Platelet derived growth factor B gene expression in the Xenopus laevis developing central nervous system

Platelet-derived growth factor B (PDGF-B) belongs to the mitogen and growth factor family and like the other members it has many roles in cell differentiation, proliferation and migration during development, adult life and in pathological conditions. Among them it has been observed that aberrant PDGF signalling is frequently linked to glioma development and progression, and Pdgf-b over-expression in mouse neural progenitors leads to the formation of gliomas. Despite this evidence, the mechanisms underlying PDGF-B driven tumorigenesis and its role during brain development are not fully understood. In order to contribute to clarifying possible new roles of pdgf-b signalling, we present here the embryonic gene expression pattern of pdgf-b, so far unknown in early vertebrate development. By using Xenopus laevis as a model system we performed qRT-PCR and whole mount in situ hybridization. Pdgf-b mRNA is expressed in discrete regions of the developing central nervous system, in the cranial nerve placodes and in the notochord. We also compared the gene expression of pdgf-b with that of its receptor pdgfr-a suggesting so far unsuspected roles for this signalling pathway during the development of specific embryonic structures

One-step rapid tracking and isolation of senescent cells in cellular systems, tissues, or animal models via GLF16

Identification and isolation of senescent cells is challenging, rendering their detailed analysis an unmet need. We describe a precise one-step protocol to fluorescently label senescent cells, for flow cytometry and fluorescence microscopy, implementing a fluorophore-conjugated Sudan Black-B analog, GLF16. Also, a micelle-based approach allows identification of senescent cells in vivo and in vitro, enabling live-cell sorting for downstream analyses and live in vivo tracking. Our protocols are applicable to cellular systems, tissues, or animal models where senescence is present. For complete details on the use and execution of this protocol, please refer to Magkouta et al.</p

Use of Xenopus laevis Neural Crest Cells to study the role of PDGF-B in gliomas tumor progression

Gliomas are the most common primary tumours of the central nervous system; one of their major features is the ability of individual cells to infiltrate the brain parenchyma, which renders these tumours also the most aggressive. Evidences gained from studies on animal models have firmly established a causal connection between aberrant PDGF-B signalling and the formation of some gliomas. The aim of my project is to unravel the mechanisms underlying PDGF-B driven gliomagenesis and tumour progression, especially focusing on alteration of motility and migration. For this purpose, I used the animal model Xenopus laevis and in particular I focused on Neural Crest Cells (NCC), considering the many similarities shared by this population with cancer cells. I demonstrated that over-expression of human PDGF-B in Xenopus embryos, obtained by mRNA microinjection, can alter NCC behaviour in terms of migration and streams segregation, validating the system. I then analysed, in PDGF-B injected embryos, the expression of some of the key players known to regulate NCC development such as: Cadherins, Chemokines and their receptors, Neuropilins and Semaphorins and Wnt/PCP pathway members. The results obtained highlighted a significant down-regulation of neuropilin-2 and of wnt11, a Wnt/PCP pathway member, suggesting for the first time a link between their expression and the PDGF signalling. The discovery of these new putative targets of the PDGF-B signalling pathway opens new possible lines of investigations aimed to identify alternative therapeutic approaches for glioma treatment. Interestingly, I also evidenced that the gene expression pattern of Xenopus pdgf-b during embryogenesis relies in domains close to NCC, suggesting a possible physiological role for it in NCC development. In this regard, I deeper characterised the spatial and temporal expression profile of Pdgf-b during Xenopus embryogenesis and I performed loss of function experiments that showed how NCC migration is affected after pdgf-b down-regulation. Furthermore, with an in vivo chemotaxis assay, I observed that PDGF-BB can act as a chemoattractant for NCC in vivo, suggesting a putative chemotactic role for it and strengthening the hypothesis that this growth factor might play a physiological role in NCC migration

Telomere shortening produces an inflammatory environment that increases tumor incidence in zebrafish

