Subsequent Terminal Differentiation Research Articles

Intracerebral Hemorrhage Leads to Anemia By Disrupting Erythropoiesis and Erythromyeloblastic Islands

Intracerebral hemorrhage (ICH), caused by the rupture of a cerebral vessel and subsequent entry of blood into the brain parenchyma, is a major form of acquired brain injury and contributes a remarkable proportion of stroke-related morbidity and mortality. Due to the few proven effective emergency and preventive treatments, patients who survived suffer varying degrees of poor prognosis that affect their quality of life. There is an urgent need to explore the cellular and molecular basis of the differences in the severity and prognosis of ICH. Anemia is a common manifestation of ICH. However, the mechanisms underlying ICH-induced anemia as we as the correlation between anemia and ICH progression/prognosis remain unclear. In this study, we found that approximately 35.1% (39 of 111) of ICH patients exhibited anemia and that decreased hemoglobin levels were positively associated with higher scores on the National Institutes of Health Stroke Scale, which quantifies the degree of neurological deficit, at admission. Furthermore, patients with decreased hemoglobin levels at admission showed higher modified Rankin scale scores at 3 months, which reflect the global disability of daily living at this time, suggesting that anemia was associated with a more severe progression and poor patient prognosis. To investigate the effects of ICH on bone marrow (BM) erythropoiesis, we conducted experiments in 12-month-old mice with collagenase-induced striatal ICH. Our results showed that ICH caused apparent anemia on day 3, associated with larger hematomas in the striatum and more severe neurologic deficits. We first evaluated the phenotype of hematopoietic progenitors and found that mice suffering ICH exhibited increased granulocyte monocyte progenitors but a significant decrease in common myeloid progenitors and megakaryocyte erythrocyte progenitors compared to control mice, suggesting a reduced source of erythroid progenitors. Further analysis revealed that the colony-forming ability of erythroid progenitors and subsequent terminal differentiation was significantly suppressed after ICH. To assess whether ICH-induced suppression of erythropoiesis is associated with erythromyeloblastic islands (EMBIs), the niche for erythropoiesis/myelopoiesis, we performed ICH using 12-month-old EpoR-tdTomato-Cre mice and found that the number of BM EMBIs and EpoR + macrophages decreased significantly. Moreover, the number and proportion of developing erythroid cells surrounding EMBIs decreased while LY6G + cells representing granulocytes increased prominently, indicating an altered microenvironment, with EMBI favoring granulopoiesis over erythropoiesis after ICH. Studies have shown that inflammation plays a crucial role in secondary brain injury after ICH. We hypothesize that inflammatory cytokines may contribute to the suppression of erythropoiesis observed in ICH mouse models. To test this, we measured inflammatory factors in both peripheral blood and BM fluid using Quantibody Mouse Inflammation Array. In particular, IL-6 concentrations in peripheral blood and BM fluid in ICH mice were significantly higher than controls. Treatment with MR16-1, an IL-6 antagonist, alleviated ICH-induced anemia and improved BM erythropoiesis in ICH mice, as evidenced by increased peripheral red blood cells and hemoglobin levels, elevated numbers of erythroid progenitors and terminal erythroblast. MR16-1 treatment also improved BM EMBI formation and increased EpoR + macrophages. In addition, MR16-1 treatment effectively improved neurologic deficits and reduced brain hematoma after ICH. Administration of recombinant IL-6 to wild-type mice resulted in decreased levels of red blood cells and hemoglobin along with suppression of erythropoiesis in the BM. Collectively, these results suggest that ICH modulates erythropoiesis and erythromyeloblastic islands function to ultimately lead to anemia and reveal a possible therapeutic opportunity to reverse ICH-induced anemia and improve ICH prognosis by targeting IL-6.

Current Strategies of Modeling Human Trophoblast Using Human Pluripotent Stem Cells in vitro.

