Stem Cell Maintenance Research Articles

Genetic variation in NOTCH1 is associated with overweight and obesity in Brazilian elderly

Excessive weight (overweight and obesity) is a common disorder involving genetic and environmental factors, associated with cardiovascular diseases, type-2 diabetes, and others. NOTCH1 is critical for the maintenance of stem cells and adult tissues, being reported as a key player in metabolism and adipogenesis in animals. Thus, we test the hypothesis that NOTCH1 Single Nucleotide Polymorphisms (SNPs) are associated with excessive weight. Participants from the census-based cohort SABE (Saúde, Bem Estar e Envelhecimento—Health, Well-Being, and Aging), carried out in the city of São Paulo-Brazil, were stratified into cases and controls according to BMI. We filter the SNPs located at the start and end positions of NOTCH1 and 50 Kb on both sides. We selected SNPs with minor allelic frequency (MAF) greater than or equal to 0.01 and Hardy–Weinberg equilibrium (p > 0.05) and r2 ≥ 0.8. We performed an association study with genotypes and haplotypes, as well as in silico functional analysis of the identified SNPs. We observed an association of the SNP rs9411207 with the risk of excessive weight, under log-additive model, and the genotype distribution showed an increased frequency of homozygous TT (OR 1.50, CI 1.20–1.88; p = 0.0002). The haplotype GAT constructed from this and other SNPs in high Linkage Disequilibrium was more frequent in excessive-weight individuals (p = 0.003). In silico analyses suggested that these SNPs are likely to affect the transcription of NOTCH1 and other genes involved in adipogenesis and metabolism. This is the first study reporting association between NOTCH1 SNPs and the risk of excessive weight. Considering the possibility of NOTCH1 modulation, additional population studies are important to replicate these data and confirm the usefulness of risk genotypes for management strategies of excessive weight.

WTAP/IGF2BP3 mediated m6A modification of the EGR1/PTEN axis regulates the malignant phenotypes of endometrial cancer stem cells

Endometrial cancer (EC) stem cells (ECSCs) are pivotal in the oncogenesis, metastasis, immune escape, chemoresistance, and recurrence of EC. However, the specific mechanism of stem cell maintenance in EC cells (ECCs) has not been clarified. We found that WTAP and m6A levels decreased in both EC and ECSCs, and that knocking down WTAP promoted ECCs and ECSCs properties, including proliferation, invasion, migration, cisplatin resistance, and self-renewal. The downregulation of WTAP leads to a decrease in the m6A modification of EGR1 mRNA, and it is difficult for IGF2BP3, as an m6A reader, to recognize and bind to EGR1 mRNA that has lost m6A modification, resulting in a decrease in the stability of EGR1 mRNA. A decrease in the EGR1 level led to a decrease of in the expression tumor suppressor gene PTEN, resulting in deregulation and loss of cellular homeostasis and thereby fostering EC stem cell traits. Notably, the enforced overexpression of WTAP, EGR1, and PTEN inhibited the oncogenic effects of ECCs and ECSCs in vivo, and the combined overexpression of WTAP + EGR1 and EGR1 + PTEN further diminished the tumorigenic potential of these cells. Our findings revealed that the WTAP/EGR1/PTEN pathway is important regulator of EC stem cell maintenance, chemotherapeutic resistance, and tumorigenesis, suggesting a novel and promising therapeutic avenue for treating EC.

Protein phosphatase 6 promotes stemness of colorectal cancer cells.

Colorectal cancer (CRC) remains a significant global health concern, demanding a more profound comprehension of its molecular foundations for the development of improved therapeutic strategies. This study aimed to elucidate the role of protein phosphatase 6 (PP6), a member of the type 2A protein phosphatase family, in CRC. Protein phosphatase 6 functions as a heterotrimer with a catalytic subunit (PP6c), regulatory subunits (PP6Rs; PP6R1, PP6R2, and PP6R3), and scaffold subunits (ANKRD28, ANKRD44, and ANKRD52). Elevated PP6c expression has been identified in CRC tissues compared to normal mucosa, aligning with its potential involvement in CRC pathogenesis. PP6c knockdown resulted in decreased colony-forming ability and invivo proliferation of various CRC cell lines. Transcriptome analysis revealed that PP6c knockdown resulted in altered expression of genes associated with cancer stemness. Notably, the PP6c-PP6R3 complex is a key player in regulating cancer stem cell (CSC) markers. Additionally, increased PP6c expression was observed in CSC-like cells induced by sphere formation, implicating the role of PP6c in CSC maintenance. This study highlights the role of PP6c in CRC and suggests that it is a potential therapeutic target disrupting a pathway critical for CRC progression and stem cell maintenance.

