ABSTRACT
In classical Hodgkin lymphoma (cHL), the highly abundant CD4+ T cells in the vicinity of tumor cells are considered essential for tumor cell survival, but are ill-defined. Although they are activated, they consistently lack expression of activation marker CD26. In this study, we compared sorted CD4+CD26- and CD4+CD26+ T cells from cHL lymph node cell suspensions by RNA sequencing and T cell receptor variable gene segment usage analysis. This revealed that although CD4+CD26- T cells are antigen experienced, they have not clonally expanded. This may well be explained by the expression of exhaustion associated transcription factors TOX and TOX2, immune checkpoints PDCD1 and CD200, and chemokine CXCL13, which were amongst the 100 significantly enriched genes in comparison with the CD4+CD26+ T cells. Findings were validated in single-cell RNA sequencing data from an independent cohort. Interestingly, immunohistochemistry revealed predominant and high frequency of staining for TOX and TOX2 in the T cells attached to the tumor cells. In conclusion, the dominant CD4+CD26- T cell population in cHL is antigen experienced, polyclonal, and exhausted. This population is likely a main contributor to the very high response rates to immune checkpoint inhibitors in cHL.
Subject(s)
CD4-Positive T-Lymphocytes , HMGB Proteins , High Mobility Group Proteins , Hodgkin Disease , Dipeptidyl Peptidase 4/immunology , HMGB Proteins/biosynthesis , HMGB Proteins/immunology , High Mobility Group Proteins/biosynthesis , High Mobility Group Proteins/immunology , Hodgkin Disease/genetics , Hodgkin Disease/immunology , Hodgkin Disease/metabolism , Humans , Lymph Nodes/pathology , Transcription Factors/geneticsABSTRACT
The endocardium is the endothelial component of the vertebrate heart and plays a key role in heart development. Where, when, and how the endocardium segregates during embryogenesis have remained largely unknown, however. We now show that Nkx2-5+ cardiac progenitor cells (CPCs) that express the Sry-type HMG box gene Sox17 from embryonic day (E) 7.5 to E8.5 specifically differentiate into the endocardium in mouse embryos. Although Sox17 is not essential or sufficient for endocardium fate, it can bias the fate of CPCs toward the endocardium. On the other hand, Sox17 expression in the endocardium is required for heart development. Deletion of Sox17 specifically in the mesoderm markedly impaired endocardium development with regard to cell proliferation and behavior. The proliferation of cardiomyocytes, ventricular trabeculation, and myocardium thickening were also impaired in a non-cell-autonomous manner in the Sox17 mutant, likely as a consequence of down-regulation of NOTCH signaling. An unknown signal, regulated by Sox17 and required for nurturing of the myocardium, is responsible for the reduction in NOTCH-related genes in the mutant embryos. Our results thus provide insight into differentiation of the endocardium and its role in heart development.
Subject(s)
Cell Differentiation , Embryo, Mammalian/embryology , Endocardium/embryology , Gene Expression Regulation, Developmental , HMGB Proteins/biosynthesis , SOXF Transcription Factors/biosynthesis , Signal Transduction , Stem Cells/metabolism , Animals , Embryo, Mammalian/cytology , Endocardium/cytology , HMGB Proteins/genetics , Mesoderm/cytology , Mesoderm/embryology , Mice , Mice, Transgenic , Receptors, Notch/genetics , Receptors, Notch/metabolism , SOXF Transcription Factors/genetics , Stem Cells/cytologyABSTRACT
The transcriptional repressor Capicua (Cic) controls tissue patterning and restricts organ growth, and has been recently implicated in several cancers. Cic has emerged as a primary sensor of signaling downstream of the receptor tyrosine kinase (RTK)/extracellular signal-regulated kinase (ERK) pathway, but how Cic activity is regulated in different cellular contexts remains poorly understood. We found that the kinase Minibrain (Mnb, ortholog of mammalian DYRK1A), acting through the adaptor protein Wings apart (Wap), physically interacts with and phosphorylates the Cic protein. Mnb and Wap inhibit Cic function by limiting its transcriptional repressor activity. Down-regulation of Cic by Mnb/Wap is necessary for promoting the growth of multiple organs, including the wings, eyes, and the brain, and for proper tissue patterning in the wing. We have thus uncovered a previously unknown mechanism of down-regulation of Cic activity by Mnb and Wap, which operates independently from the ERK-mediated control of Cic. Therefore, Cic functions as an integrator of upstream signals that are essential for tissue patterning and organ growth. Finally, because DYRK1A and CIC exhibit, respectively, prooncogenic vs. tumor suppressor activities in human oligodendroglioma, our results raise the possibility that DYRK1A may also down-regulate CIC in human cells.
