Polyploidization Process Research Articles

Transgressive and subgenome expression level dominance and co-expression network analyses at the early fiber development in allopolyploid Gossypium

Polyploidization can lead to the emergence of new species. Allotetraploid cotton arose through hybridization and whole genome duplication of its diploid A and D genome progenitors. As a prominent global fiber crop, cotton serves as an excellent model organism for exploring plant evolutionary biology and fiber development, particularly in polyploidization. This study utilized transcriptomics to analyze expression difference between diploid and allotetraploid cotton, aiming to further the understanding of transcriptomic dynamics associated at different fiber developmental stages during polyploidization process. In this study, we identified additive and non-additive genes and clarified the contributions of non-additive genes during the tetraploid formation process. Furthermore, we constructed a gene co-expression network, providing new insights into the transcriptional regulation of polyploid cotton fiber development. Among non-additive genes, more A genome-biased expression level dominance (ELD-A) than D genome (ELD-D) occurred in the tetraploids. In Gossypium hirsutum, ELD-A genes played major roles during initial elongation, exerting greater dominance in fiber development. Transgressive genes impacted early fiber development in Gossypium barbadense. While the At subgenome profoundly influences tetraploid fiber development, Dt sub-genome activation, inheritance and sub/neo-functionalization promote superior high-yield traits. By constructing co-expression networks, we identified key fiber developmental candidate genes, providing valuable resources for functional research and breeding perspectives to develop superior cotton varieties.

Impact of Polyploidy Induction for Salinity Stress Mitigation in Soybean (Glycine max L. Merrill)

Polyploidy induction is recognized as one of the major evolutionary processes leading to remarkable morphological, physiological, and genetic variations in plants. Soybean (Glycine max L.), also known as soja bean or soya bean, is an annual leguminous crop of the pea family (Fabaceae) that shares a paleopolypoidy history, dating back to approximately 56.5 million years ago with other leguminous crops such as cowpea and other Glycine specific polyploids. This crop has been documented as one of the polyploid complex species among legumes whose gene evolution and resultant adaptive growth characteristics following induced polyploidization has not been fully explored. Furthermore, no successfully established in vivo or in vitro based polyploidy induction protocols have been reported to date, particularly, with the intention to develop mutant plants showing strong resistance to abiotic salinity stress. This review, therefore, describes the role of synthetic polyploid plant production in soybean for the mitigation of high soil salt stress levels and how this evolving approach could be used to further enhance the nutritional, pharmaceutical and economic industrial value of soybeans. This review also addresses the challenges involved during the polyploidization process.

EloA promotes HEL polyploidization upon PMA stimulation through enhanced ERK1/2 activity

Megakaryocytes (MKs) are the unique non-pathological cells that undergo polyploidization in mammals. The polyploid formation is critical for understanding the MK biology, and transcriptional regulation is involved in the differentiation and maturation of MKs. However, little is known about the functions of transcriptional elongation factors in the MK polyploidization. In this study, we investigated the role of transcription elongation factor EloA in the polyploidy formation during the MK differentiation. We found that EloA was highly expressed in the erythroleukemia cell lines HEL and K562. Knockdown of EloA in HEL cell line was shown to impair the phorbol myristate acetate (PMA) induced polyploidization process, which was used extensively to model megakaryocytic differentiation. Selective over-expression of EloA mutants with Pol II elongation activity partially restored the polyploidization. RNA-sequencing revealed that knockdown of EloA decelerated the transcription of genes enriched in the ERK1/2 cascade pathway. The phosphorylation activity of ERK1/2 decreased upon the EloA inhibition, and the polyploidization process of HEL was hindered when ERK1/2 phosphorylation was inhibited by PD0325901 or SCH772984. This study evidenced a positive role of EloA in HEL polyploidization upon PMA stimulation through enhanced ERK1/2 activity.

