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The allocation of epiblast cells to ectodermal and germ-line lineages is influenced by the position of the cells in the gastrulating mouse embryo.

The developmental potency of cells in the proximal and distal regions of the epiblast of pre- and early-primitive-streak-stage mouse embryos was assessed by their differentiation in the host embryo following orthotopic and heterotopic cell transplantation. Normally, cells in the distal epiblast differentiate predominantly into neuroectoderm and surface ectoderm. However, when they were transplanted to proximal regions of the epiblast, distal epiblast cells behaved like proximal epiblast cells: they colonised the extraembryonic mesoderm and other mesodermal tissues in the posterior region of the host embryo. In addition, about 3.7% of the transplanted distal epiblast cells differentiated into primordial germ cells. This proportion is comparable to the 3.9% of orthotopically transplanted proximal epiblast cells that became primordial germ cells. When proximal epiblast cells were transplanted heterotopically to distal sites, their descendants were generally absent from the extraembryonic mesoderm and the germ cell population of the host embryo. Like cells in the distal epiblast, they mostly colonised the neural plate and surface ectoderm. This plasticity of cell fate suggests that the epiblast cells are not irreversibly allocated to any specific lineages, including the germ line. The adoption of developmental fate that is typical of the cell population at the site of transplantation suggests that the specification of cell lineages is subject to certain site-specific influences in the epiblast. Allocation of cells to the ectodermal and germ cell lineages may be subject to local tissue interactions and the restriction of morphogenetic tissue movement of different epiblast cell populations during gastrulation.

Animals↗

Notochord regulates cardiac lineage in zebrafish embryos.

We focus here upon regulation by the notochord of myocardial cell fate in zebrafish. Myocardial precursors, defined by lineage tracing in the living embryo, are in the lateral plate mesoderm adjacent to the notochord-prechordal plate junction. Interestingly, the anterior end of the notochord corresponds to the posterior extent of the heart progenitor field, defined by this lineage analysis. This suggested that the notochord might suppress, or the prechordal plate might enhance, the cardiogenic fate. Nkx2.5 expression is, in the zebrafish embryo, closely correlated with the position of myocardial precursors, which reside adjacent to the notochord-prechordal plate junction. This expression, however, is extinguished in the region posterior to this junction, a region normally not contributing cells to the heart. Laser ablation of the notochord tip between the 4-somite and 12-somite stage causes posterior expansion of the Nkx2. 5-expressing region. The ntl mutation of the notochord is associated with posterior extension of Nkx2.5 expression. Lineage tracking, by laser activation of caged fluoresceinated dextran, confirms that, normally, lateral plate cells next to the notochord do not contribute progeny to the heart. After anterior notochord ablation, these cells are redirected to a heart cell fate. These data suggest that the anterior notochord delimits the posterior extent of the heart field by suppressing the heart cell fate.

Animals↗

Programmed transformations in neuroblast gene expression during Drosophila CNS lineage development.

During Drosophila embryonic CNS development, the sequential neuroblast (NB) expression of four proteins, Hunchback (Hb), Pou-homeodomain proteins 1 and 2 (referred to collectively as Pdm), and Castor (Cas), identifies a transcription factor network regulating the temporal development of all ganglia. The Zn-finger proteins Hb and Cas, acting as repressors, confine Pdm expression to a narrow intermediate temporal window; this results in the generation of three panneural domains whose cellular constituents are marked by expression of Hb, Pdm, or Cas (R. Kambadur et al., 1998, Genes Dev. 12, 246-260). Seeking to identify the cellular mechanisms that generate these expression compartments, we studied the lineage development of isolated NBs in culture. We found that the Hb, Pdm, and Cas expression domains are generated by transitions in NB gene expression that are followed by gene product perdurance within sequentially produced sublineages. Our results also indicate that following Cas expression, many CNS NBs continue their asymmetric divisions generating additional progeny, which can be identified by the expression of the bHLH transcription factor Grainyhead (Gh). Gh appears to be a terminal embryonic CNS lineage marker. Taken together, these studies indicate that once NBs initiate lineage development, no additional signaling between NBs and the neuroectoderm and/or mesoderm is required to trigger the temporal progression of Hb --> Pdm --> Cas --> Gh expression during NB outgrowth.

