Branching Lineages Research Articles

Neofunctionalization of Androgen Receptor by Gain-of-Function Mutations in Teleost Fish Lineage.

Steroid hormone receptor family provides an example of evolution of diverse transcription factors through whole-genome duplication (WGD). However, little is known about how their functions have been evolved after the duplication. Teleosts present a good model to investigate an accurate evolutionary history of protein function after WGD, because a teleost-specific WGD (TSGD) resulted in a variety of duplicated genes in modern fishes. This study focused on the evolution of androgen receptor (AR) gene, as two distinct paralogs, ARα and ARβ, have evolved in teleost lineage after TSGD. ARα showed a unique intracellular localization with a higher transactivation response than that of ARβ. Using site-directed mutagenesis and computational prediction of protein-ligand interactions, we identified two key substitutions generating a new functionality of euteleost ARα. The substitution in the hinge region contributes to the unique intracellular localization of ARα. The substitution on helices 10/11 in the ligand-binding domain possibly modulates hydrogen bonds that stabilize the receptor-ligand complex leading to the higher transactivation response of ARα. These substitutions were conserved in Acanthomorpha (spiny-rayed fish) ARαs, but not in an earlier branching lineage among teleosts, Japanese eel. Insertion of these substitutions into ARs from Japanese eel recapitulates the evolutionary novelty of euteleost ARα. These findings together indicate that the substitutions generating a new functionality of teleost ARα were fixed in teleost genome after the divergence of the Elopomorpha lineage. Our findings provide a molecular explanation for an adaptation process leading to generation of the hyperactive AR subtype after TSGD.

Evolution of mitochondrial gene order in Annelida

Annelida is a highly diverse animal group with over 21,000 described species. As part of Lophotrochozoa, the vast majority of annelids are currently classified into two groups: Errantia and Sedentaria, together forming Pleistoannelida. Besides these taxa, Sipuncula, Amphinomidae, Chaetopteridae, Oweniidae and Magelonidae can be found branching at the base of the tree. Comparisons of mitochondrial genomes have been used to investigate phylogenetic relationship within animal taxa. Complete annelid mitochondrial genomes are available for some Sedentaria and Errantia and in most cases exhibit a highly conserved gene order. Only two complete genomes have been published from the basal branching lineages and these are restricted to Sipuncula. We describe the first complete mitochondrial genome sequences for all other basal branching annelid families: Owenia fusiformis (Oweniidae), Magelona mirabilis (Magelonidae), Eurythoe complanata (Amphinomidae), Chaetopterus variopedatus and Phyllochaetopterus sp. (Chaetopteridae). The mitochondrial gene order of all these taxa is substantially different from the pattern found in Pleistoannelida. Additionally, we report the first mitochondrial genomes in Annelida that encode genes on both strands. Our findings demonstrate that the supposedly highly conserved mitochondrial gene order suggested for Annelida is restricted to Pleistoannelida, representing the ground pattern of this group. All investigated basal branching annelid taxa show a completely different arrangement of genes than observed in Pleistoannelida. The gene order of protein coding and ribosomal genes in Magelona mirabilis differs only in two transposition events from a putative lophotrochozoan ground pattern and might be the closest to an ancestral annelid pattern. The mitochondrial genomes of Myzostomida show the conserved pattern of Pleistoannelida, thereby supporting their inclusion in this taxon.

Origin and intra-island diversification of Sulawesi endemic Adrianichthyidae

Although the family Adrianichthyidae is broadly distributed throughout East and Southeast Asia, 19 endemic species, over half of the family, are distributed in Sulawesi, which is an island in Wallacea. However, it remains unclear how this Adrianichthyidae biodiversity hotspot was shaped. In this study, we reconstructed molecular phylogenies for the Sulawesi adrianichthyids and estimated the divergence times of major lineages to infer the detailed history of their origin and subsequent intra-island diversification. The mitochondrial and nuclear phylogenies revealed that Sulawesi adrianichthyids are monophyletic, which indicates that they diverged from a single common ancestor. Species in the earliest branching lineages are currently distributed in the central and southeastern parts of the island, indicating that the common ancestor colonized Sula Spur, which is a large promontory that projects from the Australian continental margin, from Asia by oversea dispersal c.a. 20Mya. The first diversification event on Sulawesi, the split of the genus Adrianichthys, occurred c.a. 16Mya, and resulted in the nesting of Adrianichthys within Oryzias. Strong geographic structure was evident in the phylogeny; many species in the lineages branching off early are riverine and widely distributed in the southeastern and southwestern arms of Sulawesi, which suggests that oversea dispersal between tectonic subdivisions of this island during the late Miocene (7–5Mya) contributed to the distributions and diversification of the early branching lineages. In contrast, most species in the lineages branched off later are endemic to a single tectonic lake or lake system in the central Sulawesi, suggesting that habitat fragmentation due to the Pliocene collisions (c.a. 4Mya) among the tectonic subdivisions was the primary factor for diversification of the late branching, lacustrine lineages. Adrianichthys and some Oryzias in a certain late branching lineage are sympatric in Lake Poso, which indicates multiple colonizations of these distinct lineages into this tectonic lake. Thus, the diversification of Sulawesi adrianichthyids largely reflects the complex geological history of this island.

