Differentiation Of Cell Types Research Articles

Growth condition-dependent differences in methylation imply transiently differentiated DNA methylation states in Escherichia coli.

DNA methylation in bacteria frequently serves as a simple immune system, allowing recognition of DNA from foreign sources, such as phages or selfish genetic elements. However, DNA methylation also affects other cell phenotypes in a heritable manner (i.e. epigenetically). While there are several examples of methylation affecting transcription in an epigenetic manner in highly localized contexts, it is not well-established how frequently methylation serves a more general epigenetic function over larger genomic scales. To address this question, here we use Oxford Nanopore sequencing to profile DNA modification marks in three natural isolates of Escherichia coli. We first identify the DNA sequence motifs targeted by the methyltransferases in each strain. We then quantify the frequency of methylation at each of these motifs across the entire genome in different growth conditions. We find that motifs in specific regions of the genome consistently exhibit high or low levels of methylation. Furthermore, we show that there are replicable and consistent differences in methylated regions across different growth conditions. This suggests that during growth, E. coli transiently differentiate into distinct methylation states that depend on the growth state, raising the possibility that measuring DNA methylation alone can be used to infer bacterial growth states without additional information such as transcriptome or proteome data. These results show the utility of using Oxford Nanopore sequencing as an economic means to infer DNA methylation status. They also provide new insights into the dynamics of methylation during bacterial growth and provide evidence of differentiated cell states, a transient analog to what is observed in the differentiation of cell types in multicellular organisms.

CC chemokine ligand 20 (CCL20) positively regulates collagen type I production in 3D skin equivalent tissues.

Chemokines are a group of small proteins that induce chemoattraction and inflammation and contribute to the differentiation and homeostasis of various cell types. Here we explored the role of chemokines, extracellular matrix production, and myofibroblast differentiation in self-assembled skin equivalents (SASE), a three-dimensional (3D) skin-equivalent tissue model. We found that the expression of three chemokines, C-C motif chemokine ligand (CCL) 20, C-X-C motif chemokine ligand (CXCL) 5, and CXCL8, increased with differentiation to myofibroblasts. Addition of recombinant CCL20 to human skin fibroblast induced collagen Type I alpha 2 gene expression, but did not affect the expression of alpha smooth muscle actin expression. Conversely, siRNA gene knockdown of CCL20 effectively inhibited the expression of collagen Type I gene and protein. Furthermore, when the CCL20 gene in fibroblasts was knocked down in SASE, collagen Type I synthesis and stromal thickness were decreased. Taken together, these results have indicated the utility of SASE in understanding how cytokines such as CCL20 positively regulate extracellular matrix proteins such as collagen Type I production during myofibroblast differentiation in 3D tissues that mimic human skin.

TEMPO enables sequential genetic labeling and manipulation of vertebrate cell lineages

During development, regulatory factors appear in a precise order to determine cell fates over time. Consequently, to investigate complex tissue development, it is necessary to visualize and manipulate cell lineages with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here, we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labeling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation and inactivation of reporters and/or effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.

Heterozygous premature termination in zinc-finger domain of Krüppel-like factor 2 gene associates with dysregulated immunity.

Krüppel-like factor 2 (KLF2) is a transcription factor with significant roles in development, maturation, differentiation, and proliferation of several cell types. In immune cells, KLF2 regulates maturation and trafficking of lymphocytes and monocytes. KLF2 participates in regulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Although pulmonary arterial hypertension (PAH) related to KLF2 genetic variant has been suggested, genetic role of KLF2 associated with immune dysregulation has not been described. We identified a family whose members suffered from lymphopenia, autoimmunity, and malignancy. Whole exome sequencing revealed a KLF2 p.(Glu318Argfs*87) mutation disrupting the highly conserved zinc finger domain. We show a reduced amount of KLF2 protein, defective nuclear localization and altered protein-protein interactome. The phenotypically variable positive cases presented with B and T cell lymphopenia and abnormalities in B and T cell maturation including low naive T cell counts and low CD27+IgD-IgM- switched memory B cells. KLF2 target gene (CD62L) expression was affected. Although the percentage of (CD25+FOXP3+, CD25+CD127-) regulatory T cells (Treg) was high, the naive Treg cells (CD45RA+) were absent. Serum IgG1 levels were low and findings in one case were consistent with common variable immunodeficiency (CVID). Transcription of NF-κβ pathway genes and p65/RelA phosphorylation were not significantly affected. Inflammasome activity, transcription of genes related with JAK/STAT pathway and interferon signature were also comparable to controls. Evidence of PAH was not found. In conclusion, KLF2 variant may be associated with familial immune dysregulation. Although the KLF2 deficient family members in our study suffered from lymphopenia, autoimmunity or malignancy, additional study cohorts are required to confirm our observations.