We previously established a trophoblast differentiation protocol from primed human pluripotent stem cells (PSC). To induce this lineage, we use a combination of Bone Morphogenetic Protein-4 (BMP4) and the WNT inhibitor IWP2. This protocol has enabled us to obtain a pure population of trophectoderm (TE)-like cells that could subsequently be terminally differentiated into syncytiotrophoblasts (STB) and extravillous trophoblasts (EVT). However, the resulting TE-like cells could only be terminally differentiated to a variable mixture of STB and EVT, with a bias toward the STB lineage. Recently, methods have been developed for derivation and culture of self-renewing human trophoblast stem cells (TSC) from human embryos and early gestation placental tissues. These primary TSCs were further able to differentiate into either STB or EVT with high efficiency using the lineage specific differentiation protocols. Based partly on these protocols, we have developed methods for establishing self-renewing TSC-like cells from PSC, and for efficient lineage-specific terminal differentiation. Here, we describe in detail the protocols to derive and maintain PSC-TSC, from both embryonic stem cells (ESC) and patient-derived induced pluripotent stem cells (iPSC), and their subsequent terminal differentiation to STB and EVT. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Trophoblast Differentiation into TE-like Cells Basic Protocol 2: Conversion of PSC-Derived TE-like Cells to TSC Basic Protocol 3: Passaging PSC-Derived TSC in iCTB Complete Medium Basic Protocol 4: STB Differentiation from PSC-derived TSC Basic Protocol 5: EVT Differentiation from PSC-derived TSC Support Protocol 1: Geltrex-coated tissue culture plate preparation Support Protocol 2: Collagen IV-coated tissue culture plate preparation Support Protocol 3: Fibronectin-coated tissue culture plate preparation.

Crucial role of iron in epigenetic rewriting during adipocyte differentiation mediated by JMJD1A and TET2 activity.

Iron metabolism is closely associated with the pathogenesis of obesity. However, the mechanism of the iron-dependent regulation of adipocyte differentiation remains unclear. Here, we show that iron is essential for rewriting of epigenetic marks during adipocyte differentiation. Iron supply through lysosome-mediated ferritinophagy was found to be crucial during the early stage of adipocyte differentiation, and iron deficiency during this period suppressed subsequent terminal differentiation. This was associated with demethylation of both repressive histone marks and DNA in the genomic regions of adipocyte differentiation-associated genes, includingPparg, which encodes PPARγ, the master regulator of adipocyte differentiation. In addition, we identified several epigenetic demethylases to be responsible for iron-dependent adipocyte differentiation, with the histone demethylase jumonji domain-containing 1A and the DNA demethylase ten-eleven translocation 2 as the major enzymes. The interrelationship between repressive histone marks and DNA methylation was indicated by an integrated genome-wide association analysis, and was also supported by the findings that both histone and DNA demethylation were suppressed by either the inhibition of lysosomal ferritin flux or the knockdown of iron chaperone poly(rC)-binding protein 2. In summary, epigenetic regulations through iron-dependent control of epigenetic enzyme activities play an important role in the organized gene expression mechanisms of adipogenesis.

Quiescence regulation by normal haematopoietic stem cells and leukaemia stem cells.

The haematopoietic system is maintained by rare haematopoietic stem cells (HSCs), which are quiescent most of the time and only divide occasionally to self-renew and/or to undergo commitment to clonal expansion via the generation of highly proliferative progenitor cells. The latter is responsible for the generation of all mature cells of the system through subsequent lineage commitment and terminal differentiation. Cells with similar properties also exist in leukaemias and are known as leukaemia stem cells (LSCs). Quiescence provides essential protection for both HSC and LSC from cytotoxic stress and DNA damage and, in the case of LSCs, likely causes therapy resistance and disease relapse in leukaemia patients. Specific inhibition of LSC quiescence has been considered a promising strategy for eliminating LSCs and curing leukaemias. Although the understanding of mechanisms responsible for quiescence maintenance in these cells remains limited, particularly for LSCs, recent studies have suggested potential differences in their dependency on certain pathways and their levels of stress and DNA damage caused by increased cycling. Such differences likely stem from oncogenic mutations in LSCs and could be specifically exploited for the elimination of LSCs while sparing normal HSCs in the future.

Antigen cross-presentation in young tumor-bearing hosts promotes CD8+ T cell terminal differentiation.