Using Whole Genome Analysis to Examine Recombination across Diverse Sequence Types of Staphylococcus aureus.

Staphylococcus aureus is an important clinical pathogen worldwide and understanding this organism's phylogeny and, in particular, the role of recombination, is important both to understand the overall spread of virulent lineages and to characterize outbreaks. To further elucidate the phylogeny of S. aureus, 35 diverse strains were sequenced using whole genome sequencing. In addition, 29 publicly available whole genome sequences were included to create a single nucleotide polymorphism (SNP)-based phylogenetic tree encompassing 11 distinct lineages. All strains of a particular sequence type fell into the same clade with clear groupings of the major clonal complexes of CC8, CC5, CC30, CC45 and CC1. Using a novel analysis method, we plotted the homoplasy density and SNP density across the whole genome and found evidence of recombination throughout the entire chromosome, but when we examined individual clonal lineages we found very little recombination. However, when we analyzed three branches of multiple lineages, we saw intermediate and differing levels of recombination between them. These data demonstrate that in S. aureus, recombination occurs across major lineages that subsequently expand in a clonal manner. Estimated mutation rates for the CC8 and CC5 lineages were different from each other. While the CC8 lineage rate was similar to previous studies, the CC5 lineage was 100-fold greater. Fifty known virulence genes were screened in all genomes in silico to determine their distribution across major clades. Thirty-three genes were present variably across clades, most of which were not constrained by ancestry, indicating horizontal gene transfer or gene loss.

Evolutionary Trends of the Transposase-Encoding Open Reading Frames A and B (orfA and orfB) of the Mycobacterial IS6110 Insertion Sequence.

BackgroundThe IS6110 insertion sequence, a member of the IS3 family of insertion sequences, was found to be specific to the Mycobacterium tuberculosis complex (MTBC). Although IS6110 has been extensively characterized as a transposable genetic marker, the evolutionary history of its own transposase-encoding sequence has not, to the best of our knowledge, been investigated.Methodology/Principal FindingsHere we explored the evolution of the IS6110 sequence by analysing the genetic variability and the selective forces acting on its transposase-encoding open reading frames (ORFs) A and B (orfA and orfB). For this purpose, we used a strain collection consisting of smooth tubercle bacilli (STB), an early branching lineage of the MTBC, and present-day M. tuberculosis strains representing the full breadth of genetic diversity in Tunisia. In each ORF, we found a major haplotype that dominated over a flat distribution of rare descendent haplotypes, consisting mainly of single- and double-nucleotide variant singletons. The predominant haplotypes consisted of both ancestral and present-day strains, suggesting that IS6110 acquisition predated the emergence of the MTBC. There was no evidence of recombination and both ORFs were subjected to strict purifying selection, as demonstrated by their dN/dS ratios (0.29 and 0.51, respectively), as well as their significantly negative Tajima’s D statistics. Strikingly, the purifying selection acting on orfA proved much more stringent, suggesting its critical role in regulating the transpositional process. Maximum likelihood analyses further excluded any possibility of positive selection acting on single amino acid residues.Conclusions/SignificanceTaken together our data fit with an evolutionary scenario according to which the observed variability pattern of the IS6110 transposase-encoding ORFs is generated mainly through random point mutations that accrued on a functionally optimal IS6110 copy, whose acquisition predated the emergence of the MTBC complex. Background selection acting against deleterious mutations led to an excess of low-frequency variants.

Cell lineage visualisation

AbstractCell lineages describe the developmental history of Cell populations and are produced by combining time‐lapse imaging and image processing. Biomedical researchers study Cell lineages to understand fundamental processes such as Cell differentiation and the pharmacodynamic action of anticancer agents. Yet, the interpretation of Cell lineages is hindered by their complexity and insufficient capacity for visual analysis. We present a novel approach for interactive visualisation of Cell lineages. Based on an understanding of Cellular biology and live‐Cell imaging methodology, we identify three requirements: multimodality (Cell lineages combine spatial, temporal, and other properties), symmetry (related to lineage branching structure), and synchrony (related to temporal alignment of Cellular events). We address these by combining visual summaries of the spatiotemporal behaviour of an arbitrary number of lineages, including variation from average behaviour, with node‐link representations that emphasise the presence or absence of symmetry and synchrony. We illustrate the merit of our approach by presenting a real‐world case study where the cytotoxic action of the anticancer drug topotecan was determined.