Animals↗

Schistosoma mansoni: continuous variation in susceptibility of the vector snail of schistosomiasis, Biomphalaria tenagophila I. Self-fertilization-lineage.

Mascara, D., Kawano, T., Magnanelli, A. C., Silva, R. P. S., Sant' Anna, O. A., and Morgante, J. S. 1999. Schistosoma mansoni: continuous variation in susceptibility of the vector snail of schistosomiasis, Biomphalaria tenagophila I. Self-Fertilization-Lineage. Experimental Parasitology 93, 133-141. Artificial selection of Biomphalaria tenagophila snails for susceptibility to infection by Schistosoma mansoni (Brazilian SJ strain) was carried out from natural populations. After five self-fertilization generations, two lineages were isolated and were designated as SUSC (highly susceptible 93-100%) and RES (nonsusceptible 5-0%). Length of the prepatent period, cercarial production, and mortality of the hosts in postexposure were determined in all generations (F(1)-F(8)) and were analyzed as quantitative traits related to host susceptibility. Distribution patterns of frequencies were observed within snail families (samples derived from one F(0) snail), these traits showing a significant influence by selection applied to susceptibility. The multiple quantitative classes were described in terms of continuous variation. During the selection of SUSC lineage, classes with higher values of prepatent length and lower cercarial production were eliminated, and the heritability calculated for these two traits was 0.811 and 0.709, respectively. Experimental results were correlated with an increase in the level of susceptibility in the generations selected and are discussed in relation to inheritance patterns as well as the quantitative variation of susceptibility.

Animals↗

Tissue distribution and functional expression of a cDNA encoding a novel mixed lineage kinase.

Hypertrophy is an adaptive response of the heart to myocardial injury or hemodynamic overload that may progress and contribute to cardiac decompensation and eventually to heart failure. The signaling pathways controlling this response in the cardiac myocyte are poorly understood. A data mining effort of a human failed heart cDNA library was undertaken in an effort to identify novel signaling molecules involved in cardiac hypertrophy. This effort identified a novel kinase (MLK7) homologous to the mixed lineage kinase family of proteins. The mixed lineage kinases are mitogen-activated protein kinase kinase kinases (MAPKKKs) which activate stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 kinase pathways. They contain a catalytic domain with homology to both serine/threonine and tyrosine-specific kinases and a dual leucine zipper. MLK7 is identical to leucine zipper and sterile-alpha motif protein kinase (ZAK) through the leucine zipper domain but has a completely divergent COOH-terminus and shares approximately 40% homology with the other MLKs overall. Expression of MLK7 mRNA is most abundant in skeletal muscle and heart, with expression restricted to the cardiac myocyte. The recombinant histidine tagged MLK7 expressed and purified from insect cells exhibited serine/threonine kinase activity in vitro with myelin basic protein as substrate. When expressed in cardiac myocytes, MLK7 activated SAPK/JNK1, and ERK and p38 to a lesser extent. Additionally, MLK7 altered fetal gene expression and increased protein synthesis in cardiac myocytes. These data suggest that MLK7 is a new member of the mixed lineage kinase family that modulates cardiac SAPK/JNK pathway and may play a role in cardiac hypertrophy and progression to heart failure.

Adult↗

Evidence for two independent lineages of shiitake of the americas (Lentinula boryana) based on rDNA and beta-tubulin gene sequences.

Portions of ribosomal RNA genes (rDNA) were sequenced from members of the genus Lentinula and were used, along with partial beta-tubulin gene sequences, for phylogenetic reconstructions. The rDNA sequences of L. boryana were separated into two well-defined lineages. Lineage 1 was composed of isolates from Mexico and Costa Rica while lineage 2 encompassed isolates from the United States, Venezuela, and Brazil. The two South American isolates of L. boryana had nearly identical ITS sequences and very closely related beta-tubulin sequences. This high level of similarity may indicate that sexual reproduction occurs among the sampled populations, although this is difficult to reconcile with the large geographic distances (over 4000 km) that separate some of the collecting locations. An alternative explanation may be that the isolates sampled are the product of a rapid population expansion over a large geographic area. Analyses of partial beta-tubulin gene sequences that were rooted using Pleurotus spp. support the hypothesis that L. boryana is monophyletic.

