss of Vhl in single astrocytes induces their growth and pathogenic crosstalk with neighbouring endothelial cells, driving hemangioblastoma development in VHL disease. Our new somatic mosaic mouse models will also enable testing of novel drugs against this disease.

cancer biology↗

bioRxiv2025

Genetic mosaics reveal mechanisms of resistance to VEGF signaling loss during angiogenesis

The VEGF ligand and its main receptor VEGFR2 are considered to be essential for endothelial differentiation, proliferation, sprouting and survival. Blood vessels cannot form in growing embryos or tissues when VEGF/VEGFR2 signalling is compromised in all cells. The Anti-VEGF blocking antibody is one of the most widely used antibodies in the clinics, blocking angiogenesis in cancer, wound healing or in ischemic diseases. However, vascular resistance to Anti-VEGF has been reported. Here we used iFlpMosaics and iSuRe-HadCre to induce and track genetic mosaics of endothelial cells (ECs) lacking VEGFR2 during the entire embryonic and postnatal development. Surprisingly, Vegfr2KOECs adapt, proliferate normally and compete with wild-type cells over time, ultimately forming a substantial portion of the capillary network in most organs. We found that Vegfr2KO ECs are not able to sprout during the first wave of angiogenesis in most tissues, because it is highly VEGF-dependent and favours the growth and mobilization of wild-type ECs. However, due to their inability to respond to VEGF, over time Vegfr2KOECs become dominant in veins, which provide for a long term and continuous source of ECs for subsequent waves of VEGF-independent angiogenesis. Comparative scRNAseq analysis of ECs with acute and long-term loss of VEGFR2, revealed both common and organ-specific molecular mechanisms of adaptation and resistance to VEGF signalling loss. This included significant endothelial venousization and the upregulation of ligands for VEGFR1 and VEGFR3. VEGFR1 only partially compensated for VEGFR2 loss in capillary ECs, whereas VEGFR3 only compensated in arterial ECs. Loss of the three VEGF receptors did not compromise venous growth. This work changes our understanding of the role of VEGF signaling and its receptors in angiogenesis and reveals mechanisms of adaptation and resistance to their loss.

developmental biology↗

bioRxiv2025

A molecular circuit regulates fate plasticity in emerging and adult AT2 cells

Alveolar AT1 and AT2 cells are vital for lung gas exchange and become compromised in several diseases. While key differentiation signals are known, their emergence and fate plasticity are unclear. Here we show in the embryonic lung that single AT2s emerge at intermediate zones, extrude, and connect with nearby epithelium via interlumenal junctioning. We observe that AT2s retain fate plasticity until the bZIP transcription factor C/EBP suppresses Notch signaling at a novel Dlk1 enhancer. Both Dlk1 and Cebpa are regulated by the polycomb repressive complex (PRC2), which together form a "pulse generator" circuit that times Dlk1 expression and thus Notch activation, resulting in a "salt and pepper" pattern of AT1 and AT2 fate. In injured adult lungs, C/EBP downregulation is required to re-access AT2 fate plasticity and is mediated by the dominant negative C/EBP family member CHOP. Finally, Cebpa loss also activates a "defender" AT2 state, distinct from its reparative state, and we propose AT2s toggle between either state following infection to protect and repair alveoli.

developmental biology↗

bioRxiv2025

Context-dependent response of endothelial cells to PIK3CA mutation

Cancer mutations in the PIK3CA gene cause congenital disorders. The endothelium is among the most frequently affected tissues in these disorders, displaying aberrant vascular overgrowth in the form of malformations. Pathological PIK3CA vascular phenotypes are found in veins and capillaries but rarely in arteries for reasons that are unclear at present. Here, using lineage tracing, we show that expression of mutated PIK3CAH1047R in endothelial cells leads to marked clonal expansions in capillary and venous endothelial cells. In contrast, mature arterial endothelial cells are refractory to PIK3CA mutation under these conditions and never display pathological phenotypes. Moreover, PIK3CAH1047Rexpression in arterial precursors interrupts arterial differentiation, thereby driving fate switch towards venous identity. This fate rewiring offers an additional layer of protection to prevent arterial damage in response to PIK3CA genetic perturbation. Molecularly, the PIK3CAH1047R-driven arterial-to-venous fate switch is orchestrated by upregulation of the vein-specifying transcription factor Nr2f2/COUP-TFII. Our findings reveal that pathogenic responses to PIK3CAH1047Rgreatly depend on the diferentation stage and fate trajectory of the targeted cell. Arteries are thus shielded against PIK3CA mutation, solving the long-standing question on the rarity of PIK3CA-related arterial malformations observed in patients.