Diffusion maps for high-dimensional single-cell analysis of differentiation data.

Single-cell technologies have recently gained popularity in cellular differentiation studies regarding their ability to resolve potential heterogeneities in cell populations. Analyzing such high-dimensional single-cell data has its own statistical and computational challenges. Popular multivariate approaches are based on data normalization, followed by dimension reduction and clustering to identify subgroups. However, in the case of cellular differentiation, we would not expect clear clusters to be present but instead expect the cells to follow continuous branching lineages. Here, we propose the use of diffusion maps to deal with the problem of defining differentiation trajectories. We adapt this method to single-cell data by adequate choice of kernel width and inclusion of uncertainties or missing measurement values, which enables the establishment of a pseudotemporal ordering of single cells in a high-dimensional gene expression space. We expect this output to reflect cell differentiation trajectories, where the data originates from intrinsic diffusion-like dynamics. Starting from a pluripotent stage, cells move smoothly within the transcriptional landscape towards more differentiated states with some stochasticity along their path. We demonstrate the robustness of our method with respect to extrinsic noise (e.g. measurement noise) and sampling density heterogeneities on simulated toy data as well as two single-cell quantitative polymerase chain reaction datasets (i.e. mouse haematopoietic stem cells and mouse embryonic stem cells) and an RNA-Seq data of human pre-implantation embryos. We show that diffusion maps perform considerably better than Principal Component Analysis and are advantageous over other techniques for non-linear dimension reduction such as t-distributed Stochastic Neighbour Embedding for preserving the global structures and pseudotemporal ordering of cells. The Matlab implementation of diffusion maps for single-cell data is available at https://www.helmholtz-muenchen.de/icb/single-cell-diffusion-map. fbuettner.phys@gmail.com, fabian.theis@helmholtz-muenchen.de Supplementary data are available at Bioinformatics online.

FP563MICRORNA REGULATION OF MACROPHAGE PHENOTYPE IN PERITONEAL FIBROSIS

Introduction and Aims: Peritoneal fibrosis is a major cause of treatment failure in peritoneal dialysis, which is linked to repeat episodes of infection-driven peritoneal inflammation. Macrophages are a heterogeneous population of immune cells essential for immune surveillance, response to infection and the resolution of inflammation. Distinct subpopulations of macrophages are implicated in tissue repair and fibrosis. Recent data from our group defines distinct origin, function and transcriptional control of discrete macrophage subsets within the peritoneal cavity, namely tissue resident (homeostatic) and inflammatory. microRNAs are post-transcriptional regulators of gene expression that play fundamental roles in regulation of cellular phenotype. However, the role of microRNAs has not been examined in this context. Aim: To investigate microRNA regulation of macrophage phenotype in the context of peritoneal fibrosis. Methods: An in vivo study of microRNA regulation of tissue resident and inflammatory macrophage phenotype in an established murine model of peritoneal fibrosis. Results: A single episode of acute peritoneal inflammation is self-limiting with restoration of tissue homeostasis, whereas repeat episodes initiate the development of fibrosis. In response to acute inflammation two distinct macrophage lineage branches are evident, M2-like tissue resident macrophages (F4/80highCD11bhighTIM4-/+) and bone marrow derived inflammatory macrophages (F4/80+CD11b+TIM4-). We have employed both hybridization arrays and next generation sequencing to identify differential expression of microRNAs in resident and inflammatory macrophages in response to single and repeat episodes of acute inflammation. Analysis of the microRNA expression data, together with in silico prediction of putative mRNA targets, has identified several microRNAs implicated in regulation of macrophage phenotype. In order to determine the precise functions of these microRNAs we are developing both constitutive and condition lentiviral vectors for 1) microRNA expression and 2) microRNA repression based on the Tough Decoy (TuD) principle. Conclusions: Macrophages are a heterogeneous population of cells tailored by their microenvironment and tissue-specific functions. Delineating the post-transcription regulation of macrophage heterogeneity has important therapeutic applications in peritoneal fibrosis.

