Efficient GFP-labeling and analysis of spermatogenic cells using the IRG transgene and flow cytometry

AbstractSpermatogenesis is a highly ordered developmental program that produces haploid male germ cells. The study of male germ cell development in the mouse has provided unique perspectives into the molecular mechanisms that control cell development and differentiation in mammals, including tissue-specific gene regulatory programs. An intrinsic challenge in spermatogenesis research is the heterogeneity of germ and somatic cell types present in the testis. Techniques to separate and isolate distinct mouse spermatogenic cell types have great potential to shed light on molecular mechanisms controlling mammalian cell development, while also providing new insights into cellular events important for human reproductive health. Here, we detail a versatile strategy that combines Cre-lox technology to fluorescently label germ cells, with flow cytometry to discriminate and isolate germ cells in different stages of development for cellular and molecular analyses.

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Studying the Role and Molecular Mechanisms of MAP4K3 in Sorafenib Resistance of Hepatocellular Carcinoma

BioMed Research International ◽

10.1155/2020/4965670 ◽

2020 ◽

Vol 2020 ◽

pp. 1-8

Author(s):

Yi Shi ◽

Xiaofei Mo ◽

Simei Hong ◽

Tianbao Li ◽

Baozhen Chen ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Flow Cytometry ◽

Cell Proliferation ◽

Molecular Mechanisms ◽

Cellular Proliferation ◽

Resistant Cell ◽

Therapeutic Drug ◽

Specific Gene ◽

Sorafenib Treatment ◽

Proliferation And Apoptosis

Sorafenib is the first FDA-approved therapeutic drug for molecular target medication on advanced-stage hepatocellular carcinoma. It is reported that sorafenib could improve the survival of progression-free patients for 4 to 6 months; however, most of the patients developed drug resistance. Thus, it is critical to reveal the biological mechanisms behind sorafenib resistance. In this study, a sorafenib-resistant model was developed by exposing HepG2 cells to sorafenib with gradient increasing concentration, and the resistance-related genes were screened by microarray. Real-time qPCR was used to validate selected gene expression of the resistance model, and lentivirus vector-mediated RNA interference was applied for specific gene knockdown. In addition, high-throughput High Celigo Select (HCS) and flow cytometry were used to measure the effect on cellular proliferation and apoptosis. As a result, our study established a sorafenib-resistant model with IC50 of 9.988 μM. The Affymetrix expression profile of the sorafenib-resistant model showed 35 resistant-related genes, and 91.4% of the resistant genes showed upregulation in HepG2 resistance cells. In addition, 20 genes were knocked down to measure cell proliferation, and MAP4K3 with high proliferation inhibiting phenotype was chosen for further study. Meanwhile, the HCS results revealed that shMAP4K3 transfection could downregulate resistant cell proliferation, and the flow cytometry results showed that cell apoptosis was significantly increased in the MAP4K3 knockdown group. In summary, MAP4K3 is a novel molecular marker for improving the drug sensitivity of sorafenib treatment in hepatocellular carcinoma.

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Diverse patterns of cell-specific gene expression in response to glucocorticoid in the developing small intestine

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00139.2006 ◽

2006 ◽

Vol 291 (6) ◽

pp. G1041-G1050 ◽

Cited By ~ 13

Author(s):

Murat B. Yaylaoglu ◽

Barbara M. Agbemafle ◽

Thomas J. Oesterreicher ◽

Milton J. Finegold ◽

Christina Thaller ◽

...