The immune system undergoes a progressive functional remodeling with age. Understanding how age bias shapes antitumor immunity is essential in designing effective immunotherapies, especially for pediatric patients. Here, we explore antitumor CD8+ T cell responses generated in young (prepubescent) and adult (presenescent) mice. Using an MHCI-deficient tumor model, we observed that tumor-reactive CD8+ T cells expanded in young tumor-bearing (TB) mice acquired a terminally differentiated phenotype characterized by overexpression of inhibitory receptors and the transcription factor Tox1. Furthermore, tumor-infiltrating CD8+ T cells from young tumors yielded a poor cytokine response compared with CD8+ T cells infiltrating adult tumors. Young migratory dendritic cells (migDCs) from the draining lymph nodes (dLNs), and mononuclear phagocytic cells (MPCs) infiltrating young tumors, were more competent in capturing and cross-presenting tumor antigen, leading to enhanced priming of CD8+ T cells in dLNs and their subsequent terminal differentiation in the tumors. Single-cell transcriptional profiling of tumor-infiltrating MPCs demonstrated that young MPCs are polarized toward an inflammatory, effector phenotype. Consistent with our observations in young versus adult TB mice, analysis of immune infiltrates from pediatric solid tumors showed a correlation between tumor-infiltrating CD8+ T cells with an exhaustion phenotype and the frequency of PD-L1-expressing monocytes/macrophages. Collectively, these data indicate that a young tissue microenvironment contributes to the generation of an immune response skewed toward a less pliable terminal effector state, thus narrowing the window for immunotherapeutic interventions.

LKB1 Coordinates Metabolic Pathways to Maintain Homeostasis of Haematopoietic Stem Cells and Regulate Erythropoiesis

Erythropoiesis is a highly regulated multistage process by which hematopoietic stem cells (HSCs) proliferate, differentiate and eventually form mature erythrocytes. Stem-cell maintenance and cell differentiation require homeostasis and coordination of multiple metabolisms. However, underlying mechanism of this coordination is poorly identified. Liver kinase B1 (LKB1) acts as evolutionarily conserved regulator to control cellular metabolism, cell polarity and proliferation during development and stress response. Considering that all the fundamental cellular processes regulated by LKB1 are present in erythropoiesis, we hypothesize that LKB1 may involve in orchestrating this coordination.To explore the role of LKB1 in entire erythropoiesis, we firstly crossed mice carrying loxP-flanked LKB1 alleles with EpoR-tdTomato-Cre mice, which can lead to cleavage in from HSCs to erythroblasts. LKB1fl/fl EPORcre mice developed exhaustion of HSCs, progressive severe anemia and ultimately lethality. Intriguingly, HSCs of LKB1fl/fl EPORcre mice exhibited a swiftly skewness toward the erythoid lineage when LKB1 is deleted. Nevertheless, erythroblasts of LKB1fl/fl EPORcre mice were significantly reduced. Further analysis showed that the colony forming ability of the colony-forming unit-erythroid (CFU-E) cells and subsequent terminal erythoid differentiation in LKB1fl/fl EPORcre mice were seriously impaired.In order to study erythropoiesis more specifically without the influence of stem cells, we next assessed the impact of deletion of LKB1 in the mouse erythropoietic system using GYPA-eGFP-Cre mice. LKB1fl/flGYPAcre mice showed mild anemia but had the normal lifespan.The Ter119 + cells were decreased while CFU-E cells were significantly increased. However, the colony forming ability of sorted CFU-E cells from LKB1fl/flGYPAcre mice were drastically reduced. In addition, LKB1fl/flGYPAcre mice exhibited disordered terminal erythropoiesis. Taken together, these results indicate that LKB1 may play multinomial roles in regulating stem-cell homeostasis and effective erythoid differentiation.Previous studies have shown that LKB1 controls energy metabolism via phosphorylating AMPK and regulating PGC-1 transcription to maintain homeostasis of HSCs. We found that both of p-AMPK and PGC-1 of LKB1fl/fl EPORcre mice were down-regulated in Lin - cells while Ter119 + cells have comparable p-AMPK and PGC-1. In this regard, LKB1fl/flGYPAcre mice, whether in Lin - cells or in Ter119 + cells, exhibited similar p-AMPK and PGC-1 level. Moreover, administration of ZLN005, an activator of PGC-1, partly rescued the anemia in LKB1fl/fl EPORcre mice but not in LKB1fl/flGYPAcre mice. These data imply that there is other underlying mechanism of LKB1 in erythropoiesis.To gain further mechanistic insight, we carried out proteomics analysis. Gene ontology analysis of differentially expressed proteins revealed that cholesterol metabolism related genes were significantly altered under LKB1 deficiency. We then found that LKB1 ablation led to a reduction of cholesterol level and diminishment of expression levels of primary cholesterol biosynthesis related genes. Moreover, the mature active form of SREBP2, the master transcriptional regulator of cholesterol biosynthesis, was prominently reduced. Golgi apparatus, in which SREBP2 is cleaved for activation, is intumescent or dispersed in erythroid cells of LKB1fl/fl EPORcre mice and LKB1fl/flGYPAcre mice. These results supported that loss of LKB1 impaired the cholesterol metabolism in erythropoiesis.To demonstrate Golgi apparatus-dependent cholesterol metabolism is essential for erythropoiesis, we treated LKB1fl/flGYPAcre mice with 2,3-oxidosqualene, the important intermediate in cholesterol synthesis and found that the phenotypes of LKB1fl/flGYPAcre mice were effectively restored. In parallel, 2,3-oxidosqualene treatment just slightly alleviated the anemia of LKB1fl/fl EPORcre mice. However, combination treatment with 2,3-oxidosqualene and ZLN005 was more effective in restoring phenotypes in LKB1fl/fl EPORcre mice, in contrast to ZLN005 alone.Thus, LKB1 serves as an essential metabolic regulator to coordinate energy metabolism in hematopoietic stem cells and lipid metabolism in erythoid cells, thereby maintaining homeostasis of haematopoietic stem cells and functional fitness of erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

