FAK-Src signalling is important to renal collecting duct morphogenesis: discovery using a hierarchical screening technique

Summary This report describes a hierarchical screening technique for identification of pathways that control the morphogenesis of the renal collecting duct system. The multi-step screen involves a first round using a 2-dimensional, cell-line-based scrape-healing assay, then a second round using a 3-dimensional tubulogenesis assay; both of these rounds use new cell lines described in this report. The final stage is ex vivo organ culture. We demonstrate the utility of the screen by using it to identify the FAK–Src-pathway signalling as being important for collecting duct development, specifically for the cell proliferation on which this development depends

Making immortalized cell lines from embryonic mouse kidney

Immortalized cell lines derived from embryonic mouse kidneys are useful tools for exploring signaling pathways, morphogenetic mechanisms, and gene function in renal development: they also provide a means for efficient first-round screening of panels of small molecules intended to combat renal pathologies such as the development of cysts, and such cell line-based screening can allow a valuable reduction in the numbers of animals needed for a given line of research. This chapter presents a simple method for generating cell lines from the "Immortomouse," which carries a temperature-sensitive SV40 antigen, under the control of an interferon-regulated promoter

Evaluating Population Genetic Structure and Potential Genomic Signals of Natural Selection in a Migratory Songbird (Protonotaria citrea)

In this study I attempted to further resolve the population genetic structure in the Prothonotary Warbler (Protonotaria citrea), and conducted an outlier SNP analysis and exploratory gene ontology analysis to investigate potential ongoing natural selection in the species. This analysis of population structure confirms previous work by DeSaix et al. (2019), where weak population structure was observed between eastern sites along the Atlantic Coastal Plain, and western sites in the Mississippi Alluvial Valley, possibly due to a genetic discontinuity across the Appalachian Mountains. I conducted two forms of outlier SNP analyses, a principal component analysis (PCA)-based approach to identify SNPs associated with local adaptation, and a partial redundancy analysis (pRDA) to identify SNPs associated with site-specific environmental factors. I then performed an exploratory gene ontology analysis of the top ten outliers identified from both methods. I found two of the top outliers both fell within gene structures that aligned to proteins localized to the eye, which I suggest may be due to selective pressure on the visual system of the Prothonotary Warbler, perhaps due to sexual selection or adaptation to light pollution. Further investigation is needed to determine whether this signal is not a false positive, and if so, determine what selective pressures are acting on the Prothonotary Warbler visual system. There did not appear to any other discernible patterns in the gene ontologies of the top outlier SNPs, but a full gene ontology analysis may be able to reveal additional selective pressures for further investigation

An inducible CiliaGFP mouse model for in vivo visualization and analysis of cilia in live tissue

BACKGROUND: Cilia are found on nearly every cell type in the mammalian body, and have been historically classified as either motile or immotile. Motile cilia are important for fluid and cellular movement; however, the roles of non-motile or primary cilia in most tissues remain unknown. Several genetic syndromes, called the ciliopathies, are associated with defects in cilia structure or function and have a wide range of clinical presentations. Much of what we know about the formation and maintenance of cilia comes from model systems like C. elegans and Chalmydomonas. Studies of mammalian cilia in live tissues have been hampered by difficulty visualizing them. RESULTS: To facilitate analyses of mammalian cilia function we generated an inducible Cilia(GFP) mouse by targeting mouse cDNA encoding a cilia-localized protein somatostatin receptor 3 fused to GFP (Sstr3::GFP) into the ROSA26 locus. In this system, Sstr3::GFP is expressed from the ubiquitous ROSA26 promoter after Cre mediated deletion of an upstream Neo cassette flanked by lox P sites. Fluorescent cilia labeling was observed in a variety of live tissues and after fixation. Both cell-type specific and temporally regulated cilia labeling were obtained using multiple Cre lines. The analysis of renal cilia in anesthetized live mice demonstrates that cilia commonly lay nearly parallel to the apical surface of the tubule. In contrast, in more deeply anesthetized mice the cilia display a synchronized, repetitive oscillation that ceases upon death, suggesting a relationship to heart beat, blood pressure or glomerular filtration. CONCLUSIONS: The ability to visualize cilia in live samples within the Cilia(GFP) mouse will greatly aid studies of ciliary function. This mouse will be useful for in vivo genetic and pharmacological screens to assess pathways regulating cilia motility, signaling, assembly, trafficking, resorption and length control and to study cilia regulated physiology in relation to ciliopathy phenotypes

Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of Snail and E-cadherin

Epicardial epithelial-mesenchymal transition (EMT) is hypothesized to generate cardiovascular progenitor cells that differentiate into various cell types, including coronary smooth muscle and endothelial cells, perivascular and cardiac interstitial fibroblasts and cardiomyocytes. Here we show that an epicardial-specific knockout of Wt1 leads to a reduction of mesenchymal progenitor cells and their derivatives. We demonstrate that Wt1 is essential for repression of the epithelial phenotype in epicardial cells and during Embryonic Stem (ES) cell differentiation, through direct transcriptional regulation of Snail (Snai1) and E-cadherin (Cdh1), two of the major mediators of EMT. Some mesodermal lineages fail to form in Wt1 null embryoid bodies but this effect is rescued by the expression of Snai1, underlining the importance of EMT in generating these differentiated cells. These new insights into the molecular mechanisms regulating cardiovascular progenitor cells and EMT will shed light on the pathogenesis of heart diseases and may help the development of cell based therapies

Tear fluid biomarkers in ocular and systemic disease: potential use for predictive, preventive and personalised medicine

In the field of predictive, preventive and personalised medicine, researchers are keen to identify novel and reliable ways to predict and diagnose disease, as well as to monitor patient response to therapeutic agents. In the last decade alone, the sensitivity of profiling technologies has undergone huge improvements in detection sensitivity, thus allowing quantification of minute samples, for example body fluids that were previously difficult to assay. As a consequence, there has been a huge increase in tear fluid investigation, predominantly in the field of ocular surface disease. As tears are a more accessible and less complex body fluid (than serum or plasma) and sampling is much less invasive, research is starting to focus on how disease processes affect the proteomic, lipidomic and metabolomic composition of the tear film. By determining compositional changes to tear profiles, crucial pathways in disease progression may be identified, allowing for more predictive and personalised therapy of the individual. This article will provide an overview of the various putative tear fluid biomarkers that have been identified to date, ranging from ocular surface disease and retinopathies to cancer and multiple sclerosis. Putative tear fluid biomarkers of ocular disorders, as well as the more recent field of systemic disease biomarkers, will be shown

High-Resolution Genomic Profiling of Chromosomal Abnormalities in Human Stem Cells Using the 135 K StemArray

Culturing stem cells for an extended period of time can lead to acquired chromosomal aberrations. Determining the copy number variant (CNV) profile of stem cell lines is critical since CNVs can have dramatic effects on gene expression and tumorigenic potential. Here, we describe an improved version of our StemArray, a stem-cell-focused comparative genomic hybridization (aCGH) microarray, which contains 135,000 probes and covers over 270 stem cell and cancer related genes at the exon level. We have dramatically increased the median probe spacing throughout the genome in order to obtain a higher resolution genetic profile of the cell lines. To illustrate the importance of using the StemArray, we describe a karyotypically normal iPSC line in which we detected acquired chromosomal variations that could affect the cellular phenotype of the cells. Identifying adaptive chromosomal aberrations in stem cell lines is essential if they are to be used in regenerative medicine

A Glutamine-Rich Factor Affects Stem Cell Genesis in Leech

Leech embryogenesis is a model for investigating cellular and molecular processes of development. Due to the unusually large size of embryonic stem cells (teloblasts: 50–300 μm) in the glossiphoniid leech, Theromyzon tessulatum, and the presence of identifiable stem cell precursors (proteloblasts), we previously isolated a group of genes upregulated upon stem cell birth. In the current study, we show that one of these genes, designated Theromyzon proliferation (Tpr), is required for normal stem cell genesis; specifically, transient Tpr knockdown experiments conducted with antisense oligonucleotides and monitored by semiquantitative RT-PCR, caused abnormal proteloblast proliferation leading to embryonic death, but did not overtly affect neuroectodermal or mesodermal stem cell development once these cells were born. Tpr encodes a large glutamine-rich (∼34%) domain that shares compositional similarity with strong transcriptional enhancers many of which have been linked with trinucleotide repeat disorders (e.g., Huntington's)