Gardnerella exposures alter bladder gene expression and augment uropathogenic Escherichia coli urinary tract infection in mice

The anaerobic actinobacteriu

A Retrotransposon Insertion in the 5' Regulatory Domain of Ptf1a Results in Ectopic Gene Expression and Multiple Congenital Defects in Danforth’s Short Tail Mouse

Danforth's short tail mutant (Sd) mouse, first described in 1930, is a classic spontaneous mutant exhibiting defects of the axial skeleton, hindgut, and urogenital system. We used meiotic mapping in 1,497 segregants to localize the mutation to a 42.8-kb intergenic segment on chromosome 2. Resequencing of this region identified an 8.5-kb early retrotransposon (ETn) insertion within the highly conserved regulatory sequences upstream of Pancreas Specific Transcription Factor, 1a (Ptf1a). This mutation resulted in up to tenfold increased expression of Ptf1a as compared to wild-type embryos at E9.5 but no detectable changes in the expression levels of other neighboring genes. At E9.5, Sd mutants exhibit ectopic Ptf1a expression in embryonic progenitors of every organ that will manifest a developmental defect: the notochord, the hindgut, and the mesonephric ducts. Moreover, at E 8.5, Sd mutant mice exhibit ectopic Ptf1a expression in the lateral plate mesoderm, tail bud mesenchyme, and in the notochord, preceding the onset of visible defects such as notochord degeneration. The Sd heterozygote phenotype was not ameliorated by Ptf1a haploinsufficiency, further suggesting that the developmental defects result from ectopic expression of Ptf1a. These data identify disruption of the spatio-temporal pattern of Ptf1a expression as the unifying mechanism underlying the multiple congenital defects in Danforth's short tail mouse. This striking example of an enhancer mutation resulting in profound developmental defects suggests that disruption of conserved regulatory elements may also contribute to human malformation syndromes

Noninvasive Assessment of Antenatal Hydronephrosis in Mice Reveals a Critical Role for Robo2 in Maintaining Anti-Reflux Mechanism

Antenatal hydronephrosis and vesicoureteral reflux (VUR) are common renal tract birth defects. We recently showed that disruption of the Robo2 gene is associated with VUR in humans and antenatal hydronephrosis in knockout mice. However, the natural history, causal relationship and developmental origins of these clinical conditions remain largely unclear. Although the hydronephrosis phenotype in Robo2 knockout mice has been attributed to the coexistence of ureteral reflux and obstruction in the same mice, this hypothesis has not been tested experimentally. Here we used noninvasive high-resolution micro-ultrasonography and pathological analysis to follow the progression of antenatal hydronephrosis in individual Robo2-deficient mice from embryo to adulthood. We found that hydronephrosis progressed continuously after birth with no spontaneous resolution. With the use of a microbubble ultrasound contrast agent and ultrasound-guided percutaneous aspiration, we demonstrated that antenatal hydronephrosis in Robo2-deficient mice is caused by high-grade VUR resulting from a dilated and incompetent ureterovesical junction rather than ureteral obstruction. We further documented Robo2 expression around the developing ureterovesical junction and identified early dilatation of ureteral orifice structures as a potential fetal origin of antenatal hydronephrosis and VUR. Our results thus demonstrate that Robo2 is crucial for the formation of a normal ureteral orifice and for the maintenance of an effective anti-reflux mechanism. This study also establishes a reproducible genetic mouse model of progressive antenatal hydronephrosis and primary high-grade VUR

Polyploid Superficial Cells that Maintain the Urothelial Barrier Are Produced via Incomplete Cytokinesis and Endoreplication

Summary: The urothelium is an epithelia barrier lined by a luminal layer of binucleated, octoploid, superficial cells. Superficial cells are critical for production and transport of uroplakins, a family of proteins that assemble into a waterproof crystalline plaque that helps protect against infection and toxic substances. Adult urothelium is nearly quiescent, but rapidly regenerates in response to injury. Yet the mechanism by which binucleated, polyploid, superficial cells are produced remains unclear. Here, we show that superficial cells are likely to be derived from a population of binucleated intermediate cells, which are produced from mononucleated intermediate cells via incomplete cytokinesis. We show that binucleated intermediate and superficial cells increase DNA content via endoreplication, passing through S phase without entering mitosis. The urothelium can be permanently damaged by repetitive or chronic injury or disease. Identification of the mechanism by which superficial cells are produced may be important for developing strategies for urothelial repair. : Binucleated superficial cells are critical for urothelial barrier function. Wang et al. show that they derive from binucleated intermediate cells that form via incomplete cytokinesis. Both superficial and intermediate cells increase ploidy via endoreplication, a feature likely to be important for repair and response to environmental changes. Keywords: endoreplication, urothelium, polyploidy, epithelial barrier, regeneratio

Blocking Ecm1 function alters <i>Ret</i> expression and branching.