Subject(s)
Body Patterning/genetics , Drosophila Proteins/genetics , Drosophila/genetics , HMGB Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/genetics , Repressor Proteins/genetics , Adaptor Proteins, Signal Transducing/genetics , Animals , Drosophila/growth & development , Drosophila Proteins/biosynthesis , Gene Expression Regulation, Developmental , HMGB Proteins/biosynthesis , Humans , Neoplasms/genetics , Phosphorylation , Protein Serine-Threonine Kinases/biosynthesis , Repressor Proteins/biosynthesis , Wings, Animal/growth & development , Dyrk KinasesABSTRACT
Epithelial renewal in the Drosophila intestine is orchestrated by Intestinal Stem Cells (ISCs). Following damage or stress the intestinal epithelium produces ligands that activate the epidermal growth factor receptor (EGFR) in ISCs. This promotes their growth and division and, thereby, epithelial regeneration. Here we demonstrate that the HMG-box transcriptional repressor, Capicua (Cic), mediates these functions of EGFR signaling. Depleting Cic in ISCs activated them for division, whereas overexpressed Cic inhibited ISC proliferation and midgut regeneration. Epistasis tests showed that Cic acted as an essential downstream effector of EGFR/Ras signaling, and immunofluorescence showed that Cic's nuclear localization was regulated by EGFR signaling. ISC-specific mRNA expression profiling and DNA binding mapping using DamID indicated that Cic represses cell proliferation via direct targets including string (Cdc25), Cyclin E, and the ETS domain transcription factors Ets21C and Pointed (pnt). pnt was required for ISC over-proliferation following Cic depletion, and ectopic pnt restored ISC proliferation even in the presence of overexpressed dominant-active Cic. These studies identify Cic, Pnt, and Ets21C as critical downstream effectors of EGFR signaling in Drosophila ISCs.
Subject(s)
DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , ErbB Receptors/genetics , HMGB Proteins/genetics , Nerve Tissue Proteins/genetics , Proto-Oncogene Proteins c-ets/genetics , Proto-Oncogene Proteins/genetics , Receptors, Invertebrate Peptide/genetics , Repressor Proteins/genetics , Transcription Factors/genetics , Animals , Cell Proliferation/genetics , DNA-Binding Proteins/biosynthesis , Drosophila/genetics , Drosophila Proteins/biosynthesis , Gene Expression Regulation, Developmental , HMGB Proteins/biosynthesis , Intestines/cytology , Intestines/growth & development , Nerve Tissue Proteins/biosynthesis , Proto-Oncogene Proteins/biosynthesis , RNA, Messenger/biosynthesis , Repressor Proteins/biosynthesis , Signal Transduction/genetics , Stem Cells/cytology , Transcription Factors/biosynthesisABSTRACT
Gastrulation leads to three germ layers--ectoderm, mesoderm and endoderm--that are separated by two basement membranes. In the mouse embryo, the emergent gut endoderm results from the widespread intercalation of cells of two distinct origins: pluripotent epiblast-derived definitive endoderm (DE) and extra-embryonic visceral endoderm (VE). Here we image the trajectory of prospective DE cells before intercalating into the VE epithelium. We show that the transcription factor SOX17, which is activated in prospective DE cells before intercalation, is necessary for gut endoderm morphogenesis and the assembly of the basement membrane that separates gut endoderm from mesoderm. Our results mechanistically link gut endoderm morphogenesis and germ layer segregation, two central and conserved features of gastrulation.
Subject(s)
Endoderm/embryology , Germ Layers/embryology , HMGB Proteins/metabolism , Mesoderm/embryology , Morphogenesis/physiology , SOXF Transcription Factors/metabolism , Animals , Basement Membrane/cytology , Basement Membrane/embryology , Cadherins/biosynthesis , Cell Differentiation , Embryo, Mammalian , Epithelium/embryology , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/biosynthesis , Fibronectins/metabolism , Gastrulation , Green Fluorescent Proteins/genetics , HMGB Proteins/biosynthesis , Hepatocyte Nuclear Factor 3-beta/genetics , Mice , Mice, Transgenic , Morphogenesis/genetics , Optical Imaging/methods , SOXF Transcription Factors/biosynthesisABSTRACT
Cells of the inner cell mass (ICM) of the mouse blastocyst differentiate into the pluripotent epiblast or the primitive endoderm (PrE), marked by the transcription factors NANOG and GATA6, respectively. To investigate the mechanistic regulation of this process, we applied an unbiased, quantitative, single-cell-resolution image analysis pipeline to analyze embryos lacking or exhibiting reduced levels of GATA6. We find that Gata6 mutants exhibit a complete absence of PrE and demonstrate that GATA6 levels regulate the timing and speed of lineage commitment within the ICM. Furthermore, we show that GATA6 is necessary for PrE specification by FGF signaling and propose a model where interactions between NANOG, GATA6, and the FGF/ERK pathway determine ICM cell fate. This study provides a framework for quantitative analyses of mammalian embryos and establishes GATA6 as a nodal point in the gene regulatory network driving ICM lineage specification.