The Drosophila histone methyltransferase SET1 coordinates multiple signaling pathways in regulating male germline stem cell maintenance and differentiation.

Many tissue-specific adult stem cell lineages maintain a balance between proliferation and differentiation. Here, we study how the H3K4me3 methyltransferase Set1 regulates early-stage male germ cells in Drosophila. Early-stage germline-specific knockdown of Set1 results in temporally progressive defects, arising as germ cell loss and developing into overpopulated early-stage germ cells. These germline defects also impact the niche architecture and cyst stem cell lineage non-cell-autonomously. Additionally, wild-type Set1, but not the catalytically inactive Set1, rescues the Set1 knockdown phenotypes, highlighting the functional importance of the methyltransferase activity of Set1. Further, RNA-sequencing experiments reveal key signaling pathway components, such as the JAK-STAT pathway gene Stat92E and the BMP pathway gene Mad, which are upregulated upon Set1 knockdown. Genetic interaction assays support the functional relationships between Set1 and JAK-STAT or BMP pathways, as both Stat92E and Mad mutations suppress the Set1 knockdown phenotypes. These findings enhance our understanding of the balance between proliferation and differentiation in an adult stem cell lineage. The phenotype of germ cell loss followed by over-proliferation when inhibiting a histone methyltransferase also raises concerns about using their inhibitors in cancer therapy.

A regulatory loop of JAK/STAT signalling and its downstream targets represses cell fate conversion and maintains male germline stem cell niche homeostasis.

A specialised microenvironment, termed niche, provides extrinsic signals for the maintenance of residential stem cells. However, how residential stem cells maintain niche homeostasis and whether stromal niche cells could convert their fate into stem cells to replenish lost stem cells upon systemic stem cell loss remain largely unknown. Here, through systemic identification of JAK/STAT downstream targets in adult Drosophila testis, we show that Escargot (Esg), a member of the Snail family of transcriptional factors, is a putative JAK/STAT downstream target. esg is intrinsically required in cyst stem cells (CySCs) but not in germline stem cells (GSCs). esg depletion in CySCs results in CySC loss due to differentiation and non-cell autonomous GSC loss. Interestingly, hub cells are gradually lost by delaminating from the hub and converting into CySCs in esg-defective testes. Mechanistically, esg directly represses the expression of socs36E, the well-known downstream target and negative regulator of JAK/STAT signalling. Finally, further depletion of socs36E completely rescues the defects observed in esg-defective testes. Collectively, JAK/STAT target Esg suppresses SOCS36E to maintain CySC fate and repress niche cell conversion. Thus, our work uncovers a regulatory loop between JAK/STAT signalling and its downstream targets in controlling testicular niche homeostasis under physiological conditions.

Supragel-mediated efficient generation of pancreatic progenitor clusters and functional glucose-responsive islet-like clusters

