Zebrafish as a model to study the role of Peroxisome Proliferating-Activated Receptors in adipogenesis and obesity

The Peroxisome Proliferator-Activated Receptors (PPARs) PPARA and PPARD are regulators of lipid metabolism with important roles in energy release through lipid breakdown, while PPARG plays a key role in lipid storage and adipogenesis. The aim of this review is to describe the role of PPARs in lipid metabolism, adipogenesis, and obesity and evaluate the zebrafish as an emerging vertebrate model to study the function of PPARs. Zebrafish are an appropriate model to study human diseases, including obesity and related metabolic diseases, as pathways important for adipogenesis and lipid metabolism which are conserved between mammals and fish. This review synthesizes knowledge on the role of PPARs in zebrafish and focuses on the putative function of PPARs in zebrafish adipogenesis. Using in silico analysis, we confirm the presence of five PPARs (pparaa, pparab, pparda, ppardb, and pparg) in the zebrafish genome with 67–74% identity to human and mouse PPARs. During development, pparda/b paralogs and pparg show mRNA expression around the swim bladder and pancreas, the region where adipocytes first develop, whereas pparg is detectable in adipocytes at 15 days post fertilization (dpf). This review indicates that the zebrafish is a promising model to investigate the specific functions of PPARs in adipogenesis and obesity.This research is financially supported by Netherlands Organisation for Scientific Research (NWO) VIDI/864.09.005 and ASPASIA/015.006.018

Uncovering regulatory pathways that affect hematopoietic stem cell function using 'genetical genomics'

We combined large-scale mRNA expression analysis and gene mapping to identify genes and loci that control hematopoietic stem cell (HSC) function. We measured mRNA expression levels in purified HSCs isolated from a panel of densely genotyped recombinant inbred mouse strains. We mapped quantitative trait loci (QTLs) associated with variation in expression of thousands of transcripts. By comparing the physical transcript position with the location of the controlling QTL, we identified polymorphic cis-acting stem cell genes. We also identified multiple trans-acting control loci that modify expression of large numbers of genes. These groups of coregulated transcripts identify pathways that specify variation in stem cells. We illustrate this concept with the identification of candidate genes involved with HSC turnover. We compared expression QTLs in HSCs and brain from the same mice and identified both shared and tissue-specific QTLs. Our data are accessible through WebQTL, a web-based interface that allows custom genetic linkage analysis and identification of coregulated transcripts.

Altered Adipogenesis in Zebrafish Larvae Following High Fat Diet and Chemical Exposure Is Visualised by Stimulated Raman Scattering Microscopy.

Early life stage exposure to environmental chemicals may play a role in obesity by altering adipogenesis; however, robust in vivo methods to quantify these effects are lacking. The goal of this study was to analyze the effects of developmental exposure to chemicals on adipogenesis in the zebrafish (Danio rerio). We used label-free Stimulated Raman Scattering (SRS) microscopy for the first time to image zebrafish adipogenesis at 15 days post fertilization (dpf) and compared standard feed conditions (StF) to a high fat diet (HFD) or high glucose diet (HGD). We also exposed zebrafish embryos to a non-toxic concentration of tributyltin (TBT, 1 nM) or Tris(1,3-dichloroisopropyl)phosphate (TDCiPP, 0.5 µM) from 0–6 dpf and reared larvae to 15 dpf under StF. Potential molecular mechanisms of altered adipogenesis were examined by qPCR. Diet-dependent modulation of adipogenesis was observed, with HFD resulting in a threefold increase in larvae with adipocytes, compared to StF and HGD. Developmental exposure to TBT but not TDCiPP significantly increased adipocyte differentiation. The expression of adipogenic genes such as pparda, lxr and lepa was altered in response to HFD or chemicals. This study shows that SRS microscopy can be successfully applied to zebrafish to visualize and quantify adipogenesis, and is a powerful approach for identifying obesogenic chemicals in vivo.This research is financially supported by Netherlands Organisation for Scientific Research (NWO) VIDI/864.09.005, ASPASIA /015.006.018, VICI/918.10.628, NWO-Groot grant, and the European Union’s Horizon 2020 research and innovation program under grant agreement number 654148 LaserLaB Europe

The Caenorhabditis elegans HEN1 Ortholog, HENN-1, Methylates and Stabilizes Select Subclasses of Germline Small RNAs