High Throughput Epigenomic Mapping of Hematopoietic Stem and Progenitor Cells Using Low Cell Input Acoustic Enhanced Immunoprecipitation

Acute myeloid leukemia (AML) is a complex disease with several etiologies characterized by incomplete cellular differentiation and clonal proliferation of hematopoietic stem and progenitor cells (HSPCs) in place of normal hematopoiesis. Non-mutational epigenetic reprogramming is an emerging hallmark and enabling characteristic of cancer pathogenesis. Epigenetic regulation of gene activity is an essential component of chromatin stability and maintenance of proper cellular function and differentiation. Modifications of gene and histone landscape methylation and acetylation, three-dimensional chromatin structure, and protein-controlled activating and silencing switches are all regulated by epigenetic components, and thus are integral to understanding gene regulatory mechanisms during cell fate changes, disease onset, progression, as well as for optimizing therapeutic regime. In addition, this information holds immense potential for accurate identification of disease biomarkers, cell of origin, and targets for epigenetic therapy. However, existing chromatin immunoprecipitation sequencing (ChIP-Seq) workflows require significant cellular material and are very time-consuming, requiring cumbersome optimization. Moreover, cellular heterogeneity can influence accuracy of results from the perspective of sample preparation efficiency and also population-biased effects. Both normal and malignant bone marrow are heterogeneous in cell type and stage of differentiation that leads to imprecise results with regard to epigenetic determinates. In our study, we implemented Covaris's Adaptive Focused Acoustics® (AFA®) technology to miniaturize sample volumes for both chromatin shearing and immunoprecipitation in a 96-well format. This approach enables a standardized, robust, automatable, half-day workflow for both ChIP and Hi-ChIP applications. AFA ChIPseq- was applied to flow sorted hematopoietic stem and progenitor cells (HSPC) from individual mice. We utilized a range of epitopes including major histone modifications (H3K9me1/2/3, H3K27ac) and factors (CTCF, cohesin). Interestingly, we found epitope-dependent differences in AFA-enhanced binding with CTCF saturating at 30 minutes, whereas H3K27ac required 4 hours of AFA pulse to facilitate chromatin binding. Additionally, we evaluated low input in vitro and in vivo samples, with limited dilution of lineage negative HSPCs ranging from 500,000 to 5,000 cells with comparable sheared chromatin yield and immunoprecipitated material. Overlap of peaks called by MACS2 was consistent among the various cell inputs as well as when compared with gold standard CTCF ChIPseq- data previously performed on pooled samples from our previously published work. The low cell input capability permitted deconvolution of dynamic epigenetic changes in protein binding and chromatin structure during normal lineage specification of hematopoietic stem cells. Epigenetic remodeling of these sites was consistent with established gene programs essential for myeloid differentiation. Data were found to be in alignment with standard ChIPseq- and Hi-C datasets generated from similar cell populations. The simplified and significantly shortened (Hi)ChIP-seq processes showcased in this study will be highly beneficial for both research and clinical diagnostic applications owing to their requirements for using 96-well plate format and their ability to become integrated into automated workflows. Taken together, this technology and workflow represents a high throughput epigenetic system for small cell numbers of cells with a multitude of applications enabling laboratories focused in cell biology and translational research.