Subject(s)
Blastocyst Inner Cell Mass/cytology , Endoderm/embryology , GATA6 Transcription Factor/metabolism , Homeodomain Proteins/biosynthesis , Animals , Benzamides/pharmacology , Cell Differentiation , Cell Lineage , Diphenylamine/analogs & derivatives , Diphenylamine/pharmacology , Embryo Culture Techniques , Embryo, Mammalian/metabolism , Endoderm/cytology , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibroblast Growth Factor 4/metabolism , GATA6 Transcription Factor/biosynthesis , GATA6 Transcription Factor/genetics , Gene Expression Regulation, Developmental , HMGB Proteins/biosynthesis , Homeodomain Proteins/antagonists & inhibitors , MAP Kinase Signaling System , Mice , Mice, Knockout , Nanog Homeobox Protein , SOXF Transcription Factors/biosynthesisABSTRACT
RATIONALE: The Notch pathway stabilizes sprouting angiogenesis by favoring stalk cells over tip cells at the vascular front. Because tip and stalk cells have different properties in morphology and function, their transcriptional regulation remains to be distinguished. Transcription factor Sox17 is specifically expressed in endothelial cells, but its expression and role at the vascular front remain largely unknown. OBJECTIVE: To specify the role of Sox17 and its relationship with the Notch pathway in sprouting angiogenesis. METHODS AND RESULTS: Endothelial-specific Sox17 deletion reduces sprouting angiogenesis in mouse embryonic and postnatal vascular development, whereas Sox17 overexpression increases it. Sox17 promotes endothelial migration by destabilizing endothelial junctions and rearranging cytoskeletal structure and upregulates expression of several genes preferentially expressed in tip cells. Interestingly, Sox17 expression is suppressed in stalk cells in which Notch signaling is relatively high. Notch activation by overexpressing Notch intracellular domain reduces Sox17 expression both in primary endothelial cells and in retinal angiogenesis, whereas Notch inhibition by delta-like ligand 4 (Dll4) blockade increases it. The Notch pathway regulates Sox17 expression mainly at the post-transcriptional level. Furthermore, endothelial Sox17 ablation rescues vascular network from excessive tip cell formation and hyperbranching under Notch inhibition in developmental and tumor angiogenesis. CONCLUSIONS: Our findings demonstrate that the Notch pathway restricts sprouting angiogenesis by reducing the expression of proangiogenic regulator Sox17.
Subject(s)
Endothelial Cells/metabolism , HMGB Proteins/physiology , Neovascularization, Pathologic/physiopathology , Neovascularization, Physiologic/physiology , Receptors, Notch/physiology , SOXF Transcription Factors/physiology , Signal Transduction/physiology , Animals , Carcinoma, Lewis Lung/blood supply , Cell Differentiation , Cell Movement , Cytoskeleton/ultrastructure , Embryo, Mammalian/blood supply , Embryonic Stem Cells , Gene Expression Regulation , HMGB Proteins/biosynthesis , HMGB Proteins/genetics , Human Umbilical Vein Endothelial Cells , Humans , Intercellular Junctions/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Morphogenesis/genetics , Protein Structure, Tertiary , RNA, Small Interfering/pharmacology , Receptor, Notch1/genetics , Receptor, Notch1/physiology , Recombinant Fusion Proteins , Retinal Vessels/growth & development , SOXF Transcription Factors/biosynthesis , SOXF Transcription Factors/genetics , Specific Pathogen-Free Organisms , Transcription, GeneticABSTRACT
Mouse embryonic fibroblasts (MEFs) have been extensively used as feeder cells to support the in vitro propagation of human embryonic stem cells (hESCs). However, owing to the risk of cross-contamination with animal or other unknown pathogens, the use of MEFs does not meet requirements for the clinical application of hESCs. Moreover, the actual role played by the feeders in the differentiation of hESCs is still unclear. In this study, human embryonic fibroblasts (HEFs) were used as feeder cells to support the establishment and undifferentiated growth of hESCs, and the capability of HEFs to induce the differentiation of definitive endoderm (DE) was evaluated. Three new hES cell lines were derived. These cell lines exhibited and maintained the common features of traditional hESCs after prolonged culture in vitro. Furthermore, DE differentiation of the newly established hES cell lines was performed using 100 ng/ml activin A, and the effects were compared among HEFs, MEFs, and feeder-free systems. On day 5 of induction, DE (SOX17(+)) cells appeared with comparable efficiency in both human and mouse feeder systems (85.0 +/- 8.9% and 78.7 +/- 3.4%, respectively). These levels were considerably superior to that obtained in the feeder-free system (22.7 +/- 5.6%). The SOX17(+) cells tended to differentiate into an endodermal lineage in vivo and could be further induced into glucagon and C-peptide double positive islet-like clusters in vitro. Our studies suggest that, in terms of therapeutic application, HEFs can be an effective substitute for MEFs for sustaining the derivation and DE differentiation of hESCs.
