Development of a Conditional Mesd (Mesoderm Development) Allele for Functional Analysis of the Low-Density Lipoprotein Receptor-Related Family in Defined Tissues

The Low-density lipoprotein receptor-Related Protein (LRP) family members are essential for diverse processes ranging from the regulation of gastrulation to the modulation of lipid homeostasis. Receptors in this family bind and internalize a diverse array of ligands in the extracellular matrix (ECM). As a consequence, LRPs regulate a wide variety of cellular functions including, but not limited to lipid metabolism, membrane composition, cell motility, and cell signaling. Not surprisingly, mutations in single human LRPs are associated with defects in cholesterol metabolism and development of atherosclerosis, abnormalities in bone density, or aberrant eye vasculature, and may be a contributing factor in development of Alzheimer’s disease. Often, members of this diverse family of receptors perform overlapping roles in the same tissues, complicating the analysis of their function through conventional targeted mutagenesis. Here, we describe development of a mouse Mesd (Mesoderm Development) conditional knockout allele, and demonstrate that ubiquitous deletion of Mesdusing Cre-recombinase blocks gastrulation, as observed in the traditional knockout and albino-deletion phenotypes. This conditional allele will serve as an excellent tool for future characterization of the cumulative contribution of LRP members in defined tissues

Radial Construction of an Arterial Wall

SummarySome of the most serious diseases involve altered size and structure of the arterial wall. Elucidating how arterial walls are built could aid understanding of these diseases, but little is known about how concentric layers of muscle cells and the outer adventitial layer are assembled and patterned around endothelial tubes. Using histochemical, clonal, and genetic analysis in mice, here we show that the pulmonary artery wall is constructed radially, from the inside out, by two separate but coordinated processes. One is sequential induction of successive cell layers from surrounding mesenchyme. The other is controlled invasion of outer layers by inner layer cells through developmentally regulated cell reorientation and radial migration. We propose that a radial signal gradient controls these processes and provide evidence that PDGF-B and at least one other signal contribute. Modulation of such radial signaling pathways may underlie vessel-specific differences and pathological changes in arterial wall size and structure.Video Abstrac

Recapitulation of Developing Artery Muscularization in Pulmonary Hypertension

Excess smooth muscle accumulation is a key component of many vascular disorders, including atherosclerosis, restenosis, and pulmonary artery hypertension, but the underlying cell biological processes are not well defined. In pulmonary artery hypertension, reduced pulmonary artery compliance is a strong independent predictor of mortality, and pathological distal arteriole muscularization contributes to this reduced compliance. We recently demonstrated that embryonic pulmonary artery wall morphogenesis consists of discrete developmentally regulated steps. In contrast, poor understanding of distal arteriole muscularization in pulmonary artery hypertension severely limits existing therapies that aim to dilate the pulmonary vasculature but have modest clinical benefit and do not prevent hypermuscularization. Here, we show that most pathological distal arteriole smooth muscle cells, but not alveolar myofibroblasts, derive from pre-existing smooth muscle. Furthermore, the program of distal arteriole muscularization encompasses smooth muscle cell dedifferentiation, distal migration, proliferation, and then redifferentiation, thereby recapitulating many facets of arterial wall development

Assessment of <i>Mesd</i> recombination in liver in response to adCreGFP infection.

<p>(<b>A–B</b>) GFP fluorescence in <i>Mesd-Floxed</i> homozygous livers 48 hours post adCre-GFP injection. (<b>A</b>) Liver 1 was photographed at 17 ms exposure on a dissecting microscope under 535nm UV light at 11.0×magnification. (<b>B</b>) In contrast, liver 2 showed fluorescence in only sporadic cells, and was photographed at 80×magnification and at a 432 ms exposure, to capture the few fluorescing cells. (<b>C</b>) PCR amplification to assess adCre mediated excision of <i>Mesd-Floxed.</i> (Lanes 1–2) <i>Mesd-LoxP/Mesd-Wild-type</i> (LoxP/Wt) and <i>Mesd-Floxed/Mesd-Wild-type</i> (Flox/Wt) tail DNA preparations were used for size reference. (Lanes 3–4) genomic DNA prepared from livers and brains isolated from non-infected animal. (Lanes 5–12) Genomic DNA prepared from liver and brain isolated from adCreGFP infected animals 48 hours post infection (animal 1 & 2) and ten weeks post infection (animals 3 & 4).</p

The <i>Mesd-LoxP</i> allele does not complement the <i>Mesd</i> knockout and albino deletion alleles.