EMT-like Activation in CLL Provides Novel Therapeutic Target

Introduction: There is growing interest in the role of Epithelial to Mesenchymal Transition (EMT) regulators in immune cell development, but the functional role of EMT in B-cell biology and transformation remains obscured. The outcomes of the "classical" EMT activation of epithelial cells are known; committed migratory cells do not divide and become resistant to apoptotic stimuli. Importantly, EMT supports migratory cells' "stem cell-like" properties, allowing differentiation into more than one cell type and/or terminal differentiation and senescence. Chronic lymphocytic leukaemia (CLL) cells engage with antigen and microenvironment stimuli in tissue sites that lead to proliferation, followed by their egress from the tissue sites into the peripheral blood. This lifecycle implies functional changes altering CLL cell movement. Here we show EMT-like activation in CLL that correlates with disease progression and transformation. We identified a targetable candidate and show that it can be inhibited by small molecule ligands. Results: Our analysis of available expression datasets showed the expression of various EMT factors in healthy B cells. The single-cell analysis of lymph nodes confirms strong EMT-like activation in healthy B-cells (GSE159929), confirming the idea that genes involved in classical EMT would be expressed differently depending on the stage of B-cell differentiation. The following GENEVESTIGATOR analysis of B cell malignancies highlighted high EMT-like activation in CLL cases. However, differential expression (DE) analysis of peripheral blood (PB) samples from 44 CLL patients and 24 healthy B-cell donors identified six EMT factors aberrantly expressed in CLL cells compared to healthy B cells indicating significant changes in CLL EMT-like activation. At the transcription level, ZEB1 expression was higher and ZEB2 expression lower in PB CLL cells. The main stimulatory events in CLL cells appear restricted to affected tissue sites, primarily in lymph nodes (LN) where CLL cells migrate to reactivate and proliferate. Further, EMT factor protein expression was confirmed in CLL LN tissue microarrays. Strikingly, transformation into a more aggressive form (Richter syndrome) strongly correlates with an increase in ZEB2 but not ZEB1 protein expression. Interestingly, analysis of the published transcriptome data set of CLL cells co-cultured with nurse-like cells shows an increase in ZEB2 and a decrease in ZEB1 expression after 14 days (GSE13811). An apparent reliance of the EMT-like activation in CLL on the environmental stimuli has been demonstrated in CLL cells stimulated with BCR activation (anti-IgM) or mitogen (PMA), implying protection from cell death and activation of migration. Additionally, we observed higher S100A4 expression in CLL cells compared to mature B-cells from healthy individuals. Previously it has been shown that this calcium-dependent protein can control cell movement by direct interaction with non-muscle myosin and via regulation of stress fibre formation, thus facilitating cellular protrusions and increasing cell migration and invasion. Our analysis has shown the S100A4 positive correlation with CLL markers of poor prognosis, Zap70 and CD38. The S100A4 knockdown with siRNA dramatically impeded CLL migration in trans-well assay (Figure 1). Biochemical inhibitor screening for disruption of S100A4 interaction with myosin II identified several promising leads active in the low micromolar range in both biochemical and cell assays. Conclusions: Our findings improve the understanding of CLL pathobiology. Normally, healthy naïve B cells give rise to multiple B-cell subtypes with specific functions and locations. EMT-like activation likely contributes to B-cell development at different stages of differentiation and B-cell malignant transformation. Our investigation shows that the EMT-like activation is dysregulated in CLL cells compared to healthy B cells. The EMT expression signature changes upon B-cell receptor stimulation. These results suggest that targeting some of the aberrantly expressed EMT components such as S100A4 in CLL can be exploited, prolonging remission by, for example, impeding CLL returning to the tissue sites after an egress due to the treatment with Ibrutinib depriving these cells of vital microenvironmental support. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Retinoid metabolism: new insights.

Vitamin A (retinol) is a critical micronutrient required for the control of stem cell functions, cell differentiation, and cell metabolism in many different cell types, both during embryogenesis and in the adult organism. However, we must obtain vitamin A from food sources. Thus, the uptake and metabolism of vitamin A by intestinal epithelial cells, the storage of vitamin A in the liver, and the metabolism of vitamin A in target cells to more biologically active metabolites, such as retinoic acid (RA) and 4-oxo-RA, must be precisely regulated. Here, I will discuss the enzymes that metabolize vitamin A to RA and the cytochrome P450 Cyp26 family of enzymes that further oxidize RA. Because much progress has been made in understanding the regulation of ALDH1a2 (RALDH2) actions in the intestine, one focus of this review is on the metabolism of vitamin A in intestinal epithelial cells and dendritic cells. Another focus is on recent data that 4-oxo-RA is a ligand required for the maintenance of hematopoietic stem cell dormancy and the important role of RARβ (RARB) in these stem cells. Despite this progress, many questions remain in this research area, which links vitamin A metabolism to nutrition, immune functions, developmental biology, and nuclear receptor pharmacology.

HUSCH: an integrated single-cell transcriptome atlas for human tissue gene expression visualization and analyses.

