Carboxypeptidase A5 identifies a novel mast cell lineage in the zebrafish providing new insight into mast cell fate determination

Blood ◽  
2008 ◽  
Vol 112 (7) ◽  
pp. 2969-2972 ◽  
Author(s):  
J. Tristan Dobson ◽  
Jake Seibert ◽  
Evelyn M. Teh ◽  
Sahar Da'as ◽  
Robert B. Fraser ◽  
...  
Keyword(s):  
Mast Cell ◽  
Cell Fate ◽  
Cell Lineage ◽  
Cell Fate Determination ◽  
Specific Enzyme ◽  
Efficient System ◽  
And Function ◽  
Insight Into ◽  
Lysozyme C

Abstract Mast cells (MCs) play critical roles in allergy and inflammation, yet their development remains controversial due to limitations posed by traditional animal models. The zebrafish provides a highly efficient system for studying vertebrate hematopoiesis. We have identified zebrafish MCs in the gill and intestine, which resemble their mammalian counterparts both structurally and functionally. Carboxypeptidase A5 (cpa5), a MC-specific enzyme, is expressed in zebrafish blood cells beginning at 24 hours post fertilization (hpf). At 28 hpf, colocalization is observed with pu.1, mpo, l-plastin, and lysozyme C, but not fms or cepbα, identifying these early MCs as a distinct myeloid population arising from a common granulocyte/monocyte progenitor. Morpholino “knock-down” studies demonstrate that transcription factors gata-2 and pu.1, but not gata-1 or fog-1, are necessary for early MC development. These studies validate the zebrafish as an in vivo tool for studying MC ontogeny and function with future capacity for modeling human MC diseases.

Identification of Bipotent Basophil/Mast Cell Progenitors in Adult Murine Hematopoiesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 633-633
Author(s):  
Yojiro Arinobu ◽  
Hiromi Iwasaki ◽  
Michael F. Gurish ◽  
Shinichi Mizuno ◽  
Hirokazu Shigematsu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Mast Cells ◽  
Cell Fate ◽  
Bone Marrow Cells ◽  
Cell Lineage ◽  
Primary Role ◽  
Peripheral Tissues ◽  
Fate Decision

Abstract Basophils and mast cells are multifunctional hematopoietic effectors that co-operate to mount a variety of allergic and innate immune responses. Their origin and developmental relationships, however, have not yet been resolved, and remain as one of the major issues in the biology of hematopoiesis. Here we report that progenitors bipotent for basophils and mast cells(basophil/mast cell progenitors: BMCPs) are prospectively isolatable within murine spleen. We have shown that the β7-integrin(β7) is an essential molecule for tissue-specific homing of putative precursors for intestinal mast cells (J Exp Med194:1243, 2001). To identify a candidate population that seeds intestinal progenitors for mast cells, we searched for β7+ cells in the bone marrow and the spleen. Lin−c-Kit+ spleen cells contained a fraction of cells expressing β7 at high levels. They also expressed FcγRII/III, but the majority of these cells did not express FcεRIα. These Lin−c-Kit+FcγRII/III+β7hiFcεRIα−/locells exclusively differentiated into mature mast cells and basophils. Strikingly, single Lin−c-Kit+FcγRII/III+β7hiFcεRIα−/locells formed colonies containing both basophils and mast cells as well as pure mast cell or basophil colonies. We thus named these cells as BMCPs. In 2-day cultures, purified BMCPs upregulated FcεRIα, giving rise to Lin−CD34+FcεRIαhic-Kit+ and Li− CD34+FcεRIαhic-Kit− blastic cell populations, and they differentiated exclusively into mast cells and basophils, respectively. Based on this phenotype, we searched for precursors committed to either lineage in vivo. The Lin−CD34+ bone marrow cells contained FcεRIαhic-Kit−cells, which differentiated exclusively into basophils. We named this population as basophil progenitors (BaPs). Since mast cell progenitors(MCPs) were not isolatable as a distinct population in the bone marrow or the spleen, we searched for MCPs in the intestine. We newly identified CD45+Li− CD34+β7hiFcεRIαlocells in the intestine, and these cells exclusively formed pure mast cell colonies, which were named as intestinal MCPs. Since the expression of C/EBPα was dramatically increased in BaPs but was downregulated in MCPs, we hypothesized that it plays a key role in the basophil versus the mast cell lineage commitment. To test this hypothesis, we disrupted or overexpressed C/EBPα at the BMCP stage. BMCPs disrupted with C/EBPα gave rise exclusively to mast cells, while, BMCPs overexpressing C/EBPα differentiated mainly into basophils, suggesting that C/EBPα plays a primary role in deciding the basophil vs. mast cell fate at the BMCP stage. Thus, differentiation of the BMCPs into committed progeny may lead to selective migration, BaPs to the bone marrow or MCPs to peripheral tissues, and this fate decision is controlled principally by C/EBPα. These newly identified progenitors should be useful to analyze the mechanism of commitment into each of these lineages, and could also be therapeutic targets for a variety of allergic and autoimmune disorders.

