Curation of NISEED, an integrative framework for the digital representation of embryonic development

NISEED (Network for In situ Expression and Embryological Data) is a generic infrastructure for the creation, maintenance and integration of molecular and anatomical information on model organisms. We applied it to ascidians which are marine invertebrate chordates. These animals constitute model organisms of choice for developmental biology because their embryos develop with a small number of cells and an invariant lineage, allowing their study with a cellular level of resolution. In ANISEED (Ascidian NISEED), embryogenesis of ascidian is represented at the level of the genome via functional gene annotations, cis-regulatory elements or gene expression data, at the level of the cell by representing its morphology, fates, lineage, and relations with its neighbors, or at the level of the whole embryo by representing its anatomy and morphogenesis at successive developmental stages. The system provides also tool and standard to enter, annotate, curate and manage data. All results can be accessed through the ANISEED website at "http://aniseed-ibdm.univ-mrs.fr":http://aniseed-ibdm.univ-mrs.fr
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Combined drug action of 2-phenylimidazo[2,1-b]benzothiazole derivatives on cancer cells according to their oncogenic molecular signatures

The development of targeted molecular therapies has provided remarkable advances into the treatment of human cancers. However, in most tumors the selective pressure triggered by anticancer agents encourages cancer cells to acquire resistance mechanisms. The generation of new rationally designed targeting agents acting on the oncogenic path(s) at multiple levels is a promising approach for molecular therapies. 2-phenylimidazo[2,1-b]benzothiazole derivatives have been highlighted for their properties of targeting oncogenic Met receptor tyrosine kinase (RTK) signaling. In this study, we evaluated the mechanism of action of one of the most active imidazo[2,1-b]benzothiazol-2-ylphenyl moiety-based agents, Triflorcas, on a panel of cancer cells with distinct features. We show that Triflorcas impairs in vitro and in vivo tumorigenesis of cancer cells carrying Met mutations. Moreover, Triflorcas hampers survival and anchorage-independent growth of cancer cells characterized by 'RTK swapping' by interfering with PDGFRβ phosphorylation. A restrained effect of Triflorcas on metabolic genes correlates with the absence of major side effects in vivo. Mechanistically, in addition to targeting Met, Triflorcas alters phosphorylation levels of the PI3K-Akt pathway, mediating oncogenic dependency to Met, in addition to Retinoblastoma and nucleophosmin/B23, resulting in altered cell cycle progression and mitotic failure. Our findings show how the unusual binding plasticity of the Met active site towards structurally different inhibitors can be exploited to generate drugs able to target Met oncogenic dependency at distinct levels. Moreover, the disease-oriented NCI Anticancer Drug Screen revealed that Triflorcas elicits a unique profile of growth inhibitory-responses on cancer cell lines, indicating a novel mechanism of drug action. The anti-tumor activity elicited by 2-phenylimidazo[2,1-b]benzothiazole derivatives through combined inhibition of distinct effectors in cancer cells reveal them to be promising anticancer agents for further investigation

Combined drug action of 2-phenylimidazo[2,1-b]benzothiazole derivatives on cancer cells according to their oncogenic molecular signatures

International audienceThe development of targeted molecular therapies has provided remarkable advances into the treatment of human cancers. However, in most tumors the selective pressure triggered by anticancer agents encourages cancer cells to acquire resistance mechanisms. The generation of new rationally designed targeting agents acting on the oncogenic path(s) at multiple levels is a promising approach for molecular therapies. 2-phenylimidazo[2,1-b]benzothiazole derivatives have been highlighted for their properties of targeting oncogenic Met receptor tyrosine kinase (RTK) signaling. In this study, we evaluated the mechanism of action of one of the most active imidazo[2,1-b]benzothiazol-2-ylphenyl moiety-based agents, Triflorcas, on a panel of cancer cells with distinct features. We show that Triflorcas impairs in vitro and in vivo tumorigenesis of cancer cells carrying Met mutations. Moreover, Triflorcas hampers survival and anchorage-independent growth of cancer cells characterized by "RTK swapping" by interfering with PDGFRβ phosphorylation. A restrained effect of Triflorcas on metabolic genes correlates with the absence of major side effects in vivo. Mechanistically, in addition to targeting Met, Triflorcas alters phosphorylation levels of the PI3K-Akt pathway, mediating oncogenic dependency to Met, in addition to Retinoblastoma and nucleophosmin/B23, resulting in altered cell cycle progression and mitotic failure. Our findings show how the unusual binding plasticity of the Met active site towards structurally different inhibitors can be exploited to generate drugs able to target Met oncogenic dependency at distinct levels. Moreover, the disease-oriented NCI Anticancer Drug Screen revealed that Triflorcas elicits a unique profile of growth inhibitory-responses on cancer cell lines, indicating a novel mechanism of drug action. The anti-tumor activity elicited by 2-phenylimidazo[2,1-b]benzothiazole derivatives through combined inhibition of distinct effectors in cancer cells reveal them to be promising anticancer agents for further investigation

Drosophila Aversive Behavior toward Erwinia carotovora carotovora Is Mediated by Bitter Neurons and Leukokinin

International audienceThe phytopathogen Erwinia carotovora carotovora (Ecc) has been used successfully to decipher some of the mechanisms that regulate the interactions between Drosophila melanogaster and bacteria, mostly following forced association between the two species. How do Drosophila normally perceive and respond to the presence of Ecc is unknown. Using a fly feeding two-choice assay and video tracking, we show that Drosophila are first attracted but then repulsed by an Ecc-contaminated solution. The initial attractive phase is dependent on the olfactory Gr63a and Gaq proteins, whereas the second repulsive phase requires a functional gustatory system. Genetic manipulations and calcium imaging indicate that bitter neurons and gustatory receptors Gr66a and Gr33a are needed for the aversive phase and that the neuropeptide leukokinin is also involved. We also demonstrate that these behaviors are independent of the NF-kB cascade that controls some of the immune, metabolic, and behavioral responses to bacteria

