The splicing co-factor Barricade/Tat-SF1 is required for cell cycle and lineage progression in Drosophila neural stem cells.

Stem cells need to balance self-renewal and differentiation for correct tissue development and homeostasis. Defects in this balance can lead to developmental defects or tumor formation. In recent years, mRNA splicing has emerged as an important mechanism regulating cell fate decisions. Here we address the role of the evolutionarily conserved splicing co-factor Barricade (Barc)/Tat-SF1/CUS2 in Drosophila neural stem cell (neuroblast) lineage formation. We show that Barc is required for the generation of neurons during Drosophila brain development by ensuring correct neural progenitor proliferation and differentiation. Barc associates with components of the U2 small nuclear ribonucleoprotein (snRNP) complex, and its depletion causes alternative splicing in the form of intron retention in a subset of genes. Using bioinformatics analysis and a cell culture-based splicing assay, we found that Barc-dependent introns share three major traits: they are short, GC rich and have weak 3' splice sites. Our results show that Barc, together with the U2 snRNP complex, plays an important role in regulating neural stem cell lineage progression during brain development and facilitates correct splicing of a subset of introns.

Canonical NF-κB signaling in hepatocytes acts as a tumor-suppressor in hepatitis B virus surface antigen-driven hepatocellular carcinoma by controlling the unfolded protein response.

UNLABELLED: Chronic hepatitis B virus (HBV) infection remains the most common risk factor for hepatocellular carcinoma (HCC). Efficient suppression of HBV viremia and necroinflammation as a result of nucleos(t)ide analogue treatment is able to reduce HCC incidence; nevertheless, hepatocarcinogenesis can occur in the absence of active hepatitis, correlating with high HBV surface antigen (HBsAg) levels. Nuclear factor κB (NF-κB) is a central player in chronic inflammation and HCC development. However, in the absence of severe chronic inflammation, the role of NF-κB signaling in HCC development remains elusive. As a model of hepatocarcinogenesis driven by accumulation of HBV envelope polypeptides, HBsAg transgenic mice, which show no HBV-specific immune response, were crossed to animals with hepatocyte-specific inhibition of canonical NF-κB signaling. We detected prolonged, severe endoplasmic reticulum stress already at 20 weeks of age in NF-κB-deficient hepatocytes of HBsAg-expressing mice. The unfolded protein response regulator binding immunoglobulin protein/78-kDa glucose-regulated protein was down-regulated, activating transcription factor 6, and eIF2α were activated with subsequent overexpression of CCAAT/enhancer binding protein homologous protein. Notably, immune cell infiltrates and liver transaminases were unchanged. However, as a result of this increased cellular stress, insufficient hepatocyte proliferation due to G1 /S-phase cell cycle arrest with overexpression of p27 and emergence of ductular reactions was detected. This culminated in increased DNA damage already at 20 weeks of age and finally led to 100% HCC incidence due to NF-κB inhibition. CONCLUSION: The role of canonical NF-κB signaling in HCC development depends on the mode of liver damage; in the case of HBsAg-driven hepatocarcinogenesis, NF-κB in hepatocytes acts as a critical tumor suppressor by augmenting the endoplasmic reticulum stress response.

Hepatic activation of IKK/NFκB signaling induces liver fibrosis via macrophage-mediated chronic inflammation.

UNLABELLED: Liver damage in humans is induced by various insults including alcohol abuse, hepatitis B/C virus infection, autoimmune or metabolic disorders and, when persistent, leads to development of liver fibrosis. Because the nuclear factor-κB (NF-κB) system is activated in response to several of these stresses, we hypothesized that NF-κB activation in hepatocytes may contribute to fibrosis development. To activate the NF-κB signaling pathway in a time- and cell-type-specific manner in the liver, we crossed transgenic mice carrying the tetracycline-responsive transactivator under the control of the liver activator protein promotor with transgenic mice carrying a constitutively active form of the Ikbkb gene (IKK2 protein [CAIKK2]). Double-transgenic mice displayed doxycycline-regulated CAIKK2 expression in hepatocytes. Removal of doxycycline at birth led to activation of NF-κB signaling, moderate liver damage, recruitment of inflammatory cells, hepatocyte proliferation, and ultimately to spontaneous liver fibrosis development. Microarray analysis revealed prominent up-regulation of chemokines and chemokine receptors and this induction was rapidly reversed after switching off the CAIKK2 expression. Turning off the transgene expression for 3 weeks reversed stellate cell activation but did not diminish liver fibrosis. The elimination of macrophages by clodronate-liposomes attenuated NF-κB-induced liver fibrosis in a liver-injury-independent manner. CONCLUSION: Our results revealed that hepatic activation of IKK/NF-κB is sufficient to induce liver fibrosis by way of macrophage-mediated chronic inflammation. Therefore, agents controlling the hepatic NF-κB system represent attractive therapeutic tools to prevent fibrosis development in multiple chronic liver diseases.

Time-resolved transcriptomics in neural stem cells identifies a v-ATPase/Notch regulatory loop.

Drosophila melanogaster neural stem cells (neuroblasts [NBs]) divide asymmetrically by differentially segregating protein determinants into their daughter cells. Although the machinery for asymmetric protein segregation is well understood, the events that reprogram one of the two daughter cells toward terminal differentiation are less clear. In this study, we use time-resolved transcriptional profiling to identify the earliest transcriptional differences between the daughter cells on their way toward distinct fates. By screening for coregulated protein complexes, we identify vacuolar-type H+-ATPase (v-ATPase) among the first and most significantly down-regulated complexes in differentiating daughter cells. We show that v-ATPase is essential for NB growth and persistent activity of the Notch signaling pathway. Our data suggest that v-ATPase and Notch form a regulatory loop that acts in multiple stem cell lineages both during nervous system development and in the adult gut. We provide a unique resource for investigating neural stem cell biology and demonstrate that cell fate changes can be induced by transcriptional regulation of basic, cell-essential pathways.

A Combination of CRISPR/Cas9 and Standardized RNAi as a Versatile Platform for the Characterization of Gene Function.

Traditional loss-of-function studies in Drosophila suffer from a number of shortcomings, including off-target effects in the case of RNA interference (RNAi) or the stochastic nature of mosaic clonal analysis. Here, we describe minimal in vivo GFP interference (miGFPi) as a versatile strategy to characterize gene function and to conduct highly stringent, cell type-specific loss-of-function experiments in Drosophila miGFPi combines CRISPR/Cas9-mediated tagging of genes at their endogenous locus with an immunotag and an exogenous 21 nucleotide RNAi effector sequence with the use of a single reagent, highly validated RNAi line targeting this sequence. We demonstrate the utility and time effectiveness of this method by characterizing the function of the Polymerase I (Pol I)-associated transcription factor Tif-1a, and the previously uncharacterized gene MESR4, in the Drosophila female germline stem cell lineage. In addition, we show that miGFPi serves as a powerful technique to functionally characterize individual isoforms of a gene. We exemplify this aspect of miGFPi by studying isoform-specific loss-of-function phenotypes of the longitudinals lacking (lola) gene in neural stem cells. Altogether, the miGFPi strategy constitutes a generalized loss-of-function approach that is amenable to the study of the function of all genes in the genome in a stringent and highly time effective manner.