An Outer Arm Dynein Conformational Switch Is Required for Metachronal Synchrony of Motile Cilia in Planaria

Here we use the motile ventral cilia of the planarian S. mediterranea to examine the role of outer arm dynein in the generation and maintenance of metachronal synchrony. We demonstrate that a single dynein light chain plays a mechanosensory role necessary to entrain and maintain the metachronal synchrony of motile cilia

Understanding the role of growth factors in embryonic development: insights from the lens

Growth factors play key roles in influencing cell fate and behaviour during development. The epithelial cells and fibre cells that arise from the lens vesicle during lens morphogenesis are bathed by aqueous and vitreous, respectively. Vitreous has been shown to generate a high level of fibroblast growth factor (FGF) signalling that is required for secondary lens fibre differentiation. However, studies also show that FGF signalling is not sufficient and roles have been identified for transforming growth factor-β and Wnt/Frizzled families in regulating aspects of fibre differentiation. In the case of the epithelium, key roles for Wnt/β-catenin and Notch signalling have been demonstrated in embryonic development, but it is not known if other factors are required for its formation and maintenance. This review provides an overview of current knowledge about growth factor regulation of differentiation and maintenance of lens cells. It also highlights areas that warrant future study

Ocular Phenotype of Relaxin Gene Knockout (Rln(-/-)) Mice

Purpose: To test if relaxin deficiency affects ocular structure and function we investigated expression of relaxin (Rln) and RXFP receptors (Rxfp1, Rxfp2), and compared ocular phenotypes in relaxin gene knockout (Rln-/- ) and wild type (Rln+/+ ) mice. Materials and Methods: Rln, Rxfp1 and Rxfp2 mRNA expression was detected in ocular tissues of Rln+/+ mice using RT-PCR. The eyes of 11 Rln-/- and 5 Rln+/+ male mice were investigated. Corneal and retinal thickness was assessed using optical coherence tomography. Intraocular pressure was measured using a rebound tonometer. Retinal, choroidal and sclera morphology and thickness were evaluated histologically. Eyes were collected and fixed for immunofluorescence staining or used for RNA extraction to evaluate mRNA expression using real-time PCR. Results: Rln mRNA was expressed only in the retina, whereas Rxfp1 transcripts were detected in the retina, cornea and sclera/choroid. Rxfp2 was only present in the cornea. None of these genes were expressed in the lacrimal gland, eyelid or lens. Intraocular pressure was higher and central cornea of Rln-/- mice was significantly thicker and had significantly larger endothelial cells and a lower endothelial cell density than Rln+/+ mice. Immunohistochemistry demonstrated no significant difference in AQP3 and AQP5 staining in the cornea or other regions between wildtype and Rln-/- mice. mRNA expression of Aqp4 was significantly higher in Rln-/- than in Rln+/+ corneas, whereas Col1a2, Mmp9, Timp1 and Timp2 were significantly decreased. Expression of Aqp1, Aqp4, Aqp5, Vim and Tjp1 was significantly decreased in Rln-/- compared to Rln+/+ uvea. No significant differences in these genes were detected in the retina. Retinal, choroidal and scleral thicknesses were not different and morphology appeared normal. Conclusion: The findings indicate that loss of Rln affects expression of several genes in the uvea and cornea and results in thicker corneas with altered endothelial cells. Many of the gene changes suggest alterations in extracellular matrix and fluid transport between cells

Exposure of trophoblast cells to fine particulate matter air pollution leads to growth inhibition, inflammation and ER stress

