Purification of astrocyte subtype based on a double reporter mice approach for downstream transcription profiling.
Ontology highlight
ABSTRACT:
SUBMITTER: Aguirrebengoa M
PROVIDER: S-EPMC8672091 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Astrocytes are recognized to be a heterogeneous population of cells that differ morphologically, functionally, and molecularly. Whether this heterogeneity results from generation of distinct astrocyte cell lineages, each functionally specialized to perform specific tasks, remains an open question. In this study, we used RNA sequencing analysis to determine the global transcriptome profile of the Olig2-expressing astrocyte subtype (Olig2-AS), a specific spinal astrocyte subtype that segregates early during development from Olig2 progenitors and differs from other spinal astrocytes by the expression of Olig2. We identified 245 differentially expressed genes. Among them, 135 exhibit higher levels of expression when compared with other populations of spinal astrocytes, indicating that these genes can serve as a "unique" functional signature of Olig2-AS. Among them, we identify two genes, inka2 and kcnip3, as specific molecular markers of the Olig2-AS in the P7 spinal cord. Our work thus reveals that Olig2 progenitors produce a unique spinal astrocyte subtype.
| S-EPMC8281609 | biostudies-literature
Project description:The diversity of cardiac lineages contributes to the heterogeneity of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs). Here, we report the generation of a hiPSC TBX5Clover2 and NKX2-5TagRFP double reporter to delineate cardiac lineages and isolate lineage-specific subpopulations. Molecular analyses reveal that four different subpopulations can be isolated based on the differential expression of TBX5 and NKX2-5, TBX5+NKX2-5+, TBX5+NKX2-5-, TBX5-NKX2-5+, and TBX5-NKX2-5-, mimicking the first heart field, epicardial, second heart field, and endothelial lineages, respectively. Genetic and functional characterization indicates that each subpopulation differentiates into specific cardiac cells. We further identify CORIN as a cell-surface marker for isolating the TBX5+NKX2-5+ subpopulation and demonstrate the use of lineage-specific CMs for precise drug testing. We anticipate that this tool will facilitate the investigation of cardiac lineage specification and isolation of specific cardiac subpopulations for drug screening, tissue engineering, and disease modeling.
| S-EPMC6499654 | biostudies-literature
Project description:BackgroundThe development of quantitative models of signal transduction, as well as parameter estimation to improve existing models, depends on the ability to obtain quantitative information about various proteins that are part of the signaling pathway. However, commonly-used measurement techniques such as Western blots and mobility shift assays provide only qualitative or semi-quantitative data which cannot be used for estimating parameters. Thus there is a clear need for techniques that enable quantitative determination of signal transduction intermediates.ResultsThis paper presents an integrated modeling and experimental approach for quantitatively determining transcription factor profiles from green fluorescent protein (GFP) reporter data. The technique consists of three steps: (1) creating data sets for green fluorescent reporter systems upon stimulation, (2) analyzing the fluorescence images to determine fluorescence intensity profiles using principal component analysis (PCA) and K-means clustering, and (3) computing the transcription factor concentration from the fluorescence intensity profiles by inverting a model describing transcription, translation, and activation of green fluorescent proteins. We have used this technique to quantitatively characterize activation of the transcription factor NF-kappaB by the cytokine TNF-alpha. In addition, we have applied the quantitative NF-kappaB profiles obtained from our technique to develop a model for TNF-alpha signal transduction where the parameters were estimated from the obtained data.ConclusionThe technique presented here for computing transcription factor profiles from fluorescence microscopy images of reporter cells generated quantitative data on the magnitude and dynamics of NF-kappaB activation by TNF-alpha. The obtained results are in good agreement with qualitative descriptions of NF-kappaB activation as well as semi-quantitative experimental data from the literature. The profiles computed from the experimental data have been used to re-estimate parameters for a NF-kappaB model and the results of additional experiments are predicted very well by the model with the new parameter values. While the presented approach has been applied to NF-kappaB and TNF-alpha signaling, it can be used to determine the profile of any transcription factor as long as GFP reporter fluorescent profiles are available.