<p>In vitro cultured kidneys from Hoxb7-GFP mice grown either in the absence (A, C, E and G) or in the presence(B, D, F and H) of the Ecm1 blocking antibody. A–D: Kidney rudiments were subjected to insitu hybridization and probed for <i>Ret</i> expression. E–H: Hoxb7-GFP kidneys showing branching of the ureteric bud tip. C and D: Arrowheads indicate the expression of <i>Ret</i> in the ub tips (C) or in the clefts (D).</p

A) Stromal cell distribution depends on <i>Ret</i> and branching.

<p>Histological sections of Foxd1-LacZ kidneys during embryonic development showing the distribution of stromal cells (blue) in the cortical region of the kidney (a–f). (a) Ampulla stage of ureteric bud (shown in pink, ureteric bud, ub) surrounded by nephron progenitor cells (pink, condensing mesenchyme, cm) and Foxd1- lacZ stromal cells (blue). (b) Growing ampulla surrounded by nephron progenitors at the tips and cleft (yellow arrow head) at the center occupied by stromal cells. (d–e) Stromal cells occupy the cleft made by the bifurcating ureteric bud tips. (f) <i>Ret</i> expression in the ureteric bud tips (white arrows). B) In vitro cultures of Hoxb7-GFP and Foxd1-GFP embryonic kidneys grown either in the presence of RA (culture beads with 10 µg/mL of RA) (a–i) or in the absence of RA (culture beads with basal media) (j–r) for 24, 48 and 72 hours. Hoxb7-GFP kidney cultures were probed for <i>Ret</i> expression after culture at 24, 48 and 72 hours (a–c and j–l). Hoxb7-GFP kidneys showing ureteric bud branching (d–f and m–o). Foxd1-GFP kidneys showing stromal cell distribution (g–i and p–r), asterisks show the ureteric buds with stromal cells around them.</p

Microarray data depicting stromal genes regulated by retinoic acid (>1.5 fold change in expression); also shown retinoic acid suppressed genes.

<p>Microarray data depicting stromal genes regulated by retinoic acid (>1.5 fold change in expression); also shown retinoic acid suppressed genes.</p

Retinoic acid controls stromal cell fate: A–F: Histological sections of wild type, <i>Rara</i>; <i>Rarb2</i> double mutant and <i>Raldh2</i> embryonic kidneys.

<p>A and B: sections of wild type kidneys showing normal distribution of the nephrons. NZ- nephrogenic zone: region under the renal capsule where continuous branching of ub and induction of nephron occurs. DZ- differentiating zone: region containing medullary stroma, differentiating nephrons and collecting duct branches. C, D and E, F: sections of <i>Rara^−/−; Rarβ^−/−</i> and <i>Raldh2</i> kidneys respectively showing reduced NZ region. In situ hybridization in cultured kidneys (G and H): <i>Foxd1LacZ</i> in vitro cultured kidneys probed for <i>Ret</i> expression. Expression of <i>FoxD1-lacZ</i> (blue) in the stromal cells of kidneys cultured in the presence of RA (G) or in the absence of RA (H) for 72 hours. Expression of <i>Ret</i> in the ureteric buds (shown in purple in G) is absent in kidneys cultured in the absence of RA (H). The expression of the stromal marker <i>Foxd1-LacZ</i> is shown in blue (G and H) in kidneys grown on RA+ or RA− media. I–L: wild type in vitro cultured kidneys; cultured in RA+ or RA− media. I and J: kidneys cultured for 24 hours and K and L kidneys cultured for 48 hours. Immunostaining with TUNEL to detect apoptotic cells (labeled in blue, I–L). Arrowheads in G and H indicate stromal cells, which are depleted in H. Arrowheads in J and L represent TUNEL positive cells which are increased in L.</p

Validation of Microarray data. Kidneys cultured in the presence of RA (A, B, C) or absence of RA (F, G, H) and subjected to in situ hybridization of the various genes.

<p>Ecm1 is dependent on retinoic acid and regulates <i>Ret</i> expression in the ureteric bud clefts. <i>Ecm1</i> expression pattern in wild type (D wholemount and E section) and <i>Raldh2</i> kidneys (I wholemount and J section).</p