Hijacking of internal calcium dynamics by intracellularly residing viral rhodopsins.

Rhodopsins are ubiquitous light-driven membrane proteins with diverse functions, including ion transport. Widely distributed, they are also coded in the genomes of giant viruses infecting phytoplankton where their function is not settled. Here, we examine the properties of OLPVR1 (Organic Lake Phycodnavirus Rhodopsin) and two other type 1 viral channelrhodopsins (VCR1s), and demonstrate that VCR1s accumulate exclusively intracellularly, and, upon illumination, induce calcium release from intracellular IP3-dependent stores. In vivo, this light-induced calcium release is sufficient to remote control muscle contraction in VCR1-expressing tadpoles. VCR1s natively confer light-induced Ca2+ release, suggesting a distinct mechanism for reshaping the response to light of virus-infected algae. The ability of VCR1s to photorelease calcium without altering plasma membrane electrical properties marks them as potential precursors for optogenetics tools, with potential applications in basic research and medicine.

Xenopus: An in vivo model for studying skin response to ultraviolet B irradiation.

Ultraviolet B (UVB) in sunlight cause skin damage, ranging from wrinkles to photoaging and skin cancer. UVB can affect genomic DNA by creating cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidine (6-4) photoproducts (6-4PPs). These lesions are mainly repaired by the nucleotide excision repair (NER) system and by photolyase enzymes that are activated by blue light. Our main goal was to validate the use of Xenopus laevis as an in vivo model system for investigating the impact of UVB on skin physiology. The mRNA expression levels of xpc and six other genes of the NER system and CPD/6-4PP photolyases were found at all stages of embryonic development and in all adult tissues tested. When examining Xenopus embryos at different time points after UVB irradiation, we observed a gradual decrease in CPD levels and an increased number of apoptotic cells, together with an epidermal thickening and an increased dendricity of melanocytes. We observed a quick removal of CPDs when embryos are exposed to blue light versus in the dark, confirming the efficient activation of photolyases. A decrease in the number of apoptotic cells and an accelerated return to normal proliferation rate was noted in blue light-exposed embryos compared with their control counterparts. Overall, a gradual decrease in CPD levels, detection of apoptotic cells, thickening of epidermis, and increased dendricity of melanocytes, emulate human skin responses to UVB and support Xenopus as an appropriate and alternative model for such studies.

LIM Homeobox-2 Suppresses Hallmarks of Adult and Pediatric Liver Cancers by Inactivating MAPK/ERK and Wnt/Beta-Catenin Pathways.

Introduction: Hepatocellular carcinoma and hepatoblastoma are two liver cancers characterized by gene deregulations, chromosomal rearrangements, and mutations in Wnt/beta-catenin (Wnt) pathway-related genes. LHX2, a transcriptional factor member of the LIM homeobox gene family, has important functions in embryogenesis and liver development. LHX2 is oncogenic in many solid tumors and leukemia, but its role in liver cancer is unknown. Methods: We analyzed the expression of LHX2 in hepatocellular carcinoma and hepatoblastoma samples using various transcriptomic datasets and biological samples. The role of LHX2 was studied using lentiviral transduction, in vitro cell-based assays (growth, migration, senescence, and apoptosis), molecular approaches (phosphokinase arrays and RNA-seq), bioinformatics, and two in vivo models in chicken and Xenopus embryos. Results: We found a strong connection between LHX2 downregulation and Wnt activation in these two liver cancers. In hepatoblastoma, LHX2 downregulation correlated with multiple poor outcome parameters including higher patient age, intermediate- and high-risk tumors, and low patient survival. Forced expression of LHX2 reduced the proliferation, migration, and survival of liver cancer cells in vitro through the inactivation of MAPK/ERK and Wnt signals. In vivo, LHX2 impeded the development of tumors in chick embryos and repressed the Wnt pathway in Xenopus embryos. RNA-sequencing data and bioinformatic analyses confirmed the deregulation of many biological functions and molecular processes associated with cell migration, cell survival, and liver carcinogenesis in LHX2-expressing hepatoma cells. At a mechanistic level, LHX2 mediated the disassembling of beta-catenin/T-cell factor 4 complex and induced expression of multiple inhibitors of Wnt (e.g., TLE/Groucho) and MAPK/ERK (e.g., DUSPs) pathways. Conclusion: Collectively, our findings demonstrate a tumor suppressive function of LHX2 in adult and pediatric liver cancers.

HDAC inhibition induces expression of scaffolding proteins critical for tumor progression in pediatric glioma: focus on EBP50 and IRSp53.