Keyword(s):

Epithelial Cells ◽

Molecular Mechanisms ◽

Cell Types ◽

Northern Blotting ◽

Specific Gene ◽

Cellular Expression ◽

Intestinal Segments ◽

Transcript Gene ◽

Microarray Studies ◽

Glucocorticoid Action

Although glucocorticoids are known to elicit functional maturation of the gastrointestinal tract, the molecular mechanisms of glucocorticoid action on the developing intestine have not been fully elucidated. Our previous microarray studies identified 66 transcripts as being rapidly induced in the jejunum following dexamethasone (Dex) administration to suckling mice. Now we report the specific cellular location of a subset of these transcripts. Mouse pups at P8 received Dex or vehicle and intestinal segments were collected 3–4 h later. Robotic-based in situ hybridization (ISH) was performed with digoxygenin-labeled riboprobes. Transcripts studied included Ndrg1, Sgk1, Fos, and two unknown genes ( Gene 9 and Gene 36). As predicted, ISH revealed marked diversity of cellular expression. In small intestinal segments, Sgk1 mRNA was in all epithelial cells; Fos mRNA was confined to epithelial cells at the villus tip; and Ndrg1 and Gene 36 mRNAs were localized to epithelial cells of the upper crypt and villus base. The remaining transcript ( Gene 9) was induced modestly in villus stroma and strongly in the muscle layers. In the colon, Ndrg1, Sgk1, and Gene 36 were induced in all epithelial cells; Gene 9 was in muscle layers only; and Fos was not detectable. For jejunal segments, quantitation of ISH signals in tissue from Dex-treated and vehicle-treated mice demonstrated mRNA increases very similar to those measured by Northern blotting. We conclude that glucocorticoid action in the intestine reflects diverse molecular mechanisms operating in different cell types and that quantitative ISH is a valuable tool for studying hormone action in this tissue.

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STAT3 and STAT5 Activation in Solid Cancers

Cancers ◽

10.3390/cancers11101428 ◽

2019 ◽

Vol 11 (10) ◽

pp. 1428 ◽

Cited By ~ 18

Author(s):

Sebastian Igelmann ◽

Heidi Neubauer ◽

Gerardo Ferbeyre

Keyword(s):

Tumor Suppressor ◽

Molecular Mechanisms ◽

Target Genes ◽

Critical Role ◽

Control Cell ◽

Cytokine Signaling ◽

Specific Gene ◽

Mitochondrial Functions ◽

Context Dependent

The Signal Transducer and Activator of Transcription (STAT)3 and 5 proteins are activated by many cytokine receptors to regulate specific gene expression and mitochondrial functions. Their role in cancer is largely context-dependent as they can both act as oncogenes and tumor suppressors. We review here the role of STAT3/5 activation in solid cancers and summarize their association with survival in cancer patients. The molecular mechanisms that underpin the oncogenic activity of STAT3/5 signaling include the regulation of genes that control cell cycle and cell death. However, recent advances also highlight the critical role of STAT3/5 target genes mediating inflammation and stemness. In addition, STAT3 mitochondrial functions are required for transformation. On the other hand, several tumor suppressor pathways act on or are activated by STAT3/5 signaling, including tyrosine phosphatases, the sumo ligase Protein Inhibitor of Activated STAT3 (PIAS3), the E3 ubiquitin ligase TATA Element Modulatory Factor/Androgen Receptor-Coactivator of 160 kDa (TMF/ARA160), the miRNAs miR-124 and miR-1181, the Protein of alternative reading frame 19 (p19ARF)/p53 pathway and the Suppressor of Cytokine Signaling 1 and 3 (SOCS1/3) proteins. Cancer mutations and epigenetic alterations may alter the balance between pro-oncogenic and tumor suppressor activities associated with STAT3/5 signaling, explaining their context-dependent association with tumor progression both in human cancers and animal models.

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Expression Profiling of Mammalian Male Meiosis and Gametogenesis Identifies Novel Candidate Genes for Roles in the Regulation of Fertility

Molecular Biology of the Cell ◽

10.1091/mbc.e03-10-0762 ◽

2004 ◽

Vol 15 (3) ◽

pp. 1031-1043 ◽

Cited By ~ 97

Author(s):

Ulrich Schlecht ◽

Philippe Demougin ◽

Reinhold Koch ◽

Leandro Hermida ◽

Christa Wiederkehr ◽

...