Asia↗

Evolution of Hoxa-11 in lineages phylogenetically positioned along the fin-limb transition.

HOXA11 is a transcription factor implicated in paired appendage development. To identify signatures of evolutionary change in the structural, and putative functional, domains of HOXA11, we studied its evolution in tetrapod and nontetrapod lineages that represent approximately 1.5 billion years of evolutionary time. Here, Hoxa-11 gene proper sequences were determined for frog (Xenopus tropicalis), coelacanth (Latimeria chalumnae), common zebrafish (Danio rerio; Hoxa-11a and Hoxa-11b paralogs), and giant zebrafish (D. aequipinnatus; Hoxa-11b) and aligned against previously published Hoxa-11 sequences of human, mouse, chick, and newt. Based on aligned Hoxa-11 amino acid sequences, the protein was demarcated into three segments: Domains I (N-terminal) and III (homeobox + C-terminal), which varied slightly in rates and patterns of evolution, and a variable, overall hydrophilic region (HyD), which partially overlaps with Domain I. As judged by character reconstructions of HOXA11 Domains I and III, no significant changes in rates of coding sequence evolution occurred in tetrapods (frog and chick), relative to coelacanth (a lobe-finned fish), i.e., across the fin-limb transition. Accelerated rates of Hoxa-11 coding sequence evolution were observed for the mammalian and newt lineages. This was shown to be a gene-specific phenomenon. The duplicated Hoxa-11a and Hoxa-11b genes of zebrafish exhibited accelerated rates of evolution and accumulated substitutions at sites that are conserved among coelacanth and all tetrapods examined. Amino acid sequence comparisons of the HyD of HOXA11 suggested that a putative repressor subdomain, containing stretches of consecutive alanine residues, emerged within the tetrapods. A high degree of nucleotide conservation in the 5' half of the Hoxa-11 intron was observed for tetrapod and nontetrapod lineages. Using electrophoretic mobility shift assays, a 35-bp intron sequence, which is 100% conserved in all Hoxa-11 loci except for the zebrafish Hoxa-11a paralog, was found to bind protein(s) in HeLa and chick whole-cell extracts.

Alanine↗

Why does a nematode have an invariant cell lineage?

C. elegans is renowned for its invariant embryogenesis and functions as a major paradigm for a mode of development coupled to an invariant lineage. Recent work, however, suggests that the embryogenesis of the nematode is much more flexible than anticipated. The invariant premorphogenetic stage is formed from variable earlier stages through a sorting of cells. Cells do not act as individuals but already early in embryogenesis a regionalization of the embryo occurs. Cells are diversified by a binary specification of 'abstract' blastomere (regional) identities. The determination of tissues may thus be a very late event. It appears that C. elegans, although assigning cell fates in an invariant lineage pattern, uses the same strategies and mechanisms for embryogenesis as organisms with variable lineages.

Journal Article↗

The effect of constitutive CD8 expression on thymic selection; implications for the mechanism of CD4 and CD8 lineage commitment.

The choice of an immature T cell to become a CD4 cell or a CD8 cell may be the result of instructive signals generated during positive selection. Alternatively, the initial commitment to a CD4 or CD8 lineage may be made independently of the specificity of the TCR. In this article, I describe our efforts to test the latter possibility using mice expressing a constitutive CD8 transgene. If lineage commitment is initially independent of MHC specificity, expression of ectopic CD8 in CD4 cells should allow CD4 cells with class I specific TCR to mature. We find that although some mature class I specific CD4 cells can be detected in CD8 transgenic mice, they are a minor population compared to class I selected CD8 cells. The implications of these results for the mechanism of lineage commitment are discussed.