| S-EPMC2491602 | biostudies-literature
Project description:We observed that heat shock of Caenorhabditis elegans leads to the formation of nuclear double-stranded RNA (dsRNA) foci, detectable with a dsRNA-specific monoclonal antibody. These foci significantly overlap with nuclear HSF-1 granules. To investigate the molecular mechanism(s) underlying dsRNA foci formation, we used RNA-seq to globally characterize total RNA and immunoprecipitated dsRNA from control and heat shocked worms. We find a subset of both sense and antisense transcripts enriched in the dsRNA pool by heat shock overlap with dsRNA transcripts enriched by deletion of tdp-1, which encodes the C. elegans ortholog of TDP-43. Interestingly, transcripts involved in translation are over-represented in the dsRNAs induced by either heat shock or deletion of tdp-1. Also enriched in the dsRNA transcripts are sequences downstream of annotated genes (DoGs), which we globally quantified with a new algorithm. To validate these observations, we used fluorescence in situ hybridization (FISH) to confirm both antisense and downstream of gene transcription for eif-3.B, one of the affected loci we identified.
| S-EPMC6453478 | biostudies-literature
Project description:Transcription factor (TF) reporter mice have proved integral to the characterization of murine innate lymphoid cell (ILC) development and function. Here, we implemented a CRISPR/Cas9-generated combinatorial reporter approach for the simultaneous resolution of several key TFs throughout ILC development in both the fetal liver and adult bone marrow. We demonstrate that the Tcf7-expressing early innate lymphoid precursor (EILP) and the common helper ILC precursor (CHILP) both contain a heterogeneous mixture of specified ILC and lymphoid tissue inducer (LTi) precursors with restricted lineage potential rather than a shared precursor. Moreover, the earliest specified precursor to the LTi lineage was identified upstream of these populations, before Tcf7 expression. These findings match dynamic changes in chromatin accessibility associated with the expression of key TFs (i.e., GATA3 and RORγ(t)), highlighting the distinct origins of ILC and LTi lineages at the epigenetic and functional levels, and provide a revised map for ILC development.
| S-EPMC7590509 | biostudies-literature
Project description:Understanding the molecular mechanisms of gene regulation is pivotal for understanding how cells establish and modify their identities and functions. Multiple transcription factors (TFs) coordinate to alter gene expression in cells; however, a method to quantitatively analyze the activity of each TF is lacking, particularly in vivo. Here, we introduce a viral-vector-based TF reporter battery that can be used to simultaneously analyze the activity of multiple TFs, visualized as the TF activity profile (TFAP) obtained by qPCR. We show that the cells possess distinct TFAPs that dynamically change according to experimental manipulation or physiological activity. We report a practical method to obtain the TFAP of a defined cell population and their experience-dependent changes in the mouse brain in vivo. The TFAP obtained by our method will help bridge the information gap between the genome and transcriptome and aid the multi-omics view of understanding the gene regulation system.
| S-EPMC8904617 | biostudies-literature
Project description:ObjectiveAstrocytes have gained attention as important players in neurological disease. In line with their heterogeneous character, defects in specific astrocyte subtypes have been identified. Leukodystrophy vanishing white matter (VWM) shows selective vulnerability in white matter astrocytes, but the underlying mechanisms remain unclear. Induced pluripotent stem cell technology is being extensively explored in studies of pathophysiology and regenerative medicine. However, models for distinct astrocyte subtypes for VWM are lacking, thereby hampering identification of disease-specific pathways.MethodsHere, we characterize human and mouse pluripotent stem cell-derived gray and white matter astrocyte subtypes to generate an in vitro VWM model. We examined morphology and functionality, and used coculture methods, high-content microscopy, and RNA sequencing to study VWM cultures.ResultsWe found intrinsic vulnerability in specific astrocyte subpopulations in VWM. When comparing VWM and control cultures, white matter-like astrocytes inhibited oligodendrocyte maturation, and showed affected pathways in both human and mouse cultures, involving the immune system and extracellular matrix. Interestingly, human white matter-like astrocytes presented additional, human-specific disease mechanisms, such as neuronal and mitochondrial functioning.InterpretationAstrocyte subtype cultures revealed disease-specific pathways in VWM. Cross-validation of human- and mouse-derived protocols identified human-specific disease aspects. This study provides new insights into VWM disease mechanisms, which helps the development of in vivo regenerative applications, and we further present strategies to study astrocyte subtype vulnerability in neurological disease. ANN NEUROL 2019;86:780-792.