Keyword(s):

Germ Cell ◽

Germ Cells ◽

Expression Profiling ◽

Large Scale ◽

Developmental Stages ◽

Expression Patterns ◽

Control Cell ◽

Cell Types ◽

Male Meiosis ◽

Cell Expression

We report a comprehensive large-scale expression profiling analysis of mammalian male germ cells undergoing mitotic growth, meiosis, and gametogenesis by using high-density oligonucleotide microarrays and highly enriched cell populations. Among 11,955 rat loci investigated, 1268 were identified as differentially transcribed in germ cells at subsequent developmental stages compared with total testis, somatic Sertoli cells as well as brain and skeletal muscle controls. The loci were organized into four expression clusters that correspond to somatic, mitotic, meiotic, and postmeiotic cell types. This work provides information about expression patterns of ∼200 genes known to be important during male germ cell development. Approximately 40 of those are included in a group of 121 transcripts for which we report germ cell expression and lack of transcription in three somatic control cell types. Moreover, we demonstrate the testicular expression and transcriptional induction in mitotic, meiotic, and/or postmeiotic germ cells of 293 as yet uncharacterized transcripts, some of which are likely to encode factors involved in spermatogenesis and fertility. This group also contains potential germ cell-specific targets for innovative contraceptives. A graphical display of the data is conveniently accessible through the GermOnline database at http://www.germonline.org .

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Germline competent mesoderm: the substrate for vertebrate germline and somatic stem cells?

Biology Open ◽

10.1242/bio.058890 ◽

2021 ◽

Vol 10 (10) ◽

Author(s):

Aaron M. Savage ◽

Ramiro Alberio ◽

Andrew D. Johnson

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Germ Cells ◽

Molecular Mechanisms ◽

Germ Plasm ◽

Cell Types ◽

Model Organisms ◽

Developmental Potential ◽

Tissue Specific ◽

Tissue Specific Stem Cells

ABSTRACT In vitro production of tissue-specific stem cells [e.g. haematopoietic stem cells (HSCs)] is a key goal of regenerative medicine. However, recent efforts to produce fully functional tissue-specific stem cells have fallen short. One possible cause of shortcomings may be that model organisms used to characterize basic vertebrate embryology (Xenopus, zebrafish, chick) may employ molecular mechanisms for stem cell specification that are not conserved in humans, a prominent example being the specification of primordial germ cells (PGCs). Germ plasm irreversibly specifies PGCs in many models; however, it is not conserved in humans, which produce PGCs from tissue termed germline-competent mesoderm (GLCM). GLCM is not conserved in organisms containing germ plasm, or even in mice, but understanding its developmental potential could unlock successful production of other stem cell types. GLCM was first discovered in embryos from the axolotl and its conservation has since been demonstrated in pigs, which develop from a flat-disc embryo like humans. Together these findings suggest that GLCM is a conserved basal trait of vertebrate embryos. Moreover, the immortal nature of germ cells suggests that immortality is retained during GLCM specification; here we suggest that the demonstrated pluripotency of GLCM accounts for retention of immortality in somatic stem cell types as well. This article has an associated Future Leaders to Watch interview with the author of the paper.

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50 years of spermatogenesis: Sertoli cells and their interactions with germ cells

Biology of Reproduction ◽

10.1093/biolre/ioy027 ◽

2018 ◽

Vol 99 (1) ◽

pp. 87-100 ◽

Cited By ~ 27

Author(s):

Michael D Griswold

Keyword(s):