Contributions to the knowledge of Formicidae (Hymenoptera, Aculeata): a new diagnosis of the family, the first global male-based key to subfamilies, and a treatment of early branching lineages

The diagnosis of the Formicidae is revised, including five new, unreversed apomorphies, of which one is a unique synapomorphy. The first global male-based key to all subfamilies is provided and illustrated, and all ant subfamilies are diagnosed for males on a global scale for the first time. Three lineages of “basal ants” are assessed in detail: the Amblyoponinae, Leptanillinae, and Martialinae. The males of Martialis heureka (Martialinae) and Apomyrma (Amblyoponinae) are described. The Martialinae and Leptanillinae are diagnosed based on males, and additional diagnostic traits for the male of Amblyoponinae and worker of Martialis are provided. The placement of Scyphodon and Noonilla in the Formicidae and Leptanillinae is confirmed. Morphological characters of the Amblyoponinae, the Leptanillinae, and the Martialinae are contrasted, and potentially homologous apomorphies are signaled.

Phylogeny of the Alismatales (Monocotyledons) and the relationship of Acorus (Acorales?).

A phylogenetic analysis of the early branching lineages of the monocotyledons is performed using data from two plastid genes (rbcL and matK), five mitochondrial genes (atp1, ccmB, cob, mttB and nad5) and morphology. The complete matrix includes 93 terminals representing Acorus, the 14 families currently recognized within Alismatales, and numerous lineages of monocotyledons and other angiosperms. Total evidence analysis results in an almost completely resolved strict consensus tree, but all data partitions, genomic as well as morphological, are incongruent. The effects of RNA editing and potentially processed paralogous sequences are explored and discussed. Despite a decrease in incongruence length differences after exclusion of edited sites, the major data partitions remain significantly incongruent. The 14 families of Alismatales are all found to be monophyletic, but Acorus is found to be included in Alismatales rather than being the sister group to all other monocotyledons. The placement is strongly supported by the mitochondrial data, atp1 in particular, but it cannot be explained as an artifact caused by patterns of editing or by sampling of processed paralogues.

Phylogeny of world stag beetles (Coleoptera: Lucanidae) reveals a Gondwanan origin of Darwin’s stag beetle

Stag beetles (family Lucanidae Latreille, 1804) are one of the earliest branching lineages of scarab beetles that are characterized by the striking development of the male mandibles. Despite stag beetles’ popularity among traditional taxonomists and amateur collectors, there has been almost no study of lucanid relationships and evolution. Entomologists, including Jeannel (1942), have long recognized resemblance between the austral stag beetles of the tribes Chiasognathini, Colophonini, Lamprimini, Pholidotini, Rhyssonotini, and Streptocerini, but this hypothesis of their close relationship across the continents has never been tested. To gain further insight into lucanid phylogeny and biogeography, we reconstructed the first molecular phylogeny of world stag beetles using DNA sequences from mitochondrial 16S rDNA, nuclear 18S and 28S rDNA, and the nuclear protein-coding (NPC) gene wingless for 93 lucanid species representing all extant subfamilies and 24 out of the 27 tribes, together with 14 representative samples of other early branching scarabaeoid families and two staphyliniform beetle families as outgroups. Both Bayesian inference (BI) and maximum likelihood inference (MLI) strongly supported the monophyly of Lucanidae sensu lato that includes Diphyllostomatidae. Within Lucanidae sensu stricto, the subfamilies Lucaninae and Lampriminae appeared monophyletic under both methods of phylogenetic inferences; however, Aesalinae and Syndesinae were found to be polyphyletic. A time-calibrated phylogeny based on five fossil data estimated the origin of crown group Lucanidae as circa 160 million years ago (MYA). Divergence between the Neotropical and Australasian groups of the Chiasognathini was estimated to be circa 47MYA, with the South African Colophonini branching off from the ancient Chiasognathini lineage around 87MYA. Another Gondwanan relationship was recovered between the Australasian Eucarteria and the Neotropical Casignetus, which diverged circa 58MYA. Lastly, as Jeannel’s hypothesis predicted, divergence within Lampriminae between the Australasian Lamprima and the Neotropical Streptocerus was estimated to be circa 37MYA. The split of these lineages were generally concordant with the pattern of continental break-up of the super-continent Gondwana, and our biogeographic reconstructions based on the dispersal-extinction-cladogenesis model (DEC) corroborate our view that the divergences in these austral lineages were caused by vicariance events following the Gondwanan break-up. In addition, the phylogenetic position and geographic origin of the Hawaiian genus Apterocyclus was revealed for the first time. Overall, our results provide the framework toward studying lucanid relationships and divergence time estimates, which allowed for more accurate biogeographic explanations and discussions on ancestral lucanids and the evolutionary origin of the enlarged male mandibles.