Pax5 determines B- versus T-cell fate and does not block early myeloid-lineage development

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4342-4346 ◽  
Author(s):  
Claudiu V. Cotta ◽  
Zheng Zhang ◽  
Hyung-Gyoon Kim ◽  
Christopher A. Klug
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Nk Cell ◽  
Cell Lineage ◽  
Blood Analysis ◽  
Hematopoietic Stem ◽  
Peripheral Blood Analysis

Abstract Progenitor B cells deficient in Pax5 are developmentally multipotent, suggesting that Pax5 is necessary to maintain commitment to the B-cell lineage. Commitment may be mediated, in part, by Pax5 repression of myeloid-specific genes. To determine whether Pax5 expression in multipotential cells is sufficient to restrict development to the B-cell lineage in vivo, we enforced expression of Pax5 in hematopoietic stem cells using a retroviral vector. Peripheral blood analysis of all animals reconstituted with Pax5-expressing cells indicated that more than 90% of Pax5-expressing cells were B220+ mature B cells that were not malignant. Further analysis showed that Pax5 completely blocked T-lineage development in the thymus but did not inhibit myelopoiesis or natural killer (NK) cell development in bone marrow. These results implicate Pax5 as a critical regulator of B- versus T-cell developmental fate and suggest that Pax5 may promote commitment to the B-cell lineage by mechanisms that are independent of myeloid gene repression.

scholarly journals Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage

2019 ◽  
Author(s):  
Cassandra K. Hayne ◽  
Casey A. Schmidt ◽  
A. Gregory Matera ◽  
Robin E. Stanley
Keyword(s):  
Animal Cells ◽  
Trna Splicing ◽  
Polynucleotide Kinase ◽  
Genes Encoding ◽  
Intron Removal ◽  
And Function ◽  
Insight Into ◽  
Negative Modulator

ABSTRACTThe splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34, and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1’s role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.

scholarly journals Annotation and Expression of IDN2-like and FDM-like Genes in Sexual and Aposporous Hypericum perforatum L. accessions

Plants ◽  
2019 ◽  
Vol 8 (6) ◽  
pp. 158
Author(s):  
Andrea Basso ◽  
Gianni Barcaccia ◽  
Giulio Galla
Keyword(s):  
Cell Fate ◽  
Hypericum Perforatum ◽  
Seed Set ◽  
Regulation Of Gene Expression ◽  
Cell Fate Determination ◽  
Functional Aspects ◽  
Hypericum Perforatum L ◽  
Potential Interactions ◽  
Insight Into ◽  
Reproductive Phenotype