Understanding gene expression patterns across different human cell types is crucial for investigating mechanisms of cell type differentiation, disease occurrenceand progression. The recent development of single-cell RNA-seq (scRNA-seq) technologies significantly boosted the characterization of cell type heterogeneities in different human tissues. However, the huge number of datasets in the public domain also posed challenges in data integration and reuse. We present Human Universal Single Cell Hub (HUSCH, http://husch.comp-genomics.org), an atlas-scale curated database that integrates single-cell transcriptomic profiles of nearly 3 million cells from 185 high-quality human scRNA-seq datasets from 45 different tissues. All the data in HUSCH were uniformly processed and annotated with a standard workflow. In the single dataset module, HUSCH provides interactive gene expression visualization, differentially expressed genes, functional analyses, transcription regulatorsand cell-cell interaction analyses for each cell type cluster. Besides, HUSCH integrated different datasets in the single tissue module and performs data integration, batch correction, and cell type harmonization. This allows a comprehensive visualization and analysis of gene expression within each tissue based on single-cell datasets from multiple sources and platforms. HUSCH is a flexible and comprehensive data portal that enables searching, visualizing, analyzing, and downloading single-cell gene expression for the human tissue atlas.

Chromatin Accessibility and Transcriptional Differences in Human Stem Cell-Derived Early-Stage Retinal Organoids.

Retinogenesis involves the specification of retinal cell types during early vertebrate development. While model organisms have been critical for determining the role of dynamic chromatin and cell-type specific transcriptional networks during this process, an enhanced understanding of the developing human retina has been more elusive due to the requirement for human fetal tissue. Pluripotent stem cell (PSC) derived retinal organoids offer an experimentally accessible solution for investigating the developing human retina. To investigate cellular and molecular changes in developing early retinal organoids, we developed SIX6-GFP and VSX2-tdTomato (or VSX2-h2b-mRuby3) dual fluorescent reporters. When differentiated as 3D organoids these expressed GFP at day 15 and tdTomato (or mRuby3) at day 25, respectively. This enabled us to explore transcriptional and chromatin related changes using RNA-seq and ATAC-seq from pluripotency through early retina specification. Pathway analysis of developing organoids revealed a stepwise loss of pluripotency, while optic vesicle and retina pathways became progressively more prevalent. Correlating gene transcription with chromatin accessibility in early eye field development showed that retinal cells underwent a clear change in chromatin landscape, as well as gene expression profiles. While each dataset alone provided valuable information, considering both in parallel provided an informative glimpse into the molecular nature eye development.

Relationship between total and differential quarter somatic cell counts at dry-off and early lactation.

Mastitis is a most common disease of dairy cows and causes tremendous economic loss to the dairy industry worldwide. Somatic cell counts (SCC) reflect the inflammatory response to infections and is a metric used as key indicator in mastitis screening programs, typically within the framework of national milk recording schemes. Besides the determination of total SCC, the differentiation of cell types has been described to be beneficial for a more definite description of the actual udder health status of dairy cows. Differential somatic cell count (DSCC) represents the combined proportion of polymorphonuclear leukocytes (PMN) and lymphocytes expressed as a percentage of the total. The aim of this study was to investigate the relationship between SCC and differential somatic cell count (DSCC) in individual quarter milk samples collected at different time points: at dry-off, after calving and at the lactation peak. We used individual quarter data from farms representing the specialized production system of Parmigiano Reggiano cheese in Northern Italy. Average DSCC values ranged between 44.9% and 56.3%, with higher values (60.4%-72.1%) in milk samples with ≥ 1 million SCC/ml (where the proportion of samples with DSCC > 70% can be as high as 0.73). Moderate overall correlations between DSCC and log(SCC) were estimated (Pearson = 0.42, Spearman = 0.38), with a clear increasing trend with parity and around the lactation peak (e.g. Pearson = 0.59 at 60 DIM in parity 4). Taking SCC values as indicators of subclinical mastitis, DSCC would diagnose mastitis with 0.75 accuracy. Data editing criteria do have an impact on results, with stricter filtering leading to lower correlations between log(SCC) and DSCC. In conclusion DSCC and SCC provide different descriptions of the udder health status of dairy cows which, at least to some extent, are independent. DSCC alone doesn’t provide more accurate information than SCC at quarter level but, used in combination with SCC, can be of potential interest within the framework of milk recording programs, especially in the context of selective dry-cow therapy (SDCT). However, this needs further investigation and updated threshold values need to be selected and validated.