| S-EPMC6856690 | biostudies-literature
Project description:The innovation in recombinant protein technology has brought forth a host of challenges related to the purification of these therapeutic proteins. This article delves into the intricate landscape of developing purification processes for artificially designed therapeutic proteins. The key hurdles include controlling protein reduction, protein capture, ensuring stability, eliminating aggregates, removing host cell proteins and optimizing protein recovery. In this review, we outline the purification strategies in order to obtain products of high purity, highlighting the corresponding solutions to circumvent the unique challenges presented by recombinant therapeutic proteins, and exemplify the practical applications by case studies. Finally, a perspective towards future purification process development is provided.
| S-EPMC10791043 | biostudies-literature
Project description:Intestinal hormones are key regulators of digestion and energy homeostasis secreted by rare enteroendocrine cells. These cells produce over ten different hormones including GLP-1 and GIP peptides known to promote insulin secretion. To date, the molecular mechanisms controlling the specification of the various enteroendocrine subtypes from multipotent Neurog3(+) endocrine progenitor cells, as well as their number, remain largely unknown. In contrast, in the embryonic pancreas, the opposite activities of Arx and Pax4 homeodomain transcription factors promote islet progenitor cells towards the different endocrine cell fates. In this study, we thus investigated the role of Arx and Pax4 in enteroendocrine subtype specification. The small intestine and colon of Arx- and Pax4-deficient mice were analyzed using histological, molecular, and lineage tracing approaches. We show that Arx is expressed in endocrine progenitors (Neurog3(+)) and in early differentiating (ChromograninA(-)) GLP-1-, GIP-, CCK-, Sct- Gastrin- and Ghrelin-producing cells. We noted a dramatic reduction or a complete loss of all these enteroendocrine cell types in Arx mutants. Serotonin- and Somatostatin-secreting cells do not express Arx and, accordingly, the differentiation of Serotonin cells was not affected in Arx mutants. However, the number of Somatostatin-expressing D-cells is increased as Arx-deficient progenitor cells are redirected to the D-cell lineage. In Pax4-deficient mice, the differentiation of Serotonin and Somatostatin cells is impaired, as well as of GIP and Gastrin cells. In contrast, the number of GLP-1 producing L-cells is increased concomitantly with an upregulation of Arx. Thus, while Arx and Pax4 are necessary for the development of L- and D-cells respectively, they conversely restrict D- and L-cells fates suggesting antagonistic functions in D/L cell allocation. In conclusion, these finding demonstrate that, downstream of Neurog3, the specification of a subset of enteroendocrine subtypes relies on both Arx and Pax4, while others depend only on Arx or Pax4.
| S-EPMC3343025 | biostudies-literature
Project description:BackgroundProteins are used as reagents in a broad range of scientific fields. The reliability and reproducibility of experimental data will largely depend on the quality of the (recombinant) proteins and, consequently, these should undergo thorough structural and functional controls. Depending on the downstream application and the biochemical characteristics of the protein, different sets of specific features will need to be checked.ResultsA number of examples, representative of recurrent issues and previously published strategies, has been reported that illustrate real cases of recombinant protein production in which careful strategy design at the start of the project combined with quality controls throughout the production process was imperative to obtain high-quality samples compatible with the planned downstream applications. Some proteins possess intrinsic properties (e.g., prone to aggregation, rich in cysteines, or a high affinity for nucleic acids) that require certain precautions during the expression and purification process. For other proteins, the downstream application might demand specific conditions, such as for proteins intended for animal use that need to be endotoxin-free.ConclusionsThis review has been designed to act as a practical reference list for researchers who wish to produce and evaluate recombinant proteins with certain specific requirements or that need particular care for their preparation and storage.
| S-EPMC8991485 | biostudies-literature