Subject(s)
Cell Differentiation/physiology , Embryo, Mammalian/cytology , Embryonic Stem Cells/cytology , Fibroblasts/cytology , Activins/pharmacology , Animals , C-Peptide/pharmacology , Cell Differentiation/drug effects , Cells, Cultured , Coculture Techniques , DNA-Binding Proteins/biosynthesis , Embryo, Mammalian/metabolism , Embryonic Stem Cells/metabolism , Endoderm/cytology , Endoderm/metabolism , Fibroblasts/metabolism , Glucagon/pharmacology , HMGB Proteins/biosynthesis , High Mobility Group Proteins/biosynthesis , Humans , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , Mice , Mice, Inbred ICR , SOXF Transcription Factors , Transcription Factors/biosynthesisABSTRACT
Previous studies have demonstrated that the transcription factor Sox2 is essential during the early stages of development. Furthermore, decreasing the expression of Sox2 severely interferes with the self-renewal and pluripotency of embryonic stem (ES) cells. Other studies have shown that Sox2, in conjunction with the transcription factor Oct-3/4, stimulates its own transcription as well as the expression of a growing list of genes (Sox2:Oct-3/4 target genes) that require the cooperative action of Sox2 and Oct-3/4. Remarkably, recent studies have shown that overexpression of Sox2 decreases expression of its own gene, as well as four other Sox2:Oct-3/4 target genes (Oct-3/4, Nanog, Fgf-4, and Utf1). This finding led to the prediction that overexpression of Sox2 in ES cells would trigger their differentiation. In the current study, we initially engineered mouse ES cells for inducible overexpression of Sox2. Using this model system, we demonstrate that small increases (twofold or less) in Sox2 protein trigger the differentiation of ES cells into cells that exhibit markers for a wide range of differentiated cell types, including neuroectoderm, mesoderm, and trophectoderm but not endoderm. We also demonstrate that elevating the levels of Sox2 quickly downregulates several developmentally regulated genes, including Nanog, and a newly identified Sox2:Oct-3/4 target gene, Lefty1. Together, these data argue that the self-renewal of ES cells requires that Sox2 levels be maintained within narrow limits. Thus, Sox2 appears to function as a molecular rheostat that controls the expression of a critical set of embryonic genes, as well as the self-renewal and differentiation of ES cells.
Subject(s)
Cell Differentiation/physiology , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/physiology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , HMGB Proteins/biosynthesis , HMGB Proteins/physiology , Transcription Factors/biosynthesis , Transcription Factors/physiology , Animals , Cell Differentiation/genetics , Cell Line , DNA-Binding Proteins/genetics , Directed Molecular Evolution/methods , Gene Expression Regulation, Developmental/physiology , HMGB Proteins/genetics , Mice , SOXB1 Transcription Factors , Transcription Factors/geneticsABSTRACT
SOX2 is a key neurodevelopmental gene involved in maintaining the pluripotency of stem cells and proliferation of neural progenitors and astroglia. Two evolutionally conserved enhancers, SRR1 and SRR2, are involved in controlling SOX2 expression during neurodevelopment; however, the molecular mechanisms regulating their activity are not known. We have examined DNA methylation and histone H3 acetylation at both enhancers in NT2-D1 progenitors, neurons and astrocytes, to establish the role of epigenetic mechanisms in cell-type-specific SOX2 expression. This study showed that 1) unmethylated DNA and acetylated histones at both enhancers correlated with a high level of SOX2 expression in proliferating neural progenitors and 2) reversible modifications of the SRR1 element were observed during gene reexpression in astrocytes, whereas permanent epigenetic marks on the SRR2 enhancer were seen in neurons where the gene was silenced. Taken together, these results are clear illustrations of cell-type-specific epigenomes and suggest mechanisms by which they may be created and maintained.