Ambient air pollution is considered a major environmental health threat to pregnant women. Our previous work has shown an association between exposure to airborne particulate matter (PM) and an increased risk of developing pre-eclamspia. It is now recognized that many pregnancy complications are due to underlying placental dysfunction, and this tissue plays a pivotal role in pre-eclamspia. Recent studies have shown that PM can enter the circulation and reach the human placenta but the effects of PM on human placental function are still largely unknown. In this work we investigated the effects of airborne PM on trophoblast cells. Human, first trimester trophoblast cells (HTR-8/SV) were exposed to urban pollution particles (Malmö PM2.5; Prague PM10) for up to seven days in vitro and were analysed for uptake, levels of hCGβ and IL-6 secretion and proteomic analysis. HTR-8/SVneo cells rapidly endocytose PM within 30 min of exposure and particles accumulate in the cell in perinuclear vesicles. High doses of Prague and Malmö PM (500–5000 ng/ml) significantly decreased hCGβ secretion and increased IL-6 secretion after 48 h exposure. Exposure to PM (50 ng/ml) for 48h or seven days led to reduced cellular growth and altered protein expression. The differentially expressed proteins are involved in networks that regulate cellular processes such as inflammation, endoplasmic reticulum stress, cellular survival and molecular transport pathways. Our studies suggest that trophoblast cells exposed to low levels of urban PM respond with reduced growth, oxidative stress, inflammation and endoplasmic reticulum stress after taking up the particles by endocytosis. Many of the dysfunctional cellular processes ascribed to the differentially expressed proteins in this study, are similar to those described in PE, suggesting that low levels of urban PM may disrupt cellular processes in trophoblast cells. Many of the differentially expressed proteins identified in this study are involved in inflammation and may be potential biomarkers for PE

<i>Esrp1</i> is a marker of mouse fetal germ cells and differentially expressed during spermatogenesis

<div><p>ESRP1 regulates alternative splicing, producing multiple transcripts from its target genes in epithelial tissues. It is upregulated during mesenchymal to epithelial transition associated with reprogramming of fibroblasts to iPS cells and has been linked to pluripotency. Mouse fetal germ cells are the founders of the adult gonadal lineages and we found that <i>Esrp1</i> mRNA was expressed in both male and female germ cells but not in gonadal somatic cells at various stages of gonadal development (E12.5-E15.5). In the postnatal testis, <i>Esrp1</i> mRNA was highly expressed in isolated cell preparations enriched for spermatogonia but expressed at lower levels in those enriched for pachytene spermatocytes and round spermatids. Co-labelling experiments with PLZF and c-KIT showed that ESRP1 was localized to nuclei of both Type A and B spermatogonia in a speckled pattern, but was not detected in SOX9⁺ somatic Sertoli cells. No co-localization with the nuclear speckle marker, SC35, which has been associated with post-transcriptional splicing, was observed, suggesting that ESRP1 may be associated with co-transcriptional splicing or have other functions. RNA interference mediated knockdown of <i>Esrp1</i> expression in the seminoma-derived Tcam-2 cell line demonstrated that ESRP1 regulates alternative splicing of mRNAs in a non-epithelial cell germ cell tumour cell line.</p></div

<i>Esrp1</i> is highly expressed in spermatogonia in the adult testis.

<p>RT-ddPCR of isolated germ cells from postnatal mouse testes show that <i>Esrp1</i> is highly expressed in spermatogonia (S), weakly expressed in gonocytes/spermatogonia (G) and barely detectable in pachytene spermatocytes (PS) and round spermatids (RS). Expression data are expressed as copies/μl, normalised against <i>Cyclophilin A</i> expression, and as mean ± S.E.M of three independent samples (except for Round Spermatids n = 2). Individual data are shown as black circles, mean as a red line and the error bars in black. Asterisks indicate significance differences by ANOVA and Tukey’s post-hoc analysis (**, p<0.01; ***, p<0.001).</p

ESRP1-mediated alternative splicing in TCam-2 cells.

<p><b>A.</b> TCam-2 cells transfected with siRNA constructs for <i>ESRP1</i> displayed a dramatic (~85%) and significant (n = 3 each group; p<0.0001, Student’s <i>t</i>-test) decrease in expression of <i>ESRP1</i> mRNA. Similar data were obtained in two independent experiments. <b>B.</b> By western blot analysis, a distinct 75kD band for ESRP1 was detected in non-transfected and control siRNA-transfected TCam2 cells but was not detectable in cells transfected with either siRNA construct. Similar data were obtained in two independent experiments. <b>C.</b> RT-PCR using primers specific to splice variants of <i>CTNND1</i> and <i>DOCK7</i> showed that loss of ESRP1 expression promoted inclusion of exons 2–3 in <i>CTNND1</i> transcripts and inclusion of exon 23 in <i>DOCK7</i> transcripts. By contrast no changes were detected for the constant exons in these genes. The splicing pattern and band size are illustrated for each transcript variant.</p

Digital PCR primer and probe sequences.

<p>Digital PCR primer and probe sequences.</p