Functional EGF domain of the human neuregulin 1α produced in Escherichia coli with accurate disulfide bonds.

Neuregulins comprise a large family of growth factors containing an epidermal growth factor (EGF) domain. NRG1 acts in signaling pathways involved in proliferation, apoptosis, migration, differentiation, and adhesion of many normal cell types and in human diseases. The EGF domain of NRG1 mediates signaling by interaction with members of the ErbB family of receptors. Easy access to correctly folded hNRG1α EGF domain can be a valuable tool to investigate its function in different cell types. The EGF domain of hNRG1α was produced in Escherichia coli in fusion with TrxA and purified after cleavage of TrxA. Conformation and stability analyses were performed by using biophysical methods and the disulfide bonds were mapped by mass spectrometry. The activity of the hNRG1α EGF domain was demonstrated in cell proliferation and migration assays. Approximately 3.3mg of hNRG1α EGF domain were obtained starting from a 0.5L of E. coli culture. Correct formation of the three disulfide bonds was demonstrated by mass spectrometry with high accuracy. Heat denaturation assays monitored by circular dichroism and dynamic light scattering revealed that it is a highly stable protein. The recombinant EGF domain of hNRG1α purified in this work is highly active, inducing cell proliferation at concentration as low as 0.05 ng/mL. It induces also cell migration as demonstrated by a gap closure assay. The EGF domain of hNRG1α was produced in E. coli with the correct disulfide bonds and presented high stimulation of HeLa cell proliferation and NDFH cell migration.

Nucleosome proteostasis and histone turnover.

Maintenance of protein folding homeostasis, or proteostasis is critical for cell survival as well as for execution of cell type specific biological processes such as muscle cell contractility, neuronal synapse and memory formation, and cell transition from a mitotic to post-mitotic cell type. Cell type specification is driven largely by chromatin organization, which dictates which genes are turned off or on, depending on cell needs and function. Loss of chromatin organization can have catastrophic consequences either on cell survival or cell type specific function. Chromatin organization is highly dependent on organization of nucleosomes, spatiotemporal nucleosome assembly and disassembly, and histone turnover. In this review our goal is to highlight why nucleosome proteostasis is critical for chromatin organization, how this process is mediated by histone chaperones and ATP-dependent chromatin remodelers and outline potential and established mechanisms of disrupted nucleosome proteostasis during disease. Finally, we highlight how these mechanisms of histone turnover and nucleosome proteostasis may conspire with unfolded protein response programs to drive histone turnover in cell growth and development.

Effects of Notch signaling pathway inhibition by dibenzazepine in acute experimental toxoplasmosis

Notch signaling pathway plays a crucial role in cellular fate across species, being important for the differentiation and development of several cell types. The aim of this study was to evaluate the effect of Notch inhibition pathway by dibenzazepine (DBZ) in histological and inflammatory alterations and, tissue parasitism in acute Toxoplasma gondii infection. For this, C57BL/6 mice were treated with DBZ before infection with T. gondii, and the small intestine, lungs and liver were analyzed. The genes related to Notch signaling pathway were assayed through qPCR in the organs, and cytokine measurement was performed in serum samples. In the small intestine, T. gondii infection impaired the Hes1 and Math1 mRNA expressions, increased the inflammation and decreased goblet and Paneth cell numbers. The DBZ-treatment was able to partially preserve these cells, however, the parasitism and inflammation were not altered. In parallel, the high IL-2, IL-6, TNF and, IFN-γ levels induced by infection were not changed with the DBZ treatment, with the IFN-γ levels even higher. In contrast, in the liver and lungs, the DBZ-treatment diminished parasitism and inflammation. Our results highlight that Notch pathway inhibition in T.gondii infection results in different parasitological and inflammatory outcomes depending on the organ analyzed.

TGFβ superfamily signaling regulates the state of human stem cell pluripotency and capacity to create well-structured telencephalic organoids.

