Morphology and body weights of <i>Kcnj13</i> null mutant mice.

<p>a. Analysis of Kir7.1 expression in WT, heterozygous and null mutant mice; cyclophilin A (Cyc1) is used as constitutively expressed control gene. b. Gross morphology of WT, and heterozygous and homozygous <i>Kcnj13</i> null mutant newborn pups. c. Body weight vs. embryonic stage for WT (circles), <i>Kcnj13</i>^<i>+/-</i> (triangles), and <i>Kcnj13</i>^-/- (squares) embryos. Results are expressed as mean ± S.E.M. of the following numbers of embryos: 12.5 dpc: WT 3, <i>Kcnj13</i>^<i>+/-</i> 9, <i>Kcnj13</i>^-/- 4; 13.5 dpc: WT 9, <i>Kcnj13</i>^<i>+/-</i> 14, <i>Kcnj13</i>^-/- 2; n 14.5 dpc: WT 3, <i>Kcnj13</i>^<i>+/-</i> 11, <i>Kcnj13</i>^-/- 6; 15.5 dpc: WT 7, <i>Kcnj13</i>^<i>+/-</i> 20, <i>Kcnj13</i>^-/- 14; 16.5 dpc: WT 7, <i>Kcnj13</i>^<i>+/-</i> 5, <i>Kcnj13</i>^-/- 4; 17.5 dpc:WT 5, <i>Kcnj13</i>^<i>+/-</i> 12, <i>Kcnj13</i>^-/- 6; 18.5 dpc: WT 4, <i>Kcnj13</i>^<i>+/-</i> 5, <i>Kcnj13</i>^-/- 6; P0: WT 7, <i>Kcnj13</i>^<i>+/-</i> 15, <i>Kcnj13</i>^-/- 10. * p< 0.001; ** p <0.05 for the differences between <i>Kcnj13</i>^-/- and <i>Kcnj13</i>^<i>+/+</i> data (ANOVA).</p

Basolateral expression of Kir7.1 channel in the epithelium of the airways.

<p>Immunohistochemical detection of Kir7.1 channel in trachea (left) and bronchiole (right) in adult <i>Kcnj13</i>^+/+, newborn <i>Kcnj13</i>^+/+ or newborn <i>Kcnj13</i>^-/- mice. Tissue sections were treated with anti-Kir7.1 antibody (1:15,000). Kir7.1 expression was restricted to the basolateral membrane of airway epithelium in adult and newborn <i>Kcnj13</i>^+/+ mice. Staining in <i>Kcnj13</i>^-/- tissues shows complete absence of specific immunoreactive signal. Nuclei were counterstained with Fast Red. Scale bar represents 50 μm.</p

Pulmonary abnormalities in embryonic lungs from <i>Kcnj13</i>^-/- mice.

<p>a. Hematoxylin and eosin stained lung sections taken at various gestational stages as indicated. Morphological differences in KO lungs were observed at E18.5 and P0. Null mutant mice show a lower air space and thicker walls at lung terminal sacs compared to WT and heterozygous mice. No differences were visible between <i>Kcnj13</i>^+/+ and <i>Kcnj13</i>^+/- genotypes. Scale bars represent 100 μm. b. Morphometric analysis of terminal sac spaces in lungs at various gestational stages. Significant reduction in spaces was observed in Kir7.1 deficient mice from E18.5 onwards. Results are expressed as mean ± S.E.M, # p<0.05 and * p<0.01 for the difference with WT by ANOVA. c. Graphical representation of newborn lung flotation test. Grey sections of columns correspond to percent of floating lungs, with black being the percent sinking lungs.</p

Survival of <i>Kcnj13</i>^- mutant mice.

<p>Survival of <i>Kcnj13</i>^- mutant mice.</p

Expression of receptor-type protein tyrosine phosphatase in developing and adult renal vasculature

<div><p>Renal vascular development is a coordinated process that requires ordered endothelial cell proliferation, migration, intercellular adhesion, and morphogenesis. In recent decades, studies have defined the pivotal role of endothelial receptor tyrosine kinases (RPTKs) in the development and maintenance of renal vasculature. However, the expression and the role of receptor tyrosine phosphatases (RPTPs) in renal endothelium are poorly understood, though coupled and counterbalancing roles of RPTKs and RPTPs are well defined in other systems. In this study, we evaluated the promoter activity and immunolocalization of two endothelial RPTPs, VE-PTP and PTPμ, in developing and adult renal vasculature using the heterozygous LacZ knock-in mice and specific antibodies. In adult kidneys, both VE-PTP and PTPμ were expressed in the endothelium of arterial, glomerular, and medullary vessels, while their expression was highly limited in peritubular capillaries and venous endothelium. VE-PTP and PTPμ promoter activity was also observed in medullary tubular segments in adult kidneys. In embryonic (E12.5, E13.5, E15.5, E17.5) and postnatal (P0, P3, P7) kidneys, these RPTPs were expressed in ingrowing renal arteries, developing glomerular microvasculature (as early as the S-shaped stage), and medullary vessels. Their expression became more evident as the vasculatures matured. Peritubular capillary expression of VE-PTP was also noted in embryonic and postnatal kidneys. Compared to VE-PTP, PTPμ immunoreactivity was relatively limited in embryonic and neonatal renal vasculature and evident immunoreactivity was observed from the P3 stage. These findings indicate 1) VE-PTP and PTPμ are expressed in endothelium of arterial, glomerular, and medullary renal vasculature, 2) their expression increases as renal vascular development proceeds, suggesting that these RPTPs play a role in maturation and maintenance of these vasculatures, and 3) peritubular capillary VE-PTP expression is down-regulated in adult kidneys, suggesting a role of VE-PTP in the development of peritubular capillaries.</p></div

VE-PTP promoter activity in adult mouse kidney.