<p>Animals heterozygous for the Cre-recombined <i>Mesd</i> (<i>Mesd-LoxP</i>) allele were mated to animals heterozygous for the <i>Mesd</i> conventional knockout (<i>Mesd-KO</i>) (A–B), the albino deletion (<i>Mesd-3YPSD</i>) deletion (C–D), or the <i>Mesd-LoxP</i> allele (E–F). Embryos were dissected at embryonic day (E) 7.5 or E 8.5 and photographed at the indicated magnifications (5× or 2.5×). (<b>A</b>) At E 7.5, gastrulation was near completion in wild-type embryos and the epiblast (ep), amnion (am) and chorion (ch) were clearly visible. (<b>B, B’</b>) In contrast, in the <i>Mesd</i> mutant littermate gastrulation was blocked, the extra-embryonic ectoderm (ex) and epiblast (ep) were organized as epithelia, and Reichert’s membrane and trophoblast (rm/tb) were expanded in comparison to the epiblast. In panel B’, Reichert’s membrane was removed to facilitate better visualization of the <i>Mesd</i> egg-cylinder. (<b>C</b> and <b>E</b>) By E 8.5 the size of the wild-type embryo had increased considerably, and the brain (br), heart (he), and somites (so) were clearly visible. (<b>D–F’’</b>) In contrast, the mutant littermates had only doubled in size, and the epiblast (ep) remained undifferentiated, and Reichert’s membrane and trophoblast (rm/tb) were greatly expanded; this layer was removed in panel D’ and F’. The embryo shown in panel F’ was rotated 90 degrees (<b>F’’</b>) to allow visualization of the shortened anterior/posterior axis and characteristic indentation on the left side of the pictured <i>Mesd</i> mutant. Previously, we demonstrated that the indentation was coincident with expression of Hex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075782#pone.0075782-Hsieh1" target="_blank">[1]</a>.</p

The <i>Mesd</i> conditional allele.

<p>(<b>A</b>) Comparison of the <i>Mesd Wild-type, Floxed,</i> and <i>LoxP Mesd</i> alleles and targeting vector. The <i>Wild-type Mesd</i> allele (top map) has three exons (1–3) that are designated by grey rectangles. The first exon encodes the signal peptide that directs the MESD protein into the ER as well as the N-terminal helical region essential for maturation of LRPs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075782#pone.0075782-Hsieh1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075782#pone.0075782-Kohler1" target="_blank">[3]</a>. The 3′ untranslated portion of the third exon is indicated by light grey. In the <i>Mesd</i> targeting vector, we introduced the <i>Neomycin resistance</i> gene (<i>Neo</i>) (white rectangle) flanked by Flp-recombinase sites (green triangles) into the <i>Mesd</i> first intron. In addition, we introduced three LoxP sites (red triangles) flanking the first and second exons and the <i>Neo</i> gene.The targeting construct was introduced into C57BL/6 embryonic stem cells (<i>Ozgene</i>), and cell lines heterozygous for the <i>Floxed</i> allele (middle map) were identified by Southern analysis using the 5P, enP, and P3 probes, whose positions are indicated by black rectangles (described in Materials and Methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075782#pone-0075782-t001" target="_blank">Table 1</a>). Based on Southern analysis using these probes, we determined that only the first two LoxP sites were incorporated into ES cells; the recombination sites are indicated by dashed lines. Expression of Cre-recombinase will trigger recombination between LoxP sites to generate the <i>LoxP</i> allele (bottom map). (<b>B</b>) Heterozygous <i>Floxed</i> mice were mated to C57BL/6 mice (left) for strain propagation or to animals homozygous for a ubiquitously expressed Cre-recombinase (right) to generate the recombined <i>LoxP</i> (knockout) allele. Progeny were genotyped by Southern analysis of <i>BamH</i>I digested DNA using the enP probe. This probe clearly distinguished the <i>Wt</i> (20 kb) fragment from the <i>Floxed</i> (7.4 kb) or <i>LoxP</i> (4 kb) fragments.</p

p53 Regulates the Extent of Fibroblast Proliferation and Fibrosis in Left Ventricle Pressure Overload

BACKGROUND: Cardiomyopathy is characterized by the pathological accumulation of resident cardiac fibroblasts that deposit ECM (extracellular matrix) and generate a fibrotic scar. However, the mechanisms that control the timing and extent of cardiac fibroblast proliferation and ECM production are not known, hampering the development of antifibrotic strategies to prevent heart failure. METHODS: We used the Tcf21 (transcription factor 21)MerCreMer mouse line for fibroblast-specific lineage tracing and p53 (tumor protein p53) gene deletion. We characterized cardiac physiology and used single-cell RNA-sequencing and in vitro studies to investigate the p53-dependent mechanisms regulating cardiac fibroblast cell cycle and fibrosis in left ventricular pressure overload induced by transaortic constriction. RESULTS: Cardiac fibroblast proliferation occurs primarily between days 7 and 14 following transaortic constriction in mice, correlating with alterations in p53-dependent gene expression. p53 deletion in fibroblasts led to a striking accumulation of Tcf21-lineage cardiac fibroblasts within the normal proliferative window and precipitated a robust fibrotic response to left ventricular pressure overload. However, excessive interstitial and perivascular fibrosis does not develop until after cardiac fibroblasts exit the cell cycle. Single-cell RNA sequencing revealed p53 null fibroblasts unexpectedly express lower levels of genes encoding important ECM proteins while they exhibit an inappropriately proliferative phenotype. in vitro studies establish a role for p53 in suppressing the proliferative fibroblast phenotype, which facilitates the expression and secretion of ECM proteins. Importantly, Cdkn2a (cyclin-dependent kinase inhibitor 2a) expression and the p16Ink4a-retinoblastoma cell cycle control pathway is induced in p53 null cardiac fibroblasts, which may eventually contribute to cell cycle exit and fulminant scar formation. CONCLUSIONS: This study reveals a mechanism regulating cardiac fibroblast accumulation and ECM secretion, orchestrated in part by p53-dependent cell cycle control that governs the timing and extent of fibrosis in left ventricular pressure overload