A live-imaging protocol to track cell movement in the Xenopus embryo

International audienceTracking individual cell movement during development is challenging, particularly in tissues subjected to major remodeling. Currently, most live imaging techniques in Xenopus are limited to tissue explants and/or to superficial cells. We describe here a protocol to track immature multiciliated cells (MCCs) moving within the inner epidermal layer of a whole embryo. In addition, we present a data processing protocol to uncouple the movements of individual cells from the coplanar drifts of the tissue in which they are embedded.For complete details on the use and execution of this protocol, please refer to Chuyen et al. (2021)

A Quantitative Approach to the Study of Cell Shapes and Interactions during Early Chordate Embryogenesis

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Endogenous neural stem cells modulate microglia and protect from demyelination

In response to corpus callosum (CC) demyelination, subventricular zone-derived neural progenitors (SVZdNP) are mobilized and generate new myelinating oligodendrocytes. Here, we examine the putative immunomodulatory properties of endogenous SVZdNP during demyelination in the cuprizone model. We observed that SVZdNP density is higher in the lateral and rostral CC regions that show weaker demyelination and is inversely correlated with activated microglia density and pro-inflammatory cytokines levels. Single-cell RNA-sequencing further revealed CC areas with high SVZdNP mobilization are enriched in a microglial cell subpopulation with immunomodulatory signature. We identified ligand/receptor couple MFGE8 (milk fat globule-epidermal growth factor-8)/integrin β3 as a ligand/receptor couple implicated in SVZdNP/microglia dialog. MFGE8 is highly enriched in immature SVZdNP mobilized to the demyelinated CC and promotes myelin debris phagocytosis in vitro. Altogether these results demonstrate that beyond their cell replacement capacity endogenous progenitors display immunomodulatory properties highlighting a new role for endogenous SVZdNP in myelin regeneration

Creating 3D Digital Replicas of Ascidian Embryos from Stacks of Confocal Images

During embryonic development, cell behaviors that are tightly coordinated both spatially and temporally integrate at the tissue level and drive embryonic morphogenesis. Over the past 20 years, advances in imaging techniques, in particular, the development of confocal imaging, have opened a new world in biology, not only giving us access to a wealth of information, but also creating new challenges. It is sometimes difficult to make the best use of the recordings of the complex, inherently three-dimensional (3D) processes we now can observe. In particular, these data are often not directly suitable for even simple but conceptually fundamental quantifications. This article describes a process whereby image stacks gathered from live or fixed ascidian embryos are digitalized and segmented to produce 3D embryo replicas. These replicas can then be interfaced via a 3D Virtual Embryo module to a model organism database (Aniseed) that allows one to relate the geometrical properties of cells and cell contacts to additional parameters such as cell lineage, cell fates, or the underlying genetic program. Such an integrated system can serve several general purposes. First, it makes it possible to quantify and better understand the dynamics of cell behaviors during embryonic development, including, for instance, the automatic detection of asymmetric cell divisions or the evolution of cell contacts. Second, the 3D Virtual Embryo software proposes a panel of mathematical shape descriptors to precisely quantify cellular geometries and generate a 3D identity card for each embryonic cell. Such reconstructions open the door to a detailed 3D simulation of morphogenesis

Imaging of Fixed Ciona Embryos for Creating 3D Digital Replicas

During embryonic development, cell behaviors that are tightly coordinated both spatially and temporally integrate at the tissue level and drive embryonic morphogenesis. Over the past 20 years, advances in imaging techniques, in particular, the development of confocal imaging, have opened a new world in biology, not only giving us access to a wealth of information, but also creating new challenges. It is sometimes difficult to make the best use of the recordings of the complex, inherently three-dimensional (3D) processes we now can observe. In particular, these data are often not directly suitable for even simple but conceptually fundamental quantifications. This article presents a method for imaging embryonic development with cellular resolution in fixed ascidian embryos. A large fraction of the ascidian community primarily studies the development of the cosmopolitan ascidian Ciona intestinalis . Because the embryos of this species are insufficiently transparent and show significant autofluorescence, live imaging is difficult. Thus, whole embryos are fixed and optically cleared. They are then stained and imaged on a regular or two-photon confocal microscope. The resulting image stacks can subsequently be digitalized and segmented to produce 3D embryo replicas that can be interfaced to a model organism database and used to quantify cell shapes

Time-Lapse Imaging of Live Phallusia Embryos for Creating 3D Digital Replicas.

International audienceDuring embryonic development, cell behaviors that are tightly coordinated both spatially and temporally integrate at the tissue level and drive embryonic morphogenesis. Over the past 20 years, advances in imaging techniques, in particular, the development of confocal imaging, have opened a new world in biology, not only giving us access to a wealth of information, but also creating new challenges. It is sometimes difficult to make the best use of the recordings of the complex, inherently three-dimensional (3D) processes we now can observe. In particular, these data are often not directly suitable for even simple but conceptually fundamental quantifications. This article provides a method to fluorescently label and image structures of interest that will subsequently be reconstructed, such as cell membranes or nuclei. The protocol describes live imaging of Phallusia mammillata embryos, which are robust, colorless, and optically transparent with negligible autofluorescence. Their diameter ranges from 100 µm to 120 µm, which allows time-lapse microscopy of whole embryos using two-photon microscopy with a high-resolution objective. Although two-photon imaging is described in detail, any imaging technology that results in a z -stack may be used. The resulting image stacks can subsequently be digitalized and segmented to produce 3D embryo replicas that can be interfaced to a model organism database and used to quantify cell shapes