developmental biology↗

bioRxiv2024

Resolving the design principles that control postnatal vascular growth and scaling

After birth, tissues grow continuously until reaching adult size, with each organ exhibiting unique cellular dynamics, growth patterns, and (stem or non-stem) cell sources. Using a suite of experimental and computational multiscale approaches, we found that aortic expansion is guided by specific biological principles and scales with the vertebral column rather than animal body weight. Expansion proceeds via two distinct waves of arterial cell proliferation along blood flow that are spatially stochastic, yet temporally coordinated. Each wave exhibits unique cell cycle kinetics and properties, with the first wave exhibiting cell cycle durations as fast as 6 hours. Single-cell RNA sequencing showed changes in fatty acid metabolism concomitant with an increase in cell size. Mathematical modeling and experiments indicated endothelial cell extrusion is essential for homeostatic aortic growth and balancing excess proliferation. In a genetic model of achondroplasia, the aorta achieves proper scaling through enhanced cell extrusion while maintaining normal proliferation dynamics. Collectively, these results provide a blueprint of the principles that orchestrate aortic growth which depends entirely on differentiated cell proliferation rather than resident stem cells.

developmental biology↗

bioRxiv2023

iFlpMosaics: A method for the ratiometric induction and high-throughput comparative analysis of mutant and wildtype cells

To understand gene function, it is necessary to compare cells carrying the mutated target gene with normal cells. In most biomedical studies, the cells being compared are in different mutant and control animals and therefore do not experience the same epigenetic changes and tissue microenvironment. The experimental induction of genetic mosaics is essential to determine a gene cell-autonomous function and to model the etiology of diseases caused by somatic mutations. Current technologies used to induce genetic mosaics in mice lack either accuracy, throughput or barcoding diversity. Here, we present a large set of new genetic tools and mouse lines that enable Flp recombinase-dependent ratiometric induction and single-cell clonal tracking of multiple fluorescently labeled wildtype and Cre-mutant cells within the same time window and tissue microenvironment. The labeled cells can be profiled by multispectral imaging or by FACS and scRNA-seq. This technology facilitates the induction and analysis of genetic mosaics in any cell type and for any given single or combination of floxed genes. iFlpMosaics enables a more accurate understanding of how induced genetic mutations affect the biology of single cells during tissue development, homeostasis, and disease.

cell biology↗

bioRxiv2023

Radical fringe facilitates NOTCH1 and JAG1 cis interactions to sustain Hematopoietic stem cell fate

Hematopoietic stem cells (HSCs) develop within a short time window from the hemogenic endothelium in the aorta- gonads-and mesonephros (AGM) region during embryonic development. The first HSCs reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). The signalling mechanisms that divert HSCs from HPCs are unknown. Notch signaling is essential for arterial specification, IAHC formation and HSC activity, but current studies on how Notch drives these different fates are inconsistent. To determine the role of Notch in the specification of hemogenic endothelium, HSC and/or HPCs, we extensively analyzed Notch dynamics in the period of HSC generation. We defined the expression pattern of Notch signalling molecules at the gene and protein level and established a molecular mechanism that reconcile previous studies demonstrating the loss of HSC activity in NOTCH1, JAG1 and RBPJ null mutants, the enhanced HSC generation by blocking specific Notch activities or the abrogation of emerging HSCs by high Notch activation. We now demonstrate that Notch activity is highest in a subset of Gfi1+ hemogenic endothelial cells and is gradually lost with HSC maturation. We uncover that the HSC phenotype is maintained through loss of Notch activity due to increasing levels of NOTCH1 and JAG1 interactions on the surface of the same cell (cis) that renders the NOTCH1 receptor from being activated. Forcing activation of the NOTCH1 receptor in IAHC cells activates a hematopoietic differentiation program and supports a cis-inhibitory function for JAG1 and NOTCH1. Furthermore, we demonstrate that this cis-inhibitory interaction is enabled by RADICAL FRINGE (RFNG), a glycosyltransferase that enhances the affinity of NOTCH1 to JAG1 in cis. Finally, our results indicate that NOTCH1-JAG1 cis-inhibition is necessary for preserving the HSC phenotype in the hematopoietic clusters of the aorta.

