Analysis of placental labyrinth vasculature at E14.5.

<p>Frozen sections of placenta were stained with antibodies to LM-111 to label all basement membranes, to cytokeratin 8 (CK8) to label trophoblasts (green in A–C, A′–C′), and to PECAM to label endothelial cells (green in D–F, D′–F′). The reduced vascular complexity in the <i>LMα5−/−</i> labyrinth (B, E) was rescued and made similar to normal (A, D) by hLMα5 secretion from <i>LMα5−/−;ROSA26TA;hLMα5;Tie2cre</i> endothelial cells (C, F) exposed to doxycycline.</p

Mosaic placental labyrinths containing <i>LMα5</i>−/− trophoblasts and hLMα5-expressing endothelial cells show hLMα5 deposition and normal vascularization.

<p>(A) Schematic diagram of the strategy for forcing expression of hLMα5 in endothelial cells on the <i>LMα5−/−</i> background. Cre recombinase driven by the Tie2 promoter removes a floxed STOP located between the <i>Rosa26</i> promoter and the reverse tetracycline transactivator (rtTA). rtTA binds and activates the tetracycline-inducible TetO₇ promoter in the presence of doxycycline, thereby driving transcription of the hLMα5 cDNA in endothelial cells. (B–G) <i>LMα5−/−;ROSA26TA;hLMα5;Tie2cre</i> embryos (top panels) were compared with <i>LMα5−/−</i> embryos (bottom panels). Mouse LMα5 was undetectable in kidney (B, B′) or placenta (E, E′). Human LMα5 was detected in both kidney and placental vasculatures of <i>LMα5−/−;ROSA26TA;hLMα5;Tie2cre</i> embryos (C, F) but not of <i>LMα5−/−</i> embryos (C′,F′), both of which show the typical <i>LMα5</i> null phenotype (D, D′). Expression of hLMα5 in endothelial cells was associated with a normalized placental labyrinth architecture, demonstrated by the LM-111 antibody staining pattern (compare G and G′).</p

Mosaic placental labyrinths containing wild-type trophoblasts and <i>LMα5</i>−/− endothelial cells show LMα5 deposition and normal vascularization.

<p>(A, B) Schematic diagrams of the strategy for conditional mouse <i>LMα5</i> mutation. Using the Cre/loxP system, we generated <i>LMα5^flox/ko</i>; Sox2Cre embryos. Sox2cre, when inherited from a male, is active in epiblast, but not in trophectoderm. Thus, epiblast-derived cells (A), which include the embryo proper as well as extraembryonic endothelial cells, are not able to synthesize LMα5, but trophoblasts, which derive from trophectoderm (B), can. (C–H; C′–H′) Analysis of LMα5 expression and tissue architecture in control (top rows) and <i>LMα5^flox/ko</i>; Sox2cre mutant (bottom rows) embryos. LMα5 was not expressed in the kidney of <i>LMα5^flox/ko</i>; Sox2cre embryos (C′; counterstained with anti-nidogen in D′; compare with control, C and D), which show developmental abnormalities typical of <i>LMα5</i>−/− embryos (E′; arrows indicate exencephaly and syndactyly) not seen in control (E). In contrast, LMα5 was present in the placental labyrinth of <i>LMα5^flox/ko</i>; Sox2cre embryos (F′) and of control (F), and placental LM-111 and PECAM expression and localization were similar to those observed in control <i>LMα5+/</i>− placenta (G–H, G′–H′). Cytokeratin 8 (CK8) was used to identify trophoblasts (G, G′).</p

LMα5 is expressed in both endothelial cells and trophoblasts in the normal placenta.

