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

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

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

Localization of hemidesmosomal components is restored in rescued <i>Lamc2</i> KO mice.

<p>Frozen skin sections of <i>Lamc2</i> KO (A–D), rescued <i>Lamc2</i> KO (E–H), and <i>Lamc2</i> WT (I–L) newborn mice were immunostained for skin hemidesmosomal components plectin (A, E, I), BP180/Col XVII (B, F, J), and integrin chains α6 (C, G, K) and ß4 (D, H, L). The immunostaining pattern for all hemidesmosomal proteins in the <i>Lamc2</i> KO mice appeared discontinuous, whereas the staining patterns in rescued <i>Lamc2</i> KO and <i>Lamc2</i> WT mice appeared more linear.</p

Schematic diagrams of the <i>Lamc2</i> allele and transgenes used in these studies and genotyping.

<p>(A) <i>Lamc2</i> null allele is generated by Cre recombinase, which removes exon 8 and the Neo-TK insert. Primer locations are indicated. (B) The K14-rtTA transgene contains a human keratin 14 (K14) promoter driving the reverse tetracycline transactivator (rtTA) and a SV40 poly A signal sequence. (C) The TetO-HuLamC2 transgene contains seven copies of the tetracycline operator (tetO) with a CMV minimal promoter driving the human laminin γ2 cDNA and a bovine growth hormone polyA signal sequence. The binding of doxycycline (Dox) to the rtTA promotes recruitment and binding to the tetO and activation of the promoter. (D) PCR analysis of genomic tail DNA of the <i>Lamc2</i> allele was performed using primers P1, P2, and P3. The mutant allele was detected with primer pair P1–P3, and the wild-type (WT) allele was detected using primer pair P2–P3. The K14-rtTA transgene was detected using K14- and rtTA-specific primers. The human laminin γ2 transgene was detected using primers specific to human laminin γ2. Mice that were a knockout for the <i>Lamc2</i> allele and carried both the K14-rtTA and the TetO-HuLamC2 transgenes (#9) were “rescued” <i>Lamc2</i> KO mice.</p

Expression of human laminin γ2 facilitates assembly of hemidesmosomes in rescued <i>Lamc2</i> KO mice.

<p>Transmission electron microscopic images of newborn skin of <i>Lamc2</i> KO (A), rescued <i>Lamc2</i> KO (B), and <i>Lamc2</i> WT (C) mice are shown. Hemidesmosomes of newborn <i>Lamc2</i> KO skin are poorly formed, devoid of lamina densa and anchoring filaments, and containing few anchoring fibrils (A). In contrast, rescued <i>Lamc2</i> KO (B) and <i>Lamc2</i> WT (C) mice had well-organized hemidesmosomes with electron dense plaques, anchoring filaments, anchoring fibrils, and darkened areas of lamina densa abutting the hemidesmosomes (arrows). All images are of the same magnification. Bar represents 500 nm.</p

Adult tissues of rescued <i>Lamc2</i> KO mice appear grossly similar to <i>Lamc2</i> WT controls.

<p>Paraffin-embedded tissue sections of adult <i>Lamc2</i> WT (A–H) and rescued <i>Lamc2</i> KO (A'–H') mice were stained with H&E. Despite a lack of laminin γ2 expression, the brain (A, A'), heart (B, B'), intestine (C, C'), kidney (D, D'), liver (E, E'), lung (F, F'), spleen (G, G'), and stomach (H, H') appear grossly similar between the <i>Lamc2</i> WT and rescued <i>Lamc2</i> KO mice.</p

Alterations in Lm-332 expression do not alter skin differentiation.

<p>Frozen skin sections of <i>Lamc2</i> KO (A–C), rescued <i>Lamc2</i> KO (D–F), and <i>Lamc2</i> WT (G–I) newborn mice were immunostained for skin differentiation markers loricrin (A, D, G), K10 (B, E, H), and K14 (C, F, I). No significant differences were detected in the staining patterns of these skin differentiation markers in <i>Lamc2</i> KO, the rescued <i>Lamc2</i> KO, and <i>Lamc2</i> WT mice. The epidermis of each of these mice displayed loricrin in the granular layer, K10 in the spinous layer, and K14 in the basal layer.</p

Expression of human laminin γ2 under the K14 promoter prevented blistering of rescued <i>Lamc2</i> KO mice.

<p>The paws (A, D, G), skin (B, E, H), and mouth (C, F, I) of <i>Lamc2</i> KO (A–C), rescued <i>Lamc2</i> KO (D–F), and <i>Lamc2</i> WT (G–I) newborn mice were examined. Skin blistering was most evident on the paws of <i>Lamc2</i> KO (A), but epidermal detachment (B) and separation of the oral mucosa of the roof palate and tongue (arrows in C) were detected microscopically after H&E staining. Blistering was not observed in the rescued <i>Lamc2</i> KO (D–F) or <i>Lamc2</i> WT (G–I) mice.</p