Small RNAs regulate diverse biological processes by directing effector proteins called Argonautes to silence complementary mRNAs. Maturation of some classes of small RNAs involves terminal 2′-O-methylation to prevent degradation. This modification is catalyzed by members of the conserved HEN1 RNA methyltransferase family. In animals, Piwi-interacting RNAs (piRNAs) and some endogenous and exogenous small interfering RNAs (siRNAs) are methylated, whereas microRNAs are not. However, the mechanisms that determine animal HEN1 substrate specificity have yet to be fully resolved. In Caenorhabditis elegans, a HEN1 ortholog has not been studied, but there is evidence for methylation of piRNAs and some endogenous siRNAs. Here, we report that the worm HEN1 ortholog, HENN-1 (HEN of Nematode), is required for methylation of C. elegans small RNAs. Our results indicate that piRNAs are universally methylated by HENN-1. In contrast, 26G RNAs, a class of primary endogenous siRNAs, are methylated in female germline and embryo, but not in male germline. Intriguingly, the methylation pattern of 26G RNAs correlates with the expression of distinct male and female germline Argonautes. Moreover, loss of the female germline Argonaute results in loss of 26G RNA methylation altogether. These findings support a model wherein methylation status of a metazoan small RNA is dictated by the Argonaute to which it binds. Loss of henn-1 results in phenotypes that reflect destabilization of substrate small RNAs: dysregulation of target mRNAs, impaired fertility, and enhanced somatic RNAi. Additionally, the henn-1 mutant shows a weakened response to RNAi knockdown of germline genes, suggesting that HENN-1 may also function in canonical RNAi. Together, our results indicate a broad role for HENN-1 in both endogenous and exogenous gene silencing pathways and provide further insight into the mechanisms of HEN1 substrate discrimination and the diversity within the Argonaute family

Bmi1 Confers Resistance to Oxidative Stress on Hematopoietic Stem Cells

The polycomb-group (PcG) proteins function as general regulators of stem cells. We previously reported that retrovirus-mediated overexpression of Bmi1, a gene encoding a core component of polycomb repressive complex (PRC) 1, maintained self-renewing hematopoietic stem cells (HSCs) during long-term culture. However, the effects of overexpression of Bmi1 on HSCs in vivo remained to be precisely addressed.In this study, we generated a mouse line where Bmi1 can be conditionally overexpressed under the control of the endogenous Rosa26 promoter in a hematopoietic cell-specific fashion (Tie2-Cre;R26Stop(FL)Bmi1). Although overexpression of Bmi1 did not significantly affect steady state hematopoiesis, it promoted expansion of functional HSCs during ex vivo culture and efficiently protected HSCs against loss of self-renewal capacity during serial transplantation. Overexpression of Bmi1 had no effect on DNA damage response triggered by ionizing radiation. In contrast, Tie2-Cre;R26Stop(FL)Bmi1 HSCs under oxidative stress maintained a multipotent state and generally tolerated oxidative stress better than the control. Unexpectedly, overexpression of Bmi1 had no impact on the level of intracellular reactive oxygen species (ROS).Our findings demonstrate that overexpression of Bmi1 confers resistance to stresses, particularly oxidative stress, onto HSCs. This thereby enhances their regenerative capacity and suggests that Bmi1 is located downstream of ROS signaling and negatively regulated by it

Haematopoietic stem cells in perisinusoidal niches are protected from ageing.

With ageing, intrinsic haematopoietic stem cell (HSC) activity decreases, resulting in impaired tissue homeostasis, reduced engraftment following transplantation and increased susceptibility to diseases. However, whether ageing also affects the HSC niche, and thereby impairs its capacity to support HSC function, is still widely debated. Here, by using in-vivo long-term label-retention assays we demonstrate that aged label-retaining HSCs, which are, in old mice, the most quiescent HSC subpopulation with the highest regenerative capacity and cellular polarity, reside predominantly in perisinusoidal niches. Furthermore, we demonstrate that sinusoidal niches are uniquely preserved in shape, morphology and number on ageing. Finally, we show that myeloablative chemotherapy can selectively disrupt aged sinusoidal niches in the long term, which is linked to the lack of recovery of endothelial Jag2 at sinusoids. Overall, our data characterize the functional alterations of the aged HSC niche and unveil that perisinusoidal niches are uniquely preserved and thereby protect HSCs from ageing

EZH2 and BMI1 inversely correlate with prognosis and TP53 mutation in breast cancer

Introduction PolycombGroup (PcG) proteins maintain gene repression through histone modifications and have been implicated in stem cell regulation and cancer. EZH2 is part of Polycomb Repressive Complex 2 (PRC2) and trimethylates H3K27. This histone mark recruits the BMI1-containing PRC1 that silences the genes marked by PRC2. Based on their role in stem cells, EZH2 and BMI1 have been predicted to contribute to a poor outcome for cancer patients. Methods We have analysed the expression of EZH2 and BMI1 in a well-characterised dataset of 295 human breast cancer samples. Results Interestingly, although EZH2 overexpression correlates with a poor prognosis in breast cancer, BMI1 overexpression correlates with a good outcome. Although this may reflect transformation of different cell types, we also observed a functional difference. The PcG-target genes INK4A and ARF are not expressed in tumours with high BMI1, but they are expressed in tumours with EZH2 overexpression. ARF expression results in tumour protein P53 (TP53) activation, and we found a significantly higher proportion of TP53 mutations in tumours with high EZH2. This may explain why tumours with high EZH2 respond poorly to therapy, in contrast to tumours with high BMI1. Conclusions Overall, our data highlight that whereas EZH2 and BMI1 may function in a 'linear' pathway in normal development, their overexpression has different functional consequences for breast tumourigenesi