The protein IDN2, together with the highly similar interactors FDM1 and FDM2, is required for RNA-directed DNA methylation (RdDM) and siRNA production. Epigenetic regulation of gene expression is required to restrict cell fate determination in A. thaliana ovules. Recently, three transcripts sharing high similarity with the A. thaliana IDN2 and FDM1-2 were found to be differentially expressed in ovules of apomictic Hypericum perforatum L. accessions. To gain further insight into the expression and regulation of these genes in the context of apomixis, we investigated genomic, transcriptional and functional aspects of the gene family in this species. The H. perforatum genome encodes for two IDN2-like and 7 FDM-like genes. Differential and heterochronic expression of FDM4-like genes was found in H. perforatum pistils. The involvement of these genes in reproduction and seed development is consistent with the observed reduction of the seed set and high variability in seed size in A. thaliana IDN2 and FDM-like knockout lines. Differential expression of IDN2-like and FDM-like genes in H. perforatum was predicted to affect the network of potential interactions between these proteins. Furthermore, pistil transcript levels are modulated by cytokinin and auxin but the effect operated by the two hormones depends on the reproductive phenotype.

scholarly journals Analysis of the myogenin promoter reveals an indirect pathway for positive autoregulation mediated by the muscle-specific enhancer factor MEF-2.

1992 ◽  
Vol 12 (9) ◽  
pp. 3665-3677 ◽  
Author(s):  
D G Edmondson ◽  
T C Cheng ◽  
P Cserjesi ◽  
T Chakraborty ◽  
E N Olson
Keyword(s):  
Cell Fate ◽  
Consensus Sequence ◽  
Cell Lineage ◽  
Mammalian Development ◽  
Indirect Pathway ◽  
Binding Factor ◽  
Nonmuscle Cells ◽  
And Function ◽  
Myod Gene

Transcriptional cascades that specify cell fate have been well described in invertebrates. In mammalian development, however, gene hierarchies involved in determination of cell lineage are not understood. With the recent cloning of the MyoD family of myogenic regulatory factors, a model system has become available with which to study the dynamics of muscle determination in mammalian development. Myogenin, along with other members of the MyoD gene family, possesses the apparent ability to redirect nonmuscle cells into the myogenic lineage. This ability appears to be due to the direct activation of an array of subordinate or downstream genes which are responsible for formation and function of the muscle contractile apparatus. Myogenin-directed transcription has been shown to occur through interaction with a DNA consensus sequence known as an E box (CANNTG) present in the control regions of numerous downstream genes. In addition to activating the transcription of subordinate genes, members of the MyoD family positively regulate their own expression and cross-activate one another's expression. These autoregulatory interactions have been suggested as a mechanism for induction and maintenance of the myogenic phenotype, but the molecular details of the autoregulatory circuits are undefined. Here we show that the myogenin promoter contains a binding site for the myocyte-specific enhancer-binding factor, MEF-2, which can function as an intermediary of myogenin autoactivation. Since MEF-2 can be induced by myogenin, these results suggest that myogenin and MEF-2 participate in a transcriptional cascade in which MEF-2, once induced by myogenin, acts to amplify and maintain the myogenic phenotype by acting as a positive regulator of myogenin expression.

scholarly journals Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage

2020 ◽  
Vol 48 (14) ◽  
pp. 7609-7622 ◽  
Author(s):  
Cassandra K Hayne ◽  
Casey A Schmidt ◽  
Maira I Haque ◽  
A Gregory Matera ◽  
Robin E Stanley
Keyword(s):  
Animal Cells ◽  
Trna Splicing ◽  
Polynucleotide Kinase ◽  
Genes Encoding ◽  
Intron Removal ◽  
And Function ◽  
Insight Into ◽  
Negative Modulator

Abstract The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34 and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1’s role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.

scholarly journals Stochastic antagonism between two proteins governs a bacterial cell fate switch

Science ◽  
2019 ◽  
Vol 366 (6461) ◽  
pp. 116-120 ◽  
Author(s):  
Nathan D. Lord ◽  
Thomas M. Norman ◽  
Ruoshi Yuan ◽  
Somenath Bakshi ◽  
Richard Losick ◽  
...  
Keyword(s):  
Cell Fate ◽  
Regulatory Networks ◽  
Cell Types ◽  
Feedback Loops ◽  
Cell Fate Determination ◽  
Cell Fate Decision ◽  
A Cell ◽  
Fate Decision ◽  
Antagonistic Interactions