Cell lineage branching as a strategy for proliferative control.

BackgroundHow tissue and organ sizes are specified is one of the great unsolved mysteries in biology. Experiments and mathematical modeling implicate feedback control of cell lineage progression, but a broad understanding of what lineage feedback accomplishes is lacking.ResultsBy exploring the possible effects of various biologically relevant disturbances on the dynamic and steady state behaviors of stem cell lineages, we find that the simplest and most frequently studied form of lineage feedback - which we term renewal control - suffers from several serious drawbacks. These reflect fundamental performance limits dictated by universal conservation-type laws, and are independent of parameter choice. Here we show that introducing lineage branches can circumvent all such limitations, permitting effective attenuation of a wide range of perturbations. The type of feedback that achieves such performance - which we term fate control - involves promotion of lineage branching at the expense of both renewal and (primary) differentiation. We discuss the evidence that feedback of just this type occurs in vivo, and plays a role in tissue growth control.ConclusionsRegulated lineage branching is an effective strategy for dealing with disturbances in stem cell systems. The existence of this strategy provides a dynamics-based justification for feedback control of cell fate in vivo.See commentary article: http://dx.doi.org/10.1186/s12915-015-0123-7.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0122-8) contains supplementary material, which is available to authorized users.

Evo-devo of non-bilaterian animals.

The non-bilaterian animals comprise organisms in the phyla Porifera, Cnidaria, Ctenophora and Placozoa. These early-diverging phyla are pivotal to understanding the evolution of bilaterian animals. After the exponential increase in research in evolutionary development (evo-devo) in the last two decades, these organisms are again in the spotlight of evolutionary biology. In this work, I briefly review some aspects of the developmental biology of nonbilaterians that contribute to understanding the evolution of development and of the metazoans. The evolution of the developmental genetic toolkit, embryonic polarization, the origin of gastrulation and mesodermal cells, and the origin of neural cells are discussed. The possibility that germline and stem cell lineages have the same origin is also examined. Although a considerable number of non-bilaterian species are already being investigated, the use of species belonging to different branches of non-bilaterian lineages and functional experimentation with gene manipulation in the majority of the non-bilaterian lineages will be necessary for further progress in this field.

Regarding the origin of Nguyen Cuu lineage branch and the chief captain of the power system Nguyen Cuu Van

Regarding the origin of Nguyen Cuu lineage branch: During the early period of land clearance in the South, many lineage branches from Tonkin organized themselves into groups of migrants to cultivate the new lands in Cochin. Nguyen Cuu lineage branch was one of them. However, this lineage branch didn’t exist in the Nguyen Lords time. Nguyen CuuKieu’s Nguyen lineage branch originally came from Quy rural district (i.e. Tong Son rural district). Thanks to his great contributions, Lord Nguyen Phuc Nguyen married Princess Ngoc Dinh to him, then granted national status Nguyen Phuc to his lineage branch. Later, to distinguish the hierarchy of lineage branches in Nguyen Phuoc clan system, in 1820 King Minh Mang changed his lineage branch into Nguyen Cuu, and Nguyen CuuKieu was the first to get into the historical record of this lineage branch. The chief captain of the power system Nguyen Cuu Van: There is no documented record of the year of birth and death of Nguyen Cuu Van. About his background, Nguyen Cuu Van was Nguyen CuuDuc's son; Nguyen CuuDuc was Nguyen CuuKieu’s son, i.e. Nguyen Cuu Van was Nguyen CuuKieu’s grandson. The chief captain of the power system Nguyen Cuu Van contributed a great deal to the early period of land clearance in the western part of the South. He was a famous general who repelled the Siamese army whose intention was to impose influences on the Chenla kingdom; thereby helped Nguyen Lords to legally acquire part of the land which is today the western part of the South. At the same time, with the outstanding political and economic vision, he held land clearance, digging for rivers and building forts, both for defence and fertile land in the areas bordering the provinces which now are Long An, TienGiang and Vinh Long. In addition, it was he who left cultural imprints through the construction of temples and pagodas in the new land to next generations.

