The <i>mRx-Cre</i> activity in the eye, forebrain and hypothalamus analyzed using the <i>ROSA26R</i> reporter line.

<p>Whole-mounts or sections were stained with X-gal at indicated stages to show the <i>mRx-Cre</i>-mediated Cre activity. (A) The X-gal⁺ cells were first observed in the optic sulcus of E8.5 embryo. (B–D’) The Cre activity in developing brain. Whole-mounts (C, D, D’), coronal sections (C’, D’) and transversal section (C’) showing Cre activity in embryonic brain. (F) Coronal section of adult brain showing Cre activity in the hypothalamus and cortex. (E–E’) Sections through the adult eye showing strong uniform Cre activity in all layers of the retina. OS-optic sulcus; F-forebrain; GE-ganglionic eminences; H-hypothalamus; OB-olfactory bulbs, C-cortex; RPE-retinal pigmented epithelium; ONL-outer nuclear layer; INL-inner nuclear layer; GCL-ganglion cell layer.</p

Schematic representation of <i>Cre</i> recombinase-expressing transgenic mouse lines used in this study.

<p>(A) The <i>Rx-Cre</i>; with a 4-kb DNA fragment upstream of the medaka <i>Rx3</i> gene driving <i>Cre</i> expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063029#pone.0063029-Swindell1" target="_blank">[12]</a>. (B) To generate <i>MB31-Cre</i>, the 4-kb DNA fragment upstream of the medaka <i>Rx3</i> gene was linked to the coding region of <i>Cre</i> recombinase. The <i>IRES</i> sequence was used to connect <i>Cre</i> expression with EGFP to monitor the transgene expression. (C) To generate <i>mRx-Cre BAC</i>, a BAC containing 200 kb covering the <i>Rx</i> locus was modified by BAC recombineering. The <i>Cre</i> coding region (Cre-pA) was inserted into the <i>Rx</i> translational initiation start site (ATG). The exons are indicated with black boxes.</p

Activity of <i>Rx-Cre</i>, <i>MB31-Cre</i> and <i>mRx-Cre</i> in the eye primordium analyzed using the <i>ROSA26R-EYFP</i> reporter line.

<p>(A–C) Whole-mounts showing <i>EYFP</i> expression (green) in the overall embryo at E10.5. (A’–C’) Coronal sections through the eye region co-stained with DAPI (blue) showing Cre activity in the retina, retinal pigmented epithelium and invaginating lens pit (dashed line) at E10.5.</p

<i>In vivo</i> activity of <i>Rx-Cre</i>, <i>MB31-Cre</i> and <i>mRx-Cre</i> transgene products assessed using the <i>ROSA26R</i> line.

<p>Whole-mounts (A–F) or coronal sections (A’–F’) were stained with X-gal at indicated stages to show the Cre activity in the eye primordium (white arrowheads). Surface ectoderm and developing lens are indicated with dashed lines. SE – surface ectoderm; OV – optic vesicle; RPE – retinal pigmented epithelium; RE – retina; vf – ventral forebrain.</p

Hepatoprotective Effect of MMP-19 Deficiency in a Mouse Model of Chronic Liver Fibrosis

<div><p>Liver fibrosis is characterized by the deposition and increased turnover of extracellular matrix. This process is controlled by matrix metalloproteinases (MMPs), whose expression and activity dynamically change during injury progression. MMP-19, one of the most widely expressed MMPs, is highly expressed in liver; however, its contribution to liver pathology is unknown. The aim of this study was to elucidate the role of MMP-19 during the development and resolution of fibrosis by comparing the response of MMP-19-deficient (MMP19KO) and wild-type mice upon chronic liver CCl₄-intoxication. We show that loss of MMP-19 was beneficial during liver injury, as plasma ALT and AST levels, deposition of fibrillar collagen, and phosphorylation of SMAD3, a TGF-ß1 signaling molecule, were all significantly lower in MMP19KO mice. The ameliorated course of the disease in MMP19KO mice likely results from a slower rate of basement membrane destruction and ECM remodeling as the knockout mice maintained significantly higher levels of type IV collagen and lower expression and activation of MMP-2 after 4 weeks of CCl₄-intoxication. Hastened liver regeneration in MMP19KO mice was associated with slightly higher IGF-1 mRNA expression, slightly increased phosphorylation of Akt kinase, decreased TGF-ß1 mRNA levels and significantly reduced SMAD3 phosphorylation. In addition, primary hepatocytes isolated from MMP19KO mice showed impaired responsiveness towards TGF-ß1 stimulation, resulting in lower expression of Snail1 and vimentin mRNA. Thus, MMP-19-deficiency improves the development of hepatic fibrosis through the diminished replacement of physiological extracellular matrix with fibrotic deposits in the beginning of the injury, leading to subsequent changes in TGF-ß and IGF-1 signaling pathways.</p> </div

Generation of Spink5/Klk5/Klk7 mutant lines.

