Mouse Primordial Germ Cells: In Vitro Culture and Conversion to Pluripotent Stem Cell Lines.

Primordial germ cells (PGCs) are the embryonic precursors of the gametes. Despite decades of research, in vitro culture of PGCs remains a major challenge and has previously relied on undefined components such as serum and feeders. Notably, PGCs cultured for extended periods do not maintain their lineage identity but instead undergo conversion to form pluripotent stem cell lines called embryonic germ (EG) cells in response to LIF/STAT3 signaling. Here we report both established and new methodologies to derive EG cells, in a range of different conditions. We show that basic fibroblast growth factor is not required for EG cell conversion. We detail the steps taken in our laboratory to systematically remove complex components and establish a fully defined protocol that allows efficient conversion of isolated PGCs to pluripotent EG cells. In addition, we demonstrate that PGCs can adhere and proliferate in culture without the support of feeder cells or serum. This may well suggest novel approaches to establishing short-term culture of PGCs in defined conditions.

The role of disaccharidases in the digestion - diagnosis and significance of their deficiency in children and adults.

Disaccharidases are a group of enzymes of the small intestinal brush border, that are essential for degradation of disaccharides (sucrose, lactose, maltose, isomaltose, trehalose) into monosaccharides, which are then absorbed from the gastrointestinal tract. Their deficiency may occur at any stage of human life and have a genetic basis or be a secondary to ongoing gastrointestinal disease. Disaccharidase deficiencies cause disorders of digestion and absorption leading to occurrence of clinical symptoms such as abdominal pain, flatulence, diarrhea. For more than fifty years disaccharidase activity (DA) measurements in the small intestine biopsy samples are still considered the "gold standard" in the diagnostics for disaccharide deficiency. The aim of this review was to emphasize the role of disaccharidases in the digestion. Moreover, the significance of their deficiency in children and adults based on the current knowledge was described. It was showed that deficiency or inactivity of disaccharidases may lead to gastrointestinal intolerance symptoms. Early diagnostics allows the initiation of appropriate treatment, which contribute to reduction or complete resolution of clinical symptoms.

SLIC-CAGE: high-resolution transcription start site mapping using nanogram-levels of total RNA.

Cap analysis of gene expression (CAGE) is a methodology for genome-wide quantitative mapping of mRNA 5' ends to precisely capture transcription start sites at a single nucleotide resolution. In combination with high-throughput sequencing, CAGE has revolutionized our understanding of the rules of transcription initiation, led to discovery of new core promoter sequence features, and discovered transcription initiation at enhancers genome-wide. The biggest limitation of CAGE is that even the most recently improved version (nAnT-iCAGE) still requires large amounts of total cellular RNA (5 µg), preventing its application to scarce biological samples such as those from early embryonic development or rare cell types. Here, we present SLIC-CAGE, a Super-Low Input Carrier-CAGE approach to capture 5' ends of RNA polymerase II transcripts from as little as 5-10 ng of total RNA. This dramatic increase in sensitivity is achieved by specially designed, selectively degradable carrier RNA. We demonstrate the ability of SLIC-CAGE to generate data for genome-wide promoterome with 1000-fold less material than required by existing CAGE methods, by generating a complex, high-quality library from mouse embryonic day 11.5 primordial germ cells.

Synthesis and characterization of novel classes of PDE10A inhibitors - 1H-1,3-benzodiazoles and imidazo[1,2-a]pyrimidines.

New compounds containing [1,2,4]triazolo [1,5-a]pyridine (I), pyrazolo [1,5-a]pyridine (II), 1H-1,3-benzodiazole (III) and imidazo [1,2-a]pyrimidine (IV) backbones were designed and synthesized for PDE10A interaction. Among these compounds, 1H-1,3-benzodiazoles and imidazo [1,2-a]pyrimidines showed the highest affinity for PDE10A enzyme as well as good metabolic stability. Both classes of compounds were identified as selective and potent PDE10A enzyme inhibitors.

Epigenetic reprogramming enables the transition from primordial germ cell to gonocyte.

Gametes are highly specialized cells that can give rise to the next generation through their ability to generate a totipotent zygote. In mice, germ cells are first specified in the developing embryo around embryonic day (E) 6.25 as primordial germ cells (PGCs). Following subsequent migration into the developing gonad, PGCs undergo a wave of extensive epigenetic reprogramming around E10.5-E11.5, including genome-wide loss of 5-methylcytosine. The underlying molecular mechanisms of this process have remained unclear, leading to our inability to recapitulate this step of germline development in vitro. Here we show, using an integrative approach, that this complex reprogramming process involves coordinated interplay among promoter sequence characteristics, DNA (de)methylation, the polycomb (PRC1) complex and both DNA demethylation-dependent and -independent functions of TET1 to enable the activation of a critical set of germline reprogramming-responsive genes involved in gamete generation and meiosis. Our results also reveal an unexpected role for TET1 in maintaining but not driving DNA demethylation in gonadal PGCs. Collectively, our work uncovers a fundamental biological role for gonadal germline reprogramming and identifies the epigenetic principles of the PGC-to-gonocyte transition that will help to guide attempts to recapitulate complete gametogenesis in vitro.

Altered Disrupted-in-Schizophrenia-1 Function Affects the Development of Cortical Parvalbumin Interneurons by an Indirect Mechanism.

Disrupted-in-Schizophrenia-1 (DISC1) gene has been linked to schizophrenia and related major mental illness. Mouse Disc1 has been implicated in brain development, mainly in the proliferation, differentiation, lamination, neurite outgrowth and synapse formation and maintenance of cortical excitatory neurons. Here, the effects of two loss-of-function point mutations in the mouse Disc1 sequence (Q31L and L100P) on cortical inhibitory interneurons were investigated. None of the mutations affected the overall number of interneurons. However, the 100P, but not the 31L, mutation resulted in a significant decrease in the numbers of interneurons expressing parvalbumin mRNA and protein across the sensory cortex. To investigate role of Disc1 in regulation of parvalbumin expression, mouse wild-type Disc-1 or the 100P mutant form were electroporated in utero into cortical excitatory neurons. Overexpression of wild-type Disc1 in these cells caused increased densities of parvalbumin-expressing interneurons in the electroporated area and in areas connected with it, whereas expression of Disc1-100P did not. We conclude that the 100P mutation prevents expression of parvalbumin by a normally sized cohort of interneurons and that altering Disc1 function in cortical excitatory neurons indirectly affects parvalbumin expression by cortical interneurons, perhaps as a result of altered functional input from the excitatory neurons.

DISC1 genetics, biology and psychiatric illness.

Psychiatric disorders are highly heritable, and in many individuals likely arise from the combined effects of genes and the environment. A substantial body of evidence points towards DISC1 being one of the genes that influence risk of schizophrenia, bipolar disorder and depression, and functional studies of DISC1 consequently have the potential to reveal much about the pathways that lead to major mental illness. Here, we review the evidence that DISC1 influences disease risk through effects upon multiple critical pathways in the developing and adult brain.