Although several synthetic hydrogels with defined stiffness have been developed to facilitate the proliferation and maintenance of human pluripotent stem cells (hPSCs), the influence of biochemical cues in lineage-specific differentiation and functional cluster formation has been rarely reported. Here, we present the application of Supragel, a supramolecular hydrogel formed by synthesized biotinylated peptides, for islet-like cluster differentiation. We observed that Supragel, with a peptide concentration of 5 mg/mL promoted spontaneous hPSCs formation into uniform clusters, which is mainly attributable to a supporting stiffness of ∼1.5 kPa as provided by the Supragel matrix. Supragel was also found to interact with the hPSCs and facilitate endodermal and subsequent insulin-secreting cell differentiation, partially through its components: the sequences of RGD and YIGSR that interacts with cell membrane molecules of integrin receptor. Compared to Matrigel and suspension culturing conditions, more efficient differentiation of the hPSCs was also observed at the stages 3 and 4, as well as the final stage toward generation of insulin-secreting cells. This could be explained by 1) suitable average size of the hPSCs clusters cultured on Supragel; 2) appropriate level of cell adhesive sites provided by Supragel during differentiation. It is worth noting that the Supragel culture system was more tolerance in terms of the initial seeding densities and less demanding, since a standard static cell culture condition was sufficient for the entire differentiation process. Our observations demonstrate a positive role of Supragel for hPSCs differentiation into islet-like cells, with additional potential in facilitating germ layer differentiation.

Identification of De Novo Dividing Stem Cells.

The ability to alternate between quiescent and proliferating states is a remarkable feature of many types of somatic stem cells. The balance between quiescent and proliferating states is vital for maintenance of stem cells over the lifespan, and its disturbance may lead to premature depletion of the stem cell pool and loss of the tissue regenerative or renewal capacity at later stages of life. The question on how this balance is regulated is of critical importance in stem cell research and biology of aging. Assessment of the balance between quiescent and proliferating states has remained challenged until recently due to the lack of approaches for robust determination of the rate at which stem cells exit reversible cell cycle arrest. Here, we propose a simple method for detection of those stem cells that have entered the division cycle after a prolonged period of quiescence.The method combines cumulative and pulse labeling with thymidine analogues 5-bromo-2'-deoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU). In the discussed labeling scheme, cells that have incorporated only the second label, EdU, are de novo dividing cells. The suggested double labeling method provides quantitative assessment of the rate at which stem cells exit the quiescent state and allows the fates of de novo dividing stem cells to be traced.

Human Neural Stem Cell Expansion in Natural Polymer Scaffolds Under Chemically Defined Condition.

The maintenance and expansion of human neural stem cells (hNSCs) in 3D tissue scaffolds is a promising strategy in producing cost-effective hNSCs with quality and quantity applicable for clinical applications. A few biopolymers have been extensively used to fabricate 3D scaffolds, including hyaluronic acid, collagen, alginate, and chitosan, due to their bioactive nature and availability. However, these polymers are usually applied in combination with other biomolecules, leading to their responses difficult to ascribe to. Here, scaffolds made of chitosan, alginate, hyaluronic acid, or collagen, are explored for hNSC expansion under xeno-free and chemically defined conditions and compared for hNSC multipotency maintenance. This study shows that the scaffolds made of pure chitosan support the highest adhesion and growth of hNSCs, yielding the most viable cells with NSC marker protein expression. In contrast, the presence of alginate, hyaluronic acid, or collagen induces differentiation toward immature neurons and astrocytes even in the maintenance medium and absence of differentiation factors. The cells in pure chitosan scaffolds preserve the level of transmembrane protein profile similar to that of standard culture. These findings point to the potential of using pure chitosan scaffolds as a base scaffolding material for hNSC expansion in 3D.

Roles of long non‑coding RNAs in esophageal cell squamous carcinoma (Review).

Esophageal squamous cell carcinoma (ESCC) is a prevalent and deadly malignancy of the digestive tract. Recent research has identified long non‑coding RNAs (lncRNAs) as crucial regulators in the pathogenesis of ESCC. These lncRNAs, typically exceeding 200 nucleotides, modulate gene expression through various mechanisms, including the competing endogenous RNA (ceRNA) pathway and RNA‑protein interactions. The current study reviews the multifaceted roles of lncRNAs in ESCC, highlighting their involvement in processes such as proliferation, migration, invasion, epithelial‑mesenchymal transition, cell cycle progression, resistance to radiotherapy and chemotherapy, glycolysis, apoptosis, angiogenesis, autophagy, tumor growth, metastasis and the maintenance of cancer stem cells. Specific lncRNAs like HLA complex P5, LINC00963 and non‑coding repressor of NFAT have been shown to enhance resistance to radio‑ and chemotherapy by modulating pathways such as AKT signaling and microRNA interaction, which promote cell survival and proliferation under therapeutic stress. Furthermore, lncRNAs like family with sequence similarity 83, member A antisense RNA 1, zinc finger NFX1‑type containing 1 antisense RNA 1 and taurine upregulated gene 1 are implicated in enhancing invasive and proliferative capabilities of ESCC cells through the ceRNA mechanism, while interactions with RNA‑binding proteins further influence cancer cell behavior. The comprehensive analysis underscores the potential of lncRNAs as biomarkers for prognosis and therapeutic targets in ESCC, suggesting avenues for future research focused on elucidating the detailed molecular mechanisms and clinical applications of lncRNAs in ESCC management.

