In inbred mouse strains, permissiveness to intracellular replication of Legionella pneumophila is controlled by a single locus (Lgn1), which maps to a region within distal Chromosome 13 that contains multiple copies of the gene baculoviral IAP repeat-containing 1 (Birc1, also called Naip; refs. 1-3). Genomic BAC clones from the critical interval were transferred into transgenic mice to functionally complement the Lgn1-associated susceptibility of A/J mice to L. pneumophila. Here we report that two independent BAC clones that rescue susceptibility have an overlapping region of 56 kb in which the entire Lgn1 transcript must lie. The only known full-length transcript coded in this region is Birc1e (also called Naip5).
Citrobacter rodentium is a natural mouse pathogen widely used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections in humans. While C. rodentium causes self-limiting colitis in most inbred mouse strains, it induces fatal diarrhea in susceptible strains. The physiological pathways as well as the genetic determinants leading to susceptibility have remained largely uncharacterized. Here we use a forward genetic approach to identify the R-spondin2 gene (Rspo2) as a major determinant of susceptibility to C. rodentium infection. Robust induction of Rspo2 expression during infection in susceptible mouse strains causes a potent Wnt-mediated proliferative response of colonic crypt cells, leading to the generation of an immature and poorly differentiated colonic epithelium with deficiencies in ion-transport components. Our data demonstrate a previously unknown role of R spondins and Wnt signaling in susceptibility to infectious diarrhea and identify Rspo2 as a key molecular link between infection and intestinal homeostasis.
Legionella pneumophila is an intracellular pathogen that causes Legionnaires’ disease in humans. Inbred mouse strains are uniformly resistant to L. pneumophila infection with the notable exception of A/J, where the chromosome 13 locus Lgn1 renders A/J macrophages permissive to L. pneumophila replication. The mouse Lgn1 region is syntenic with the spinal muscular atrophy (SMA) locus on human chromosome 5 and includes several copies of the neuronal apoptosis inhibitory protein (Naip) gene. We have analyzed a possible link among Lgn1, Naip, and macrophage function. RNA expression studies show that Naip (mostly copy 2) mRNA transcripts are expressed in macrophage-rich tissues, such as spleen, lung, and liver and are abundant in primary macrophages. Immunoblotting and immunoprecipitation analyses identify Naip protein expression in mouse macrophages and in macrophage cell lines RAW 264.7 and J774A. Interestingly, macrophages from permissive A/J mice express significantly less Naip protein than their nonpermissive C57BL/6J counterpart. Naip protein expression is increased after phagocytic events. Naip protein levels during infection with either virulent or avirulent strains of L. pneumophila increase during the first 6 h postinfection and remain elevated during the 48-h observation period. This enhanced expression is also observed in macrophages infected with Salmonella typhimurium. Likewise, an increase in Naip protein levels in macrophages is observed 24 h after phagocytosis of Latex beads. The cosegregation of Lgn1 and Naip together with the detected Naip protein expression in host macrophages as well as its modulation after phagocytic events and during intracellular infection make it an attractive candidate for the Lgn1 locus.
Expression of the Endothelin-2 (Edn2) mRNA is greatly increased in the photoreceptors (PRs) of mouse models of inherited PR degeneration (IPD). To examine the role of Edn2 in mutant PR survival, we generated Edn2−/− mice carrying homozygous Pde6brd1 alleles or the Tg(RHO P347S) transgene. In the Edn2−/− background, PR survival increased 110% in Pde6brd1/rd1 mice at post-natal (PN) day 15, and 60% in Tg(RHO P347S) mice at PN40. In contrast, PR survival was not increased in retinal explants of Pde6brd1/rd1; Edn2−/− mice. This finding, together with systemic abnormalities in Edn2−/− mice, suggested that the increased survival of mutant PRs in the Edn2−/− background resulted at least partly from the systemic EDN2 loss of function. To examine directly the role of EDN2 in mutant PRs, we used a scAAV5-Edn2 cDNA vector to restore Edn2 expression in Pde6brd1/rd1; Edn2−/− PRs and observed an 18% increase in PR survival at PN14. Importantly, PR survival was also increased after injection of scAAV5-Edn2 into Pde6brd1/rd1 retinas, by 31% at PN15. Together, these findings suggest that increased Edn2 expression is protective to mutant PRs. To begin to elucidate Edn2-mediated mechanisms that contribute to PR survival, we used microarray analysis and identified a cohort of 20 genes with >4-fold increased expression in Tg(RHO P347S) retinas, including Fgf2. Notably, increased expression of the FGF2 protein in Tg(RHO P347S) PRs was ablated in Tg(RHO P347S); Edn2−/− retinas. Our findings indicate that the increased expression of PR Edn2 increases PR survival, and suggest that the Edn2-dependent increase in PR expression of FGF2 may contribute to the augmented survival.
Retinal bipolar (BP) cells mediate the earliest steps in image processing in the visual system, but the genetic pathways that regulate their development and function are incompletely known. We identified PRDI-BF1 and RIZ homology domain containing 8 (PRDM8) as a highly conserved transcription factor that is abundantly expressed in mouse retina. During development and in maturity, PRDM8 is expressed strongly in BP cells and a fraction of amacrine and ganglion cells. To determine whether Prdm8 is essential to BP cell development or physiology, we targeted the gene in mice. Prdm8 EGFP/EGFP mice showed nonprogressive b-wave deficits on electroretinograms, consistent with compromised BP cell function or circuitry resembling the incomplete form of human congenital stationary night blindness (CSNB). BP cell specification was normal in Prdm8 EGFP/EGFP retina as determined by VSX2 + cell numbers and retinal morphology at postnatal day 6. BP subtype differentiation was impaired, however, as indicated by absent or diminished expression of BP subtype-specific markers, including the putative PRDM8 regulatory target PKCα (Prkca) and its protein. By adulthood, rod bipolar (RB) and type 2 OFF-cone bipolar (CB) cells were nearly absent from Prdm8-null mice. Although no change was detected in total amacrine cell (AC) numbers, increased PRKCA + and cholinergic ACs and decreased GABAergic ACs were seen, suggesting an alteration in amacrine subtype identity. These findings establish that PRDM8 is required for RB and type 2 OFF-CB cell survival and amacrine subtype identity, and they present PRDM8 as a candidate gene for human CSNB.retina | bipolar cell | amacrine cell | genetics | development R etinal bipolar (BP) cells are the first interneurons in the mammalian visual signaling pathway, connecting photoreceptors (PRs) to ganglion cells and then, through the optic nerve, to the brain. In mouse retina, 13 BP subtypes are distinguished by their (i) predominant presynaptic (rod or cone) input, (ii) morphology (axon length and terminal field width), (iii) functional response to increased illumination (depolarizing or ON-BP cells and hyperpolarizing or OFF-BP cells), and (iv) molecular markers (1). Mouse retina has only one type of rod BP (RB) cell, primarily postsynaptic to rod PRs, which extends its axon to the innermost sublamina of the inner plexiform layer (IPL) and depolarizes in response to increments in illumination. The axons of cone BP (CB) cells ramify throughout the IPL, but those that terminate in the outer sublaminae are functionally OFF-CB cells that hyperpolarize to light increments, whereas those that terminate in the inner sublaminae are ON-CB cells (1). Moreover, ON-and OFF-BP cells make direct and indirect synaptic connections with corresponding ON-and OFF-retinal ganglion cells (RGCs) in the IPL. The ON and OFF properties of BP cells result from differences in glutamate receptor activity on BP cell dendrites (1). Hence, the early integration of visual signals in the mammalian retina is determined by the specific syn...
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