A GFP-Tagged Gross Deletion on Chromosome 1 Causes Malignant Peripheral Nerve Sheath Tumors and Carcinomas in Zebrafish

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft-tissue sarcomas, characterized by complex karyotypes. The molecular bases of such malignancy are poorly understood and efficient targeted molecular therapies are currently lacking. Here we describe a novel zebrafish model of MPNSTs, represented by the transgenic mutant line Tg(-8.5nkx2.2a:GFP)ia2. ia2 homozygous animals displayed embryonic lethality by 72 hpf, while the heterozygotes develop visible tumor masses with high frequency in adulthood. Histological and immunohistochemical examination revealed aggressive tumors with either mesenchymal or epithelial features. The former (54% of the cases) arose either in the abdominal cavity, or as intrathecal/intraspinal lesions and is composed of cytokeratin-negative spindle cells with fascicular/storiform growth pattern consistent with zebrafish MPNSTs. The second histotype was composed by polygonal or elongated cells, immunohistochemically positive for the pan-cytokeratin AE1/AE3. The overall histologic and immunohistochemical features were consistent with a malignant epithelial neoplasm of possible gastrointestinal/pancreatic origin. With an integrated approach, based on microsatellite (VNTR) and STS markers, we showed that ia2 insertion, in Tg(-8.5nkx2.2a:GFP)ia2 embryos, is associated with a deletion of 15.2 Mb in the telomeric portion of chromosome 1. Interestingly, among ia2 deleted genes we identified the presence of the 40S ribosomal protein S6 gene that may be one of the possible drivers for the MPNSTs in ia2 mutants.Thanks to the peculiar features of zebrafish as animal model of human cancer (cellular and genomic similarity, transparency and prolificacy) and the GFP tag, the Tg(-8.5nkx2.2a:GFP)ia2 line provides a manageable tool to study in vivo with high frequency MPNST biology and genetics, and to identify, in concert with the existing zebrafish MPNST models, conserved relevant mechanisms in zebrafish and human cancer development

Cryogenic cave minerals recorded the 1889 CE melt event in northeastern Greenland

The investigation of cryogenic cave minerals (CCMs) has developed in recent decades to be a particularly valuable proxy for palaeo-permafrost reconstruction. Due to difficulties, however, in obtaining reliable chronologies with the so-called “fine” form of these minerals, such studies have thus far utilised the “coarse” form. In this study, we successfully investigate the northernmost-known deposit of fine-grained CCMs, which are situated in Cove Cave (Greenlandic translation: Eqik Qaarusussuaq), a low-elevation permafrost cave in northeastern Greenland (80∘ N). The Cove Cave CCMs display a complex mineralogy that consists of fine-grained cryogenic cave carbonates and sulfate minerals (gypsum, eugsterite, mirabilite, and löweite). Until now, previous attempts to date fine-grained CCMs have been unsuccessful; however, here we demonstrate that precise dating is possible with both isochron-based 230Th / U dating and 14C dating if the dead carbon fraction is reliably known. The dating result (65±17 a BP; 1885±17 CE) shows that the Cove Cave CCMs formed during the late Little Ice Age, a time interval characterised by cold temperatures and abundant permafrost in northeastern Greenland, making water infiltration into Cove Cave dependent on the water amount and latent heat. We relate the CCM formation to a combination of black carbon deposition and anomalously high temperatures, which led to widespread melting over large areas of the Greenland ice sheet in the course of a few days. We propose that the anomalous weather conditions of 1889 CE also affected northeastern Greenland, where the enhanced melting of a local ice cap resulted in water entering the cave and rapidly freezing. While calcite and gypsum likely precipitated concurrently with freezing, the origin of the other sulfate minerals might not be purely cryogenic but could be linked to the subsequent sublimation of this ice accumulation in a very dry cave environment.</p

Nmnat1-Rbp7 Is a Conserved Fusion-Protein That Combines NAD+ Catalysis of Nmnat1 with Subcellular Localization of Rbp7