Relationships between Hematopoiesis and Hepatogenesis in the Midtrimester Fetal Liver Characterized by Dynamic Transcriptomic and Proteomic Profiles

In fetal hematopoietic organs, the switch from hematopoiesis is hypothesized to be a critical time point for organogenesis, but it is not yet evidenced. The transient coexistence of hematopoiesis will be useful to understand the development of fetal liver (FL) around this time and its relationship to hematopoiesis. Here, the temporal and the comparative transcriptomic and proteomic profiles were observed during the critical time points corresponding to the initiation (E11.5), peak (E14.5), recession (E15.5), and disappearance (3 ddp) of mouse FL hematopoiesis. We found that E11.5-E14.5 corresponds to a FL hematopoietic expansion phase with distinct molecular features, including the expression of new transcription factors, many of which are novel KRAB (Kruppel-associated box)-containing zinc finger proteins. This time period is also characterized by extensive depression of some liver functions, especially catabolism/utilization, immune and defense, classical complement cascades, and intrinsic blood coagulation. Instead, the other liver functions increased, such as xenobiotic and sterol metabolism, synthesis of carbohydrate and glycan, the alternate and lectin complement cascades and extrinsic blood coagulation, and etc. Strikingly, all of the liver functions were significantly increased at E14.5-E15.5 and thereafter, and the depression of the key pathways attributes to build the hematopoietic microenvironment. These findings signal hematopoiesis emigration is the key to open the door of liver maturation

EZH2 Depletion Blocks the Proliferation of Colon Cancer Cells

The Enhancer of Zeste 2 (EZH2) protein has been reported to stimulate cell growth in some cancers and is therefore considered to represent an interesting new target for therapeutic intervention. Here, we investigated a possible role of EZH2 for the growth control of colon cancer cells. RNA interference (RNAi)-mediated intracellular EZH2 depletion led to cell cycle arrest of colon carcinoma cells at the G1/S transition. This was associated with a reduction of cell numbers upon transient transfection of synthetic EZH2-targeting siRNAs and with inhibition of their colony formation capacity upon stable expression of vector-borne siRNAs. We furthermore tested whether EZH2 may repress the growth-inhibitory p27 gene, as reported for pancreatic cancer. However, expression analyses of colon cancer cell lines and colon cancer biopsies did not reveal a consistent correlation between EZH2 and p27 levels. Moreover, EZH2 depletion did not re-induce p27 expression in colon cancer cells, indicating that p27 repression by EZH2 may be cell- or tissue-specific. Whole genome transcriptome analyses identified cellular genes affected by EZH2 depletion in colon cancer cell lines. They included several cancer-associated genes linked to cellular proliferation or invasion, such as Dag1, MageD1, SDC1, Timp2, and Tob1. In conclusion, our results demonstrate that EZH2 depletion blocks the growth of colon cancer cells. These findings might provide benefits for the treatment of colon cancer

Increased Expression of PcG Protein YY1 Negatively Regulates B Cell Development while Allowing Accumulation of Myeloid Cells and LT-HSC Cells

Ying Yang 1 (YY1) is a multifunctional Polycomb Group (PcG) transcription factor that binds to multiple enhancer binding sites in the immunoglobulin (Ig) loci and plays vital roles in early B cell development. PcG proteins have important functions in hematopoietic stem cell renewal and YY1 is the only mammalian PcG protein with DNA binding specificity. Conditional knock-out of YY1 in the mouse B cell lineage results in arrest at the pro-B cell stage, and dosage effects have been observed at various YY1 expression levels. To investigate the impact of elevated YY1 expression on hematopoetic development, we utilized a mouse in vivo bone marrow reconstitution system. We found that mouse bone marrow cells expressing elevated levels of YY1 exhibited a selective disadvantage as they progressed from hematopoietic stem/progenitor cells to pro-B, pre-B, immature B and re-circulating B cell stages, but no disadvantage of YY1 over-expression was observed in myeloid lineage cells. Furthermore, mouse bone marrow cells expressing elevated levels of YY1 displayed enrichment for cells with surface markers characteristic of long-term hematopoietic stem cells (HSC). YY1 expression induced apoptosis in mouse B cell lines in vitro, and resulted in down-regulated expression of anti-apoptotic genes Bcl-xl and NFκB2, while no impact was observed in a mouse myeloid line. B cell apoptosis and LT-HSC enrichment induced by YY1 suggest that novel strategies to induce YY1 expression could have beneficial effects in the treatment of B lineage malignancies while preserving normal HSCs