LADON, a Natural Antisense Transcript of NODAL, Promotes Tumour Progression and Metastasis in Melanoma.

The TGFß family member NODAL, repeatedly required during embryonic development, has also been associated with tumour progression. Our aim was to clarify the controversy surrounding its involvement in melanoma tumour progression. We found that the deletion of the NODAL exon 2 in a metastatic melanoma cell line impairs its ability to form tumours and colonize distant tissues. However, we show that this phenotype does not result from the absence of NODAL, but from a defect in the expression of a natural antisense transcript of NODAL, here called LADON. We show that LADON expression is specifically activated in metastatic melanoma cell lines, that its transcript is packaged in exosomes secreted by melanoma cells, and that, via its differential impact on the expression of oncogenes and tumour suppressors, it promotes the mesenchymal to amoeboid transition that is critical for melanoma cell invasiveness. LADON is, therefore, a new player in the regulatory network governing tumour progression in melanoma and possibly in other types of cancer.

Addressing Critical Issues Related to Storage and Stability of the Vault Nanoparticle Expressed and Purified from Komagataella phaffi.

The vault nanoparticle is a eukaryotic assembly consisting of 78 copies of the 99-kDa major vault protein. They generate two cup-shaped symmetrical halves, which in vivo enclose protein and RNA molecules. Overall, this assembly is mainly involved in pro-survival and cytoprotective functions. It also holds a remarkable biotechnological potential for drug/gene delivery, thanks to its huge internal cavity and the absence of toxicity/immunogenicity. The available purification protocols are complex, partly because they use higher eukaryotes as expression systems. Here, we report a simplified procedure that combines human vault expression in the yeast Komagataella phaffii, as described in a recent report, and a purification process we have developed. This consists of RNase pretreatment followed by size-exclusion chromatography, which is far simpler than any other reported to date. Protein identity and purity was confirmed by SDS-PAGE, Western blot and transmission electron microscopy. We also found that the protein displayed a significant propensity to aggregate. We thus investigated this phenomenon and the related structural changes by Fourier-transform spectroscopy and dynamic light scattering, which led us to determine the most suitable storage conditions. In particular, the addition of either trehalose or Tween-20 ensured the best preservation of the protein in native, soluble form.

Evolutionary Gain of Dbx1 Expression Drives Subplate Identity in the Cerebral Cortex.

Changes in transcriptional regulation through cis-regulatory elements are thought to drive brain evolution. However, how this impacts the identity of primate cortical neurons is still unresolved. Here, we show that primate-specific cis-regulatory sequences upstream of the Dbx1 gene promote human-like expression in the mouse embryonic cerebral cortex, and this imparts cell identity. Indeed, while Dbx1 is expressed in highly restricted cortical progenitors in the mouse ventral pallium, it is maintained in neurons in primates. Phenocopy of the primate-like Dbx1 expression in mouse cortical progenitors induces ectopic Cajal-Retzius and subplate (SP) neurons, which are transient populations playing crucial roles in cortical development. A conditional expression solely in neurons uncouples mitotic and postmitotic activities of Dbx1 and exclusively promotes a SP-like fate. Our results highlight how transcriptional changes of a single fate determinant in postmitotic cells may contribute to the expansion of neuronal diversity during cortical evolution.

CXCR7 Receptor Controls the Maintenance of Subpial Positioning of Cajal-Retzius Cells.

Cajal-Retzius (CR) cells are essential for cortical development and lamination. These pioneer neurons arise from distinct progenitor sources, including the cortical hem and the ventral pallium at pallium-subpallium boundary (PSB). CXCR4, the canonical receptor for the chemokine CXCL12, controls the superficial location of hem-derived CR cells. However, recent studies showed that CXCR7, a second CXCL12 receptor, is also expressed in CR cells at early developmental stages. We thus investigated the role of CXCR7 during CR cell development using multiple loss-of-function approaches. Cxcr7 gene inactivation led to aberrant localization of Reelin-positive cells within the pallium. In addition, Cxcr7(-/-) mice were characterized by significant accumulation of ectopic CR cells in the lateral part of the dorsal pallium compared with Cxcr4 knockout mice. Loss-of-function approaches, using either gene targeting or pharmacological receptor inhibition, reveal that CXCR7 and CXCR4 act both in CR positioning. Finally, conditional Cxcr7 deletion in cells derived from Dbx1-expressing progenitors indicates an essential role of CXCR7 in controlling the positioning of a subpopulation of PSB-derived CR cells. Our data demonstrate that CXCR7 has a role in the positioning of hem and PSB-derived CR cells, CXCL12 regulating CR cell subpial localization through the combined action of CXCR4 and CXCR7.