A Two-Step Process of Effector Programming Governs CD4+ T Cell Fate Determination Induced by Antigenic Activation in the Steady State

SUMMARYVarious processes induce and maintain immune tolerance, but effector T cells still arise under minimal perturbations of homeostasis through unclear mechanisms. We report that, contrary to the model postulating primarily tolerogenic mechanisms initiated under homeostatic conditions, effector programming is an integral part of T cell fate determination induced by antigenic activation in the steady state. This effector programming depends on a two-step process starting with induction of effector precursors that express Hopx and are imprinted with multiple instructions for their subsequent terminal effector differentiation. Such molecular circuits advancing specific terminal effector differentiation upon re-stimulation include programmed expression of interferon-γ, whose production then promotes expression of T-bet in the precursors. We further show that effector programming coincides with regulatory conversion among T cells sharing the same antigen specificity. However, conventional type 2 dendritic cells (cDC2) and T cell functions of mammalian target of rapamycin complex 1 (mTORC1) increase effector precursor induction while decreasing the proportion of T cells that can become peripheral Foxp3+ regulatory T (pTreg) cells.

Chaperonin TRiC/CCT supports mitotic exit and entry into endocycle in Drosophila.

Endocycle is a commonly observed cell cycle variant through which cells undergo repeated rounds of genome DNA replication without mitosis. Endocycling cells arise from mitotic cells through a switch of the cell cycle mode, called the mitotic-to-endocycle switch (MES), to initiate cell growth and terminal differentiation. However, the underlying regulatory mechanisms of MES remain unclear. Here we used the Drosophila steroidogenic organ, called the prothoracic gland (PG), to study regulatory mechanisms of MES, which is critical for the PG to upregulate biosynthesis of the steroid hormone ecdysone. We demonstrate that PG cells undergo MES through downregulation of mitotic cyclins, which is mediated by Fizzy-related (Fzr). Moreover, we performed a RNAi screen to further elucidate the regulatory mechanisms of MES, and identified the evolutionarily conserved chaperonin TCP-1 ring complex (TRiC) as a novel regulator of MES. Knockdown of TRiC subunits in the PG caused a prolonged mitotic period, probably due to impaired nuclear translocation of Fzr, which also caused loss of ecdysteroidogenic activity. These results indicate that TRiC supports proper MES and endocycle progression by regulating Fzr folding. We propose that TRiC-mediated protein quality control is a conserved mechanism supporting MES and endocycling, as well as subsequent terminal differentiation.

Pathological ASXL1 Mutations and Protein Variants Impair Neural Crest Development.

SummaryThe neural crest (NC) gives rise to a multitude of fetal tissues, and its misregulation is implicated in congenital malformations. Here, we investigated molecular mechanisms pertaining to NC-related symptoms in Bohring-Opitz syndrome (BOS), a developmental disorder linked to mutations in the Polycomb group factor Additional sex combs-like 1 (ASXL1). Genetically edited human pluripotent stem cell lines that were differentiated to NC progenitors and then xenotransplanted into chicken embryos demonstrated an impairment of NC delamination and emigration. Molecular analysis showed that ASXL1 mutations correlated with reduced activation of the transcription factor ZIC1 and the NC gene regulatory network. These findings were supported by differentiation experiments using BOS patient-derived induced pluripotent stem cell lines. Expression of truncated ASXL1 isoforms (amino acids 1–900) recapitulated the NC phenotypes in vitro and in ovo, raising the possibility that truncated ASXL1 variants contribute to BOS pathology. Collectively, we expand the understanding of truncated ASXL1 in BOS and in the human NC.