MAST4 regulates stem cell maintenance with DLX3 for epithelial development and amelogenesis

The asymmetric division of stem cells permits the maintenance of the cell population and differentiation for harmonious progress. Developing mouse incisors allows inspection of the role of the stem cell niche to provide specific insights into essential developmental phases. Microtubule-associated serine/threonine kinase family member 4 (Mast4) knockout (KO) mice showed abnormal incisor development with low hardness, as the size of the apical bud was decreased and preameloblasts were shifted to the apical side, resulting in amelogenesis imperfecta. In addition, Mast4 KO incisors showed abnormal enamel maturation, and stem cell maintenance was inhibited as amelogenesis was accelerated with Wnt signal downregulation. Distal-Less Homeobox 3 (DLX3), a critical factor in tooth amelogenesis, is considered to be responsible for the development of amelogenesis imperfecta in humans. MAST4 directly binds to DLX3 and induces phosphorylation at three residues within the nuclear localization site (NLS) that promotes the nuclear translocation of DLX3. MAST4-mediated phosphorylation of DLX3 ultimately controls the transcription of DLX3 target genes, which are carbonic anhydrase and ion transporter genes involved in the pH regulation process during ameloblast maturation. Taken together, our data reveal a novel role for MAST4 as a critical regulator of the entire amelogenesis process through its control of Wnt signaling and DLX3 transcriptional activity.

LncRNAs and asymmetric cell division: the epigenetic mechanisms

Asymmetric cell division (ACD) plays a pivotal role in development, tissue homeostasis, and stem cell maintenance. Emerging evidence suggests that long non-coding RNAs (lncRNAs) are key regulators of ACD, orchestrating the intricate molecular machinery that governs cell fate determination. This review summarizes current literature to elucidate the diverse roles of lncRNAs in modulating ACD across various biological contexts. The regulatory mechanisms of asymmetric cell division mediated by lncRNAs, including their interactions with protein effectors, epigenetic regulation, and subcellular localization are explored. Additionally, we discuss the implications of dysregulated lncRNAs in mediating ACD that lead to tumorigenesis. By integrating findings from diverse experimental models and cell types, this review provides insights into the multifaceted roles of lncRNAs in governing asymmetric cell division, shedding light on fundamental biological processes. Further research in this area may lead to the development of novel therapies targeting dysregulated lncRNAs to restore proper cell division and function. The knowledge of lncRNAs regulating ACD could potentially revolutionize the field of regenerative medicine and cancer therapy by targeting specific lncRNAs involved in ACD. By unraveling the complex interactions between lncRNAs and cellular processes, the potential novel opportunities for precision medicine approaches may be uncovered.

Merkel cell polyomavirus pan-T antigen knockdown reduces cancer cell stemness and promotes neural differentiation independent of RB1.