Cell fate decision circuits must be variable enough for genetically identical cells to adopt a multitude of fates, yet ensure that these states are distinct, stably maintained, and coordinated with neighboring cells. A long-standing view is that this is achieved by regulatory networks involving self-stabilizing feedback loops that convert small differences into long-lived cell types. We combined regulatory mutants and in vivo reconstitution with theory for stochastic processes to show that the marquee features of a cell fate switch in Bacillus subtilis—discrete states, multigenerational inheritance, and timing of commitments—can instead be explained by simple stochastic competition between two constitutively produced proteins that form an inactive complex. Such antagonistic interactions are commonplace in cells and could provide powerful mechanisms for cell fate determination more broadly.

Evaluation of effector cell fate and function by in vivo bioluminescence imaging

Methods ◽  
2003 ◽  
Vol 31 (2) ◽  
pp. 172-179 ◽  
Author(s):  
Matthias Edinger ◽  
Petra Hoffmann ◽  
Christopher H Contag ◽  
Robert S Negrin
Keyword(s):  
Cell Fate ◽  
Effector Cell ◽  
Bioluminescence Imaging ◽  
In Vivo Bioluminescence Imaging ◽  
And Function

Combined Single Cell Lineage and Transcriptome Sequencing Unveils Cell-Autonomous Regulators of Hematopoietic Stem Cell Fate

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 446-446
Author(s):  
Alejo E Rodriguez-Fraticelli ◽  
Caleb S Weinreb ◽  
Allon Moshe Klein ◽  
Shou-Wen Wang ◽  
Fernando D Camargo
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Conflicts Of Interest ◽  
Large Fraction ◽  
Cell Lineage ◽  
Gene Signature ◽  
Hematopoietic Stem ◽  
Cell State

Blood regeneration upon transplantation relies on the activity of long-term repopulating hematopoietic stem cells (LT-HSCs). One of the major controversies in hematopoiesis relates to the apparently different properties that HSCs have in transplantation versus unperturbed settings. In unperturbed steady state hematopoiesis, the most potent HSCs appear to be mostly dormant, and only producing platelet-lineage cells. In turn, upon transplant, even a single transplanted HSC can actively divide and regenerate hundreds of millions of blood progenitors of all lineages. It would thus appear that HSCs have different fundamental properties in each study system. However, most transplantation studies have only tracked the lineage output of the transplanted HSC clones, and rarely the regeneration of the HSC compartment itself. In addition, clonal assays have not been performed at sufficient resolution to fully capture the diversity and clonal complexity of the regenerated HSC compartment. Here, we have used expressible barcodes, which can be sequenced in conventional single cell RNAseq assays, to simultaneously record the functional outcomes and transcriptional states of thousands of HSCs. Our analysis revealed multiple clonal HSC behaviors following transplantation that drastically differ in their differentiation activity, lineage-bias and self-renewal. Surprisingly, we witnessed a large fraction of clones that efficiently repopulate the HSC compartment but show limited contribution to differentiated progeny. Furthermore, these inactive clones have increased competitive multilineage serial repopulating capacity, implying that shortly after transplant a subset of clones reestablishes the native-like LT-HSC behaviors. Our results also argue that this clonal distribution of labor is controlled by cell autonomous, heritable properties (i.e. the epigenetic cell state). Then, using only our clonal readouts to segregate single HSC transcriptomes, we unveiled the transcriptional signatures that associated with unique HSC outcomes (platelet bias, clonal expansion, dormancy, etc.) and unraveled, for the first time, a gene signature for functional long-term serially repopulating clones. We interrogated the drivers of this cell state using an in vivo inducible CRISPR screening and identified 5 novel regulators that are required to regenerate the HSC compartment in a cell autonomous fashion. In conclusion, we demonstrate that functional LT-HSCs share more similar properties in native and transplantation hematopoiesis than previously expected. Consequently, we unveil a definition of the essential, common functional properties of HSCs and the molecular programs that control them. Figure 1 Disclosures No relevant conflicts of interest to declare.

