Insights into Sex Chromosome Evolution and Aging from the Genome of a Short-Lived Fish.

The killifish Nothobranchius furzeri is the shortest-lived vertebrate that can be bred in the laboratory. Its rapid growth, early sexual maturation, fast aging, and arrested embryonic development (diapause) make it an attractive model organism in biomedical research. Here, we report a draft sequence of its genome that allowed us to uncover an intra-species Y chromosome polymorphism representing-in real time-different stages of sex chromosome formation that display features of early mammalian XY evolution "in action." Our data suggest that gdf6Y, encoding a TGF-ß family growth factor, is the master sex-determining gene in N. furzeri. Moreover, we observed genomic clustering of aging-related genes, identified genes under positive selection, and revealed significant similarities of gene expression profiles between diapause and aging, particularly for genes controlling cell cycle and translation. The annotated genome sequence is provided as an online resource (http://www.nothobranchius.info/NFINgb).

The DNA Methyltransferase 1 (DNMT1) Controls the Shape and Dynamics of Migrating POA-Derived Interneurons Fated for the Murine Cerebral Cortex.

The proliferative niches in the subpallium generate a rich cellular variety fated for diverse telencephalic regions. The embryonic preoptic area (POA) represents one of these domains giving rise to the pool of cortical GABAergic interneurons and glial cells, in addition to striatal and residual POA cells. The migration from sites of origin within the subpallium to the distant targets like the cerebral cortex, accomplished by the adoption and maintenance of a particular migratory morphology, is a critical step during interneuron development. To identify factors orchestrating this process, we performed single-cell transcriptome analysis and detected Dnmt1 expression in murine migratory GABAergic POA-derived cells. Deletion of Dnmt1 in postmitotic immature cells of the POA caused defective migration and severely diminished adult cortical interneuron numbers. We found that DNA methyltransferase 1 (DNMT1) preserves the migratory shape in part through negative regulation of Pak6, which stimulates neuritogenesis at postmigratory stages. Our data underline the importance of DNMT1 for the migration of POA-derived cells including cortical interneurons.

Sensing Cardiac Electrical Activity With a Cardiac Myocyte--Targeted Optogenetic Voltage Indicator.

RATIONALE: Monitoring and controlling cardiac myocyte activity with optogenetic tools offer exciting possibilities for fundamental and translational cardiovascular research. Genetically encoded voltage indicators may be particularly attractive for minimal invasive and repeated assessments of cardiac excitation from the cellular to the whole heart level. OBJECTIVE: To test the hypothesis that cardiac myocyte-targeted voltage-sensitive fluorescence protein 2.3 (VSFP2.3) can be exploited as optogenetic tool for the monitoring of electric activity in isolated cardiac myocytes and the whole heart as well as function and maturity in induced pluripotent stem cell-derived cardiac myocytes. METHODS AND RESULTS: We first generated mice with cardiac myocyte-restricted expression of VSFP2.3 and demonstrated distinct localization of VSFP2.3 at the t-tubulus/junctional sarcoplasmic reticulum microdomain without any signs for associated pathologies (assessed by echocardiography, RNA-sequencing, and patch clamping). Optically recorded VSFP2.3 signals correlated well with membrane voltage measured simultaneously by patch clamping. The use of VSFP2.3 for human action potential recordings was confirmed by simulation of immature and mature action potentials in murine VSFP2.3 cardiac myocytes. Optical cardiograms could be monitored in whole hearts ex vivo and minimally invasively in vivo via fiber optics at physiological heart rate (10 Hz) and under pacing-induced arrhythmia. Finally, we reprogrammed tail-tip fibroblasts from transgenic mice and used the VSFP2.3 sensor for benchmarking functional and structural maturation in induced pluripotent stem cell-derived cardiac myocytes. CONCLUSIONS: We introduce a novel transgenic voltage-sensor model as a new method in cardiovascular research and provide proof of concept for its use in optogenetic sensing of physiological and pathological excitation in mature and immature cardiac myocytes in vitro and in vivo.

RepARK--de novo creation of repeat libraries from whole-genome NGS reads.

Generation of repeat libraries is a critical step for analysis of complex genomes. In the era of next-generation sequencing (NGS), such libraries are usually produced using a whole-genome shotgun (WGS) derived reference sequence whose completeness greatly influences the quality of derived repeat libraries. We describe here a de novo repeat assembly method--RepARK (Repetitive motif detection by Assembly of Repetitive K-mers)--which avoids potential biases by using abundant k-mers of NGS WGS reads without requiring a reference genome. For validation, repeat consensuses derived from simulated and real Drosophila melanogaster NGS WGS reads were compared to repeat libraries generated by four established methods. RepARK is orders of magnitude faster than the other methods and generates libraries that are: (i) composed almost entirely of repetitive motifs, (ii) more comprehensive and (iii) almost completely annotated by TEclass. Additionally, we show that the RepARK method is applicable to complex genomes like human and can even serve as a diagnostic tool to identify repetitive sequences contaminating NGS datasets.

Eag1 expression interferes with hypoxia homeostasis and induces angiogenesis in tumors.

Ether-á-go-go-1 (Eag1) is a CNS-localized voltage-gated potassium channel that is found ectopically expressed in a majority of extracranial solid tumors. While circumstantial evidence linking Eag1 to tumor biology has been well established, the mechanisms by which the channel contributes to tumor progression remain elusive. In this study, we have used in vivo and in vitro techniques to identify a candidate mechanism. A mutation that eliminates ion permeation fails to completely abolish xenograft tumor formation by transfected cells, indicating that Eag1 contributes to tumor progression independently of its primary function as an ion channel. Our data suggest that Eag1 interferes with the cellular mechanism for maintaining oxygen homeostasis, increasing HIF-1 activity, and thereby VEGF secretion and tumor vascularization.

Silencing the activity and proliferative properties of the human EagI Potassium Channel by RNA Interference.

EagI potassium channels are natively expressed in the mammalian brain as well as in many cancer cell lines and tumor tissues. The role of EagI in malignant transformation has been suggested by several experiments, but the lack of specific EagI inhibitors has made it difficult to examine the influence of the channel on oncogenesis and its potential as a therapeutic target. We have used short interfering RNA to test the effects of EagI reduction on the behavior of tumor cells in vitro. By generating and optimizing an EagI-specific short interfering RNA system, we were able to study the effects of EagI depletion on several cancer cell lines that endogenously express this protein. We show here that our short interfering RNA sequences act specifically on EagI, reproducibly induce a significant decrease in the proliferation of tumor cell lines, and do not trigger any observable nonspecific responses.