Cancer incidence increases exponentially with age when human telomeres are shorter. Similarly, telomerase reverse transcriptase (tert) mutant zebrafish have premature short telomeres and anticipate cancer incidence to younger ages. However, because short telomeres constitute a road block to cell proliferation, telomere shortening is currently viewed as a tumor suppressor mechanism and should protect from cancer. This conundrum is not fully understood. In our current study, we report that telomere shortening promotes cancer in a noncell autonomous manner. Using zebrafish chimeras, we show increased incidence of invasive melanoma when wild-type (WT) tumors are generated in tert mutant zebrafish. Tissues adjacent to melanoma lesions (skin) and distant organs (intestine) in tert mutants exhibited higher levels of senescence and inflammation. In addition, we transferred second generation (G2) tert blastula cells into WT to produce embryo chimeras. Cells with very short telomeres induced increased tumor necrosis factor1-α (TNF1-α) expression and senescence in larval tissues in a noncell autonomous manner, creating an inflammatory environment. Considering that inflammation is protumorigenic, we transplanted melanoma-derived cells into G2 tert zebrafish embryos and observed that tissue environment with short telomeres leads to increased tumor development. To test if inflammation was necessary for this effect, we treated melanoma transplants with nonsteroid anti-inflammatory drugs and show that higher melanoma dissemination can be averted. Thus, apart from the cell autonomous role of short telomeres in contributing to genome instability, we propose that telomere shortening with age causes systemic chronic inflammation leading to increased tumor incidence

One-step rapid tracking and isolation of senescent cells in cellular systems, tissues, or animal models via GLF16

Identification and isolation of senescent cells is challenging, rendering their detailed analysis an unmet need. We describe a precise one-step protocol to fluorescently label senescent cells, for flow cytometry and fluorescence microscopy, implementing a fluorophore-conjugated Sudan Black-B analog, GLF16. Also, a micelle-based approach allows identification of senescent cells in vivo and in vitro, enabling live-cell sorting for downstream analyses and live in vivo tracking. Our protocols are applicable to cellular systems, tissues, or animal models where senescence is present. For complete details on the use and execution of this protocol, please refer to Magkouta et al. 1. </p

The transcription factor HIF2α partakes in the differentiation block of acute myeloid leukemia

Abstract One of the defining features of acute myeloid leukemia (AML) is an arrest of myeloid differentiation whose molecular determinants are still poorly defined. Pharmacological removal of the differentiation block contributes to the cure of acute promyelocytic leukemia (APL) in the absence of cytotoxic chemotherapy, but this approach has not yet been translated to non‐APL AMLs. Here, by investigating the function of hypoxia‐inducible transcription factors HIF1α and HIF2α, we found that both genes exert oncogenic functions in AML and that HIF2α is a novel regulator of the AML differentiation block. Mechanistically, we found that HIF2α promotes the expression of transcriptional repressors that have been implicated in suppressing AML myeloid differentiation programs. Importantly, we positioned HIF2α under direct transcriptional control by the prodifferentiation agent all‐trans retinoic acid (ATRA) and demonstrated that HIF2α blockade cooperates with ATRA to trigger AML cell differentiation. In conclusion, we propose that HIF2α inhibition may open new therapeutic avenues for AML treatment by licensing blasts maturation and leukemia debulking

One-step rapid tracking and isolation of senescent cells in cellular systems, tissues, or animal models via GLF16

Summary: Identification and isolation of senescent cells is challenging, rendering their detailed analysis an unmet need. We describe a precise one-step protocol to fluorescently label senescent cells, for flow cytometry and fluorescence microscopy, implementing a fluorophore-conjugated Sudan Black-B analog, GLF16. Also, a micelle-based approach allows identification of senescent cells in vivo and in vitro, enabling live-cell sorting for downstream analyses and live in vivo tracking. Our protocols are applicable to cellular systems, tissues, or animal models where senescence is present.For complete details on the use and execution of this protocol, please refer to Magkouta et al.