Germ Cell ◽

Germ Cells ◽

Sertoli Cells ◽

Cultured Cells ◽

Cell Types ◽

Cell Development ◽

Specific Cell ◽

Germ Cell Development ◽

Cell Niche ◽

Transgenic Technologies

Abstract The complex morphology of the Sertoli cells and their interactions with germ cells has been a focus of investigators since they were first described by Enrico Sertoli. In the past 50 years, information on Sertoli cells has transcended morphology alone to become increasingly more focused on molecular questions. The goal of investigators has been to understand the role of the Sertoli cells in spermatogenesis and to apply that information to problems relating to male fertility. Sertoli cells are unique in that they are a nondividing cell population that is active for the reproductive lifetime of the animal and cyclically change morphology and gene expression. The numerous and distinctive junctional complexes and membrane specializations made by Sertoli cells provide a scaffold and environment for germ cell development. The increased focus of investigators on the molecular components and putative functions of testicular cells has resulted primarily from procedures that isolate specific cell types from the testicular milieu. Products of Sertoli cells that influence germ cell development and vice versa have been characterized from cultured cells and from the application of transgenic technologies. Germ cell transplantation has shown that the Sertoli cells respond to cues from germ cells with regard to developmental timing and has furthered a focus on spermatogenic stem cells and the stem cell niche. Very basic and universal features of spermatogenesis such as the cycle of the seminiferous epithelium and the spermatogenic wave are initiated by Sertoli cells and maintained by Sertoli-germ cell cooperation.

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Expression profiling reveals novel role of Hunchback in retinal glia cell development and blood-brain barrier integrity

10.1101/114363 ◽

2017 ◽

Author(s):

Montserrat Torres-Oliva ◽

Julia Schneider ◽

Gordon Wiegleb ◽

Felix Kaufholz ◽

Nico Posnien

Keyword(s):

Transcription Factor ◽

Blood Brain Barrier ◽

Molecular Mechanisms ◽

Cell Types ◽

Cell Development ◽

Brain Barrier ◽

Gene Clustering ◽

Glia Cell ◽

Blood Brain

AbstractThe development of different cell types must be tightly coordinated in different organs. The developing head of Drosophila melanogaster represents an excellent model to study the molecular mechanisms underlying this coordination because the eye-antennal imaginal discs contain the organ anlagen of nearly all adult head structures, such as the compound eyes or the antennae. We studied the genome wide gene expression dynamics during eye-antennal disc development in D. melanogaster to identify new central regulators of the underlying gene regulatory network. Expression based gene clustering and transcription factor motif enrichment analyses revealed a central regulatory role of the transcription factor Hunchback (Hb). We confirmed that hb is expressed in two polyploid retinal subperineurial glia cells (carpet cells). Our functional analysis shows that Hb is necessary for carpet cell development and loss of Hb function results in abnormal glia cell migration and photoreceptor axon guidance patterns. Additionally, we show for the first time that the carpet cells are an integral part of the blood-brain barrier.

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Developmental and evolutionary dynamics of cis-regulatory elements in mouse cerebellar cells

Science ◽

10.1126/science.abg4696 ◽

2021 ◽

pp. eabg4696

Author(s):

Ioannis Sarropoulos ◽

Mari Sepp ◽

Robert Frömel ◽

Kevin Leiss ◽

Nils Trost ◽

...

Keyword(s):

Regulatory Networks ◽

Evolutionary Dynamics ◽

Cell Types ◽

Regulatory Elements ◽

Specific Gene ◽

Organ Development ◽

Development And Differentiation ◽

Cerebellar Cell ◽

Time Specific ◽

Cerebellar Cells

Organ development is orchestrated by cell- and time-specific gene regulatory networks. In this study, we investigated the regulatory basis of mouse cerebellum development from early neurogenesis to adulthood. By acquiring snATAC-seq profiles for ~90,000 cells spanning eleven stages, we mapped cerebellar cell types and identified candidate cis-regulatory elements (CREs). We detected extensive spatiotemporal heterogeneity among progenitor cells and a gradual divergence in the regulatory programs of cerebellar neurons during differentiation. Comparisons to vertebrate genomes and snATAC-seq profiles for ∼20,000 cerebellar cells from the marsupial opossum revealed a shared decrease in CRE conservation during development and differentiation, but also differences in constraint between cell types. Our work delineates the developmental and evolutionary dynamics of gene regulation in cerebellar cells and provides insights into mammalian organ development.