Affinity separation and subsequent terminal differentiation of acute myeloid leukemia cells using the human transferrin receptor (CD71) as a capture target.

The microfluidic detection of myeloblasts in blood via the human transferrin receptor (CD71) can serve as a diagnostic marker for acute myeloid leukemia (AML). Furthermore, CD71 expression is present in all proliferating cells and can capture target cells without prior knowledge of AML subtype. The use of anti-CD71 as the affinity ligand for AML detection in this work yields a capture efficiency and purity during peak CD71 expression of 92% and 62%, respectively. Additionally, target cells were isolated from lysed, preserved blood samples with a capture purity of 32% at a concentration of 10% myeloblasts in blood, half of the current diagnosis threshold determined by the World Health Organization. Cells isolated using this capture ligand were then subjected to post-separation differentiation therapy. HL60 cells were differentiated into non-proliferating, neutrophil-like cells. After 48 hours of incubation with 1.5% DMSO, there was a decrease in the CD71 antigen expression in differentiated cells. This separation approach can be used to screen blood samples for AML cells, and the effluent of the separation is available for post-separation analyses.

Notch Signaling Regulates Lgr5+ Olfactory Epithelium Progenitor/Stem Cell Turnover and Mediates Recovery of Lesioned Olfactory Epithelium in Mouse Model.

The Notch signaling pathway regulates stem cell proliferation and differentiation in multiple tissues and organs, and is required for tissue maintenance. However, the role of Notch in regulation of olfactory epithelium (OE) progenitor/stem cells to maintain tissue function is still not clear. A recent study reported that leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is expressed in globose basal cells (GBCs) localized in OE. Through lineage tracing in vivo, we found that Lgr5+ cells act as progenitor/stem cells in OE. The generation of daughter cells from Lgr5+ progenitor/stem cells is delicately regulated by the Notch signaling pathway, which not only controls the proliferation of Lgr5+ cells and their immediate progenies but also affects their subsequent terminal differentiation. In conditionally cultured OE organoids in vitro, inhibition of Notch signaling promotes neuronal differentiation. Besides, OE lesion through methimazole administration in mice induces generation of more Notch1+ cells in the horizontal basal cell (HBC) layer, and organoids derived from lesioned OE possesses more proliferative Notch1+ HBCs. In summary, we concluded that Notch signaling regulates Lgr5+ GBCs by controlling cellular proliferation and differentiation as well as maintaining epithelial cell homeostasis in normal OE. Meanwhile, Notch1 also marks HBCs in lesioned OE and Notch1+ HBCs are transiently present in OE after injury. This implies that Notch1+ cells in OE may have dual roles, functioning as GBCs in early development of OE and HBCs in restoring the lesioned OE. Stem Cells 2018;36:1259-1272.

Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation.

Generation of precisely patterned neural cells from human pluripotent stem cells (hPSCs) is instrumental in developing disease models and stem cell therapies. Here, we provide a detailed 16-d protocol for obtaining high-purity ventral midbrain (VM) dopamine (DA) progenitors for intracerebral transplantation into animal models and for in vitro maturation into neurons. We have successfully transplanted such cells into the rat; however, in principle, the cells can be used for transplantation into any animal model, and the protocol is designed to also be compatible with clinical transplantation into humans. We show how to precisely set the balance of patterning factors to obtain specifically the caudal VM progenitors that give rise to DA-rich grafts. By specifying how to perform quality control (QC), troubleshooting and adaptation of the procedure, this protocol will facilitate implementation in different laboratories and with a variety of hPSC lines. To facilitate reproducibility of experiments and enable shipping of cells between centers, we present a method for cryopreservation of the progenitors for subsequent direct transplantation or terminal differentiation into DA neurons. This protocol is free of xeno-derived products and can be performed under good manufacturing practice (GMP) conditions.