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer associated with integration of Merkel cell polyomavirus (MCPyV). MCPyV-encoded T-antigens (TAs) are pivotal for sustaining MCC's oncogenic phenotype, i.e., repression of TAs results in reactivation of the RB pathway and subsequent cell cycle arrest. However, the MCC cell line LoKe, characterized by a homozygous loss of the RB1 gene, exhibits uninterrupted cell cycle progression after shRNA-mediated TA repression. This unique feature allows an in-depth analysis of the effects of TAs beyond inhibition of the RB pathway, revealing the decrease in expression of stem cell-related genes upon panTA-knockdown. Analysis of gene regulatory networks identified members of the E2F family (E2F1, E2F8, TFDP1) as key transcriptional regulators that maintain stem cell properties in TA-expressing MCC cells. Furthermore, minichromosome maintenance (MCM) genes, which encodes DNA-binding licensing proteins essential for stem cell maintenance, were suppressed upon panTA-knockdown. The decline in stemness occurred simultaneously with neural differentiation, marked by the increased expression of neurogenesis-related genes such as neurexins, BTG2, and MYT1L. This upregulation can be attributed to heightened activity of PBX1 and BPTF, crucial regulators of neurogenesis pathways. The observations in LoKe were confirmed in an additional MCPyV-positive MCC cell line in which RB1 was silenced before panTA-knockdown. Moreover, spatially resolved transcriptomics demonstrated reduced TA expression in situ in a part of a MCC tumor characterized by neural differentiation. In summary, TAs are critical for maintaining stemness of MCC cells and suppressing neural differentiation, irrespective of their impact on the RB-signaling pathway.

Circulating plasma fibronectin affects tissue insulin sensitivity, adipocyte differentiation, and transcriptional landscape of adipose tissue in mice.

Plasma fibronectin (pFN) is a hepatocyte-derived circulating extracellular matrix protein that affects cell morphology, adipogenesis, and insulin signaling of adipocytes invitro. In this study, we show pFN accrual to adipose tissue and its contribution to tissue homeostasis in mice. Hepatocyte-specific conditional Fn1 knockout mice (Fn1-/-ALB) show a decrease in adipose tissue FN levels and enhanced insulin sensitivity of subcutaneous (inguinal), visceral (epididymal) adipose tissue on a normal diet. Diet-induced obesity model of the Fn1-/-ALB mouse showed normal weight gain and whole-body fat mass, and normal adipose tissue depot volumes and unaltered circulating leptin and adiponectin levels. However, Fn1-/-ALB adipose depots showed significant alterations in adipocyte size and gene expression profiles. The inguinal adipose tissue on a normal diet, which had alterations in fatty acid metabolism and thermogenesis suggesting browning. The presence of increased beige adipocyte markers Ucp1 and Prdm16 supported this. In the inguinal fat, the obesogenic diet resulted in downregulation of the browning markers and changes in gene expression reflecting development, morphogenesis, and mesenchymal stem cell maintenance. Epididymal adipose tissue showed alterations in developmental and stem cell gene expression on both diets. The data suggests a role for pFN in adipose tissue insulin sensitivity and cell profiles.

PiRNAs and PIWI-like proteins in cancer and their future as biomarkers and therapy targets in breast cancer

Globally, breast cancer (BC) is the most common type of cancer, with 2.296.840 new cases in 2020 with almost 700.000 related deaths. Due to the high burden this represents, it is imperative to expand horizons to be able to find novel biomarkers and therapeutic targets that can be used to improve prognosis, treatment, and survival of breast cancer patients. In recent years, numerous studies have been conducted looking for the association between piRNAs (PIWI – interacting RNAs) and the development, pathogenesis, metastasis, and progression of different types of cancer, including BC. PiRNAs are small molecules (24-31 nucleotides) that interact with the PIWI-protein complex (PIWI-like proteins - HIWI/HILI in humans) performing regulatory functions by inducing transcriptional, post-transcriptional, translational, and post-translational epigenetic changes, which has been observed to contribute to cancer development through modifications in cell proliferation, transposon silencing, genome rearrangement, epigenetic regulation, protein regulation, and stem cell maintenance. In breast cancer, a strong association has been found between the expression of some piRNAs, PIWI-like proteins and tumor development. If a specific piRNA could be associated with a distinct type of cancer, it could then be used as an early biomarker which would allow for a better prognosis. Findings surrounding these molecular mechanisms could also spark interest in studies focusing on the modification of the expression of piRNAs in cancer cells. In this article, we intend to review in a straightforward manner the current information about piRNAs/PIWI-like proteins focusing on their expression in BC.

Hypoxia within the glioblastoma tumor microenvironment: a master saboteur of novel treatments.