Subject(s)
Calcium-Binding Proteins/physiology , Cell Differentiation/physiology , DNA-Binding Proteins/biosynthesis , Enhancer Elements, Genetic/physiology , Epigenesis, Genetic/physiology , HMGB Proteins/biosynthesis , Membrane Glycoproteins/physiology , Neurons/cytology , Receptors, Cytoplasmic and Nuclear/physiology , Receptors, Peptide/physiology , Transcription Factors/biosynthesis , Acetylation , Astrocytes/cytology , Astrocytes/metabolism , Base Sequence , Calcium-Binding Proteins/genetics , Cells, Cultured , DNA Methylation , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , HMGB Proteins/genetics , HMGB Proteins/metabolism , Humans , Membrane Glycoproteins/genetics , Molecular Sequence Data , Neurons/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Peptide/genetics , SOXB1 Transcription Factors , Stem Cells/cytology , Stem Cells/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
In vertebrate embryos, the earliest definitive marker for the neural plate, which will give rise to the entire central nervous system, is the transcription factor Sox2. Although some of the extracellular signals that regulate neural plate fate have been identified, we know very little about the mechanisms controlling Sox2 expression and thus neural plate identity. Here, we use electroporation for gain- and loss-of-function in the chick embryo, in combination with bimolecular fluorescence complementation, two-hybrid screens, chromatin immunoprecipitation, and reporter assays to study protein interactions that regulate expression of N2, the earliest enhancer of Sox2 to be activated and which directs expression to the largest part of the neural plate. We show that interactions between three coiled-coil domain proteins (ERNI, Geminin, and BERT), the heterochromatin proteins HP1alpha and HP1gamma acting as repressors, and the chromatin-remodeling enzyme Brm acting as activator control the N2 enhancer. We propose that this mechanism regulates the timing of Sox2 expression as part of the process of establishing neural plate identity.
Subject(s)
DNA-Binding Proteins/biosynthesis , HMGB Proteins/biosynthesis , Neural Plate/metabolism , Transcription Factors/biosynthesis , Amino Acid Sequence , Animals , Avian Proteins/metabolism , Cell Cycle Proteins/metabolism , Chick Embryo , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/genetics , Enhancer Elements, Genetic , Gene Expression Regulation, Developmental , HMGB Proteins/genetics , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Neural Plate/embryology , Protein Binding , SOXB1 Transcription Factors , Transcription Factors/geneticsABSTRACT
Sox2 is a key transcription factor that maintains the proliferation of neuroglial stem cells and inhibits neuronal fate commitment. Moreover, it was recently found that brain tumors contain stem cells that resemble normal neuroglial stem cells in many respects. This study was undertaken to describe Sox2 expression in various brain tumors, and to determine whether Sox2 expression is a universal feature of brain tumors, or whether its expression is limited to a specific lineage of brain tumors. Sox2 immunohistochemistry was performed on 194 brain tumor tissues of various kinds. Fetal and adult normal brain tissues obtained by autopsy and brain tissues of epilepsy patients with cortical dysplasia were used as controls. Semiquantitative reverse transcription polymerase chain reaction was used to confirm the immunohistochemical results. Double immunofluorescence was performed to characterize the lineage of Sox2-positive cells. Sox2 was found to be expressed in various glial tumors, including those with astroglial, oligodendroglial, and ependymal lineages, and in the glial components of mixed neuroglial tumors, regardless of pathologic grade. In brain tumors of embryonal origin, supratentorial primitive neuroectodermal tumors showed robust Sox2 expression, whereas medulloblastomas and pineoblastomas did not. The majority of Sox2-positive tumor cells coexpressed glial fibrillary acidic protein, and most Sox2-negative cells in medulloblastomas and pineoblastomas showed neuronal differentiation. This study suggest that Sox2 may be a tumor marker of glial lineages rather than a universal brain tumor stem cell marker, because its expression pattern was found to correspond to differentiation pathways. On the other hand, the aberrant coexpressions of Sox2 and of a neuronal marker were widely observed in glioblastomas, which reflects a disorganized differentiation pattern that characterizes highly malignant tumors.
Subject(s)
Biomarkers, Tumor/analysis , Brain Neoplasms/metabolism , HMGB Proteins/biosynthesis , Neuroglia/cytology , Neuroglia/metabolism , Transcription Factors/biosynthesis , Brain/cytology , Brain/embryology , Brain/metabolism , Brain Neoplasms/pathology , Cell Differentiation , Cell Lineage , Fluorescent Antibody Technique , Glial Fibrillary Acidic Protein/metabolism , Humans , Immunohistochemistry , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , SOXB1 Transcription FactorsABSTRACT
Sox5 is a member of the SoxD group of HMG-box transcription factors that, during the early stages of development, promotes neural crest generation. However, little is known about Sox5 function in neural crest derivatives such as the peripheral sensory nervous system. We have analysed the embryonic expression of Sox5 during chick cranial ganglia development, from the stages of ganglia condensation to those of differentiation. During this period, Sox5 expression is maintained in the crest-derived satellite glial cells in all the cranial ganglia. In contrast, Sox5 is only transiently expressed in a subpopulation of differentiating neurons of both neural crest and placode origin. This detailed analysis provides a good base to dissect the possible role of Sox5 in neural cell fate determination by future functional approaches.