The insulin receptor is required for the development of the Drosophila peripheral nervous system.

The Insulin Receptor (InR) in Drosophila presents features conserved in its mammalian counterparts. InR is required for growth; it is expressed in the central and embryonic nervous system and modulates the time of differentiation of the eye photoreceptor without altering cell fate. We show that the InR is required for the formation of the peripheral nervous system during larval development and more particularly for the formation of sensory organ precursors (SOPs) on the fly notum and scutellum. SOPs arise in the proneural cluster that expresses high levels of the proneural proteins Achaete (Ac) and Scute (Sc). The other cells will become epidermis due to lateral inhibition induced by the Notch (N) receptor signal that prevents its neighbors from adopting a neural fate. In addition, misexpression of the InR or of other components of the pathway (PTEN, Akt, FOXO) induces the development of an abnormal number of macrochaetes that are Drosophila mechanoreceptors. Our data suggest that InR regulates the neural genes ac, sc and sens. The FOXO transcription factor which is localized in the cytoplasm upon insulin uptake, displays strong genetic interaction with the InR and is involved in Ac regulation. The genetic interactions between the epidermal growth factor receptor (EGFR), Ras and InR/FOXO suggest that these proteins cooperate to induce neural gene expression. Moreover, InR/FOXO is probably involved in the lateral inhibition process, since genetic interactions with N are highly significant. These results show that the InR can alter cell fate, independently of its function in cell growth and proliferation.

Integration of differentiation signals during indirect flight muscle formation by a novel enhancer of Drosophila vestigial gene.

The gene vestigial (vg) plays a key role in indirect flight muscle (IFM) development. We show here that vg is controlled by the Notch anti-myogenic signaling pathway in myoblasts and is regulated by a novel 822 bp enhancer during IFM differentiation. Interestingly, this muscle enhancer is activated in developing fibers and in a small number of myoblasts before the fusion of myoblasts with the developing muscle fibers. Moreover, we show that this enhancer is activated by Drosophila Myocyte enhancing factor 2 (MEF2), Scalloped (SD) and VG but repressed by Twist, demonstrating a sensitivity to differentiation in vivo. In vitro experiments reveal that SD can directly bind this enhancer and MEF2 can physically interact with both SD and TWI. Cumulatively, our data reveal the interplay between different myogenic factors responsible for the expression of an enhancer activated during muscle differentiation.

Cell cycle genes regulate vestigial and scalloped to ensure normal proliferation in the wing disc of Drosophila melanogaster.

In Drosophila, the Vestigial-Scalloped (VG-SD) dimeric transcription factor is required for wing cell identity and proliferation. Previous results have shown that VG-SD controls expression of the cell cycle positive regulator dE2F1 during wing development. Since wing disc growth is a homeostatic process, we investigated the possibility that genes involved in cell cycle progression regulate vg and sd expression in feedback loops. We focused our experiments on two major regulators of cell cycle progression: dE2F1 and the antagonist dacapo (dap). Our results reinforce the idea that VG/SD stoichiometry is critical for correct development and that an excess in SD over VG disrupts wing growth. We reveal that transcriptional activity of VG-SD and the VG/SD ratio are both modulated by down-expression of cell cycle genes. We also detected a dap-induced sd up-regulation that disrupts wing growth. Moreover, we observed a rescue of a vg hypomorphic mutant phenotype by dE2F1 that is concomitant with vg and sd induction. This regulation of the VG-SD activity by dE2F1 is dependent on the vg genetic background. Our results support the hypothesis that cell cycle genes fine-tune wing growth and cell proliferation, in part, through control of the VG/SD stoichiometry and activity. This points to a homeostatic feedback regulation between proliferation regulators and the VG-SD wing selector.

In vivo analysis of Drosophila deoxyribonucleoside kinase function in cell cycle, cell survival and anti-cancer drugs resistance.

In vitro studies have shown that Drosophila melanogaster has a highly efficient single deoxyribonucleoside kinase (dNK) multisubstrate enzyme. dNK is related to the mammalian Thymidine Kinase 2 (TK2) group involved in the nucleotide synthesis salvage pathway. To study the dNK function in vivo, we constructed transgenic Drosophila strains and impaired the nucleotide de novo synthesis pathway, using antifolates such as aminopterin. Our results show that dNK overexpression rescues both cell death and cell cycle arrest triggered by this anti-cancer drug, and confers global resistance on the fly. Moreover, we show that fly viability and growth depend on the exquisite ratio between dNK expression and its substrate thymidine (dT) in the medium, and that increased dT concentrations trigger apoptosis and a decrease in body mass when dNK is mis-expressed. Finally, dNK expression, unlike that of TK2, is cell cycle dependent and under the control of CyclinE and the dE2F1 transcription factor involved in the G1/S transition. dNK is therefore functionally more closely related to mammalian TK1 than to TK2. This strongly suggests that dNK plays a role in cell proliferation in physiological conditions.