Deciphering ploidal levels of Lippia alba by using proteomics

Lippia alba (Mill.) N.E. Brown (Verbenaceae), popularly known as “lemon balm” or “bushy matgrass”, is widely used in folk medicine due to its anti-inflammatory, antispasmodic, analgesic, and digestive properties. It was described as an autopolyploid complex with five cytotypes (2n = 30, 38, 45, 60 and 90). To enhance our understanding of the biological variation of the species, we investigated, comparatively, the proteomic profile of all ploidal levels (diploid, aneuploid, triploid, tetraploid, and hexaploid). Leaf proteins were extracted with subsequent separation by two-dimensional electrophoresis, spot analysis, and protein identification by mass spectrometry. By comparing the proteomic profile of diploid accession to the profile of the other ploidal levels we identified differential expression between the analysed spots. We identified 34 proteins with differential expression between the ploidal levels in comparison with the diploid. The identified proteins seem to play relevant roles in the primary metabolism of L. alba suggesting that a specific set of proteins was selected during the polyploidization process, being the triploid the most different one. Given that protein composition can substantially affect the desired therapeutic effect, we posit that further combination of proteomic and metabolomic studies may help to unravel genetic variations and phenotypic profiles in L. alba.

Cytotype distribution and chloroplast phylogeography of the Actinidia chinensis complex

BackgroundPlant phylogeographic studies of species in subtropical China have mainly focused on rare and endangered species, whereas few studies have been conducted on taxa with relatively wide distribution, especially polyploid species. We investigated the cytotype and haplotype distribution pattern of the Actinidia chinensis complex, a widespread geographically woody liana with variable ploidy in subtropical China comprising two varieties, with three chloroplast fragments DNA (ndhF-rpl132, rps16-trnQ and trnE-trnT). Macroevolutionary, microevolutionary and niche modeling tools were also combined to disentangle the origin and the demographic history of the species or cytotypes.ResultsThe ploidy levels of 3338 individuals from 128 populations sampled throughout the species distribution range were estimated with flow cytometry. The widespread cytotypes were diploids followed by tetraploids and hexaploids, whereas triploids and octoploids occurred in a few populations. Thirty-one chloroplast haplotypes were detected. The genetic diversity and genetic structure were found to be high between varieties (or ploidy races) chinensis and deliciosa. Our results revealed that these two varieties inhabit significantly different climatic niche spaces. Ecological niche models (ENMs) indicate that all varieties’ ranges contracted during the Last Inter Glacial (LIG), and expanded eastward or northward during the Last Glacial Maximum (LGM).ConclusionsPliocene and Plio-Pleistocene climatic fluctuations and vicariance appear to have played key roles in shaping current population structure and historical demography in the A. chinensis complex. The polyploidization process also appears to have played an important role in the historical demography of the complex through improving their adaptability to environmental changes.

Evolutionary Divergence and Biased Expression of NAC Transcription Factors in Hexaploid Bread Wheat (Triticum aestivum L.).

The NAC genes, a large plant-specific family of transcription factors, regulate a wide range of pathways involved in development and response to biotic and abiotic stress. In this study, the NAC transcription factors were identified in 27 green plants, and the results showed that NAC transcription factors in plants undergo an appearance stage from water to land and a number expansion stage from gymnosperm to angiosperm. Investigating the evolutionary process of the NAC transcription factors from diploid species to hexaploid wheat revealed that tandem replications during the polyploidization process is an important event for increasing the number of NAC transcription factors in wheat. Then, the molecular characteristics, phylogenetic relationships, and expression patterns of 462 NAC transcription factors of hexaploid wheat (TaNACs) were analyzed. The protein structure results showed that TaNAC was relatively conservative at the N-terminal that contains five subdomains. All these TaNACs were divided into Group I and Group II by phylogenetic analysis, and the TaNACs in Group I should undergo strong artificial selection based on single nucleotide polymorphism (SNP) analysis. Through genome synteny and phylogenetic analysis, these TaNACs were classified into 88 groups and 9 clusters. The biased expression results of these TaNACs showed that there are 24 groups and 67 groups of neofunctionalization genes under biotic and abiotic stress, respectively, and 16 groups and 59 groups of subfunctionalization genes. This shows that neofunctionalization plays an important role in coping with different stresses. Our study provides new insights into the evolution of NAC transcription factors in hexaploid wheat.

Polyploidization is accompanied by synonymous codon usage bias in the chloroplast genomes of both cotton and wheat.