Subject(s)
Brain/embryology , Ganglia/embryology , Gene Expression Regulation, Developmental , HMGB Proteins/biosynthesis , HMGB Proteins/genetics , Neuroglia/metabolism , Animals , Cell Differentiation , Cell Lineage , Chick Embryo , DNA-Binding Proteins/biosynthesis , Gene Expression Profiling , High Mobility Group Proteins/biosynthesis , Immunohistochemistry , Neural Crest/embryology , SOXE Transcription Factors , Transcription Factors/biosynthesis , Trigeminal Ganglion/embryologyABSTRACT
The amnion is the inner of two membranes surrounding the fetus. That it arises from embryonic epiblast cells prior to gastrulation suggests that it may retain a reservoir of stem cells throughout pregnancy. We found that human amniotic epithelial cells (hAECs) harvested from term-delivered fetal membranes express mRNA and proteins present in human embryonic stem cells (hESCs), including POU domain, class 5, transcription factor 1; Nanog homeobox; SRY-box 2; and stage-specific embryonic antigen-4. In keeping with possible stem cell-like activity, hAECs were also clonogenic, and primary hAEC cultures could be induced to differentiate into cardiomyocytic, myocytic, osteocytic, adipocytic (mesodermal), pancreatic, hepatic (endodermal), neural, and astrocytic (neuroectodermal) cells in vitro, as defined by phenotypic, mRNA expression, immunocytochemical, and/or ultrastructural characteristics. However, unlike hESCs, hAECs did not form teratomas upon transplantation into severe combined immunodeficiency mice testes. Last, using flow cytometry we have shown that only a very small proportion of primary hAECs contain class IA and class II human leukocyte antigens (HLAs), consistent with a low risk of tissue rejection. However, following differentiation into hepatic and pancreatic lineages, significant proportions of cells contained class IA, but not class II, HLAs. These observations suggest that the term amnion, an abundant and easily accessible tissue, may be a useful source of multipotent stem cells that possess a degree of immune privilege.
Subject(s)
Amnion/physiology , Cell Differentiation/physiology , Fetal Stem Cells/cytology , Multipotent Stem Cells/cytology , Animals , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Female , Fetal Stem Cells/metabolism , Flow Cytometry , Glycosphingolipids/biosynthesis , Glycosphingolipids/genetics , HMGB Proteins/biosynthesis , HMGB Proteins/genetics , Histocompatibility Antigens Class I/biosynthesis , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class II/biosynthesis , Histocompatibility Antigens Class II/genetics , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/genetics , Humans , Immunohistochemistry , Male , Mice , Mice, SCID , Multipotent Stem Cells/metabolism , Nanog Homeobox Protein , Octamer Transcription Factor-3/biosynthesis , Octamer Transcription Factor-3/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , SOXB1 Transcription Factors , Stage-Specific Embryonic Antigens , Stem Cell Transplantation , Transcription Factors/biosynthesis , Transcription Factors/geneticsABSTRACT
Mechanosensory hair cells are essential for audition in vertebrates, and in many species, have the capacity for regeneration when damaged. Regeneration is robust in the fish lateral line system as new hair cells can reappear after damage induced by waterborne aminoglycoside antibiotics, platinum-based drugs, and heavy metals. Here, we characterize the loss and reappearance of lateral line hair cells induced in zebrafish larvae treated with copper sulfate using diverse molecular markers. Transgenic fish that express green fluorescent protein in different cell types in the lateral line system have allowed us to follow the regeneration of hair cells after different damage protocols. We show that conditions that damage only differentiated hair cells lead to reappearance of new hair cells within 24 h from nondividing precursors, whereas harsher conditions are followed by a longer recovery period that is accompanied by extensive cell division. In order to characterize the cell population that gives rise to new hair cells, we describe the expression of a neural stem cell marker in neuromasts. The zebrafish sox2 gene is strongly expressed in neuromast progenitor cells, including those of the migrating lateral line primordium, the accessory cells that underlie the hair cells in neuromasts, and in interneuromastic cells that give rise to new neuromasts. Moreover, we find that most of the cells that proliferate within the neuromast during regeneration express this marker. Thus, our results describe the dynamics of hair cell regeneration in zebrafish and suggest the existence of at least two mechanisms for recovery of these cells in neuromasts.
