Paths and pathways that generate cell-type heterogeneity and developmental progression in hematopoiesis.

Mechanistic studies of lymph gland hematopoiesis are limited by the availability of cell-type-specific markers. Using a combination of bulk RNA-Seq of FACS-sorted cells, single-cell RNA-Seq, and genetic dissection, we identify new blood cell subpopulations along a developmental trajectory with multiple paths to mature cell types. This provides functional insights into key developmental processes and signaling pathways.

Metabolic plasticity drives development during mammalian embryogenesis.

Mammalian preimplantation embryos follow a stereotypic pattern of development from zygotes to blastocysts. Here, we use labeled nutrient isotopologue analysis of small numbers of embryos to track downstream metabolites. Combined with transcriptomic analysis, we assess the capacity of the embryo to reprogram its metabolism through development.

Glycolysis-Independent Glucose Metabolism Distinguishes TE from ICM Fate during Mammalian Embryogenesis.

The mouse embryo undergoes compaction at the 8-cell stage, and its transition to 16 cells generates polarity such that the outer apical cells are trophectoderm (TE) precursors and the inner cell mass (ICM) gives rise to the embryo. Here, we report that this first cell fate specification event is controlled by glucose. Glucose does not fuel mitochondrial ATP generation, and glycolysis is dispensable for blastocyst formation.

Extracellular Matrix Remodeling Regulates Glucose Metabolism through TXNIP Destabilization.

The metabolic state of a cell is influenced by cell-extrinsic factors, including nutrient availability and growth factor signaling. Here, we present extracellular matrix (ECM) remodeling as another fundamental node of cell-extrinsic metabolic regulation. Unbiased analysis of glycolytic drivers identified the hyaluronan-mediated motility receptor as being among the most highly correlated with glycolysis in cancer.

Nuclear Localization of Mitochondrial TCA Cycle Enzymes as a Critical Step in Mammalian Zygotic Genome Activation.

Transcriptional control requires epigenetic changes directed by mitochondrial tricarboxylic acid (TCA) cycle metabolites. In the mouse embryo, global epigenetic changes occur during zygotic genome activation (ZGA) at the 2-cell stage. Pyruvate is essential for development beyond this stage, which is at odds with the low activity of mitochondria in this period.

Mouse mtDNA mutant model of Leber hereditary optic neuropathy.

An animal model of Leber hereditary optic neuropathy (LHON) was produced by introducing the human optic atrophy mtDNA ND6 P25L mutation into the mouse. Mice with this mutation exhibited reduction in retinal function by elecroretinogram (ERG), age-related decline in central smaller caliber optic nerve fibers with sparing of larger peripheral fibers, neuronal accumulation of abnormal mitochondria, axonal swelling, and demyelination. Mitochondrial analysis revealed partial complex I and respiration defects and increased reactive oxygen species (ROS) production, whereas synaptosome analysis revealed decreased complex I activity and increased ROS but no diminution of ATP production.

Heteroplasmy of mouse mtDNA is genetically unstable and results in altered behavior and cognition.

Maternal inheritance of mtDNA is the rule in most animals, but the reasons for this pattern remain unclear. To investigate the consequence of overriding uniparental inheritance, we generated mice containing an admixture (heteroplasmy) of NZB and 129S6 mtDNAs in the presence of a congenic C57BL/6J nuclear background. Analysis of the segregation of the two mtDNAs across subsequent maternal generations revealed that proportion of NZB mtDNA was preferentially reduced.

Mitochondrial DNA variant associated with Leber hereditary optic neuropathy and high-altitude Tibetans.

The distinction between mild pathogenic mtDNA mutations and population polymorphisms can be ambiguous because both are homoplasmic, alter conserved functions, and correlate with disease. One possible explanation for this ambiguity is that the same variant may have different consequences in different contexts. The NADH dehydrogenase subunit 1 (ND1) nucleotide 3394 T > C (Y30H) variant is such a case.

Reduction of hydrophilic ubiquinones by the flavin in mitochondrial NADH:ubiquinone oxidoreductase (Complex I) and production of reactive oxygen species.

NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a complicated, energy-transducing, membrane-bound enzyme that contains 45 different subunits, a non-covalently bound flavin mononucleotide, and eight iron-sulfur clusters. The mechanisms of NADH oxidation and intramolecular electron transfer by complex I are gradually being defined, but the mechanism linking ubiquinone reduction to proton translocation remains unknown. Studies of ubiquinone reduction by isolated complex I are problematic because the extremely hydrophobic natural substrate, ubiquinone-10, must be substituted with a relatively hydrophilic analogue (such as ubiquinone-1).

Interaction of the mitochondria-targeted antioxidant MitoQ with phospholipid bilayers and ubiquinone oxidoreductases.

MitoQ(10) is a ubiquinone that accumulates within mitochondria driven by a conjugated lipophilic triphenylphosphonium cation (TPP(+)). Once there, MitoQ(10) is reduced to its active ubiquinol form, which has been used to prevent mitochondrial oxidative damage and to infer the involvement of reactive oxygen species in signaling pathways. Here we show MitoQ(10) is effectively reduced by complex II, but is a poor substrate for complex I, complex III, and electron-transferring flavoprotein (ETF):quinone oxidoreductase (ETF-QOR).

The inhibition of mitochondrial complex I (NADH:ubiquinone oxidoreductase) by Zn2+.

NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a highly complicated, membrane-bound enzyme. It is central to energy transduction, an important source of cellular reactive oxygen species, and its dysfunction is implicated in neurodegenerative and muscular diseases and in aging. Here, we describe the effects of Zn2+ on complex I to define whether complex I may contribute to mediating the pathological effects of zinc in states such as ischemia and to determine how Zn2+ can be used to probe the mechanism of complex I.

Interactions between phospholipids and NADH:ubiquinone oxidoreductase (complex I) from bovine mitochondria.

NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a highly complicated, energy transducing, membrane-bound enzyme. It contains 46 different subunits and nine redox cofactors: a noncovalently bound flavin mononucleotide and eight iron-sulfur clusters. The mechanism of complex I is not known.