Synonymous codon usage bias (SCUB) of both nuclear and organellar genes can mirror the evolutionary specialization of plants. The polyploidization process exposes the nucleus to genomic shock, a syndrome which promotes, among other genetic variants, SCUB. Its effect on organellar genes has not, however, been widely addressed. The present analysis targeted the chloroplast genomes of two leading polyploid crop species, namely cotton and bread wheat. The frequency of codons in the chloroplast genomes ending in either adenosine (NNA) or thymine (NNT) proved to be higher than those ending in either guanidine or cytosine (NNG or NNC), and this difference was conserved when comparisons were made between polyploid and diploid forms in both the cotton and wheat taxa. Preference for NNA/T codons was heterogeneous among genes with various numbers of introns and was also differential among the exons. SCUB patterns distinguished tetraploid cotton from its diploid progenitor species, as well as bread wheat from its diploid/tetraploid progenitor species, indicating that SCUB in the chloroplast genome partially mirrors the formation of polyploidies.

Genome-Wide Identification of the Gossypium hirsutum NHX Genes Reveals that the Endosomal-Type GhNHX4A is Critical for the Salt Tolerance of Cotton.

Soil salinization, which is primarily due to excessive Na+ levels, is a major abiotic stress adversely affecting plant growth and development. The Na+/H+ antiporter (NHX) is a transmembrane protein mediating the transport of Na+ or K+ and H+ across the membrane to modulate the ionic balance of plants in response to salt stress. Research regarding NHXs has mainly focused on the vacuolar-type NHX family members. However, the biological functions of the endosomal-type NHXs remain relatively uncharacterized. In this study, 22 NHX family members were identified in Gossypium hirsutum. A phylogenetic analysis divided the GhNHX genes into two categories, with 18 and 4 in the vacuolar and endosomal groups, respectively. The chromosomal distribution of the NHX genes revealed the significant impact of genome-wide duplication during the polyploidization process on the number of GhNHX genes. Analyses of gene structures and conserved motifs indicated that GhNHX genes in the same phylogenetic cluster are conserved. Additionally, the salt-induced expression patterns confirmed that the expression levels of most of the GhNHX genes are affected by salinity. Specifically, in the endosomal group, GhNHX4A expression was substantially up-regulated by salt stress. A yeast functional complementation test proved that GhNHX4A can partially restore the salt tolerance of the salt-sensitive yeast mutant AXT3. Silencing GhNHX4A expression decreased the resistance of cotton to salt stress because of an increase in the accumulation of Na+ in stems and a decrease in the accumulation of K+ in roots. The results of this study may provide the basis for an in-depth characterization of the regulatory functions of NHX genes related to cotton salt tolerance, especially the endosomal-type GhNHX4A. Furthermore, the presented data may be useful for selecting appropriate candidate genes for the breeding of new salt-tolerant cotton varieties.

Flag leaf vein traits and their correlation with photosynthesis and grain yield in wheat genotypes of differing ploidy

Leaf venation and coupled physiological function of wild plants co-evolve during the natural selection. How artificial selection affects leaf vein traits and coordinated physiological functions of main crops are largely unknown. This study examined the changes of leaf vein traits and their correlation with gas exchange of flag leaves and yield in eight wheat genotypes of differing ploidy under the same growing conditions. The results indicate that flag leaf vein density (VLA), major-vein density (VLAmajor), and minor-vein density (VLAminor) decreased whereas the proportion of minor-vein length and interveinal distance between small longitudinal veins (IVD) increased during the polyploidization process, and the major advance occurred from the period from diploids to tetraploids. The VLA, VLAmajor, and VLAminor were closely coordinated with maximum net photosynthetic rate (PN) and photosynthetic N use efficiency (PNUE), but not with stomatal conductance. The proportion of minor-vein length and IVD were negatively related with PN and PNUE but positively related with N content per area (Narea) during wheat evolution. A higher proportion of minor-vein length and IVD, and a lower VLAmajor in flag leaves along with a larger Narea were largely responsible for the increased yield in modern cultivars. The decreased redundancy of leaf vein density and increased minor-vein proportion in modern cultivars can confer a yield advantage during wheat evolution.

Induction of Synthetic Polyploids and Assessment of Genomic Stability in Lippia alba.