Subject(s)
DNA-Binding Proteins/biosynthesis , HMGB Proteins/biosynthesis , Hair Cells, Auditory/physiology , Lateral Line System/physiology , Neurons/physiology , Stem Cells/physiology , Transcription Factors/biosynthesis , Animals , Animals, Genetically Modified , Antimetabolites , Bromodeoxyuridine , Cell Proliferation/drug effects , Copper/toxicity , DNA-Binding Proteins/genetics , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , HMGB Proteins/genetics , Hair Cells, Auditory/drug effects , Image Processing, Computer-Assisted , Immunohistochemistry , In Situ Hybridization , Mitosis/physiology , Nerve Regeneration/physiology , SOXB1 Transcription Factors , Stem Cells/drug effects , Transcription Factors/genetics , ZebrafishABSTRACT
Sox2 is a transcription factor that plays a critical role in the maintenance of the self-renewal capability of neural stem cells. This study was undertaken to investigate the expression pattern of Sox2 in mature and immature teratomas of the central nervous system. Sox2 immunohistochemistry was performed in 14 cases of central nervous system teratoma: five mature, five immature teratomas, and four mixed germ cell tumors with a prominent teratoma component. Fetal brain tissue was used as a normal control. Immunofluorescence with double labeling of stem cells and neuroglial cell markers was used for phenotyping of Sox2-positive cells. In all cases of immature teratomas, positive reactivity to Sox2 was observed in primitive neuroepithelial tissues. Sox2 was not expressed in mature tissues, except in some cuboidal or columnar epithelium of endodermal origin. In mature teratomas, Sox2 expression was limited to some endodermal epithelium in two cases, and no Sox2 expression was observed in the other three cases. The majority of Sox2-positive neuroepithelial cells also expressed neural stem cell markers, nestin and vimentin. Sox2 and neuronal and oligodendroglial markers were expressed in a mutually exclusive manner. However, mature astroglial cells coexpressed Sox2 and GFAP. In fetal brain, Sox2 was mainly expressed in ventricular and subventricular zones. Since Sox2 is strongly expressed in the primitive neuroepithelial tissues of central nervous system immature teratomas, it may be a useful biomarker for the diagnosis and quantitative grading of central nervous system immature teratomas.
Subject(s)
Central Nervous System Neoplasms/pathology , HMGB Proteins/biosynthesis , Teratoma/pathology , Transcription Factors/biosynthesis , Adolescent , Adult , Central Nervous System Neoplasms/metabolism , Child , Child, Preschool , Female , Fluorescent Antibody Technique , Glial Fibrillary Acidic Protein/analysis , Humans , Immunohistochemistry , Infant , Male , Microscopy, Confocal , SOXB1 Transcription Factors , Teratoma/metabolismABSTRACT
The zebrafish caudal fin provides a simple model to study molecular mechanisms of dermal bone regeneration. We previously showed that misexpression of Bone morphogenetic protein 2b (Bmp2b) induces ectopic bone formation within the regenerate. Here we show that in addition to bmp2b and bmp4 another family member, bmp6, is involved in fin regeneration. We further investigated the function of BMP signaling by ectopically expressing the BMP signaling inhibitor Chordin which caused: (1) inhibition of regenerate outgrowth due to a decrease of blastema cell proliferation and downregulation of msxb and msxC expression and (2) reduced bone matrix deposition resulting from a defect in the maturation and function of bone-secreting cells. We then identified targets of BMP signaling involved in regeneration of the bone of the fin rays. runx2a/b and their target col10a1 were downregulated following BMP signaling inhibition. Unexpectedly, the sox9a/b transcription factors responsible for chondrocyte differentiation were detected in the non-cartilaginous fin rays, sox9a and sox9b were not only differentially expressed but also differentially regulated since sox9a, but not sox9b, was downregulated in the absence of BMP signaling. Finally, this analysis revealed the surprising finding of the expression, in the fin regenerate, of several factors which are normally the signatures of chondrogenic elements during endochondral bone formation although fin rays form through dermal ossification, without a cartilage intermediate.
Subject(s)
Bone Morphogenetic Proteins/physiology , Cell Differentiation , Osteoblasts/cytology , Regeneration , Zebrafish/anatomy & histology , Zebrafish/physiology , Animals , Bone Morphogenetic Protein 2 , Bone Morphogenetic Protein 4 , Bone Morphogenetic Protein 6 , Bone Morphogenetic Proteins/metabolism , Cell Proliferation , Chondrocytes/cytology , Down-Regulation , Glycoproteins/biosynthesis , HMGB Proteins/biosynthesis , Homeodomain Proteins/metabolism , Intercellular Signaling Peptides and Proteins/biosynthesis , Osteogenesis , SOX9 Transcription Factor , Signal Transduction , Transcription Factors/metabolism , Zebrafish/metabolism , Zebrafish Proteins/biosynthesis , Zebrafish Proteins/metabolismABSTRACT
Eye development initiates as an evagination of the early neural plate, before the closure of the neural tube. Structural malformations of the eye such as anophthalmia and microphthalmia arise very early in development. It is not surprising therefore that three of the genes currently identified to play a significant role in these developmental eye anomalies are also major players in brain development and regionalization. However, as has been emerging for a high proportion of transcriptional regulators studied, these genes have evolved to play multiple roles throughout development, and perhaps even in adult tissue maintenance. This complex spatiotemporal expression pattern requires elaborate regulatory systems which we are beginning to unravel. A major component of these complex regulatory networks is a series of cis-acting elements, highly conserved through evolution, which spread large distances from the coding region of each gene. We describe how cross regulation for PAX6, SOX2 and perhaps OTX2 has now been uncovered, pointing to the mechanisms that can fine-tune the expression of such essential developmental components. These interactions also help us understand why there is significant phenotypic overlap between mutations at these three loci.