Comprehensive Clonal Mapping of Hematopoiesis in Vivo in Humans By Retroviral Vector Insertional Barcoding

▪Hematopoietic stem cells (HSC) are endowed with the unique role of generating an adequate and efficient pool of blood cells throughout human life. Data derived from clonal tracking of HSC activity and hematopoietic dynamics directly in vivo in humans would be of paramount importance for the design of therapies for hematological disorders and cancers. Our gene therapy (GT) clinical trials for adenosine deaminase (ADA) deficient-SCID and Wiskott-Aldrich Syndrome (WAS) based on the infusion of genetically engineered HSC, constitute unique clinical settings where each vector-marked progenitors and its blood cell progeny is traceable being univocally barcoded by a vector integration site (IS). To study early dynamics of hematopoietic reconstitution in humans, we collected by LAM-PCR + Illumina-Miseq sequencing 14.807.407 sequence reads corresponding to 71.981 IS tagging clones belonging to 13 different cell types purified from the bone marrow and the peripheral blood of 4 WAS patients up to 36 months after GT. We firstly identified and quantified identical IS shared among CD34+ progenitors, and mature Myeloid/Lymphoid cells as marker of the real-time clonal output of individual vector-marked HSC clones in vivo. We unraveled the timing of short, intermediate and long term HSC output showing that CD34+ clones active at 3-6 months after GT are not detectable at later follow up. By unsupervised clustering of IS similarities among lineages we unveiled diverse input of HSPC clonal differentiation towards lymphoid, myeloid and megakaryo-erythroid cells and found that NK cells have a distinct relationship with HSPC as compared to T and B cells. We also profiled the level of HSPC output overtime showing that early reconstitution is markedly skewed towards myeloid production. Importantly, clonogenic progenitors generated in vitro from ex vivo purified CD34+ patients’ cells, showed a IS profile coherent with that of freshly purified BM and PB cell types from the same time-point.We also studied population clonal entropy through 7 different diversity indexes and uncovered that progenitor output occurs in distinct waves during the first 6-9 months after transplantation reaching a “homeostatic equilibrium” only by 12 months after GT. At steady state we estimated by mark-recapture mathematical approaches that 1900-7000 transduced HSC clones were stably contributing to the progenitors repertoire for up to 3 years after infusion of gene corrected CD34+ cells. To evaluate the long-term preservation of activity by transplanted HSC we exploited data derived from the IS-based tracking of 4.845 clones in ADA-SCID patients performed for up to 6 years after GT. We showed that identical IS are consistently detected at multiple lineages level even several years after GT. Strikingly, by semi-quantitative PCRs on specific vector-genome junctions we tracked a fluctuating but consistent output of marked HSC over a period of 5 years without the manifestation of clonal quiescence phases. Additionally, since the gamma-retroviral vector used in ADA-SCID HSC-GT trial is able to transduce only actively replicating cells, we provided the first evidence that in vitro activated HSC, “awaken” from dormancy, can still, once infused, retain in vivo long-term activity in humans. We exploited IS similarities among the lineages for both WAS and ADA-SCID datasets to reconstruct the hematopoietic hierarchy by combining conditional probability distributions and static/dynamic graphical models of dependencies. Notably, preliminary data unveiled a link between myeloid progenitors and mature lymphoid cells that supports the recently suggested model of hematopoiesis based on a delayed branching of myeloid and lymphoid lineages. Further mathematical models are being applied to specifically study population dynamics and single HSPC contribution to hematopoiesis including stochastic models of neutral clonal drift. More detailed analysis are also being performed on IS collected from 7 distinct CD34+ subtypes isolated from GT patients and FACS sorted according to the most recent markers of HSPC differentiation. Overall our work constitute the first molecular tracking of individual hematopoietic clones in humans providing an unprecedented detailed analysis of HSC activity and dynamics in vivo. The information gathered will be crucial for the design of therapeutic approaches for a broad spectrum of hematological diseases and tumors. DisclosuresNeduva:GSK: Employment. Dow:GSK: Employment.