Polyploidy is widely recognized as a major evolutionary force in plants and has been reported in the genus Lippia (Verbenaceae). Lippia alba, the most studied species, has been documented as a polyploid complex involving at least four ploidal levels. L. alba presents remarkable chemical and genetic variation and represents a model for understanding genome organization. Although the economic and medicinal importance of the species has been widely described, no established polyploid induction protocol has been reported so far. Here, we describe the production of synthetic polyploid plants of L. alba using colchicine. The ploidal levels were estimated by flow cytometry and chromosome counting. In addition, FISH and molecular markers approaches were used to confirm the stability of the synthetic polyploids. The major component of the essential oils was estimated by GCMS to compare with the natural individuals. Tetraploids and triploids were produced providing new opportunities for investigating medicinal, pharmacological, and economic applications as well as addressing intrinsic questions involved in the polyploidization process in tropical plants.

Effect of polyploidy on the leaf epidermis structure of Cynodon dactylon (L.) Pers. (Poaceae)

Cynodon dactylon has different ploidy levels. The ‘gigas effect’ in morphoanatomic traits caused by the increase in the genetic material is poorly studied in the species. Thus, the present research aimed at carrying out a quantitative and descriptive study of the leaf epidermis structure in PI291169–04 (2n = 2× = 18); PI477004–26 (2n = 3× = 27); PI291721–22 (2n = 4× = 36); PI289750–10 (2n = 5× = 45) accessions, in order to investigate the consequences of the polyploidy. Adult leaves were fixed in FAA and the paradermal sections obtained through the scraping technique were stained in 5% safranin. The measurements of the stomata characteristics (polar diameters – PD, equatorial diameters – QD, stomatal density – SD, stomatal functionality – SF, and stomatal index – SI) were taken by means of the software ImageJ. For analysis in scanning electron microscopy, leaf fragments were fixed in Karnovsky, dehydrated in an acetone series, subjected to a critical point and covered with gold. Stomata were classified as paracytic, dumbbell-shaped and partially covered by papillae. Tector trichomes were classified as uniseriate with pointed tips. In general, as ploidy level increased, there was an increase in stomata dimension and a decrease in SD. Diploid and triploid accessions were easily differentiated in relation to PD and QD, which makes it an indirect method for determining the variation in ploidy level. The variables SD and SI were not efficient to discriminate the accessions. Leaf epidermis has characteristics that emphasize the effect of polyploidization process and its analysis may be used to screening of genotypes in breeding programs.

Aryl Hydrocarbon Receptor Promotes Liver Polyploidization and Inhibits PI3K, ERK, and Wnt/β-Catenin Signaling

SummaryAryl hydrocarbon receptor (AhR) deficiency alters tissue homeostasis. However, how AhR regulates organ maturation and differentiation remains mostly unknown. Liver differentiation entails a polyploidization process fundamental for cell growth, metabolism, and stress responses. Here, we report that AhR regulates polyploidization during the preweaning-to-adult mouse liver maturation. Preweaning AhR-null (AhR−/−) livers had smaller hepatocytes, hypercellularity, altered cell cycle regulation, and enhanced proliferation. Those phenotypes persisted in adult AhR−/− mice and correlated with compromised polyploidy, predominance of diploid hepatocytes, and enlarged centrosomes. Phosphatidylinositol-3-phosphate kinase (PI3K), extracellular signal-regulated kinase (ERK), and Wnt/β-catenin signaling remained upregulated from preweaning to adult AhR-null liver, likely increasing mammalian target of rapamycin (mTOR) activation. Metabolomics revealed the deregulation of mitochondrial oxidative phosphorylation intermediates succinate and fumarate in AhR−/− liver. Consistently, PI3K, ERK, and Wnt/β-catenin inhibition partially rescued polyploidy in AhR−/− mice. Thus, AhR may integrate survival, proliferation, and metabolism for liver polyploidization. Since tumor cells tend to be polyploid, AhR modulation could have therapeutic value in the liver.