Abstract 3915: PBA2, a dual BCR-ABL and GSK3βkinases inhibitor, represses a wide array of Imatinib resistant cell lines in chronic myeloid leukemia

Abstract Despite the clinical efficacy of the BCR-ABL (Tyrosine Kinase Inhibitors) TKIs for the treatment of Chronic Myeloid Leukemia (CML), BCR-ABL-T315I mutation still confers higher-level resistance to imatinib and the second generation BCR-ABL TKIs. Ponatinib, as the third generation BCR-ABL TKIs, has the potential activity against CML with T315I mutant BCR-ABL whereas high frequency of serious adverse events limits its clinical use. Through a compound library screen, we identified a novel compound PBA2, a dual kinases inhibitor of GSK3β and BCR-ABL, as a promising agent for the treatment of imatinib-resistant CML. Not only can PBA2 inhibit cell growth but also induce terminal cell differentiation in a series of kinase mutant CML cell lines as well as T315I mutation. Remarkably, the mechanism of action of this compound involved the simultaneous inhibition effect of BCR-ABL and GSK3β, which could be a fruitful approach to suppress the β-catenin/CBP signal pathway that promoted the interaction of β-catenin/p300, leading to growth inhibition and subsequent terminal differentiation. More importantly, we found a novel molecular link between CBP and BCR-ABL. We showed that CBP could regulate BCR-ABL expression through activating CREB which binds to the BCR-ABL promoter in CML cells to start transcription, and the BCR-ABL in turns affect the expression level of CBP. This positive feedback pathway between CBP and BCR-ABL represents a novel way for the treatment of CML, and the novel compound PBA2 targeting at this loop deserves further investigation in clinical trial. Citation Format: Ke Yang, li-Wu Fu. PBA2, a dual BCR-ABL and GSK3βkinases inhibitor, represses a wide array of Imatinib resistant cell lines in chronic myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3915.

Role of the Retinoblastoma protein, Rb, during adult neurogenesis in the olfactory bulb.

Adult neural stem cells (aNSCs) are relatively quiescent populations that give rise to distinct neuronal subtypes throughout life, yet, at a very low rate and restricted differentiation potential. Thus, identifying the molecular mechanisms that control their cellular expansion is critical for regeneration after brain injury. Loss of the Retinoblastoma protein, Rb, leads to several defects in cell cycle as well as neuronal differentiation and migration during brain development. Here, we investigated the role of Rb during adult neurogenesis in the olfactory bulb (OB) by inducing its temporal deletion in aNSCs and progenitors. Loss of Rb was associated with increased proliferation of adult progenitors in the subventricular zone (SVZ) and the rostral migratory stream (RMS) but did not alter self-renewal of aNSCs or neuroblasts subsequent migration and terminal differentiation. Hence, one month after their birth, Rb-null neuroblasts were able to differentiate into distinct subtypes of GABAergic OB interneurons but were gradually lost after 3 months. Similarly, Rb controlled aNSCs/progenitors proliferation in vitro without affecting their differentiation capacity. This enhanced SVZ/OB neurogenesis associated with loss of Rb was only transient and negatively affected by increased apoptosis indicating a critical requirement for Rb in the long-term survival of adult-born OB interneurons.

Med14 cooperates with brg1 in the differentiation of skeletogenic neural crest.

BackgroundAn intricate gene regulatory network drives neural crest migration and differentiation. How epigenetic regulators contribute to this process is just starting to be understood.ResultsWe found that mutation of med14 or brg1 in zebrafish embryos resulted in a cluster of neural crest cell-related defects. In med14 or brg1 mutants, neural crest cells that form the jaw skeleton were specified normally and migrated to target sites. However, defects in their subsequent terminal differentiation were evident. Transplantation experiments demonstrated that med14 and brg1 are required directly in neural crest cells. Analysis of med14; brg1 double mutant embryos suggested the existence of a strong genetic interaction between members of the Mediator and BAF complexes.ConclusionsThese results suggest a critical role for Mediator and BAF complex function in neural crest development, and may also clarify the nature of defects in some craniofacial abnormalities.

Simultaneous isolation of enriched myoblasts and fibroblasts for migration analysis within a novel co-culture assay.

Skeletal muscle injury elicits the activation of satellite cells and their migration to the wound area for subsequent terminal differentiation and tissue integration. However, interstitial fibroblasts recruited to the site of injury promote deposition of fibrotic tissue, which hampers myoblast-mediated muscle regeneration. Currently, analysis of myoblast migration in vitro can be accomplished using chemotactic, cell-exclusion, or wound healing assays. Yet, to investigate cell motility following skeletal muscle damage more accurately, migration assays need to better simulate the repair process. Here we present a protocol for the simultaneous isolation of myoblasts and fibroblasts from the same muscle tissue, ensuring the consistent generation of enriched, purified, and matched cell populations at a low passage number. We then describe a wound assay that uses a novel approach to the co-culture of myoblasts and fibroblasts to mimic the injured environment more closely than other established methods. Using this assay, we demonstrate that fibroblasts are able to increase myoblast migration significantly, validating our new in vitro method. As the observed effect on migration is most likely mediated by secreted factors, our assay could easily be extended to include antibody-based protein analysis of secreted factors in animal or human systems.