Real-time imaging of transcriptional activation in live cells reveals rapid up-regulation of the cyclin-dependent kinase inhibitor gene CDKN1A in replicative cellular senescence.

Cellular replicative senescence is a permanent growth arrest state that can be triggered by telomere shortening. The cyclin-dependent kinase (Cdk) inhibitor p21(CIP1/WAF1) (p21), encoded by the CDKN1A gene, is a critical cell cycle regulator whose expression increases as cells approach senescence. Although the pathways responsible for its up-regulation are not well understood, compelling evidence indicates that the upstream triggering event is telomere dysfunction. Studies of replicative senescence have been complicated by the asynchrony of its onset, which is caused by the continuous and stochastic variability in individual cell lifespans. In fact, the actual entry into senescence has never been observed in a single unperturbed cell. We report here a new in vitro human model system that allows entry into senescence to be monitored in real-time in individual viable cells. We used homologous recombination to generate non-immortalized fibroblast cells with the enhanced yellow fluorescence protein (EYFP) gene knocked into one CDKN1A gene copy, allowing promoter activity to be visualized as fluorescence intensity. Gamma irradiation, DNA-damaging drugs, expression of p14(ARF) or oncogenic Ras, and replicative exhaustion all resulted in elevated EYFP expression, demonstrating its proper control by physiological signalling circuits. Analysis by time-lapse microscopy of cultures approaching replicative senescence revealed that p21 levels rise abruptly in individual aging cells and remain elevated for extended periods of time.

Loss of retinoblastoma but not p16 function allows bypass of replicative senescence in human fibroblasts.

Current models envision replicative senescence to be under dual control by the p53 and retinoblastoma (RB) tumour-suppressor pathways. The role of the p16(INK4a)-RB pathway is controversial, and the function of RB in human cells has not been tested directly. We used targeted homologous recombination to knock out one copy of RB in presenescent human fibroblasts. During entry into senescence, RB+/- cells underwent spontaneous loss of heterozygosity and the resultant RB-/- clones bypassed senescence. The extended lifespan phase was eventually terminated by a crisis-like state. The same phenotype was documented for p21(CIP1/WAF1) and p53 heterozygous cells, indicating that loss of function of all three genes results in failure to establish senescence. By contrast, the abolition of p16 function by the expression of a p16-insensitive cyclin-dependent kinase 4 protein or siRNA-mediated knockdown provided only minimal lifespan extension that was terminated by senescence. We propose that p53, p21 and RB act in a linear genetic pathway to regulate cell entry into replicative senescence.

Interaction between apterous and early expression of vestigial in formation of the dorso-ventral compartments in the Drosophila wing disc.

BACKGROUND: Compartment formation is a developmental process that requires the existence of barriers against intermixing between cell groups. In the Drosophila wing disc, the dorso-ventral (D/V) compartment boundary is defined by the expression of the apterous (ap) selector gene in the dorsal compartment. AP activity is under control of dLMO which destabilizes the formation of the AP-CHIP complex. RESULTS: We report that D/V boundary formation in the wing disc also depends on early expression of vestigial (vg). Our data suggest that vg is already required for wing cell proliferation before D/V compartmentalization. In addition, we show that over-expression of vg can, to some extent, rescue the effect of the absence of ap on D/V boundary formation. Early VG product regulates AP activity by inducing dLMO and thus indirectly regulating ap target genes such as fringe and the PSalpha1 and PSalpha2 integrins. CONCLUSION: Normal cell proliferation is necessary for ap expression at the level of the D/V boundary. This would be mediated by vg, which interacts in a dose-dependent way with ap.

Targeted inactivation of p53 in human cells does not result in aneuploidy.

Because p53 mutation and aneuploidy usually coexist, it has been suggested that p53 inactivation leads to aneuploidy. We have rigorously tested this hypothesis in diploid human cell lines in which p53 was experimentally inactivated by targeted homologous recombination. Cells completely deficient in p53 did not become aneuploid, although a slight tendency toward tetraploidization was observed. No increased rates of numerical or structural chromosomal instabilities were observed in the p53-deficient cells. Rates of sister chromatid exchange and homologous recombination were also unaffected by p53 status. These results show that inactivation of p53 does not, in and of itself, lead to the development of aneuploidy.