<p>(A) Fluorescence-activated cell sorting was performed on dissociated E18.5 wild-type labyrinth cells after staining with a phycoerythrin (PE)-conjugated CD31/PECAM antibody. CD31+ (endothelial cell) and CD31− (trophoblast; indicated as baseline) populations were collected. (B) RT-PCR using RNA prepared from the two cell types showed that LMα5 was expressed in both: Lane 1, DNA marker; 2 and 3, LMα5 in CD31(−) and (+) cells, respectively; 4, negative control; 5 and 6, GAPDH in CD31(−) and (+) cells, respectively. (C) RNA was subjected to real time RT-PCR to quantitate the levels of laminin α1 (lama1), α5 (lama5), β1 (lamb1), and β2 (lamb2) mRNAs. Error bars represent standard deviations.</p

<i>Pdx1-Cre;LSL-Kras</i>^{<i>G12D/+</i>}<i>;Trp53</i>^{<i>flox/wt</i>}<i>;Ksr1</i>^<i>-/-</i> mice develop PanIN lesions at similar rates and severity to <i>Ksr1</i>^<i>+/-</i> and <i>Ksr1</i>^<i>+/+</i> mice.

<p>H&E staining of pancreatic tissues from <i>Pdx1-Cre</i>;<i>LSL</i>-<i>Kras</i>^{<i>G12D/</i>+}<i>;Trp53</i>^{<i>flox/wt</i>}<i>;Ksr1</i>^<i>+/+</i>, <i>Ksr1</i>^{<i>+/ -</i>}, and <i>Ksr1</i>^<i>-/-</i> mice sacrificed at 3 months of age highlights PanIN lesions surrounded by normal tissue (bar = 40μm).</p

<i>Pdx1-Cre</i>;<i>LSL</i>-<i>Kras</i>^{<i>G12D/</i>+}<i>;Trp53</i>^{<i>flox/wt</i>}<i>;Ksr1</i>^<i>-/-</i> mice have a modest but statistically significant decrease in all-cause morbidity.

<p>A. Kaplan-Meier curves for <i>Pdx1-Cre</i>;<i>LSL</i>-<i>Kras</i>^{<i>G12D/</i>+}<i>;Trp53</i>^{<i>flox/wt</i>}<i>;Ksr1</i>^<i>+/+</i> and <i>Ksr1</i>^<i>+/-</i> mice based on age at sacrifice or death. 8 <i>Ksr1</i>^<i>+/-</i> and 10 <i>Ksr1</i>^<i>+/+</i> mice had to be censored. Median age at sacrifice or death was 152 days for <i>Ksr1</i>^<i>+/-</i> mice and 160 days for <i>Ksr1</i>^<i>+/+</i>mice; there was no statistically significant difference between the two groups (p = 0.4683 by log-rank test). B. Kaplan-Meier curves for <i>Pdx1-Cre</i>;<i>LSL</i>-<i>Kras</i>^{<i>G12D/</i>+}<i>;Trp53</i>^{<i>flox/wt</i>}<i>;Ksr1</i>^<i>-/-</i>, and <i>Ksr1</i>^<i>+/-</i> combined with <i>Ksr1</i>^<i>+/+</i> mice based on age at sacrifice or death. 3 <i>Ksr1</i>^<i>-/-</i> were censored. There was a modest but statistically significant difference between median age at sacrifice or death for <i>Ksr1</i>^<i>-/-</i> mice and the control group (191 and 159 days, p = 0.0344 by log-rank test).</p

<i>Pdx1-Cre;LSL-Kras</i>^{<i>G12D/+</i>}<i>;Trp53</i>^{<i>flox/wt</i>}<i>;Ksr1</i>^<i>-/-</i> mice develop pancreatic tumors.

<p>H&E (top), pERK (middle) and Ki67 (bottom) staining of tumors harvested from <i>Pdx1-Cre</i>;<i>LSL</i>-<i>Kras</i>^{<i>G12D/</i>+}<i>;Trp53</i>^{<i>flox/wt</i>}<i>;Ksr1</i>^<i>+/+</i>, <i>Ksr1</i>^<i>+/-</i>, and <i>Ksr1</i>^<i>-/-</i> mice (bar = 100μm). Panels to the right show magnification of indicated region (bar = 40μm). Tumors have moderately differentiated ductal morphology that stains strongly for pERK, accompanied by stromal desmoplasia. Ki67 stains some ductal and surrounding cells.</p

<i>Scribble^Δpodocyte</i> mice develop normal podocyte foot processes and show no increased susceptibility to glomerular stress.