<p><b>(A)</b> Klk5^-/- line (depicted as Klk5^-/-Klk7^+/+Spink5^+/+), was used for preparation of Klk5^-/-Klk7^-/- mice (depicted as Klk5^-/-Klk7^-/-Spink5^+/+) by TALEN mutagenesis. Obtained Klk5^-/-Klk7^-/- mice were further crossed to a Flippase (FLPe) expressing mouse line to allow conditionally expressed Klk5, thus generating Klk7^-/- mice (depicted as Klk5^+/+(loxP)Klk7^-/-Spink5^+/+). Klk5^-/-, Klk7^-/- and Klk5^-/-Klk7^-/- lines were subsequently crossed with Spink5^+/- line to obtain Klk5^-/-Sp5^A135X/A135X, Klk7^-/-Sp5^A135X/A135X, and Klk5^-/-Klk7^-/-Sp5^A135X/A135X, i.e. double and triple KO lines. <b>(B)</b> Expression of Klk5, Klk7 and Spink5 at the mRNA level was quantified using qRT-PCR, n≥4 for each genotype, error bars represent standard deviations from mean.</p

Analysis of skin-barrier abnormalities.

<p><b>(A)</b> Newborn (P0) pups were analysed by barrier penetration assay using toluidine blue (TB). Remaining barrier-defect in the area of nostrils of Klk5^-/-Klk7^-/-Sp5^A135X/A135X pups in the vicinity of nostrils is marked by black arrowhead <b>(B)</b> TEWL analysis of P0 pups as a reduction of body-weight over time, n≥4 for each genotype. Error bars represent standard errors of mean; Klk/Spink5 mutant lines were compared with the wt line using Mann-Whitney U-test at 3h and 4h, ns means “not significant“; * p < 0.05 <b>(C)</b> Epidermal barrier in P5 Klk5^-/-Sp5^A135X/A135X pups was compromised in the proximity of hair follicles <b>(D)</b> Vibrissae hair (upper panel) and dorsal skin (lower panel) obtained from P5 pups analysed using scanning electron microscopy. Defective separation of hair shafts from the root sheath in Klk5^-/-Sp5^A135X/A135X is marked by white arrowhead. Dorsal skin of Klk5^-/-Sp5^A135X/A135X mice showed complete absence of hair shafts and exposed upper parts of hair follicles (yellow arrowheads); Scale bar, 300 μm.</p

6 week CCl₄ treatment results in lower liver damage in MMP19KOs compare to WTs.

<p>(A) After 4 and 6 week treatment with CCl₄, serum ALT and AST levels were significantly lower in MMP19KO than in WT mice. WT mice treated with olive oil are shown as control mice. (B) H&E staining showed larger bridging necrosis areas in WTs than in MMP19KOs. Sirius red staining showed more fibrillar collagen deposition in WTs than in MMP19KOs. (C) Liver hydroxyproline (OH-proline) content and image analysis of necrosis areas in H&E stained liver sections showed significantly more extensive damage in WTs than in MMP19KOs. Images were originally taken at 100× (H&E) and 50× (Sirius Red) magnification, scale bars = 200 µm. *p<0.05; **p<0.01; ***p<0.001 MMP19 vs. WT mice; n = 6 olive oil controls, 7 WT and 9 MMP19KO mice.</p

MMP19KOs show faster recovery from fibrosis than WTs.

<p>Mice treated with CCl₄ for 6 weeks were allowed 10 or 15 days for recovery. (A) After 15 days of recovery, significant parenchymal regeneration with less visible necrosis and fibrosis was observed in both WT and MMP19KO livers. (B) Fibrotic area (based on image analysis of Sirius red staining) was significantly lower in MMP19KOs after 15 days of recovery. (D) αSMA mRNA expression was slightly higher in WTs than in MMP19KOs throughout the course of liver damage progression but failed to drop during the recovery. Images were originally taken at 100× (H&E) and 50× (Sirius Red) magnification, scale bars = 200 µm. *p<0.05, MMP19s vs. WTs; 5–9 animals of each strain were analyzed at each time point.</p

Isolated hepatocytes from MMP19KO livers show impaired responsiveness to treatment with TGF-ß1.

<p>Primary hepatocytes were isolated from WT and MMP19KO livers, seeded on collagen I and stimulated with TGF-ß1. (A, B) mRNA levels of vimentin and Snail1 were measured in control, untreated hepatocytes, and hepatocytes stimulated with TGF-ß1 for 6, 24, or 48 h. (C) Phosphorylation of Akt was slightly increased in MMP19KO compared to WT hepatocytes. Noncontiguous lanes from one Western blot are shown. n = 5 different hepatocyte isolations from both WTs and MMP19KOs. (D) mRNA levels of Snail1 in livers from animals treated with CCl₄ for 4 weeks showed slightly lower expression of Snail1 in MMP19KOs than in WTs. 4–7 mice from each strain were analyzed for each time point. *p<0.05 MMP19KOs vs. WTs.</p