Stability in chromosome number and DNA content in synthetic tetraploids of Lolium multiflorum after two generations of selection

ABSTRACT: Chromosome doubling of Italian ryegrass genotypes ( Lolium multiflorum Lam.) adapted to the brazilian edaphoclimatic conditions is an important strategy used by breeders and aims to obtain more vigorous genotypes with better forage quality and disease resistance. The effectiveness of chromosome doubling can be measured by genetic stability and fertility rates of plants over generations. However, a common problem in the polyploidization process is the regeneration of mixoploid plants that have impaired fertility and genetic stability. The objective of this study was to verify if progenies of recently tetraploidized plants remain stable regarding DNA content and chromosome number, over two generations. Progenies of L. multiflorum plants artificially tetraploidized with colchicine treatment were evaluated. Chromosome counting and estimates of the DNA content were used to evaluate the genetic stability. The percentage of tetraploid plants (4X) increased over generations (18%, 34% and 91% in cycle 0, 1 and 2, respectively). All progenies identified as tetraploid by flow citometry showed variation in chromosome number (mixoploidy), but produced viable seeds. Results showed that stabilization in chromosome number and DNA content in tetraploidized plant progenies requires time and that the success of this procedure depends on a continuous and accurate screening and selection.

Formation of diploid and triploid hybrid groupers (hybridization of Epinephelus coioides ♀ × Epinephelus lanceolatus ♂) and their 5S gene analysis.

BackgroundInterspecies hybridization is widely used to achieve heterosis or hybrid vigor, which has been observed and harnessed by breeders for centuries. Natural allopolyploid hybrids generally exhibit more superior heterosis than both the diploid progenies and their parental species. However, polyploid formation processes have been long ignored, the genetic basis of heterosis in polyploids remains elusive.ResultsIn the present study, triploid hybrids had been demonstrated to contain two sets of chromosomes from mother species and one set from father species. Cellular polyploidization process in the embryos had been traced. The triploid hybrids might be formed by failure formation of the second polarized genome during the second meiosis stage. Four spindle centers were observed in anaphase stage of the first cell division. Three spindle centers were observed in side of cell plate after the first cell division.The 5S rDNA genes of four types of groupers were cloned and analyzed. The diploid and triploid hybrids had been proved to contain the tandem chimera structures which were recombined by maternal and paternal monomer units. The results indicated that genome re-fusion had occurred in the hybrid progenies.To further elucidate the genetic patterns of diploid and triploid hybrids, fluorescence chromosome location had been carried out, maternal 5S gene (M-386) were used as the probe. The triploid hybrids contained fewer fluorescence loci numbers than the maternal species. The results indicated that participation of paternal 5S gene in the triploid hybrid genome had degraded the match rates of M-386 probe.ConclusionsOur study is the first to investigate the cellular formation processes of natural allopolyploids in hybrid fish, the cellular polyploidization process may be caused by failure formation of the second polarized genome during the meiosis, and our results will provide the molecular basis of hybrid vigor in interspecies hybridization.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-016-0443-9) contains supplementary material, which is available to authorized users.

Landfill leachate sludge use as soil additive prior and after electrocoagulation treatment: A cytological assessment using CHO-k1 cells

Electrocoagulation has recently attracted attention as a potential technique for treating toxic effluents due to its versatility and environmental compatibility, generating a residue chemically suitable to be used as a soil additive. In the present study, landfill leachate sludge hazardous effects were investigated prior and after electrocoagulation process using in vitro assays with the mammalian cells CHO-k1. An integrated strategy for risk assessment was used to correctly estimate the possible adverse landfill leachate sludge effects on human health and ecosystem. Electrocoagulation process proved to be an effective treatment due to possibility to improve effluent adverse characteristics and produce sludge with potential to be used as soil additive. Despite low cytoxicity, the residue presented genotoxic and mutagenic effects, indicating a capacity to induce genetic damages, probably due to induction of polyploidization process in cells. The observed effects demand an improvement of waste management methods for reduce negative risks of landfill leachate sludge application.

Identification of the Human Unipotent Megakaryocyte Progenitor, and Its Pathophysiological Roles in Human Thrombopoietic Disorders