developmental biology↗

bioRxiv2023

Incongruence between transcriptional and vascular pathophysiological cell states

The Notch pathway is a major regulator of transcriptional specification and vascular biology. Previous studies have suggested that targeting the ligand Dll4 or the Notch-receptors results in similar molecular and angiogenesis outcomes. Here, we analyzed single and compound genetic mutants for all Notch signaling members and found very significant differences in the way ligands and receptors regulate vascular homeostasis. Loss of Notch receptors, leads to minor vascular pathology featuring hypermitogenic MAPK-driven cell-cycle arrest and senescence. In contrast, loss of Dll4 triggers a strong Myc-driven switch towards cell proliferation and sprouting and major organ pathology. Targeting of Myc completely suppressed the proliferative and tip-cell angiogenic states induced by Dll4 loss-of-function, however, this did not avoid vascular pathology. Only VEGF blockade prevented the pathology induced by Dll4 loss, but without fully suppressing its transcriptional and metabolic programs. This study shows incongruence between single-cell transcriptional states and adult vascular phenotypes and related pathophysiology.

physiology↗

bioRxiv2020

Endothelial-to-hematopoietic transition is induced by Notch glycosylation and upregulation of Mycn

A better understanding of the molecular mechanisms driving hematopoietic stem cell (HSC) specification and expansion may enable better pharmacological strategies to produce them in sufficient numbers for transplantation. In the embryo, HSCs arise from a defined subset of arterial endothelial cells (ECs) located in the aorta-gonad-mesonephros (AGM) region that undergo endothelial-to-hematopoietic transition (EHT). Arterialization and HSC development are generally believed to require the action of Notch. Here we show that although Notch activity is initially required for arterialization, it is detrimental to subsequent EHT. Mechanistically, we show that effective EHT depends on a Mfng-induced decrease in Jag1-Notch signaling in hemogenic ECs. This causes upregulation of Mycn, an important metabolic and cell-cycle regulator that we found to be required for EHT. During the subsequent development of hematopoietic lineages, Mycn expression decreases and its function is taken on by the homologous Myc gene.

developmental biology↗

bioRxiv2020

Notch controls arterialization by regulating the cell cycle and not differentiation

Arteries are thought to be formed by the induction of a highly conserved arterial genetic program in a subset of vessels experiencing an increase in pulsatile and oxygenated blood flow. Both VEGF and Notch signalling have been shown to be essential for the initial steps of arterial specification. Here, we combined inducible genetic mosaics and transcriptomics to modulate and understand the function of these signalling pathways on cell proliferation, arterial-venous differentiation and mobilization. We observed that endothelial cells with high VEGF or Notch signalling are not genetically pre-determined and can form both arteries and veins. Importantly, cells completely lacking the Notch-Rbpj transcriptional activator complex can form arteries when the Myc-dependent metabolic and cell-cycle activity is suppressed. Thus, arterial development does not require the induction of a Notch-dependent arterial differentiation program, but rather the timely suppression of the endothelial metabolism and cell-cycle, a process preceding arterial mobilization and complete differentiation.

developmental biology↗

bioRxiv2020

Distinct Vegfa isoforms control endothelial cell proliferation through PI3 kinase signalling mediated regulation of cdkn1a/p21

The formation of appropriately patterned blood vessel networks requires endothelial cell migration and proliferation. Signaling through the Vascular Endothelial Growth Factor A (VEGFA) pathway is instrumental in coordinating these processes. mRNA splicing generates short (diffusible) and long (extracellular matrix bound) Vegfa isoforms. The differences between these isoforms in controlling cellular functions are not understood. In zebrafish, vegfaa generates short and long isoforms, while vegfab only generates long isoforms. We found that mutations in vegfaa affected endothelial cell migration and proliferation. Surprisingly, mutations in vegfab specifically reduced endothelial cell proliferation. Analysis of downstream signaling revealed no change in MAPK (ERK) activation, while inhibiting PI3 kinase signaling phenocopied vegfab mutants. The cell cycle inhibitor cdkn1a/p21 was upregulated in vegfab deficient embryos. Accordingly, reducing cdkn1a/p21 restored endothelial cell proliferation. Together, these results suggest that extracellular matrix bound Vegfa acts through PI3K signaling to specifically control endothelial cell proliferation during angiogenesis independently of MAPK (ERK) regulation.

developmental biology↗

bioRxiv2018

Transitions in cell potency during early mouse development are driven by Notch

The Notch signalling pathway plays fundamental roles in diverse developmental processes in metazoans, where it is important in driving cell fate and directing differentiation of various cell types. However, we still have limited knowledge about the role of Notch in early preimplantation stages of mammalian development, or how it interacts with other signalling pathways active at these stages such as Hippo. By using genetic and pharmacological tools in vivo, together with image analysis of single embryos and pluripotent cell culture, we have found that Notch is active from the 4-cell stage. Transcriptomic analysis in single morula identified novel Notch targets, such as early naive pluripotency markers or transcriptional repressors such as TLE4. Our results reveal a previously undescribed role for Notch in driving transitions during the gradual loss of potency that takes place in the early mouse embryo prior to the first lineage decisions.

developmental biology↗