Subject(s)
DNA-Binding Proteins/genetics , Eye Abnormalities/embryology , Eye Abnormalities/genetics , Eye Proteins/genetics , HMGB Proteins/genetics , Homeodomain Proteins/genetics , Otx Transcription Factors/genetics , Paired Box Transcription Factors/genetics , Repressor Proteins/genetics , Transcription Factors/genetics , Animals , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/physiology , Eye Proteins/biosynthesis , Eye Proteins/physiology , HMGB Proteins/biosynthesis , HMGB Proteins/physiology , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/physiology , Humans , Otx Transcription Factors/biosynthesis , Otx Transcription Factors/physiology , PAX6 Transcription Factor , Paired Box Transcription Factors/biosynthesis , Paired Box Transcription Factors/physiology , Repressor Proteins/biosynthesis , Repressor Proteins/physiology , SOXB1 Transcription Factors , Transcription Factors/biosynthesis , Transcription Factors/physiologyABSTRACT
Identification of biomarkers could lead to the development of effective screening tests for colorectal cancer. A previous study from our laboratory showed specific alterations of nuclear structure in colon cancer. In an effort to characterize these biomarkers, protein spots were selected from separations made by two-dimensional gel electrophoresis, which were analyzed by mass spectrometry. The sequences obtained from the isolated spots revealed that they have close similarity to creatine kinase B (CKB) isoforms, heterogeneous nuclear ribonucleoprotein F (hnRNP F) and high mobility group box 1 protein (HMGB1) isoforms. To determine the expression of these proteins in colon cancer, expression was studied in 9 tumor and matched adjacent normal pairs, 5 donor colons, 16 polyps, 4 metastatic liver lesions and matched adjacent normal pairs, and 3 liver donors. CKB and hnRNP F were expressed in 78% and 89% of colon tumors, respectively. hnRNP F had a higher frequency of expression than CKB in premalignant polyps. With the establishment of differential expression of the proteins in colon cancer, their subcellular localization was analyzed. The subcellular fractions studied both showed high protein levels of hnRNP F in colon tumors compared with normal colon tissues. Surprisingly, subcellular levels of CKB were decreased in colon tumors, suggesting that the observed high CKB levels in nuclear matrix extracts are caused by the enhanced localization of CKB to the nuclear matrix during colon tumorigenesis. These results suggest an involvement of hnRNP F and CKB in colorectal cancer. Additionally, they suggest that hnRNP F is a potential marker for colorectal cancer progression.
Subject(s)
Colonic Neoplasms/genetics , Creatine Kinase, BB Form/biosynthesis , Gene Expression Profiling , HMGB Proteins/biosynthesis , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/biosynthesis , Liver Neoplasms/secondary , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Adult , Aged , Amino Acid Sequence , Biomarkers, Tumor/analysis , Case-Control Studies , Cell Transformation, Neoplastic , Colonic Neoplasms/pathology , Colonic Polyps/genetics , Colonic Polyps/pathology , Creatine Kinase, BB Form/analysis , Creatine Kinase, BB Form/genetics , Electrophoresis, Gel, Two-Dimensional , Female , HMGB Proteins/analysis , HMGB Proteins/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/analysis , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/genetics , Humans , Liver Neoplasms/genetics , Male , Middle Aged , Molecular Sequence DataABSTRACT
The identification of neural stem cells (NSCs) in situ has been prevented by the inability to identify a marker consistently expressed in all adult NSCs and is thus generally accomplished using the in vitro neurosphere-forming assay. The high-mobility group transcription factor Sox2 is expressed in embryonic neural epithelial stem cells; because these cells are thought to give rise to the adult NSC population, we hypothesized that Sox2 may continue to be expressed in adult NSCs. Using Sox2:EGFP transgenic mice, we show that Sox2 is expressed in neurogenic regions along the rostral-caudal axis of the central nervous system throughout life. Furthermore, all neurospheres derived from these neurogenic regions express Sox2, suggesting that Sox2 is indeed expressed in adult NSCs. We demonstrate that NSCs are heterogeneous within the adult brain, with differing capacities for cell production. In vitro, all neurospheres express Sox2, but the expression of markers common to early progenitor cells within individual neurospheres varies; this heterogeneity of NSCs is mirrored in vivo. For example, both glial fibrillary acidic protein and NG2 are expressed within individual neurospheres, but their expression is mutually exclusive; likewise, these two markers show distinct staining patterns within the Sox2+ regions of the brain's neurogenic regions. Thus, we propose that the expression of Sox2 is a unifying characteristic of NSCs in the adult brain, but that not all NSCs maintain the ability to form all neural cell types in vivo.