In human hematopoiesis, the megakaryocyte (Meg) lineage is known to diverge from bipotent megakaryocytic/erythroid progenitor (MEP), which resides downstream of common myeloid progenitor (CMP). However, the definition of unipotent Meg progenitor (MegP) is still controversial.To shed light on the heterogeneity of hematopoietic stem/progenitor cells (HSPCs), we analyzed the expression level of 96 lineage-specific genes in a single cell. While most of MEPs exclusively expressed erythroid- but not Meg-specific genes, a fraction of CMP cells (6.25%) showed distinct and exclusive expression pattern of platelet or Meg-specific genes. Among them were the cell surface molecules including CD41, the contents of alpha-granules of platelets and Meg-lineage specific transcription factors. Based on these data, we hypothesized that MegP might exist within the CD41+ CMP fraction.In fact, a fraction (7.9 ± 3.2 %) of CMPs expressed significant level of CD41 in FACS analysis, but none of the other CD34+ cells including hematopoietic stem cells (HSCs), MEPs and granulocyte-macrophage progenitors (GMPs) expressed CD41. Purified CD41+CMPs exhibited myeloblast-like immature morphological characteristics. Interestingly, cells with 4N-nuclei were exclusively found in CD41+CMPs (1%), indicating the initiation of endomitosis and polyploidization process has already taken place at this early stage of hematopoiesis.A conventional in vitro culture condition revealed that CD41+CMPs completely lacked erythroid or granulocyte/monocyte (GM) lineage potentials, whereas CD41-CMPs generated all types of myeloid colonies. To analyze Meg-lineage potential, we cultured them at a serum-free culture condition that is highly-optimized for Meg differentiation. The majority of single CD41+CMPs gave rise to pure megakaryocyte colonies.In addition, the novel immunodeficient mouse strain, which was recently developed by us and could support human Meg-lineage hematopoiesis much more than any strains available today, enabled us to evaluate in vivo differentiation potential of lineage-restricted progenitors. As a result, CD41+CMPs showed unipotent and robust Meg-lineage differentiation potential compared to MEPs.In order to elucidate gene expression profiling of CD41+CMPs more precisely, we performed cDNA microarray experiment. GSEA (Gene Set Enrichment Analysis) revealed that CD41+CMPs up-regulated Meg lineage-specific genes related to megakaryocyte development, platelet production, platelet activation and aggregation, and down-regulated erythroid and GM lineage-affiliated genes, clearly reflecting their lineage potentials.We also evaluated lineal relationship of these populations in vitro. While MEP did not give rise to CD41+CMPs, CD41-CMPs successfully generated MegPs, suggesting that CD41+CMPs could be the progeny of CD41-CMPs.Therefore, we defined the CD41+CMP as a human MegP. The Meg colony-producing potential of MegPs was almost thirty times as strong as that of MEPs on per-cell-basis, indicating that this population is the major source of Megs rather than MEP.Finally, to evaluate the pathophysiological significance of MegPs in human diseases, we analyzed population frequencies of each stem/progenitor population in essential thrombocythemia (ET) patients' bone marrow. The MegP pool dramatically expanded in ET BM as compared to that in normal BM (24.2 ± 8.6 % vs. 7.9 ± 3.2 % of CMP). We also evaluated JAK2 mutation (V617F) burden in each stem/progenitor population by utilizing the digital PCR method with allele specific probes and the amplicon sequencing-based detection. As a result, JAK2 mutation is highly concentrated to MegP population in accordance with expansion of MegP, suggesting that this population should play an important role in pathogenesis of ET.In summary, we have prospectively identified the unipotent MegP population, which was endowed with robust Meg potential and emerged directly from CMPs bypassing MEPs. The newly-identified MegPs robustly contribute to physiological and pathological human megakaryopoiesis including ET. DisclosuresNo relevant conflicts of interest to declare.

Identification of the human unipotent megakaryocyte progenitor, and its pathophysiological roles in human thrombopoietic disorders