G9a Histone Methyltransferase Activity in Retinal Progenitors Is Essential for Proper Differentiation and Survival of Mouse Retinal Cells

In vertebrate retinal development, various transcription factors are known to execute essential activities in gene regulation. Although epigenetic modification is considered to play a pivotal role in retinal development, the exact in vivo role of epigenetic regulation is still poorly understood. We observed that G9a histone methyltransferase, which methylates histone H3 at lysine 9 (H3K9), is substantially expressed in the mouse retina throughout development. To address in vivo G9a function in the mouse retina, we ablated G9a in retinal progenitor cells by conditional gene knock-out (G9a Dkk3 CKO). The G9a Dkk3 CKO retina exhibited severe morphological defects, including photoreceptor rosette formation, a partial loss of the outer nuclear layer, elevated cell death, and persistent cell proliferation. Progenitor cell-related genes, including several cyclins, Hes1, Chx10, and Lhx2, are methylated on histone H3K9 in the wild-type retina, but they were defective in H3K9 methylation and improperly upregulated at late developmental stages in the G9a Dkk3 CKO retina. Notably, conditional depletion of G9a in postmitotic photoreceptor precursors (G9a Crx CKO) led to the development of an almost normal retina, indicating that G9a activity mainly in retinal progenitor cells, but not in photoreceptor precursors, is essential for normal terminal differentiation of and survival of the retina. Our results suggest that proper epigenetic marks in progenitor cells are important for subsequent appropriate terminal differentiation and survival of retinal cells by repressing progenitor cell-related genes in differentiating retinal cells.

Enhancer Profiling Reveals SOX9 As a Novel Transcription Regulator of B Cell Activation and DLBCL Transformation

Abstract 527Antigen stimulation of naïve B cells (NBC) induces differentiation with a phenotype characterized by robust proliferation and genomic instability tolerance to enable activated germinal center B cells (GCB) to undergo immunoglobulin affinity maturation. Aberrant genetic events resulting from this process lead to malignant transformation and diffuse large B cell lymphoma (DLBCL). Phenotypic progression from quiescent NBC to activated GCB and malignant DLBCL involves major shifts in gene expression. Recent studies suggest that enhancers play a key role in mediating cell type-specific gene regulation. We therefore postulated that enhancers are involved in dictating the gene expression programs that govern normal and malignant B cell phenotypes; and systematic discovery of enhancers coupled with bioinformatic analysis would uncover key enhancer-binding transcription factors (TFs) that regulate these cell states.To test this hypothesis, we performed ChIP-seq on enhancer histone marks, i.e. H3K4me2, H3K27Ac, and H3K4me3, in primary NBC and GCB, and in DLBCL cell lines in biological replicates. We defined enhancers by the criterion of H3K4me2hiH3K4me3low. We observed a striking pattern of enhancer re-organization between cell types. First, we found a larger number of enhancers in primary B-cells (∼20,000) than in DLBCL (∼12,000). Second, we confirmed that enhancers are cell type-specific. For example, 11,492 out of 20,173 NBC enhancers were lost during transition to GCB (loss of H3K4me2 enrichment), while 13,088 new enhancers were gained in GCB. A similar phenomenon was also observed in DLBCL when compared to either NBC or GCB. This re-organization of enhancers suggests that cells may have dynamic gene regulatory programs during differentiation or malignant transformation. To discover TFs that act through enhancers, we used bioinformatic analyses, including FIRE and MEME, to search for TF consensus binding sequences within enhancers. Over-represented DNA motifs included motifs of SPI1, RUNX1, STAT3, RELA and SOX9, etc. SOX9 motif was significantly enriched in GCB specific enhancers (p=3.07e-15). SOX9 belongs to the SOX family TFs and plays an important role in cartilage development, sex determination, and intestinal differentiation but has not been implicated in B cell development.To investigate the role of SOX9 in B cell activation and malignant transformation, we first examined the expression of SOX9 in these cells. RNA-seq performed on human tonsilar NBC and GCB showed more than 20-fold increase of SOX9 mRNA in GCB as compared to NBC (6.75±0.80 vs 0.29±0.14, RPKM, p=0.0002). In addition, SOX9 expression was maintained in plasma B cells (2.88±0.49, RPKM). To understand how SOX9 regulates transcriptional programming in GCB, we performed SOX9 ChIP-seq in GCB to look for its targets. We found that SOX9 binds to 1,668 upstream distal enhancer regions (-5 to -100 kb of TSS) associated with 963 genes. These target genes were significantly enriched in many important pathways including cell cycle regulation (CCND2, CDC25B, CDK1), transcription regulation (BCOR, NCOR2), epigenetic regulation (BMI1, DNMT3A, MLL2, SUZ12, TET3), and MAPK signaling (MAP2K3, MAP3K7) (p<0.001). One of the SOX9 targets is PRMD1, a TF that controls the transition from GCB to plasma cells, suggesting that SOX9 may be involved in B cell terminal differentiation. To our surprise, we did not detect SOX9 mRNA in 10 out of 12 DLBCL cell lines by RNA-seq. Moreover, SOX9 was not expressed in the majority of primary malignant non-Hodgkin’s lymphoma cases studied by IHC in the Human Protein Atlas project. To examine whether reduced SOX9 expression could induce malignant transformation, we used shRNA to knockdown Sox9 in mouse BCL1 lymphoma cells and subjected them to colony forming assay in semi-solid methylcellulose. Knockdown of Sox9 increased BCL1 colony forming ability by 50% as compared to scramble, suggesting that loss of SOX9 expression maybe important for lymphomagenesis.In summary, we identified a novel germinal center TF, SOX9, by examining enrichment of TF motifs within enhancer regions uncovered by ChIP-seq. Our current data suggest that SOX9 may play an important role in germinal center reaction and subsequent terminal differentiation by regulating key factors, such as PRDM1, and that loss of SOX9 may contribute to DLBCL malignant transformation by potentially blocking the terminal differentiation of mature GCB. Disclosures:No relevant conflicts of interest to declare.