<div><p>Retinol binding proteins (Rbps) are known as carriers for transport and targeting of retinoids to their metabolizing enzymes. Rbps are also reported to function in regulating the homeostatic balance of retinoid metabolism, as their level of retinoid occupancy impacts the activities of retinoid metabolizing enzymes. Here we used zebrafish as a model to study <i>rbp7a</i> function and regulation. We find that early embryonic <i>rbp7a</i> expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on <i>aldh1a2</i>, which encodes the major enzyme for early embryonic retinoic acid production. The data are consistent with a Nodal-dependent coordination of the allocation of retinoid precursors to processing enzymes with the catalysis of retinoic acid formation. Further, we describe a novel <i>nmnat1-rbp7</i> transcript encoding a fusion of Rbp7 and the NAD⁺ (<i>Nicotinamide adenine dinucleotide</i>) synthesizing enzyme Nmnat1. We show that <i>nmnat1-rbp7</i> is conserved in fish, mouse and chicken, and that in zebrafish regulation of <i>nmnat1-rbp7a</i> is distinct from that of <i>rbp7a</i> and <i>nmnat1</i>. Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD⁺ catalyzing activities but a different subcellular localization. HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD⁺ catalyzing enzyme. These studies, taken together with previously documented NAD⁺ dependent interaction of RBPs with ER-associated enzymes of retinal catalysis, implicate functions of this newly described NMNAT1-Rbp7 fusion protein in retinol oxidation.</p></div

<i>rbp7a</i> is a direct target of FoxH1.

<p>[A] Schematic drawing of the <i>rbp7a</i> gene highlighting 4 potential FoxH1 binding sites (triangles: S1, S2, S3, and S4). Exons are indicated by boxes, the numbers correspond to the distance form the transcriptional start site. [B] Sequence comparison of the mouse FoxH1 consensus log [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143825#pone.0143825.ref037" target="_blank">37</a>] with the four potential sites in <i>rbp7a</i>. [C] <i>In vitro</i> EMSA studies with translated FoxH1 protein with oligonucleotides containing the four potential FoxH1 binding sites (sequences and positions were shown in [A, B]). Competition experiments with unspecific (unsp.comp) and specific (self.comp) unlabeled oligonucleotides were added to verify the binding specificity. [D] <i>In vivo</i> chromatin immunoprecipitation (ChIP-qPCR) experiments performed with 6hpf <i>eGFP-foxH1</i> mRNA injected MZ<i>sur</i> embryos. Bars show the enrichments of DNA fragments in the regions of FoxH1 binding sites in relation to a negative control region (<i>rhodopsin</i> promoter region) that was lacking FoxH1 binding sites (*P<0.05; error bars indicated the SEM). [E] Induced and depleted <i>rbp7a</i> expression in 5hpf wild type embryos after injection of <i>fkh-vp16</i> and <i>fkh-en</i> mRNA, respectively.</p

Subcellular localization of Nmnat1, Rbp7a and Nmnat1-Rbp7a proteins.

<p>[A] PSORT based predictions for sub-cellular location of Nmnat1, Rbp7a and Nmnat1-Rbp7a fusion. [B] Confocal image scans of HEK cells transfected with indicated fusion proteins. Note the strictly cytoplasmic and nuclear GFP-signals for Rbp7a-GFP and Nmnat1-Rbp7a-GFP, respectively. Nmnat1-Rbp7a-GFP transfected cells show weak GFP signals throughout the cytoplasm and stronger focal signals in association with the nuclei and cellular protrusions. [C] Confocal image scans of 6hpf embryo injected with mRNA encoding indicated GFP-tagged proteins. Co-injected mRNA encoding H2B-RFP (nuclear RFP, middle column) was used as loading control and to outline nuclei. Control injections of H2B-GFP and memGFP (membrane GFP) were used to document GFP/RFP co-expression. Rbp7a-GFP and Nmnat1-Rbp7a-GFP both showed nuclear and cytoplasmic localizations; but Nmnat1-GFP was only found in the nucleus.</p

<i>rbp7a</i> forms a conserved fusion gene with <i>nmnat1</i>.