![Graphic][1] In human hematopoiesis, the megakaryocyte (Meg) lineage is known to diverge from bipotent megakaryocytic/erythroid progenitor (MEP; CD34+ CD38+ IL3Ra- CD45RA-), which resides downstream of common myeloid progenitor (CMP; CD34+ CD38+ IL3Ra+ CD45RA-). However, the definition of unipotent Meg progenitor (MegP) is still controversial and, in the hierarchical map, where Meg potential mainly resides remains unclear. Here, we report prospective isolation of unipotent MegP, which is endowed with much more robust Meg potential than MEP, and emerges directly from CMP bypassing MEP. Because CD41 is known as a Meg lineage-specific marker, we first searched for the CD41-expressing cell population in the CD34 positive stem/progenitor fraction of healthy individuals. A fraction (7.9 ± 3.2 %) of CMPs expressed CD41, but none of the other CD34+ subpopulation including hematopoietic stem cells (HSCs; CD34+ CD38- CD45RA-), MEPs and granulocyte-macrophage progenitors (GMPs; CD34+ CD38+ IL3Ra+ CD45RA+) expressed significant levels of CD41. Purified CD41+ CMPs retained myeloblast-like immature morphological characteristics with large and round nucleus and basophilic cytoplasm, closely resembling those of CD41- CMPs and more immature HSCs. Interestingly, cells with immature 4N-nuclei were exclusively found in CD41+ CMPs (about 1%), indicating that a minor fraction of this population is initiating endomitosis and polyploidization process, an important characteristic of megakaryoblasts. A conventional In vitro culture condition with fetal bovine serum and a myeloerythroid cytokine cocktail revealed that CD41+ CMPs completely lacked erythroid or granulocyte/monocyte (GM) lineage potentials, whereas CD41- CMPs generated all types of myeloid colonies. These unexpected observations led us to hypothesize that CD41+ CMPs were committed to the Meg lineage. We then cultured them at a serum-free culture condition that is highly-optimized for Meg differentiation. In this condition, the majority of single CD41+ CMPs gave rise to pure megakaryocyte colonies, without forming any other myeloerythroid colonies. In addition, in cDNA microarray analysis, CD41+ CMPs possessed a distinct gene expression profile with up-regulation of Meg lineage-specific genes related to megakaryocyte development, platelet production, platelet activation and aggregation, and down-regulation of erythroid and GM lineage-affiliated genes, reflecting their lineage potential. Therefore, we defined the CD41+ CMP as a human MegP. The Meg colony-producing potential of MegPs was almost thirty­­ times as strong as that of MEPs on per-cell-basis (672 ± 232 vs. 20 ± 6.2 colonies per 1000 cells) and MegP-derived Meg colonies accounted for more than ninety percent of those from whole CD34+ CD38+ progenitor populations, indicating that this population is the major source of megakaryocytes and platelets production rather than MEP. The MegP population was found in 7 day culture of CD41- CMPs but not of MEPs, suggesting that MegPs are downstream of CMPs, but are independent of MEP. More importantly, MegP pool dramatically expanded in essential thrombocythemia (ET) patients’ bone marrow, constituting three times larger part of CMP than that in normal BM (24.2 ± 8.6 % vs. 7.9 ± 3.2 % of CMP). We are currently further analyzing ET patients’ bone marrow to obtain data regarding V617F JAK2 mutation frequency, for example. Thus, the newly-identified MegP robustly contributes to physiological and pathological human megakaryopoiesis, thus could be a therapeutic target in various thrombopoietic disorders. Disclosures No relevant conflicts of interest to declare. [1]: /embed/inline-graphic-2.gif

Oxidative stress promotes pathologic polyploidization in nonalcoholic fatty liver disease.

Polyploidization is one of the most dramatic changes that can occur in the genome. In the liver, physiological polyploidization events occur during both liver development and throughout adult life. Here, we determined that a pathological polyploidization takes place in nonalcoholic fatty liver disease (NAFLD), a widespread hepatic metabolic disorder that is believed to be a risk factor for hepatocellular carcinoma (HCC). In murine models of NAFLD, the parenchyma of fatty livers displayed alterations of the polyploidization process, including the presence of a large proportion of highly polyploid mononuclear cells, which are rarely observed in normal hepatic parenchyma. Biopsies from patients with nonalcoholic steatohepatitis (NASH) revealed the presence of alterations in hepatocyte ploidy compared with tissue from control individuals. Hepatocytes from NAFLD mice revealed that progression through the S/G2 phases of the cell cycle was inefficient. This alteration was associated with activation of a G2/M DNA damage checkpoint, which prevented activation of the cyclin B1/CDK1 complex. Furthermore, we determined that oxidative stress promotes the appearance of highly polyploid cells, and antioxidant-treated NAFLD hepatocytes resumed normal cell division and returned to a physiological state of polyploidy. Collectively, these findings indicate that oxidative stress promotes pathological polyploidization and suggest that this is an early event in NAFLD that may contribute to HCC development.