Runx1 Is Required for Notch-Induced Specification of Megakaryocyte Development from Early Hematopoietic Stem and Progenitor Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1370-1370
Author(s):  
Melanie G Cornejo ◽  
Thomas Mercher ◽  
Joseph D. Growney ◽  
Jonathan Jesneck ◽  
Ivan Maillard ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Cell Fate ◽  
Stromal Cells ◽  
Gfp Expression ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Insight Into ◽  
Megakaryocyte Differentiation

Abstract The Notch signaling pathway is involved in a broad spectrum of cell fate decisions during development, and in the hematopoietic system, it is known to favor T cell- vs B cell lineage commitment. However, its role in myeloid lineage development is less well understood. We have shown, using heterotypic co-cultures of murine primary hematopoietic stem cells (Lin-Sca-1+ckit+ HSCs) and OP9 stromal cells expressing the Notch ligand Delta1 (OP9-DL1), that Notch signaling derived from cell non-autonomous cues acts as a positive regulator of megakaryocyte fate from LSK cells. Bone marrow transplantation experiments with a constitutively active Notch mutant resulted in enhanced megakaryopoiesis in vivo, with increased MEP numbers and megakaryocyte colony formation. In contrast, expression of dnMAML using a conditional ROSA26 knock-in mouse model significantly impaired megakaryopoiesis in vivo, with a marked decrease in megakaryocyte progenitors. In order to understand the cellular differentiation pathways controlled by Notch, we first examined the ability of various purified progenitor populations to differentiate toward megakaryocytes upon Notch stimulation in vitro. We observed that CMP and MEP, but not GMP, can engage megakaryopoiesis upon Notch stimulation. Our results were consistent with expression analysis of Notch signaling genes in these purified progenitors and were supported by the observation that transgenic Notch reporter mice display higher levels of reporter (i.e. GFP) expression in HSC and MEP, vs. CMP and GMP in vivo. Furthermore, purified progenitors with high GFP expression gave rise to increased numbers of megakarocyte-containing colonies when plated in vitro compared to GFP-negative progenitors. In addition, further purification of the HSC population into long-term (LT), short-term (ST), and lymphoid-primed myeloid progenitors (LMPP) before plating on OP9-DL1 stroma showed that LMPP have a reduced ability to give rise to megakaryocytes compared to the other two populations. These data support the hypothesis that there is an early commitment to erythro/megakaryocytic fate from HSC prior to lymphoid commitment. To gain insight into the molecular mechanism underlying Notch-induced megakaryopoiesis, we performed global gene expression analysis that demonstrated the engagement of a megakaryopoietic transcriptional program when HSC were co-cultured with OP9-DL1 vs. OP9 stroma or OP9-DL1 treated with gamma-secretase inhibitor. Of interest, Runx1 was among the most upregulated genes in HSC co-cultured on OP9-DL1 stroma. To assess whether Notch signaling engages megakaryocytic fate through induction of Runx1, we plated HSC from Runx1 −/− mice on OP9-DL1 stroma. Compared to WT cells, Runx1 −/− HSC had a severely reduced ability to develop into CD41+ cells. In contrast, overexpression of Runx1 in WT HSC was sufficient to induce megakaryocyte fate on OP9 stroma without Notch stimulation. Together, our results indicate that Notch pathway activation induced by stromal cells is an important regulator of cell fate decisions in early progenitors. We show that Notch signaling is upstream of Runx1 during Notch-induced megakaryocyte differentiation and that Runx1 is an essential target of Notch signaling. We believe that these results provide important insight into the pathways controlling megakaryocyte differentiation, and may have important therapeutic potential for megakaryocyte lineage-related disorders.

Sign in / Sign up

Export Citation Format

Share Document