BACKGROUND: Diffuse midline glioma (DMG) is a pediatric malignancy with poor prognosis. Most children die less than one year after diagnosis. Recently, mutations in histone H3 have been identified and are believed to be oncogenic drivers. Targeting this epigenetic abnormality using histone deacetylase (HDAC) inhibitors such as panobinostat (PS) is therefore a novel therapeutic option currently evaluated in clinical trials. METHODS: BH3 profiling revealed engagement in an irreversible apoptotic process of glioma cells exposed to PS confirmed by annexin-V/propidium iodide staining. Using proteomic analysis of 3 DMG cell lines, we identified 2 proteins deregulated after PS treatment. We investigated biological effects of their downregulation by silencing RNA but also combinatory effects with PS treatment in vitro and in vivo using a chick embryo DMG model. Electron microscopy was used to validate protein localization. RESULTS: Scaffolding proteins EBP50 and IRSp53 were upregulated by PS treatment. Reduction of these proteins in DMG cell lines leads to blockade of proliferation and migration, invasion, and an increase of apoptosis. EBP50 was found to be expressed in cytoplasm and nucleus in DMG cells, confirming known oncogenic locations of the protein. Treatment of glioma cells with PS together with genetic or chemical inhibition of EBP50 leads to more effective reduction of cell growth in vitro and in vivo. CONCLUSION: Our data reveal a specific relation between HDAC inhibitors and scaffolding protein deregulation which might have a potential for therapeutic intervention for cancer treatment.

Leukemia inhibitory factor signaling in Xenopus embryo: Insights from gain of function analysis and dominant negative mutant of the receptor.

Leukemia inhibitory factor (LIF) is a cytokine member of the interleukin 6 family (IL6) of cytokines. It signals through a heterodimer receptor complex that consists of the LIF receptor (or LIFR formerly known as gp190) and the Interleukin 6 signal transducer (or IL6ST formerly known as gp130). LIF signaling is mediated mainly by signal transducer and activator of transcription 3 (STAT3) and has a wide variety of biological activities with pleiotropic effects on many cell types and organs among which are stem cell renewal and implantation process in mammalian embryo. Despite the wealth of data on LIF in mammalian cells, there is a paucity of information on its functions in lower vertebrates. Here, we provide information on the status and the function of LIF signaling in Xenopus amphibian. The IL6 cytokine family is highly conserved in Xenopus genome both at ligands and receptors levels. All cytokines and receptors of the family, except oncostatin M (OSM) and IL27, can be identified in the genome including the orthologs of LIF, cardiotrophin 1 (CTF1), ciliary neurotrophic factor (CNTF), cardiotrophin like cytokine factor 1 (CLCF1), LIFR, IL6ST, IL6R, IL11RA and CNTFR. Lif mRNA is zygotically expressed after midblastula transition while lifr and il6st are maternally expressed. We have investigated the functions of LIF in Xenopus early development with a gain-of-function analysis combined to the use of a dominant negative form of the receptor. The overexpression of Xenopus lif in embryo activates STAT3 phosphorylation and induces a dramatic phenotype where embryos are ventralised and show a reduction of anterior structures with microcephaly. This results mainly from BMP signal stimulation and antagonism towards IGF signals. In addition, most embryos develop tumor-like cell masses according to both autonomous and non-autonomous processes. Through the use of a dominant negative form of the receptor, we demonstrate for the first time that a functional LIF signaling is required for normal vertebrate kidney development. Owing to its experimental advantages, the Xenopus embryo constitutes a useful model to identify the molecular actors that may account for the pleiotropic functions of LIF and their role in vertebrate development.

Vestigial-like 3 is a novel Ets1 interacting partner and regulates trigeminal nerve formation and cranial neural crest migration.

Drosophila Vestigial is the founding member of a protein family containing a highly conserved domain, called Tondu, which mediates their interaction with members of the TEAD family of transcription factors (Scalloped in Drosophila). In Drosophila, the Vestigial/Scalloped complex controls wing development by regulating the expression of target genes through binding to MCAT sequences. In vertebrates, there are four Vestigial-like genes, the functions of which are still not well understood. Here, we describe the regulation and function of vestigial-like 3 (vgll3) during Xenopus early development. A combination of signals, including FGF8, Wnt8a, Hoxa2, Hoxb2 and retinoic acid, limits vgll3 expression to hindbrain rhombomere 2. We show that vgll3 regulates trigeminal placode and nerve formation and is required for normal neural crest development by affecting their migration and adhesion properties. At the molecular level, vgll3 is a potent activator of pax3, zic1, Wnt and FGF, which are important for brain patterning and neural crest cell formation. Vgll3 interacts in the embryo with Tead proteins but unexpectedly with Ets1, with which it is able to stimulate a MCAT driven luciferase reporter gene. Our findings highlight a critical function for vgll3 in vertebrate early development.