Glioblastoma (GBM) tumors are the most aggressive primary brain tumors in adults that, despite maximum treatment, carry a dismal prognosis. GBM tumors exhibit tissue hypoxia, which promotes tumor aggressiveness and maintenance of glioma stem cells and creates an overall immunosuppressive landscape. This article reviews how hypoxic conditions overlap with inflammatory responses, favoring the proliferation of immunosuppressive cells and inhibiting cytotoxic T cell development. Immunotherapies, including vaccines, immune checkpoint inhibitors, and CAR-T cell therapy, represent promising avenues for GBM treatment. However, challenges such as tumor heterogeneity, immunosuppressive TME, and BBB restrictiveness hinder their effectiveness. Strategies to address these challenges, including combination therapies and targeting hypoxia, are actively being explored to improve outcomes for GBM patients. Targeting hypoxia in combination with immunotherapy represents a potential strategy to enhance treatment efficacy.

MDB-76. ONC201 INHIBITS THE CHARACTERISTICS OF CANCER STEM CELLS IN GROUP 3 MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma (MB), the most frequent childhood brain tumor, originates in the cerebellum. Despite treatment advances, recurrence affects 40%, with a mortality rate of 30%. Cancer stem cells drive tumor initiation, maintenance, and therapy resistance, in several tumors including MB (MBSC). In response to stress, stem cells activate mechanisms like the unfolded protein response (UPR) to maintain homeostasis and survive adverse conditions. The UPR restores endoplasmic reticulum balance, supporting cell function and survival. Recent studies have shown that the UPR plays an important role in tumorigenesis, however, its function in MBSC remains unknown. METHODS To understand the involvement of the UPR in stem cell maintenance, we have investigated the biological effects of its pharmacological inhibition on Group 3 (G3) MBSC. We selected three G3 MB cell lines (D283-Med, D341-Med and Med411), for in vitro studies. Cell line selection was based on their fidelity to the expected methylation class, corresponding to the subgroup of the parental tumors, and were grown in “stem-like” conditions as neurospheres. We evaluated drugs targeting the UPR stress program, identifying ONC201 as the most effective against MBSC. ONC201 is an imipridone compound that activates p53-independent apoptosis causing changes in gene expression similar to those caused by UPR. RESULTS Our findings revealed that ONC201 modulates protein synthesis via ATF4, a pivotal UPR molecule, with stronger induction observed in MB cells cultured under “stem-like” conditions. This ATF4 upregulation triggers the activation of pro-apoptotic programs. Additionaly, ONC201 was effective on reducing the stem-like features of MBSC, including self-renewal, stem cell markers expression, migration, and invasion potential. Notably, ONC201 demonstrates synergy with vincristine and methotrexate chemotherapy. CONCLUSIONS These results propose ONC201 as a promising agent for targeting G3 medulloblastoma by compromising the stem cell compartment. Further investigation is warranted to confirm its efficacy and potential clinical application.

C-terminally phosphorylated p27 activates self-renewal driver genes to program cancer stem cell expansion, mammary hyperplasia and cancer

In many cancers, a stem-like cell subpopulation mediates tumor initiation, dissemination and drug resistance. Here, we report that cancer stem cell (CSC) abundance is transcriptionally regulated by C-terminally phosphorylated p27 (p27pT157pT198). Mechanistically, this arises through p27 co-recruitment with STAT3/CBP to gene regulators of CSC self-renewal including MYC, the Notch ligand JAG1, and ANGPTL4. p27pTpT/STAT3 also recruits a SIN3A/HDAC1 complex to co-repress the Pyk2 inhibitor, PTPN12. Pyk2, in turn, activates STAT3, creating a feed-forward loop increasing stem-like properties in vitro and tumor-initiating stem cells in vivo. The p27-activated gene profile is over-represented in STAT3 activated human breast cancers. Furthermore, mammary transgenic expression of phosphomimetic, cyclin-CDK-binding defective p27 (p27CK-DD) increases mammary duct branching morphogenesis, yielding hyperplasia and microinvasive cancers that can metastasize to liver, further supporting a role for p27pTpT in CSC expansion. Thus, p27pTpT interacts with STAT3, driving transcriptional programs governing stem cell expansion or maintenance in normal and cancer tissues.

