High-throughput discovery of genetic determinants of circadian misalignment.

Circadian systems provide a fitness advantage to organisms by allowing them to adapt to daily changes of environmental cues, such as light/dark cycles. The molecular mechanism underlying the circadian clock has been well characterized. However, how internal circadian clocks are entrained with regular daily light/dark cycles remains unclear. By collecting and analyzing indirect calorimetry (IC) data from more than 2000 wild-type mice available from the International Mouse Phenotyping Consortium (IMPC), we show that the onset time and peak phase of activity and food intake rhythms are reliable parameters for screening defects of circadian misalignment. We developed a machine learning algorithm to quantify these two parameters in our misalignment screen (SyncScreener) with existing datasets and used it to screen 750 mutant mouse lines from five IMPC phenotyping centres. Mutants of five genes (Slc7a11, Rhbdl1, Spop, Ctc1 and Oxtr) were found to be associated with altered patterns of activity or food intake. By further studying the Slc7a11tm1a/tm1a mice, we confirmed its advanced activity phase phenotype in response to a simulated jetlag and skeleton photoperiod stimuli. Disruption of Slc7a11 affected the intercellular communication in the suprachiasmatic nucleus, suggesting a defect in synchronization of clock neurons. Our study has established a systematic phenotype analysis approach that can be used to uncover the mechanism of circadian entrainment in mice

Transforming growth factor-ß (TGF-ß) signaling in hematopoiesis and tumorigenesis

Transforming growth factor β (TGF-β) signaling regulates numerous cellular and physiological processes. Dysfunction of components of this signaling pathway leads to a wide range of diseases ranging from malignant hematopoiesis, cardiovascular disease, immunity abnormalities, connective tissue disease, reproductive disorders, metabolic disorders, skeleton and muscular disorders, to developmental defects. We focused on the role of Smad4 and TBRII genes in the hematopoiesis and studied a conditional Smad4 knockout mouse. Mice with homozygous Smad4 deletion (Smad4Δ/Δ) developed severe anemia 6-8 weeks after induction (mean hemoglobin 70g/L). The anemia was not transplantable, as wild type mice reconstituted with Smad4Δ/Δ bone marrow cells had normal peripheral blood counts. In contrast, lethally irradiated Smad4Δ/Δ mice transplanted with wild type bone marrow cells developed anemia similar to non-transplanted Smad4Δ/Δ mice. Liver iron stores were decreased and blood was present in stool, indicating that the anemia was due to blood loss. Multiple polyps in stomach and colon represent a likely source of the bleeding. We conclude that Smad4 is not required for adult erythropoiesis and that anemia is solely the consequence of blood loss. Regulation of hepcidin related genes (Atoh8, Id1 and Bmp6) responded to acute bleeding in the absence of Smad4, TBRII and/or histone deacetylase 1 (HDAC1) genes. Smad4Δ/Δ mice did not develop an inflammatory disease typical for mice deficient in TGF-b receptors I and II (TBRI and TBRII), suggesting that suppression of inflammation by TGF-β is Smad4 independent. The same results were obtained when Smad4 alleles were deleted selectively in hematopoietic cells using the VavCre transgenic mice. Mice with a double knockout (Smad4Δ/Δ and TBRIIΔ/Δ) did not display the TBRIIΔ/Δ -driven lethal inflammation suggesting that Smad4 signaling is required to mediate the inflammatory phenotype. Smad4/TBRII was dispensable for the megakaryopoiesis and erythropoiesis. Finally, we confirmed that the Smad4-signaling pathway is required to suppress tumorigenesis in the gastrointestinal tract and loss of Smad4-signaling in hematopoietic cells is sufficient to cause polyp formation in the gut

A Bubble-Free Microfluidic Device for Easy-to-Operate Immobilization, Culturing and Monitoring of Zebrafish Embryos

The development of miniaturized devices for studying zebrafish embryos has been limited due to complicated fabrication and operation processes. Here, we reported on a microfluidic device that enabled the capture and culture of zebrafish embryos and real-time monitoring of dynamic embryonic development. The device was simply fabricated by bonding two layers of polydimethylsiloxane (PDMS) structures replicated from three-dimensional (3D) printed reusable molds onto a flat glass substrate. Embryos were easily loaded into the device with a pipette, docked in traps by gravity, and then retained in traps with hydrodynamic forces for long-term culturing. A degassing chamber bonded on top was used to remove air bubbles from the embryo-culturing channel and traps so that any embryo movement caused by air bubbles was eliminated during live imaging. Computational fluid dynamics simulations suggested this embryo-trapping and -retention regime to exert low shear stress on the immobilized embryos. Monitoring of the zebrafish embryogenesis over 20 h during the early stages successfully verified the performance of the microfluidic device for culturing the immobilized zebrafish embryos. Therefore, this rapid-prototyping, low-cost and easy-to-operate microfluidic device offers a promising platform for the long-term culturing of immobilized zebrafish embryos under continuous medium perfusion and the high-quality screening of the developmental dynamics

Targeted Gene Delivery to Macrophages by Biodegradable Star-Shaped Polymers

In this report, two biodegradable star-shaped polyasparamide derivatives and four analogues modified with either mannose or folic acid moiety for preferential targeting of a difficult-to-transfect immune cell type, i.e., macrophage, have been synthesized. Each of the prepared star polymers complexes with plasmid DNA to form nanosized particles featuring a core–shell-like morphology. Mannose or folate functionalized star polymers can greatly improve the transfection performance on a macrophage cell line RAW 264.7. As a result, a combination of targeting ligand modification and topological structures of gene carriers is a promising strategy for immune cells-based gene therapy

Post-transcriptional Regulation of Nkx2-5 by RHAU in Heart Development

RNA G-quadruplexes (G4s) play important roles in RNA biology. However, the function and regulation of mRNA G-quadruplexes in embryonic development remain elusive. Previously, we identified RHAU (DHX36, G4R1) as an RNA helicase that resolves mRNA G-quadruplexes. Here, we find that cardiac deletion of Rhau leads to heart defects and embryonic lethality in mice. Gene expression profiling identified Nkx2-5 mRNA as a target of RHAU that associates with its 5′ and 3′ UTRs and modulates its stability and translation. The 5′ UTR of Nkx2-5 mRNA contains a G-quadruplex that requires RHAU for protein translation, while the 3′ UTR of Nkx2-5 mRNA possesses an AU-rich element (ARE) that facilitates RHAU-mediated mRNA decay. Thus, we uncovered the mechanisms underlying Nkx2-5 post-transcriptional regulation during heart development. Meanwhile, this study demonstrates the function of mRNA 5′ UTR G-quadruplex-mediated protein translation in organogenesis