<p>[A] Schematic representation of the exon usage in <i>nmnat1</i>, <i>rbp7a</i> and <i>nmnat1-rbp7a</i> transcripts. Size of exons (boxes) and distances corresponds to the position in the genome. [B] Semi-quantitative RT-PCR analyses of <i>rbp7a</i>, <i>nmnat1</i>, and <i>fusion</i> isoform; mRNA generated from indicated embryonic stages and adult tissues. <i>β-actin</i> was used as a control for cDNA input levels. [C-D] Competitive PCRs reveal the ratios of different isoforms expression level during developmental stages. [C] Schematic indicating primers used for competitive amplification of <i>rbp7a</i> (Pr1+Pr3) versus <i>nmnat1-rbp7a</i> (Pn2+Pr3) in the upper panel and <i>nmnat1</i> (Pn2+Pn5) versus <i>nmnat1-rbp7a</i> in the lower panel. The different isoform exons are colored respectively; purple: <i>nmnat1</i>, green: <i>rbp7a</i>; the unique exon which belongs to <i>nmnat1</i> or <i>rbp7a</i> is shown in red and brown. [D] Competition RT-PCR in different embryonic stages, note the temporal changes in ratios of long (<i>nmnat1-rbp7a</i>) versus short (<i>rbp7a</i> and <i>nmnat1</i>) PCR products, which were indicated the different dominated expression time windows for isoforms. [E, F] Schematic representation of exon usage of <i>Rbp7</i>, <i>Nmnat1</i>, and the <i>Nmnat1-rbp7</i> transcriptions in mouse [E] and chicken [F]. [G] RT-PCR analyses of embryonic mRNA form mouse (E14) and chicken (d9) confirming expression of <i>Nmnat1</i>, <i>Rbp7</i> and <i>Nmnat1-rbp7</i> transcripts.</p

Retinoid and NAD⁺ levels in mRNA in Morpholino injected embryos.

<p>[A] HPLC measurements show no significant changes in of ROL, RE and RA levels in 14hpf embryos injected with either RNA encoding Rbp7a, Nmnat1-Rbp7, or morpholinos blocking <i>rbp7a</i> translation (MO-ATG) and proper splicing of rbp7a exon 2 (MO-Δex2). For each dataset three independent treatments were performed and 100 embryos each were collected at 14hpf from each group of mRNA injected, morphant, and non-injected embryos. [B] NAD⁺ levels increase after over-expressions of <i>nmnat1</i> and <i>nmnat1-rbp7a</i> fusion mRNA. Spectral peaks of HPLC measurements shows the peak characteristic of NAD⁺ reference. For each experiment, three times 100 embryos at 6hpf were collected in different treatment or wild type groups Error bars indicated the SEM (**P<0.01).</p

Complementary regulation of <i>rbp7a</i> and <i>aldh1a2</i> by Nodal signaling.

<p>[A-F] Lateral views of whole mount <i>in situ</i> stains of <i>rbp7a</i> in wild type embryos at 4hpf [A, C] 6hpf [B,D], 9hpf [E] and 10hpf [F]; [C-D] vibratome sections of 4hpf and 6hpf embryos. <i>rbp7a</i> signals are found in marginal cell [A-D], YSL, and in forerunner cells (arrowhead in [D-F]). [G-J] Marginal expression of <i>aldh1a2</i> is strongly reduced in 6hpf MZ<i>sur</i> [H] and MZ<i>oep</i> [I] embryos; [J] RT-qPCR verified reduced expression levels of <i>aldh1a2</i> in MZ<i>sur</i>, MZ<i>oep</i> as compared to wild type embryos. [K-P] Whole mount <i>in situ</i> hybridization for <i>rbp7a</i> (arrow heads) and <i>goosecoid</i> (<i>gsc</i>) (white asterisks) performed at 6 and 8 hpf in M<i>sur</i>^<i>+/-</i> as control [K, N], MZ<i>sur</i> [L, O] and MZ<i>oep</i> [M, P] embryos. Note that expression levels of <i>rbp7a</i> around the YSL were similar among the <i>gsc</i> positive embryos and the <i>gsc</i> negative MZ<i>sur</i> [B, E] and MZ<i>oep</i> [C, F] mutants. [Q-S] <i>rbp7a</i> expression at 24hpf. Arrows mark <i>rbp7a</i> signals in the posterior midbrain that were present in MZ<i>sur</i> [R] and MZ<i>oep</i> embryos [S] but not in control embryo [Q]. [T] RT-qPCR results for <i>rbp7a</i> in of 6hpf and 24hpf embryos. Graphed is the mean and SEM from triplicate experiments. Error bars indicated the SEM. Unpaired T-test was used to test the significance (*P<0.05).</p