<p>(<b>A, B</b>) No obvious histological abnormalities can be detected in PAS staining of <i>Scribble^Δpodocyte</i> kidney sections at P1 and at 6 weeks of age compared to control littermates. (<b>C, D</b>) Transmission electron micrographs display normal podocyte architecture with foot processes without any obvious ultrastructural defect. (<b>E</b>) No difference in the number of podocytes per sectioned glomerulus could be detected (ns, not significant, n = 3 each, 30 glomeruli for each mouse were analyzed). (<b>F</b>) During a one year follow up <i>Scribble^Δpodocyte</i> mice develop no significant albuminuria. (<b>G</b>) No difference in albuminuria can be detected between <i>Scribble^Δpodocyte</i> mice and control littermates in the BSA-overload model (n = 5 each) and (<b>H</b>) the ADR model (n = 6 each). Scale bars: 20 µm in (A) and (B), 1 µm in (C) and (D).</p

Migration of apical and basolateral polarity proteins during podocyte differentiation.

<p>Frozen kidney sections of newborn Wistar rat (P0) were stained using antibodies against the apical membrane protein Podocalyxin, the apical polarity protein Par3 and the basolateral polarity protein Scribble and were subjected to confocal laser microscopy. Since glomerular development is asynchronous, kidneys of newborn rats display various glomerular developmental stages. Each panel displays the expression pattern of the accordant proteins during glomerular development (from left to right): Developmental stages ranging from comma-shaped body (I), s-shaped body (II), capillary loop stage (III to IV), to a maturing glomerulus (V). (<b>A</b>) Whereas Par3 is expressed during comma-shaped body stage and localizes to the apical sited cell-cell junctions, expression of Podocalyxin starts during s-shaped body stage, when Par3 and the cell-cell contacts translocate along the lateral side of immature podocytes to basal. During this translocation the apical membrane area, marked by Podocalyxin, increases while the basolateral membrane area shrinks relatively. Arrows indicate translocation of Par3 from the apical cell-cell contacts in I to the developing foot processes in V. (<b>B</b>) Scribble localizes basal of Par3 at the cell-cell junctions and at the basolateral membrane during comma-shaped body stage (I) and translocates like Par3 during podocyte differentation to the developing foot processes in V. (<b>C</b>) While Podocalyxin and Par3 as well as Par3 and Scribble display an partial overlap of their localization (yellow in A and B), no overlap of Podocalyxin and Scribble can be detected indicating a localization to completely distinct membrane areas with Podocalyxin as an apical membrane marker and Scribble as a basolateral marker protein. Scale bars: 5 µm.</p

Generation of podocyte-specific Scribble knockout mice, <i>Scribble^Δpodocyte</i>.

<p><i>Scribble^flox/flox</i> mice were crossed with <i>NPHS2.Cre</i> mice to generate podocyte-specific <i>Scribble</i> knockout mice <i>Scribble^flox/flox; NPHS2.Cre</i> (<i>Scribble^Δpodocyte</i>). (<b>A</b>) Generation of tissue-specific <i>Scribble</i> knockout mice. (<b>B</b>) PCR analysis of genomic DNA from isolated glomeruli confirmed genomic deletion of exon 2–8 in <i>Scribble^Δpodocyte</i> mice. (<b>C, D</b>) Frozen kidney sections of control and <i>Scribble^Δpodocyte</i> mice were stained using antibodies against Scribble and the podocyte foot process marker Nephrin and were subjected to confocal laser microscopy. Arrows indicate the podocyte foot process compartment. While Scribble localizes to the podocyte foot process compartment of control mice, no Scribble expression can be detected in podocytes of <i>Scribble^Δpodocyte</i> mice. Scale bars: 5 µm.</p