SummaryTelencephalic organoids generated from human pluripotent stem cells (hPSCs) are a promising system for studying the distinct features of the developing human brain and the underlying causes of many neurological disorders. While organoid technology is steadily advancing, many challenges remain, including potential batch-to-batch and cell-line-to-cell-line variability, and structural inconsistency. Here, we demonstrate that a major contributor to cortical organoid quality is the way hPSCs are maintained prior to differentiation. Optimal results were achieved using particular fibroblast-feeder-supported hPSCs rather than feeder-independent cells, differences that were reflected in their transcriptomic states at the outset. Feeder-supported hPSCs displayed activation of diverse transforming growth factor β (TGFβ) superfamily signaling pathways and increased expression of genes connected to naive pluripotency. We further identified combinations of TGFβ-related growth factors that are necessary and together sufficient to impart broad telencephalic organoid competency to feeder-free hPSCs and enhance the formation of well-structured brain tissues suitable for disease modeling.

Prospects of exogenous inositols in maintaining of skin, hair and nails condition: A review

Myoinositol (MI) and D-chiroinositol (DCI), used in the therapy of menstrual disorders, polycystic ovarian syndrome (PCOS), hirsutism, acne, gestational diabetes, and other diseases, are required for intracellular signal transduction from insulin and other hormone receptors. Clinical practice shows that, for instance, MI and DCI in the treatment of PCOS improve the condition of skin, hair, and nails. These effects of MI and DCI are related to the normalization of insulin signaling and support of differentiation and growth of various skin cell types (keratinocytes, fibroblasts, epitheliocytes, etc.). The effects of MI and DCI on the skin and its appendages, including in wound healing, can be enhanced by manganese, which provides an antioxidant effect and improves the connective tissue matrix of the skin, and folic acid, which is involved in amino acid metabolism, proliferation, and differentiation of dividing cells.

Cellular Biogenetic Law and Its Distortion by Protein Interactions: A Possible Unified Framework for Cancer Biology and Regenerative Medicine.

The biogenetic law (recapitulation law) states that ontogenesis recapitulates phylogenesis. However, this law can be distorted by the modification of development. We showed the recapitulation of phylogenesis during the differentiation of various cell types, using a meta-analysis of human single-cell transcriptomes, with the control for cell cycle activity and the improved phylostratigraphy (gene dating). The multipotent progenitors, differentiated from pluripotent embryonic stem cells (ESC), showed the downregulation of unicellular (UC) genes and the upregulation of multicellular (MC) genes, but only in the case of those originating up to the Euteleostomi (bony vertebrates). This picture strikingly resembles the evolutionary profile of regulatory gene expansion due to gene duplication in the human genome. The recapitulation of phylogenesis in the induced pluripotent stem cells (iPSC) during their differentiation resembles the ESC pattern. The unipotent erythroblasts differentiating into erythrocytes showed the downregulation of UC genes and the upregulation of MC genes originating after the Euteleostomi. The MC interactome neighborhood of a protein encoded by a UC gene reverses the gene expression pattern. The functional analysis showed that the evolved environment of the UC proteins is typical for protein modifiers and signaling-related proteins. Besides a fundamental aspect, this approach can provide a unified framework for cancer biology and regenerative/rejuvenation medicine because oncogenesis can be defined as an atavistic reversal to a UC state, while regeneration and rejuvenation require an ontogenetic reversal.

Phospholipase D1 promotes astrocytic differentiation through the FAK/AURKA/STAT3 signaling pathway in hippocampal neural stem/progenitor cells

Phospholipase D1 (PLD1) plays a crucial role in cell differentiation of different cell types. However, the involvement of PLD1 in astrocytic differentiation remains uncertain. In the present study, we investigate the possible role of PLD1 and its product phosphatidic acid (PA) in astrocytic differentiation of hippocampal neural stem/progenitor cells (NSPCs) from hippocampi of embryonic day 16.5 rat embryos. We showed that overexpression of PLD1 increased the expression level of glial fibrillary acidic protein (GFAP), an astrocyte marker, and the number of GFAP-positive cells. Knockdown of PLD1 by transfection with Pld1 shRNA inhibited astrocytic differentiation. Moreover, PLD1 deletion (Pld1−/−) suppressed the level of GFAP in the mouse hippocampus. These results indicate that PLD1 plays a crucial role in regulating astrocytic differentiation in hippocampal NSPCs. Interestingly, PA itself was sufficient to promote astrocytic differentiation. PA-induced GFAP expression was decreased by inhibition of signal transducer and activation of transcription 3 (STAT3) using siRNA. Furthermore, PA-induced STAT3 activation and astrocytic differentiation were regulated by the focal adhesion kinase (FAK)/aurora kinase A (AURKA) pathway. Taken together, our findings suggest that PLD1 is an important modulator of astrocytic differentiation in hippocampal NSPCs via the FAK/AURKA/STAT3 signaling pathway.