MS4A3 Marks Early Myeloid Differentiation in Human Hematopoiesis

Understanding lineage specific markers contributes to investigation into lineage commitment processes in hematopoiesis. Particularly in the human study, information about hematopoietic lineage divergence is essential to refine hematopoietic lineage tree. Lineage markers are also potentially useful for therapeutic target, such as CD20 in B-cell lymphoma, and CD33 in acute myeloid leukemia. We have recently reported that special AT-rich sequence-binding protein 1 (SATB1), a global chromatin organizer, promotes lymphocyte production from hematopoietic stem cells (HSCs) (Immunity 38;1105, 2013). Expression level of SATB1 increases with early lymphoid differentiation, whereas it is shut off in committed myeloid progenitors. To search a novel cell surface molecule that marks the point of branching lineage along early myeloid and lymphoid differentiation, we performed microarray analyses comparing SATB1-overexpressed HSCs with mock-transduced HSCs. The results drew our attention to membrane-spanning 4-domains, subfamily A, member 3 (MS4A3). MS4A3, also called hematopoietic cell-specific transmembrane 4 (HTm4), is a member of the MS4A family. CD20, encoded by MS4A1 gene, belongs to the same family. We observed that expression level of MS4A3 in SATB1-overexpressed HSCs was decreased almost one tenth of that of mock HSCs.To confirm the relationship of SATB1 and MS4A3 in human hematopoietic cells, we first used chronic myeloid leukemia cell line K562, which was found to clearly express MS4A3 on their cell surface. While SATB1 expression was undetectable in original K562 cells, the exogenous expression of SATB1 significantly reduced their MS4A3 expression level, suggesting that SATB1 negatively regulates MS4A3 expression in human cells.Next, we analyzed MS4A3 expression pattern in primary human hematopoietic stem/progenitor cells. Bone marrow (BM) cells were obtained from healthy donors or patients with acute myeloid leukemia. The Institutional Review Board of Osaka University School of Medicine approved all of protocols, and written informed consents were obtained from all participants. Mononuclear cells were separated from the BM samples by density gradient centrifugation, and subsequently applied to cell sorting for Lineage marker-negative (Lin-) CD34+ CD38- HSCs, Lin- CD34+ CD38+ IL-3 receptor α (IL-3Rα)+ CD45RA- common myeloid progenitors (CMPs), Lin- CD34+ CD38+ IL3-Rα+ CD45RA+ granulocyte-macrophage progenitors (GMPs) and Lin- CD34+ CD38+ IL-3Rα- CD45RA-megakaryocyte-erythroid progenitors (MEPs). MS4A3 expression levels of the sorted cells were analyzed with real-time RT-PCR. We detected more than 10-fold amount of MS4A3 transcripts in CMPs than HSCs. Furthermore, its expression level continuously increased along myeloid lineage differentiation to GMP. On the other hand, megakaryocyte-erythroid lineage differentiation was not accompanied by MS4A3 expression and the amount of MS4A3 transcripts in MEPs was minimum as in HSCs.Flow cytometry analyses confirmed that HSCs and MEPs do not express MS4A3 on their cell surface whereas the MS4A3 expression on CMPs and GMPs is detectable. Further, the Lin- CD34+ CD38+ CD33+ cells could be fractionated according to the intensity of cell surface MS4A3 expression. To investigate the significance of cell surface MS4A3 expression for functional analyses of myeloid progenitor cells, we performed methylcellulose colony-forming assays. We found that MS4A3+ cells in Lin- CD34+ CD38+ CD33+ fraction only produced granulocyte/macrophage colonies, losing erythroid colony- and mixed colony-forming capacity. These results suggest that cell surface expression of MS4A3 is useful to distinguish granulocyte/macrophage lineage-committed progenitors from other lineage-related ones in early human hematopoiesis. We also analyzed MS4A3 expression in BM cells obtained from patients with acute leukemia. Flow cytometry analyses revealed that leukemia cells of some patients expressed substantial amount of cell surface MS4A3.In conclusion, MS4A3 is useful to monitor early stage of myeloid differentiation in human hematopoiesis. In addition, our findings of MS4A3 expression on myeloid leukemia cells, while no expression on normal HSCs, imply that MS4A3 might be a therapeutic target molecule in myelogenous leukemia. Further studies would clarify the application of MS4A3 to anti-leukemia therapy. DisclosuresNo relevant conflicts of interest to declare.

Molecular Phylogeny and Morphology of Leannia veloxifera n. gen. et sp. Unveils a New Lineage of Monothalamous Foraminifera.

Monothalamous (single-chambered) foraminifera have long been considered as the "poor cousins" of multichambered species, which calcareous and agglutinated tests dominate in the fossil record. This view is currently changing with environmental DNA surveys showing that the monothalamids may be as diverse as hard-shelled foraminifera. Yet, the majority of numerous molecular lineages revealed by eDNA studies remain anonymous. Here, we describe a new monothalamous species and genus isolated from the sample of sea grass collected in Gulf of Eilat (Red Sea). This new species, named Leannia veloxifera, is characterized by a tiny ovoid theca (about 50-100 μm) composed of thin organic wall, with two opposite apertures. The examined individuals are multinucleated and show very active reticulopodial movement. Phylogenetic analyses of SSU rDNA, actin, and beta-tubulin (ß-tubulin) show that the species represents a novel lineage branching separately from other monothalamous foraminifera. Interestingly, the SSU rDNA sequence of the new species is very similar to an environmental foraminiferal sequence from Bahamas, suggesting that the novel lineage may represent a group of shallow-water tropical allogromiids, poorly studied until now.

