Specific cell ablation is a useful method for analyzing the in vivo function of cells. We have developed a simple and sensitive method for conditional cell ablation in transgenic mice, called "toxin receptor-mediated cell knockout." We expressed the diphtheria toxin (DT) receptor in transgenic mice using a hepatocyte-specific promoter and found that injection of DT caused fulminant hepatitis. Three independently established transgenic lines demonstrated a good correlation between the sensitivity of hepatocytes to DT and the expression level of the DT receptors. Moreover, the degree of hepatocyte damage was easily controlled over a wide range of doses of injected DT without any obvious abnormalities in other cells or tissues. This system is useful for generating mouse models of disease and for studying the recovery or regeneration of tissues from cell damage or loss. As DT is a potent inhibitor of protein synthesis in both growing and non-growing cells, the method is applicable to a wide range of cells and tissues in mice or in other DT-insensitive animals.

The recruitment of basophils into the sites of allergic inflammation is often observed. However, no definitive evidence has been provided that basophils are crucially involved in the pathogenesis of chronic allergic disorders. Here, we show that basophils are responsible for the development of IgE-mediated chronic allergic inflammation independently of T cells and mast cells. A single subcutaneous injection of multivalent antigens elicited not only immediate- and late-phase ear swelling but also delayed-onset ear swelling with massive eosinophil infiltration in mice sensitized with antigen-specific IgE. Mast cells were essential for the immediate- and late-phase ear swelling but dispensable for the delayed one. T cells were also dispensable for the latter. Transfer of FcRI-expressing basophils into FcRI-deficient mice restored the development of the delayed-onset allergic inflammation. These findings indicate a novel mechanism of development of chronic allergic inflammation that is induced by basophils through the interaction of antigen, IgE, and FcRI.

Observations of rapid shifts in mitochondrial DNA (mtDNA) variants between generations prompted the creation of the bottleneck theory. A prevalent hypothesis is that a massive reduction in mtDNA content during early oogenesis leads to the bottleneck. To test this, we estimated the mtDNA copy number in single germline cells and in single somatic cells of early embryos in mice. Primordial germ cells (PGCs) show consistent, moderate mtDNA copy numbers across developmental stages, whereas primary oocytes demonstrate substantial mtDNA expansion during early oocyte maturation. Some somatic cells possess a very low mtDNA copy number. We also demonstrated that PGCs have more than 100 mitochondria per cell. We conclude that the mitochondrial bottleneck is not due to a drastic decline in mtDNA copy number in early oogenesis but rather to a small effective number of segregation units for mtDNA in mouse germ cells. These results provide new information for mtDNA segregation models and for understanding the recurrence risks for mtDNA diseases.

Defects in myosin VIIa, harmonin (a PDZ domain protein), cadherin 23, protocadherin 15 and sans (a putative scaffolding protein), underlie five forms of Usher syndrome type I (USH1). Mouse mutants for all these proteins exhibit disorganization of their hair bundle, which is the mechanotransduction receptive structure of the inner ear sensory cells, the cochlear and vestibular hair cells. We have previously demonstrated that harmonin interacts with cadherin 23 and myosin VIIa. Here we address the extent of interactions between the five known USH1 proteins. We establish the previously suggested sans-harmonin interaction and find that sans also binds to myosin VIIa. We show that sans can form homomeric structures and that harmonin b can interact with all harmonin isoforms. We reveal that harmonin also binds to protocadherin 15. Molecular characterization of these interactions indicates that through its binding to four of the five USH1 proteins, the first PDZ domain (PDZ1) of harmonin plays a central role in this network. We localize sans in the apical region of cochlear and vestibular hair cell bodies underneath the cuticular plate. In contrast to the other four known USH1 proteins, no sans labeling was detected within the stereocilia. We propose that via its binding to myosin VIIa and/or harmonin, sans controls the hair bundle cohesion and proper development by regulating the traffic of USH1 proteins en route to the stereocilia.

Proper neutrophil migration into inflammatory sites ensures host defense without tissue damage. Phosphoinositide 3-kinase (PI(3)K) and its lipid product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) regulate cell migration, but the role of PtdIns(3,4,5)P(3)-degrading enzymes in this process is poorly understood. Here, we show that Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1), a PtdIns(3,4,5)P(3) phosphatase, is a key regulator of neutrophil migration. Genetic inactivation of SHIP1 led to severe defects in neutrophil polarization and motility. In contrast, loss of the PtdIns(3,4,5)P(3) phosphatase PTEN had no impact on neutrophil chemotaxis. To study PtdIns(3,4,5)P(3) metabolism in living primary cells, we generated a novel transgenic mouse (AktPH-GFP Tg) expressing a bioprobe for PtdIns(3,4,5)P(3.) Time-lapse footage showed rapid, localized binding of AktPH-GFP to the leading edge membrane of chemotaxing ship1(+/+)AktPH-GFP Tg neutrophils, but only diffuse localization in ship1(-/-)AktPH-GFP Tg neutrophils. By directing where PtdIns(3,4,5)P(3) accumulates, SHIP1 governs the formation of the leading edge and polarization required for chemotaxis.