Here we have identified 'triple D' (3d), a recessive N-ethyl-N-nitrosourea-induced mutation and phenotype in which no signaling occurs via the intracellular Toll-like receptors 3, 7 and 9 (sensors for double-stranded RNA, single-stranded RNA and unmethylated DNA, respectively). The 3d mutation also prevented cross-presentation and diminished major histocompatibility complex class II presentation of exogenous antigen; it also caused hypersusceptibility to infection by mouse cytomegalovirus and other microbes. By positional identification, we found 3d to be a missense allele of Unc93b1, which encodes the 12-membrane-spanning protein UNC-93B, a highly conserved molecule found in the endoplasmic reticulum with multiple paralogs in mammals. Innate responses to nucleic acids and exogenous antigen presentation, which both initiate in endosomes, thus seem to depend on an endoplasmic reticulum-resident protein, which suggests communication between these organellar systems.

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At the first auditory synapse in mammals, one ribbon-type AZ of the IHC drives one postsynaptic spiral ganglion neuron (SGN) to spike at rates exceeding 100 Hz in silence and 1 kHz upon sound onset 1, 2 . Moreover, SGNs sustain firing rates of several hundred Hz during ongoing acoustic stimulation. In such a steady state, vesicle replenishment has to balance vesicle fusion at the IHC AZ. Accordingly, high rates of initial and sustained exocytosis have been found in hair cells [3][4][5][6][7][8] . Ribbon-type AZs of IHCs replenish readily releasable vesicles at hundreds of Hz over several seconds of stimulation, faster than ribbon synapses in the eye 9-15 and most non-ribbon-type AZs 16(but see ref. 17). This efficient vesicle re-supply maintains a large standing pool of fusion competent synaptic vesicles, which appears to be critical for reliable and temporally precise sound encoding [18][19][20] . and Otof +/+ mice ( Fig. 2a-b The observations of normal RRP size after resting the synapse for more than 30 seconds and of reduced vesicle re-supply during ongoing stimulation prompted us to explore RRP recovery from depletion in paired-pulse experiments (Fig. 3a). RRP recovery, assessed as the paired-pulse ratio for different inter-pulse intervals, was impaired in Otof Pga/Pga mice (Fig. 3b). This indicated a deficit in vesicle replenishment also in the rest period between stimuli. The ΔC m pattern elicited by trains of ten short (10 ms) depolarizations demonstrates the exocytosis phenotype found after a period of rest (30 s voltage-clamp at -84 mV): normal RRP exocytosis but subsequent failure (Fig. 3c). Studying the C m decline after exocytosis we observed normal endocytic membrane retrieval (Fig. 3d). Normal synaptic ultrastructure in Otof Pga/Pga IHCsIn order to explore whether a docking or a priming defect underlies the impairment of vesicle replenishment at Otof Pga/Pga IHC synapses, we studied their ultrastructure using electron microscopy (EM). Both, EM of single ultrathin sections (perpendicular to the 7 plasma membrane and the long axis of the ribbon; Supplementary Fig. 2) as well as EM tomography (Fig. 3e-f High-resolution EM tomography ( Fig. 3e-f) was used to measure the distance of membrane-proximal synaptic vesicles (labeled orange in Fig. 3f) from the plasma membrane under both conditions. The average membrane-membrane distance was approximately 6 nm regardless of condition and genotype (Supplementary Reduced rates but maintained size variability of EPSCsThe stark contrast between the absence of auditory neuron population responses in vivo (Fig. 1) (Fig. 4a-b). We pooled the data from recordings in 5. Fig. 4h-i; Kolmogorov-Smirnov test, p = 0.14). Moreover, we detected action potential generation by recording action currents in the loose-patch configuration (Fig. 4g). Together these results suggest that Otof Pga/Pga synapses should be capable of encoding sound into spiking activity in auditory nerve fibers, albeit at lower rates.In addition, we recorded from SGNs of Otof -/-mice of the same age a...

The Collaborative Cross Consortium reports here on the development of a unique genetic resource population. The Collaborative Cross (CC) is a multiparental recombinant inbred panel derived from eight laboratory mouse inbred strains. Breeding of the CC lines was initiated at multiple international sites using mice from The Jackson Laboratory. Currently, this innovative project is breeding independent CC lines at the University of North Carolina (UNC), at Tel Aviv University (TAU), and at Geniad in Western Australia (GND). These institutions aim to make publicly available the completed CC lines and their genotypes and sequence information. We genotyped, and report here, results from 458 extant lines from UNC, TAU, and GND using a custom genotyping array with 7500 SNPs designed to be maximally informative in the CC and used a novel algorithm to infer inherited haplotypes directly from hybridization intensity patterns. We identified lines with breeding errors and cousin lines generated by splitting incipient lines into two or more cousin lines at early generations of inbreeding. We then characterized the genome architecture of 350 genetically independent CC lines. Results showed that founder haplotypes are inherited at the expected frequency, although we also consistently observed highly significant transmission ratio distortion at specific loci across all three populations. On chromosome 2, there is significant overrepresentation of WSB/EiJ alleles, and on chromosome X, there is a large deficit of CC lines with CAST/EiJ alleles. Linkage disequilibrium decays as expected and we saw no evidence of gametic disequilibrium in the CC population as a whole or in random subsets of the population. Gametic equilibrium in the CC population is in marked contrast to the gametic disequilibrium present in a large panel of classical inbred strains. Finally, we discuss access to the CC population and to the associated raw data describing the genetic structure of individual lines. Integration of rich phenotypic and genomic data over time and across a wide variety of fields will be vital to delivering on one of the key attributes of the CC, a common genetic reference platform for identifying causative variants and genetic networks determining traits in mammals.

Precise control of hematopoietic stem cell (HSC) proliferation and differentiation is needed to maintain a lifetime supply of blood cells. Using genome-wide ENU mutagenesis and phenotypic screening, we have identified a mouse line that harbors a point mutation in the transactivation (TA) domain of the transcription factor c-Myb (M303V), which reduces c-Myb-dependent TA by disrupting its interaction with the transcriptional coactivator p300. The biological consequences of the c-Myb(M303V/M303V) mutation include thrombocytosis, megakaryocytosis, anemia, lymphopenia, and the absence of eosinophils. Detailed analysis of hematopoiesis in c-Myb(M303V/M303V) mice reveals distinct blocks in T cell, B cell, and red blood cell development, as well as a remarkable 10-fold increase in the number of HSCs. Cell cycle analyses show that twice as many HSCs from c-Myb(M303V/M303V) animals are actively cycling. Thus c-Myb, through interaction with p300, controls the proliferation and differentiation of hematopoietic stem and progenitor cells.