Morphogenesis of the female reproductive tract along antero-posterior and dorso-ventral axes is dependent on Amhr2+ mesenchyme in mice†.

Morphogenesis of the female reproductive tract is regulated by the mesenchyme. However, the identity of the mesenchymal lineage that directs the morphogenesis of the female reproductive tract has not been determined. Using in vivo genetic cell ablation, we identified Amhr2+ mesenchyme as an essential mesenchymal population in patterning the female reproductive tract. After partial ablation of Amhr2+ mesenchymal cells, the oviduct failed to develop its characteristic coiling due to decreased epithelial proliferation and tubule elongation during development. The uterus displayed a reduction in size and showed decreased cellular proliferation in both epithelial and mesenchymal compartments. More importantly, in the uterus, partial ablation of Amhr2+ mesenchyme caused abnormal lumen shape and altered the direction of its long axis from the dorsal-ventral axis to the left-right axis (i.e., perpendicular to the dorsal-ventral axis). Despite these morphological defects, epithelia underwent normal differentiation into secretory and ciliated cells in the oviduct and glandular epithelial cells in the uterus. These results demonstrated that Amhr2+ mesenchyme can direct female reproductive tract morphogenesis by regulating epithelial proliferation and lumen shape without affecting the differentiation of epithelial cell types.

MRNA isoform balance in neuronal development and disease.

Balanced mRNA isoform diversity and abundance are spatially and temporally regulated throughout cellular differentiation. The proportion of expressed isoforms contributes to cell type specification and determines key properties of the differentiated cells. Neurons are unique cell types with intricate developmental programs, characteristic cellular morphologies, and electrophysiological potential. Neuron-specific gene expression programs establish these distinctive cellular characteristics and drive diversity among neuronal subtypes. Genes with neuron-specific alternative processing are enriched in key neuronal functions, including synaptic proteins, adhesion molecules, and scaffold proteins. Despite the similarity of neuronal gene expression programs, each neuronal subclass can be distinguished by unique alternative mRNA processing events. Alternative processing of developmentally important transcripts alters coding and regulatory information, including interaction domains, transcript stability, subcellular localization, and targeting by RNA binding proteins. Fine-tuning of mRNA processing is essential for neuronal activity and maintenance. Thus, the focus of neuronal RNA biology research is to dissect the transcriptomic mechanisms that underlie neuronal homeostasis, and consequently, predispose neuronal subtypes to disease. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development.

Crosstalk between Ca2+ Signaling and Cancer Stemness: The Link to Cisplatin Resistance.

In the fight against cancer, therapeutic strategies using cisplatin are severely limited by the appearance of a resistant phenotype. While cisplatin is usually efficient at the beginning of the treatment, several patients endure resistance to this agent and face relapse. One of the reasons for this resistant phenotype is the emergence of a cell subpopulation known as cancer stem cells (CSCs). Due to their quiescent phenotype and self-renewal abilities, these cells have recently been recognized as a crucial field of investigation in cancer and treatment resistance. Changes in intracellular calcium (Ca2+) through Ca2+ channel activity are essential for many cellular processes such as proliferation, migration, differentiation, and survival in various cell types. It is now proved that altered Ca2+ signaling is a hallmark of cancer, and several Ca2+ channels have been linked to CSC functions and therapy resistance. Moreover, cisplatin was shown to interfere with Ca2+ homeostasis; thus, it is considered likely that cisplatin-induced aberrant Ca2+ signaling is linked to CSCs biology and, therefore, therapy failure. The molecular signature defining the resistant phenotype varies between tumors, and the number of resistance mechanisms activated in response to a range of pressures dictates the global degree of cisplatin resistance. However, if we can understand the molecular mechanisms linking Ca2+ to cisplatin-induced resistance and CSC behaviors, alternative and novel therapeutic strategies could be considered. In this review, we examine how cisplatin interferes with Ca2+ homeostasis in tumor cells. We also summarize how cisplatin induces CSC markers in cancer. Finally, we highlight the role of Ca2+ in cancer stemness and focus on how they are involved in cisplatin-induced resistance through the increase of cancer stem cell populations and via specific pathways.