Overexpression of Leap2 impairs Xenopus embryonic development and modulates FGF and activin signals.

Besides its widely described function in the innate immune response, no other clear physiological function has been attributed so far to the Liver-Expressed-Antimicrobial-Peptide 2 (LEAP2). We used the Xenopus embryo model to investigate potentially new functions for this peptide. We identified the amphibian leap2 gene which is highly related to its mammalian orthologues at both structural and sequence levels. The gene is expressed in the embryo mostly in the endoderm-derived tissues. Accordingly it is induced in pluripotent animal cap cells by FGF, activin or a combination of vegT/ß-catenin. Modulating leap2 expression level by gain-of-function strategy impaired normal embryonic development. When overexpressed in pluripotent embryonic cells derived from blastula animal cap explant, leap2 stimulated FGF while it reduced the activin response. Finally, we demonstrate that LEAP2 blocks FGF-induced migration of HUman Vascular Endothelial Cells (HUVEC). Altogether these findings suggest a model in which LEAP2 could act at the extracellular level as a modulator of FGF and activin signals, thus opening new avenues to explore it in relation with cellular processes such as cell differentiation and migration.

MRAS GTPase is a novel stemness marker that impacts mouse embryonic stem cell plasticity and Xenopus embryonic cell fate.

Pluripotent mouse embryonic stem cells (mESCs), maintained in the presence of the leukemia inhibitory factor (LIF) cytokine, provide a powerful model with which to study pluripotency and differentiation programs. Extensive microarray studies on cultured cells have led to the identification of three LIF signatures. Here we focus on muscle ras oncogene homolog (MRAS), which is a small GTPase of the Ras family encoded within the Pluri gene cluster. To characterise the effects of Mras on cell pluripotency and differentiation, we used gain- and loss-of-function strategies in mESCs and in the Xenopus laevis embryo, in which Mras gene structure and protein sequence are conserved. We show that persistent knockdown of Mras in mESCs reduces expression of specific master genes and that MRAS plays a crucial role in the downregulation of OCT4 and NANOG protein levels upon differentiation. In Xenopus, we demonstrate the potential of Mras to modulate cell fate at early steps of development and during neurogenesis. Overexpression of Mras allows gastrula cells to retain responsiveness to fibroblast growth factor (FGF) and activin. Collectively, these results highlight novel conserved and pleiotropic effects of MRAS in stem cells and early steps of development.

Comparative functional analysis of ZFP36 genes during Xenopus development.

ZFP36 constitutes a small family of RNA binding proteins (formerly known as the TIS11 family) that target mRNA and promote their degradation. In mammals, ZFP36 proteins are encoded by four genes and, although they show similar activities in a cellular RNA destabilization assay, there is still a limited knowledge of their mRNA targets and it is not known whether or not they have redundant functions. In the present work, we have used the Xenopus embryo, a model system allowing gain- and loss-of-function studies, to investigate, whether individual ZFP36 proteins had distinct or redundant functions. We show that overexpression of individual amphibian zfp36 proteins leads to embryos having the same defects, with alteration in somites segmentation and pronephros formation. In these embryos, members of the Notch signalling pathway such as hairy2a or esr5 mRNA are down-regulated, suggesting common targets for the different proteins. We also show that mouse Zfp36 protein overexpression gives the same phenotype, indicating an evolutionary conserved property among ZFP36 vertebrate proteins. Morpholino oligonucleotide-induced loss-of-function leads to defects in pronephros formation, reduction in tubule size and duct coiling alterations for both zfp36 and zfp36l1, indicating no functional redundancy between these two genes. Given the conservation in gene structure and function between the amphibian and mammalian proteins and the conserved mechanisms for pronephros development, our study highlights a potential and hitherto unreported role of ZFP36 gene in kidney morphogenesis.

Vestigial like gene family expression in Xenopus: common and divergent features with other vertebrates.

The Drosophila Vestigial and Scalloped proteins form heterodimers that control wing development and are involved in muscle differentiation. Four vestigial like genes have been described in mammals. Similar to the Drosophila vestigial gene, they encode a short conserved domain (TONDU) required for interaction with the mammalian paralogues of Drosophila Scalloped (i.e., TEAD proteins). We previously identified two TEAD genes in Xenopus laevis and we report here the expression of four distinct vestigial like genes in Xenopus (vgll1-4) that represent amphibian orthologs of the mammalian vestigial like genes. Vgll1 has a unique expression pattern which is restricted to epidermal cells, both in the embryo and in the adult. Vgll2 is expressed in the skeletal muscle lineage downstream of myogenic factors and in the embryonic brain similar to the avian and mammalian orthologues. Vgll3 expression is transient, identifies embryonic hindbrain rhombomere 2, and is negatively regulated by en2, but not by egr2. Vgll4 is mainly expressed in anterior neural structures. In summary, the four Xenopus vgll genes have unique/complex expression profiles and they are differently expressed during embryogenesis. Moreover, these amphibian vestigial like genes display distinct responses to the major signaling pathways (i.e., activin, FGF or BMP) that orchestrate pattern-formation during early development.

