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

Evolution of a dispensable mouse beta-globin gene.

Evolution of a dispensable mouse beta-globin gene

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</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

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

Conditional knockout mice for the distal appendage protein CEP164 reveal its essential roles in airway multiciliated cell differentiation.

Multiciliated cells of the airways, brain ventricles, and female reproductive tract provide the motive force for mucociliary clearance, cerebrospinal fluid circulation, and ovum transport. Despite their clear importance to human biology and health, the molecular mechanisms underlying multiciliated cell differentiation are poorly understood. Prior studies implicate the distal appendage/transition fiber protein CEP164 as a central regulator of primary ciliogenesis; however, its role in multiciliogenesis remains unknown. In this study, we have generated a novel conditional mouse model that lacks CEP164 in multiciliated tissues and the testis. These mice show a profound loss of airway, ependymal, and oviduct multicilia and develop hydrocephalus and male infertility. Using primary cultures of tracheal multiciliated cells as a model system, we found that CEP164 is critical for multiciliogenesis, at least in part, via its regulation of small vesicle recruitment, ciliary vesicle formation, and basal body docking. In addition, CEP164 is necessary for the proper recruitment of another distal appendage/transition fiber protein Chibby1 (Cby1) and its binding partners FAM92A and FAM92B to the ciliary base in multiciliated cells. In contrast to primary ciliogenesis, CEP164 is dispensable for the recruitment of intraflagellar transport (IFT) components to multicilia. Finally, we provide evidence that CEP164 differentially controls the ciliary targeting of membrane-associated proteins, including the small GTPases Rab8, Rab11, and Arl13b, in multiciliated cells. Altogether, our studies unravel unique requirements for CEP164 in primary versus multiciliogenesis and suggest that CEP164 modulates the selective transport of membrane vesicles and their cargoes into the ciliary compartment in multiciliated cells. Furthermore, our mouse model provides a useful tool to gain physiological insight into diseases associated with defective multicilia