The Endosomal Sorting Complex, ESCRT, has diverse roles in blood progenitor maintenance, lineage choice and immune response.

Most hematological malignancies are associated with reduced expression of one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT). However, the roles of ESCRT in stem cell and progenitor maintenance are not resolved. Parsing signaling pathways in relation to the canonical role of ESCRT poses a challenge. The Drosophila hematopoietic organ, the larval lymph gland, provides a path to dissect the roles of cellular trafficking pathways such as ESCRT in blood development and maintenance. Drosophila has 13 core ESCRT components. Knockdown of individual ESCRTs showed that only Vps28 and Vp36 were required in all lymph gland progenitors. Using the well-conserved ESCRT-II complex as an example of the range of phenotypes seen upon ESCRT depletion, we show that ESCRTs have cell-autonomous as well as non-autonomous roles in progenitor maintenance and differentiation. ESCRT depletion also sensitized posterior lobe progenitors to respond to immunogenic wasp infestation. We also identify key heterotypic roles for ESCRT in position-dependent control of Notch activation to suppress crystal cell differentiation. Our study shows that the cargo sorting machinery determines the identity of progenitors and their adaptability to the dynamic microenvironment. These mechanisms for control of cell fate may tailor developmental diversity in multiple contexts.

Molecular Interactions of Selective Agonists and Antagonists with the Retinoic Acid Receptor γ.

All-trans retinoic acid (ATRA), the major active metabolite of all-trans retinol (vitamin A), is a key hormonal signaling molecule. In the adult organism, ATRA has a widespread influence on processes that are crucial to the growth and differentiation of cells and, in turn, the acquisition of mature cell functions. Therefore, there is considerable potential in the use of retinoids to treat diseases. ATRA binds to the retinoic acid receptors (RAR) which, as activated by ATRA, selectively regulate gene expression. There are three main RAR isoforms, RARα, RARβ, and RARγ. They each have a distinct role, for example, RARα and RARγ regulate myeloid progenitor cell differentiation and hematopoietic stem cell maintenance, respectively. Hence, targeting an isoform is crucial to developing retinoid-based therapeutics. In principle, this is exemplified when ATRA is used to treat acute promyelocytic leukemia (PML) and target RARα within PML-RARα oncogenic fusion protein. ATRA with arsenic trioxide has provided a cure for the once highly fatal leukemia. Recent in vitro and in vivo studies of RARγ have revealed the potential use of agonists and antagonists to treat diseases as diverse as cancer, heterotopic ossification, psoriasis, and acne. During the final drug development there may be a need to design newer compounds with added modifications to improve solubility, pharmacokinetics, or potency. At the same time, it is important to retain isotype specificity and activity. Examination of the molecular interactions between RARγ agonists and the ligand binding domain of RARγ has revealed aspects to ligand binding that are crucial to RARγ selectivity and compound activity and key to designing newer compounds.

Red2Flpe-SCON: a versatile, multicolor strategy for generating mosaic conditional knockout mice

Image-based lineage tracing enables tissue turnover kinetics and lineage potentials of different adult cell populations to be investigated. Previously, we reported a genetic mouse model system, Red2Onco, which ectopically expressed mutated oncogenes together with red fluorescent proteins (RFP). This system enabled the expansion kinetics and neighboring effects of oncogenic clones to be dissected. We now report Red2Flpe-SCON: a mosaic knockout system that uses multicolor reporters to label both mutant and wild-type cells. We develop the Red2Flpe mouse line for red clone-specific Flpe expression, as well as the FRT-based SCON (Short Conditional IntrON) method to facilitate tunable conditional mosaic knockouts in mice. We use the Red2Flpe-SCON method to study Sox2 mutant clonal analysis in the esophageal epithelium of adult mice which reveal that the stem cell gene, Sox2, is less essential for adult stem cell maintenance itself, but rather for stem cell proliferation and differentiation.