Floral structure of Emmotum (Icacinaceae sensu stricto or Emmotaceae), a phylogenetically isolated genus of lamiids with a unique pseudotrimerous gynoecium, bitegmic ovules and monosporangiate thecae.

Icacinaceae sensu stricto consist of a group of early branching lineages of lamiids whose relationships are not yet resolved and whose detailed floral morphology is poorly known. The most bizarre flowers occur in Emmotum: the gynoecium has three locules on one side and none on the other. It has been interpreted as consisting of three fertile and two sterile carpels or of one fertile carpel with two longitudinal septa and two sterile carpels. This study focused primarily on the outer and inner morphology of the gynoecium to resolve its disputed structure, and ovule structure was also studied. In addition, the perianth and androecium were investigated. Flowers and floral buds of two Emmotum species, E. harleyi and E. nitens, were collected and fixed in the field, and then studied by scanning electron microscopy. Microtome section series were used to reconstruct their morphology. The gynoecium in Emmotum was confirmed as pentamerous, consisting of three fertile and two sterile carpels. Each of the three locules behaves as the single locule in other Icacinaceae, with the placenta of the two ovules being identical, which shows that three fertile carpels are present. In addition, it was found that the ovules are bitegmic, which is almost unique in lamiids, and that the stamens have monosporangiate thecae, which also occurs in the closely related family Oncothecaceae, but is not known from any other Icacinaceae sensu lato so far. The flowers of Emmotum have unique characters at different evolutionary levels: the pseudotrimerous gynoecium at angiosperm level, the bitegmic ovules at lamiid level and the monosporangiate thecae at family or family group level. However, in general, the floral morphology of Emmotum fits well in Icacinaceae. More comparative research on flower structure is necessary in Icacinaceae and other early branching lineages of lamiids to better understand the initial evolution of this large lineage of asterids.

Seedling mycorrhiza: a discussion of origin and evolution in Orchidaceae

Recent phylogenetic analyses confirm the monophyly of Orchidaceae as sister group to the remainder of Asparagales, and identify the sequence of early branching lineages in Orchidaceae. Orchid seedling mycorrhiza (OSM) involving rhizoctonious fungi is distributed widely in all subfamilies, including the first branching ones, and its status as a founding event is thus supported. OSM is recognized as one element in the character syndrome that distinguishes orchid biology, and we argue that OSM was the first to evolve. We also discuss the possible evolutionary origins of OSM in Asparagales. The prevalent mycobionts suggest a derivation from a pathogenic relationship, and sister group comparison offers little support for derivation from other mycorrhizal relationships. A combination of in situ sowings and molecular identification of seedling mycobionts has established that a broad range of fungi besides rhizoctonious mycelia are presently involved in OSM, presumably evolving secondarily and often in parallel in different orchid clades. Structural features and internal patterns of mycobiont behaviour appear to have remained largely the same, implying that OSM needs only minor physiological adjustment to accommodate new mycobionts. Such modifications will have involved checkpoints for recognition/rejection and the formation/breakdown of pelotons. These physiological mechanisms are so far largely unknown. The trophic versatility of the mycobionts and the apparently easy shifts could be a main factor in the ecological adaptability of orchids and proliferation of the family.

T-cell/myeloid mixed-phenotype acute leukemia with monocytic differentiation and isolated 17p deletion.

Mixed phenotype acute leukemia is a rare subtype of leukemia that probably arises from a hematopoietic pluripotent stem cell. The co-expression of two of myeloid, B- or T-lymphoid antigens is the hallmark of this disease. Herein, the case of a 28-year-old female patient is reported who presented with hemoglobin of 5.8 g/dL, white blood cell count of 138 × 109/L and platelet count of 12 × 109/L. The differential count of peripheral blood revealed 96% of blasts. Moreover, the patient presented with lymphadenopathy, splenomegaly and bone marrow infiltration by monocytoid blasts characterized as 7% positivity by Sudan Black cytochemical staining. Immunophenotyping revealed the involvement of blasts of both T- and monocytic lineages. The cytogenetic analysis showed an isolated 17p deletion. Thus, the diagnosis of T-cell/myeloid mixed phenotype acute leukemia was made with two particular rare features, that is, the monocytic differentiation and the 17p deletion as unique cytogenetic abnormalities. The possibility of concomitant expressions of T-cell and monocytic differentiation antigens in the same blast population is hard to explain using the classical model of hematopoiesis. However, recent studies have suggested that myeloid potential persists even when the lineage branches segregate toward B- and T-cells. The role of an isolated 17p deletion in the pathogenesis of this condition is unclear. At present, the patient is in complete remission after an allogeneic stem cell transplantation procedure.