<p><b>(A)</b> Whole mount X-gal staining of adult VE-PTP^tlacZ/+ mouse kidney. Evident β-galactosidase activity is observed in renal cortex (C), inner stripe (IS) of outer medulla, inner medulla (IM), and papilla (P). Right panel shows vascular distribution of β-galactosidase activity in renal cortex. C, cortex; OS, outer stripe; IS, inner stripe; IM, inner medulla; P, papilla. <b>(B)</b> β-galactosidase activity in adult VE-PTP^tlacZ/+ mouse kidney sections. In medulla, β-galactosidase activity is observed in vascular bundle (VB), subpopulations of medullary tubules (red arrows in panels c and d), and papillary cells (panel e). VB, vascular bundle. <b>(C)</b> β-galactosidase activity in cortical renal vasculature. RA, renal artery: RV, renal vein; AA, arcuate artery; AV, arcuate vein; IA, interlobular artery; IV, interlobular vein; Art, arteriole; G, glomerulus; af, afferent arteriole; ef, efferent arteriole. Note: VE-PTP promoter activity is limited in efferent arterioles, peritubular capillaries, and venous circulations. Scale bar, 200 μm in B-a and B-b; 100 μm in B-c, B-d, B-e, and C; 50 μm in an insert of C.</p

Anti-CD31 immunohistochemistry combined with β-galactosidase histochemistry of developing VE-PTP^tlacZ/+ and PTPμ^tlacZ/+ mice kidneys.

<p><b>(A through D)</b> Immunohistochemistry for CD31 was superimposed on β-galactosidase histochemistry in developing VE-PTP^tlacZ/+ (A, C) and PTPμ^tlacZ/+ (B, D) mice kidneys. Panels A and B show cortical area. Panels C and D display medullary region. In both mice, CD31 and β-galactosidase activity are colocalized in developing glomerular capillaries, including vascular clefts of S-shaped glomeruli (red arrows), and developing medullary vessels (black arrowheads in panels C and D). In VE-PTP^tlacZ/+ kidneys, β-galactosidase activity is also observed in endothelial cells forming peritubular capillaries (red arrowheads in panel A). Notes: β-galactosidase activity is observed in medullary tubular segments (black arrows) that are not labelled with CD31 in P0-stage VE-PTP^tlacZ/+ mouse kidney. Scale bar, 25 μm in A, C; 50 μm in B, D.</p

VE-PTP promoter activity in developing mouse kidneys.

<p><b>(A through L)</b> β-galactosidase histochemistry of developing VE-PTP^tlacZ/+ mouse kidneys. In embryonic kidneys, evident VE-PTP activity is observed in penetrating and ingrowing renal arteries (black arrows in panels A, B, C, D, and J) and maturing glomeruli (G) (panels D and J). VE-PTP promoter activity is also observed in developing medullar vessels (arrowheads in panel G). In postnatal kidneys, VE-PTP promoter activity is observed in renal arterial vessels (panels E and F), maturing glomeruli (G) (panels E, F, and K), and medullary vessels (arrow heads in panels H and I) and tubular subpopulations (arrows in panels H and I). Note: VE-PTP promoter activity is observed in the cells intermittently distributed around developing glomeruli and tubules (red arrowheads in panel J) and in peri-tubular capillaries in postnatal kidneys (red arrows in panel K). The peritubular capillary VE-PTP promoter activity is down-regulated in 3-week old mouse kidney (panel L). RA, renal artery; G, glomerulus; IA, interlobular artery; V, venous vessel; T, developing tubules. Scale bar, 100 μm in A through I; 50 μm in J through L.</p

VE-PTP promoter activity in adult mouse kidney.

<p><b>(A)</b> Whole mount X-gal staining of adult VE-PTP^tlacZ/+ mouse kidney. Evident β-galactosidase activity is observed in renal cortex (C), inner stripe (IS) of outer medulla, inner medulla (IM), and papilla (P). Right panel shows vascular distribution of β-galactosidase activity in renal cortex. C, cortex; OS, outer stripe; IS, inner stripe; IM, inner medulla; P, papilla. <b>(B)</b> β-galactosidase activity in adult VE-PTP^tlacZ/+ mouse kidney sections. In medulla, β-galactosidase activity is observed in vascular bundle (VB), subpopulations of medullary tubules (red arrows in panels c and d), and papillary cells (panel e). VB, vascular bundle. <b>(C)</b> β-galactosidase activity in cortical renal vasculature. RA, renal artery: RV, renal vein; AA, arcuate artery; AV, arcuate vein; IA, interlobular artery; IV, interlobular vein; Art, arteriole; G, glomerulus; af, afferent arteriole; ef, efferent arteriole. Note: VE-PTP promoter activity is limited in efferent arterioles, peritubular capillaries, and venous circulations. Scale bar, 200 μm in B-a and B-b; 100 μm in B-c, B-d, B-e, and C; 50 μm in an insert of C.</p

Co-immunostaining of VE-PTP and CD31 in developing and adult mice kidneys.

<p>Kidney sections from E16.5, P0 and adult mice were double immunolabeled for VE-PTP (red) and CD31 (green) as described in the “Materials & Methods”. In developing kidneys (E16.5 and P0), VE-PTP is expressed in endothelial cells in ingrowing arteries (A) and developing glomeruli (G), which are labeled with CD31. VE-PTP is also expressed in endothelial cells (yellow arrows) that distribute around the developing nephrons. In adult kidney, VE-PTP is expressed in endothelial cells in arterial and glomerular vasculature, while its expression is limited in peritubular capillaries. A, arterial vessel; IA, interlobular artery; G, glomerulus. Scale bar, 50 μm in E16.5 and P0 kidneys; 25 μm in adult kidney.</p