Identification of the Human Unipotent Megakaryocyte Progenitor, and Its Pathophysiological Roles in Human Thrombopoietic Disorders

▪In human hematopoiesis, the megakaryocyte (Meg) lineage is known to diverge from bipotent megakaryocytic/erythroid progenitor (MEP; CD34+ CD38+ IL3Ra- CD45RA-), which resides downstream of common myeloid progenitor (CMP; CD34+ CD38+ IL3Ra+ CD45RA-). However, the definition of unipotent Meg progenitor (MegP) is still controversial and, in the hierarchical map, where Meg potential mainly resides remains unclear. Here, we report prospective isolation of unipotent MegP, which is endowed with much more robust Meg potential than MEP, and emerges directly from CMP bypassing MEP.Because CD41 is known as a Meg lineage-specific marker, we first searched for the CD41-expressing cell population in the CD34 positive stem/progenitor fraction of healthy individuals. A fraction (7.9 ± 3.2 %) of CMPs expressed CD41, but none of the other CD34+ subpopulation including hematopoietic stem cells (HSCs; CD34+ CD38- CD45RA-), MEPs and granulocyte-macrophage progenitors (GMPs; CD34+ CD38+ IL3Ra+ CD45RA+) expressed significant levels of CD41. Purified CD41+ CMPs retained myeloblast-like immature morphological characteristics with large and round nucleus and basophilic cytoplasm, closely resembling those of CD41- CMPs and more immature HSCs. Interestingly, cells with immature 4N-nuclei were exclusively found in CD41+ CMPs (about 1%), indicating that a minor fraction of this population is initiating endomitosis and polyploidization process, an important characteristic of megakaryoblasts. A conventional In vitro culture condition with fetal bovine serum and a myeloerythroid cytokine cocktail revealed that CD41+ CMPs completely lacked erythroid or granulocyte/monocyte (GM) lineage potentials, whereas CD41- CMPs generated all types of myeloid colonies. These unexpected observations led us to hypothesize that CD41+ CMPs were committed to the Meg lineage. We then cultured them at a serum-free culture condition that is highly-optimized for Meg differentiation. In this condition, the majority of single CD41+ CMPs gave rise to pure megakaryocyte colonies, without forming any other myeloerythroid colonies. In addition, in cDNA microarray analysis, CD41+ CMPs possessed a distinct gene expression profile with up-regulation of Meg lineage-specific genes related to megakaryocyte development, platelet production, platelet activation and aggregation, and down-regulation of erythroid and GM lineage-affiliated genes, reflecting their lineage potential. Therefore, we defined the CD41+ CMP as a human MegP.The Meg colony-producing potential of MegPs was almost thirty­­ times as strong as that of MEPs on per-cell-basis (672 ± 232 vs. 20 ± 6.2 colonies per 1000 cells) and MegP-derived Meg colonies accounted for more than ninety percent of those from whole CD34+ CD38+ progenitor populations, indicating that this population is the major source of megakaryocytes and platelets production rather than MEP. The MegP population was found in 7 day culture of CD41- CMPs but not of MEPs, suggesting that MegPs are downstream of CMPs, but are independent of MEP. More importantly, MegP pool dramatically expanded in essential thrombocythemia (ET) patients’ bone marrow, constituting three times larger part of CMP than that in normal BM (24.2 ± 8.6 % vs. 7.9 ± 3.2 % of CMP). We are currently further analyzing ET patients’ bone marrow to obtain data regarding V617F JAK2 mutation frequency, for example. Thus, the newly-identified MegP robustly contributes to physiological and pathological human megakaryopoiesis, thus could be a therapeutic target in various thrombopoietic disorders. DisclosuresNo relevant conflicts of interest to declare.

Development and characterization of nine microsatellite loci for Sisyrinchium micranthum (Iridaceae)

Sisyrinchium micranthum is an herb with wide distribution and morphological variation, presenting three known cytotypes (2x, 4x, and 6x), which is naturally occurring from Mexico to Argentina. The development of microsatellite markers for this species is important to provide molecular tools to investigate polyploidy origin, as well as its relationship with phenotypic and genetic variability. • Nine microsatellite markers were isolated using an enriched genomic library and characterized in a diploid population. Eight markers generated polymorphic loci in 27 plants of a population from southern Brazil. The number of alleles per locus ranged from two to nine, and the observed and expected heterozygosities varied from 0.192 to 0.741 and from 0.370 to 0.884, respectively. • These species-specific DNA markers may be useful for further investigations of the involved polyploidization process and the population genetics of S. micranthum.