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ATAC-STARR-seq v1

10.17504/protocols.io.b2nqqddw ◽

2021 ◽

Author(s):

Tyler Hansen ◽

Emily Hodges

Keyword(s):

Transcription Factor ◽

Control Cell ◽

Regulatory Region ◽

Active Regions ◽

Cell Types ◽

Regulatory Elements ◽

Transcription Factor Binding ◽

Specific Gene ◽

Factor Binding ◽

Accessible Chromatin

Transcriptional enhancers control cell-type specific gene expression in humans and dysfunction can lead to debilitating diseases, including cancer. Identifying bona-fide enhancers is difficult due to a lack of spatial or sequence constraints. In addition, only a small percentage of the genome is accessible in matured cell types; and therefore, most enhancers are inactive due to their chromatin context rather than intrinsic properties of the DNA sequence itself. For this reason, we decided to assay regulatory activity exclusively within accessible chromatin. To do this, we combined assay for transposase-accessible chromatin using sequencing (ATAC-seq) with self-transcribing active regulatory region sequencing (STARR-seq); we call this method ATAC-STARR-seq. With ATAC-STARR-seq, we identify both active and silent regulatory elements in GM12878 B cells; these active and silent elements are enriched for transcription factor motifs and histone modifications associated with activating and repressing regulation, respectively. We also show that ATAC-STARR-seq quantifies chromatin accessibility and transcription factor binding. We integrate this information and subset active regions based on transcription factor binding profiles. Depending on the transcription factors bound, subsets are enriched for distinct reactome pathways. Altogether, this highlights the power of ATAC-STARR-seq to investigate the transcriptional regulatory landscape of the human genome.

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Interaction of basal positive and negative transcription elements controls repression of the proximal rat prolactin promoter in nonpituitary cells.

Molecular and Cellular Biology ◽

10.1128/mcb.12.6.2708 ◽

1992 ◽

Vol 12 (6) ◽

pp. 2708-2719 ◽

Cited By ~ 38

Author(s):

S M Jackson ◽

C A Keech ◽

D J Williamson ◽

A Gutierrez-Hartmann

Keyword(s):

Gene Expression ◽

Inhibitory Activity ◽

Promoter Activity ◽

Molecular Mechanisms ◽

Cell Types ◽

Specific Gene ◽

Specific Cell ◽

Pituitary Cells ◽

Promoter Assay ◽

Activating Function

The proximal rat prolactin (rPRL) promoter contains three cell-specific elements, designated footprints I, III, and IV, which restrict rPRL gene expression to anterior pituitary lactotroph cells. Footprint II (-130 to -120) binds a factor, which we have termed F2F, present in pituitary and nonpituitary cell types. Here we demonstrate that a key role of the footprint II site is to inhibit rPRL promoter activity in nonpituitary cells, specifically, by interfering with the basal activating function of a vicinal element. Gene transfer analysis revealed 20-fold activation of the rPRL promoter in nonpituitary cell types when footprint II was either deleted or specifically mutated. Similar activation of the intact rPRL promoter was obtained by in vivo F2F titration studies. In GH4 rat pituitary cells, the footprint II inhibitory activity was masked by the redundant, positively acting cell-specific elements and was inhibitory only if the two upstream sites, footprints III and IV, were deleted. Deletion of the -112 to -80 region in the footprint II site-specific mutant background resulted in complete loss of rPRL promoter activity in both pituitary and nonpituitary cell types, mapping a basal activating element that is operative irrespective of cell type to this region. While the basal activating element imparted an activating function in a heterologous promoter assay, the footprint II sequence did not display any inherent repressor function and actually induced several minimal heterologous promoters. However, the inhibitory activity of the footprint II site was detected only if it was in context with the basal activating element. These data underscore the importance of ubiquitous activating and inhibitory factors in establishing cell-specific gene expression and further emphasize the complexity of the molecular mechanisms which restrict gene expression to specific cell types. We provide a novel paradigm to study rPRL promoter function and hormone responsiveness independently of lactotroph cell-specific requirements.

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