Normal kidney development in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A-B: H&E stained kidney sections from day E17.5 showing normal development both in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice and controls (X200).</p

Lung phenotype of <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A–B: Representative H&E stained sections showing normality of lung (X200) morphology in <i>UPK II-Cre;LSL-K-ras^G12D</i> when compared with controls at E14.5 (pseudoglandular stage). <b>C–H</b>: Formation of air spaces was impaired in lungs (X100) of <i>UPK II-Cre;LSL-K-ras^G12D</i> mice at E17.5 (<b>C,D</b>) and E19.5 (<b>E,F</b>), reflecting a defective septation process. P1 lung morphology was also different between controls and transgenic mice, which exhibited an enlargement and simplification of sacculi (<b>G,H</b>). <b>I–J</b>: Morphometric analysis of lung sections showed a decreased total air space (<b>I</b>) and mean alveolar (saccular) area (<b>J</b>) in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice at E17.5 (n = 5); the impairment of air space development led to an increased total air space area and mean alvelolar area in the early postnatal period (P1; n = 12) in transgenic mice when compared to controls; bars, SEM. <b>K–L</b>: Masson tri-chrome staining of day P1 <i>UPK II-Cre;LSL-K-ras^G12D</i> lungs (X100), showing absence of fibrosis both in cases and controls. <b>M</b>: The direct expression of K<i>-ras^G12D</i> was ruled out with specific PCR showing the absence of recombination between <i>K-ras</i> and the Lox sequence in lung and placenta.</p

Disorganization of extracellular matrix and blood vessels in the lungs of <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A-F: Representative images of immunohistochemistry for pan-laminin (X400) (<b>A,B</b>), and immunofluorescence for entactin (X630) (<b>C,D</b>), showed a different pattern in E17.5 lung from <i>UPK II-Cre;LSL-K-ras^G12D</i> mice, with a less organized network and stronger expression in the stroma. Immunofluorescence for collagen IV (X630) (<b>E,F</b>) showed a similar, but less prominent, pattern. <b>G-J</b>: CD34 staining (<b>G,H</b>) and CD31 immunofluorescence (<b>I,J</b>) of lung vessels at E17.5 (X400) also exhibited a disorganized distribution in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice, with more mesenchymal vessels and a disruption of the normal subepithelial double capillary network (black arrows in <b>G</b>).</p

Characterization of the <i>UPK II-Cre;Rosa-Stop-YFP</i> reporter mice.

<p>A–J: <i>UPK II-Cre;Rosa-Stop-YFP^+/+</i> reporter mice reveal fluorescence only in the bladder urothelium (x200) (<b>A,B</b>) and ureter urothelium (x200) (day 1, P1) (white arrow) (<b>C,D</b>), but not in lung (x200) (P1) (<b>E,F</b>), placenta (x200) (E19.5) (<b>G,H</b>) or yolk sac (x200) (E19.5) (<b>I,J</b>) among other negative tissues. <b>K</b>: PCR for recombinant UPK II-YFP was only positive in <i>UPK II-Cre;Rosa-Stop-YFP^+/+</i> bladder (E19.5).</p

Fragmentation of ECM components in lungs of <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A: Total protein lysates from whole lung were analyzed by Western blot for for laminin β-1 from P1 <i>UPK II-Cre;LSL-K-ras^G12D</i> mice and control (Cre-) mice, showing an additional low molecular weight (mw) band (35 kDa) which suggest fragmentation. <b>B</b>: Representative images of immunofluorescence for laminin β-1 (X200) showed a disorganized membrane pattern in E17.5 lung from <i>UPK II-Cre;LSL-K-ras^G12D</i> mice. <b>C</b>: WB for lung E-cadherin in P1 lung, with an increase in low mw bands. (53 and 32 KDa) also in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.</p

Urothelial hyperplasia in the <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A: Urothelial-restricted expression of K-ras^G12D. <b>B–E</b>: H&E analysis of bladders (X200) reveals a hyperplastic urothelium at E17.5 (<b>B,C</b>) and P1 (<b>D,E</b>) (black arrows). <b>F</b>: Differences in urothelial cellularity (cells/0.15 mm²) between <i>UPK II-Cre;LSL-K-ras^G12D</i> mice and controls were significant both at E17.5 (n = 2/group; *, <i>P</i> = 0.05) and P1 (n>6/group; **, <i>P</i><0.0001); bars, SEM. <b>G–H</b>: BRDU staining of bladder (X200) showing a higher proliferation in E17.5 <i>UPK II-Cre;LSL-K-ras^G12D</i> mice (urothelium limit is marked with a blue line). The mean number of BrdU positive nuclei/200 µm of urothelium was significantly higher than in controls (n = 10; 6±2 positive nuclei/200 µm vs 1.33±0.81; <i>P</i> = 0.01).</p

Number of Acidic Residues.

<p><b><u>Chemical characteristics of fossil peptides.</u></b> Dinosaur peptide sequences were obtained from the literature and their alpha chain location and amino acid positions on the human collagen model determined. The prevalence of acidic residues (<i>bolded, underlined</i>) in the peptides was lower than predicted for “average” peptides of comparable lengths from pepsinized human collagen <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020381#pone.0020381-Miller1" target="_blank">[38]</a>, implying that regions of collagen with a less acidic nature were preferentially preserved in the fossils.</p

The collagen fibril (A) is composed of triple-helical monomers that polymerize in an overlapping fashion (B), and are derived from proteolysis of the soluble procollagen precursor (C).

<p>Fibrils appear as periodic banded structures by electron microscopy; one D-period (expanded two-dimensional view of 67 nm segment of microfibril, box) contains the complete collagen sequence from elements of five monomers and includes an overlap and gap zone; arrow, left border of overlap zone. Image of the X-ray diffraction-derived fibril subunit structure: the microfibril (D) shows aggregates of five triple-helical, rope-like monomers; magnified view shows triple helix containing three peptide chains (two α1 and one α2 chains) (E). Many thousands of microfibrils polymerize and cross-link to form cable-like collagen fibrils of vertebrates. Modified from original research <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020381#pone.0020381-Sweeney1" target="_blank">[33]</a>.</p

X-ray diffraction model of the rat collagen microfibril <i>in situ</i>; Integrins, predominant cell-binding site; MMP, matrix metalloproteinase cleavage site; FN, fibronectin binding site; decoron, decorin proteoglycan core protein binding sites; putative cell and matrix interaction domains³⁵.

<p>X-ray diffraction model of the rat collagen microfibril <i>in situ</i>; Integrins, predominant cell-binding site; MMP, matrix metalloproteinase cleavage site; FN, fibronectin binding site; decoron, decorin proteoglycan core protein binding sites; putative cell and matrix interaction domains³⁵.</p

Dinosaur peptide sequence positions were mapped on the two dimensional human collagen fibril D-period schematic³³.

<p>Dinosaur peptide sequence positions were mapped on the two dimensional human collagen fibril D-period schematic³³.</p