Efficient generation and mapping of recessive developmental mutations using ENU mutagenesis

Treatment with N-ethyl-N-nitrosourea (ENU) efficiently generates single-nucleotide mutations in mice. Along with the renewed interest in this approach, much attention has been given recently to large screens with broad aims; however, more finely focused studies have proven very productive as well. Here we show how mutagenesis together with genetic mapping can facilitate the rapid characterization of recessive loci required for normal embryonic development. We screened third-generation progeny of mutagenized mice at embryonic day (E) 18.5 for abnormalities of organogenesis. We ascertained 15 monogenic mutations in the 54 families that were comprehensively analyzed. We carried out the experiment as an outcross, which facilitated the genetic mapping of the mutations by haplotype analysis. We mapped seven of the mutations and identified the affected locus in two lines. Using a hierarchical approach, it is possible to maximize the efficiency of this analysis so that it can be carried out easily with modest infrastructure and resources.     

In vitro neurogenesis: development and functional implications of iPSC technology

Neurogenesis is the developmental process regulating cell proliferation of neural stem cells, determining their differentiation into glial and neuronal cells, and orchestrating their organization into finely regulated functional networks. Can this complex process be recapitulated in vitro using induced pluripotent stem cell (iPSC) technology? Can neurodevelopmental and neurodegenerative diseases be modeled using iPSCs? What is the potential of iPSC technology in neurobiology? What are the recent advances in the field of neurological diseases? Since the applications of iPSCs in neurobiology are based on the capacity to regulate in vitro differentiation of human iPSCs into different neuronal subtypes and glial cells, and the possibility of obtaining iPSC-derived neurons and glial cells is based on and hindered by our poor understanding of human embryonic development, we reviewed current knowledge on in vitro neural differentiation from a developmental and cellular biology perspective. We highlight the importance to further advance our understanding on the mechanisms controlling in vivo neurogenesis in order to efficiently guide neurogenesis in vitro for cell modeling and therapeutical applications of iPSCs technology.     

Mutations in SLC20A2 link familial idiopathic basal ganglia calcification with phosphate homeostasis

Familial idiopathic basal ganglia calcification (IBGC) is a genetic condition with a wide spectrum of neuropsychiatric symptoms, including parkinsonism and dementia. Here, we identified mutations in SLC20A2, encoding the type III sodium-dependent phosphate transporter 2 (PiT2), in IBGC-affected families of varied ancestry, and we observed significantly impaired phosphate transport activity for all assayed PiT2 mutants in Xenopus laevis oocytes. Our results implicate altered phosphate homeostasis in the etiology of IBGC.     

The endonuclease activity of Mili fuels piRNA amplification that silences LINE1 elements

Piwi proteins and Piwi-interacting RNAs (piRNAs) have conserved functions in transposon silencing. The murine Piwi proteins Mili and Miwi2 (also called Piwil2 and Piwil4, respectively) direct epigenetic LINE1 and intracisternal A particle transposon silencing during genome reprogramming in the embryonic male germ line. Piwi proteins are proposed to be piRNA-guided endonucleases that initiate secondary piRNA biogenesis; however, the actual contribution of their endonuclease activities to piRNA biogenesis and transposon silencing remain unknown. To investigate the role of Piwi-catalysed endonucleolytic activity, we engineered point mutations in mice that substitute the second aspartic acid to an alanine in the DDH catalytic triad of Mili and Miwi2, generating the Mili(DAH) and Miwi2(DAH) alleles, respectively. Analysis of Mili-bound piRNAs from homozygous Mili(DAH) fetal gonadocytes revealed a failure of transposon piRNA amplification, resulting in the marked reduction of piRNA bound within Miwi2 ribonuclear particles. We find that Mili-mediated piRNA amplification is selectively required for LINE1, but not intracisternal A particle, silencing. The defective piRNA pathway in Mili(DAH) mice results in spermatogenic failure and sterility. Surprisingly, homozygous Miwi2(DAH) mice are fertile, transposon silencing is established normally and no defects in secondary piRNA biogenesis are observed. In addition, the hallmarks of piRNA amplification are observed in Miwi2-deficient gonadocytes. We conclude that cycles of intra-Mili secondary piRNA biogenesis fuel piRNA amplification that is absolutely required for LINE1 silencing.     

Human gut microbiome viewed across age and geography

Gut microbial communities represent one source of human genetic and metabolic diversity. To examine how gut microbiomes differ among human populations, here we characterize bacterial species in fecal samples from 531 individuals, plus the gene content of 110 of them. The cohort encompassed healthy children and adults from the Amazonas of Venezuela, rural Malawi and US metropolitan areas and included mono- and dizygotic twins. Shared features of the functional maturation of the gut microbiome were identified during the first three years of life in all three populations, including age-associated changes in the genes involved in vitamin biosynthesis and metabolism. Pronounced differences in bacterial assemblages and functional gene repertoires were noted between US residents and those in the other two countries. These distinctive features are evident in early infancy as well as adulthood. Our findings underscore the need to consider the microbiome when evaluating human development, nutritional needs, physiological variations and the impact of westernization.     

Suppressor of cytokine signaling-1 inhibits caspase activation and protects from cytokine-induced beta cell death

Pancreatic beta cell damage caused by pro-inflammatory cytokines interleukin-1β (IL-1β), interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) is a key event in the pathogenesis of type 1 diabetes. The suppressor of cytokine signaling-1 (SOCS-1) blocks IFNγ-induced signaling and prevents diabetes in the non-obese diabetic mouse. Here, we investigated if SOCS-1 overexpression in primary beta cells provides protection from cytokine-induced islet cell dysfunction and death. We demonstrate that SOCS-1 does not prevent increase in NO production and decrease in glucose-stimulated insulin secretion in the presence of IL-1β, IFNγ, TNFα. However, it decreases the activation of caspase-3, -8 and -9, and thereby, promotes a robust protection from cytokine-induced beta cell death. Our data suggest that SOCS-1 overexpression may not be sufficient in preventing all the biological activities of IFNγ in beta cells. In summary, we show that interference with IFNγ signal transduction pathways by SOCS-1 inhibits cytokine-stimulated pancreatic beta cell death.     

Stochastic and genetic factors influence tissue-specific decline in ageing C. elegans

The nematode Caenorhabditis elegans is an important model for studying the genetics of ageing, with over 50 life-extension mutations known so far. However, little is known about the pathobiology of ageing in this species, limiting attempts to connect genotype with senescent phenotype. Using ultrastructural analysis and visualization of specific cell types with green fluorescent protein, we examined cell integrity in different tissues as the animal ages. We report remarkable preservation of the nervous system, even in advanced old age, in contrast to a gradual, progressive deterioration of muscle, resembling human sarcopenia. The age-1(hx546) mutation, which extends lifespan by 60-100%, delayed some, but not all, cellular biomarkers of ageing. Strikingly, we found strong evidence that stochastic as well as genetic factors are significant in C. elegans ageing, with extensive variability both among same-age animals and between cells of the same type within individuals.