Conditional deletion of Tgfbr2 in hypertrophic chondrocytes delays terminal chondrocyte differentiation

Transforming growth factor β (Tgfb) signaling plays an important role in endochondral ossification. Previous studies of mice in which the Tgfb type II receptor gene (Tgfbr2) was deleted in the limb bud mesenchymal cells or differentiated chondrocytes showed defects in the development of the long bones or the axial skeleton, respectively. Here, we generated mouse embryos in which the Tgfbr2 gene was ablated in hypertrophic chondrocytes. These mice exhibited delays in both the hypertrophic conversion of proliferating chondrocytes and the subsequent terminal chondrocyte differentiation. The expression domains of Col10a1, Matrix metalloproteinase 13, and Osteopontin were small, and the expression of Vascular endothelial growth factor and Platelet endothelial cell adhesion molecule was downregulated. The calcification of the bone collar in the mutant mice was markedly delayed and the periosteum was thin, possibly because of the downregulation of Indian hedgehog expression. We conclude that Tgfb signaling in hypertrophic chondrocytes positively regulates terminal chondrocyte differentiation, angiogenesis in calcified cartilage, and osteogenesis in the bone collar, at least partly through Indian hedgehog signaling in vivo.

Kin-19/casein kinase Iα has dual functions in regulating asymmetric division and terminal differentiation in C. elegans epidermal stem cells

Casein Kinase I (CKI) is a conserved component of the Wnt signaling pathway, which regulates cell fate determination in metazoans. We show that post-embryonic asymmetric division and fate specification of C. elegans epidermal stem cells are controlled by a non-canonical Wnt/b-catenin signaling pathway, involving the b-catenins WRM-1 and SYS-1, and that C. elegans kin-19/CKIa functions in this pathway. Furthermore, we find that kin-19 is the only member of the Wnt asymmetry pathway that functions with, or in parallel to, the heterochronic temporal patterning pathway to control withdrawal from self-renewal and subsequent terminal differentiation of epidermal stem cells. We show that, except in the case of kin-19, the Wnt asymmetry pathway and the heterochronic pathway function separately and in parallel to control different aspects of epidermal stem cell fate specification. However, given the function of kin-19/CKIa in both pathways, and that CKI, Wnt signaling pathway and heterochronic pathway genes are widely conserved in animals, our findings suggest that CKIa may function as a regulatory hub through which asymmetric division and terminal differentiation are coordinated in adult stem cells of vertebrates.