Smooth muscle cell differentiation from human bone marrow: variations in cell type specific markers and Id gene expression in a new model of cell culture.

Stromal cells follow a vascular smooth muscle differentiation pathway. However, cell culture models performed from human bone marrow do not allow the obtention of a large proportion of highly differentiated smooth muscle cells (SMC) and their differentiation pathways remain unclear. We have characterized a new model of SMC differentiation from human bone marrow stromal cells by using different factors (bFGF, EGF, insulin and BMP-4). A relative homogeneous population of differentiated SMC was reproducibly obtained in short-term culture with high expression of SMC markers. Id gene expression was investigated and showed that (1) Id2 mRNA expression was upregulated during SMC differentiation without change of Id1 mRNA and (2) Id1 gene expression highly increased concomitantly with a decrease of SMC markers while Id2 mRNA was slightly modulated. Our data suggested that Id genes are potentially implicated in the differentiation pathway of human SMC from bone marrow.

Induction and modulation of smooth muscle differentiation in Xenopus embryonic cells.

By comparison with skeletal or cardiac developmental programs, little is known regarding the specific factors that promote specification and differentiation of smooth muscle cells from pluripotent cells. We have analyzed the developmental expression of a subset of smooth muscle genes during Xenopus early development and showed that similar to mammals and avians, Xenopus smooth muscle myosin heavy chain (SM-MHC) is a highly specific marker of smooth muscle differentiation. Embryonic cells from animal pole explants of Xenopus blastula can be induced by basic fibroblast growth factor, Wnt, and bone morphogenetic protein signals to adopt the smooth muscle pathway. Explants from early embryos that contain neural crest cells can also differentiate into cells expressing smooth muscle genes. We examined the interplay of several transcription factors, that is SRF, myocardin, and GATA6, that induce the expression of SM-MHC in animal cap cells and found that myocardin-dependent expression of smooth muscle genes in animal cap cells is synergized by SRF but is strongly antagonized by GATA6.

Differential expression of two TEF-1 (TEAD) genes during Xenopus laevis development and in response to inducing factors.

Transcription enhancer factors 1 (TEF-1 or TEAD) make a highly conserved family of eukaryotic DNA binding proteins that activate not only viral regulatory elements but muscle specific genes and are involved in several developmental processes. In this study, we report the identification and the expression pattern of NTEF-1 (TEAD1) and DTEF-1 (TEAD3), two members of this family in Xenopus laevis. Both X. laevis NTEF-1 (XNTEF-1 or XTEAD1) and DTEF-1 (XDTEF-1 or XTEAD3) possess a 72 amino acid TEA domain characteristic of TEF-1 proteins. XNTEF-1 is a 426 amino acid protein that has 96% identity with the avian or the mammalian NTEF-1 proteins while XDTEF-1 is a 433 amino acid protein with 77 to 80% identity with the avian and mammalian DTEF-1 sequences respectively. Temporal expression analysis by RT-PCR indicated that the two genes are expressed maternally and throughout embryonic development. In the adult, the two genes are broadly expressed although they showed differences of expression between tissues. Spatial expression analysis by whole mount in situ hybridization showed that the XNTEF-1 and XDTEF-1 mRNAS were predominantly detected in eye, embryonic brain, somites and heart. In animal cap assay, the two genes are activated by bFGF but are differently regulated by BMP4, and the muscle regulatory factor Mef2d.

Immunolocalization of long-chain acyl-CoAs in plant cells.

Long chain acyl-Coenzyme A esters (acyl-CoAs) are key substrates in many enzymic reactions of lipid metabolism. Due to their amphiphilic nature, the membrane localization of these molecules cannot be established by subcellular membrane fractionation and usual biochemical studies. We have developed another approach based on ultrastructural immunogold cytochemistry. To preserve the acyl-CoA membrane content, the plant material was freeze substituted and cryoembedded after short aldehyde fixation followed by quick freezing. Using Arabidopsis thaliana root cells and specific antibodies raised against acyl-CoAs, we show that acyl-CoAs are mainly localized in endoplasmic reticulum membranes. Our results demonstrate the value of cryo-methods for the accurate localization of labile metabolites in plant cells.