Animals↗

CD4 and CD8 in T cell lineage commitment: alterations induced by expression of a CD8/CD4 chimeric transgene.

The mechanism by which thymocytes expressing both the CD4 and CD8-coreceptor proteins differentiate into mature T cells expressing either CD4 or CD8 is not well understood. We have shown that expression of a chimeric CD8/CD4 transgene can alter T cell lineage commitment such that cells expressing a transgenic class I major histocompatibility complex-restricted T cell receptor can differentiate to the CD4 rather than those the CD8 lineage. The implications of these findings and those of others for existing instructive and stochastic models of thymocyte lineage commitment are discussed, and an alternative model is considered.

Animals↗

CD4/CD8 lineage commitment in T cell receptor transgenic mice: evidence for precommitment of CD4+ CD8+ thymocytes.

CD4+ and CD8+ mature T cells arise from CD4+ CD8+ thymic precursors by a process of positive selection that ultimately requires interaction of the T cell receptor (TCR) with self major histocompatibility complex (MHC) molecules. The mechanism of commitment of immature CD4+ CD8+ thymocytes to CD4 or CD8 lineages is controversial. Using TCR transgenic mice, we present evidence that CD4+ CD8+ thymocytes are precommitted to either the CD4 or CD8 lineage, prior to positive selection and independently of TCR specificity for MHC. This lineage precommitment model places important constraints on signaling via CD4 or CD8 coreceptor molecules.

Animals↗

Lineage- and differentiation-dependent alterations in the expression of receptors for glycoconjugates (lectins) in different human hematopoietic cell lines and low grade lymphomas.

Important biological functions and cellular recognition phenomena are supposedly governed by specific sugar-protein interactions. Human hematopoietic cell lines offer an excellent model for the study of the expression of endogenous receptors for the carbohydrate part of glycoconjugates with respect to cell lineage and modulation by differentiation. Initially, a panel of fluorescent (neo)glycoproteins was successfully employed to demonstrate cytologically the actual presence of such receptors on different cell lines: the B lymphoblast line, Daudi; the T cell lymphoblastic leukemia line, P12; the multipotent leukemic line, K562 and the promyelocytic line, HL060. Biochemical analyses were performed using affinity chromatography on supports with immobilized lactose and asialofetuin (simple or complex beta-galactosides), melibiose (alpha-galactoside), fucose, N-acetyl-D-galactosamine, maltose (alpha-glucoside), the mannose-rich yeast glycoprotein, mannan, glycopeptides containing sialic acid residues and heparin. Subsequently, sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis was used to detect cell lineage-dependent changes in theses parameters. Differentiation-dependent changes in the expression of receptors with specificity to galactose, N-acetylgalactosamine, maltose and heparin were similarly uncovered upon dimethyl sulfoxide-induced differentiation of HL60 cells. Differences in this type of cellular characteristic were also apparent for lymphoma cells from patients with various histological subtypes of lowgrade lymphomas. This initial description of lineage- and differentiation-dependent differences in various human hematopoietic cell lines and in cells from patients with lowgrade lymphomas suggests that advances in the knowledge of the composition of endogenous sugar receptors (lectins) may aid in understanding aspects of the biological behavior of hematopoietic cells and their related malignancies via participation of sugar-protein (lectin) interactions.

Cell Differentiation↗

Astrocyte cell lineage. III. The morphology of differentiating mouse astrocytes in colony culture.

Disaggregated cells of newborn DBA/1J mouse neopallium were grown in colony cultures, and colonies of cells at various stages of differentiation along the astrocyte cell lineage were examined after 3 days, 1, 2 and 4 weeks by electron microscopy and by NBD-phallacidin which demonstrates the distribution of microfilaments. The earliest astrocyte precursor cells or glioblasts are closely apposed epithelial cells that rarely have junctions. Their scanty cytoplasm contains many free ribosomes but few microfilaments. The cells in the next stages of astrocyte lineage or proastroblasts are flat and are separated from each other to a variable degree. They have intercellular junctions associated with microfilaments and contain singly dispersed intermediate filaments. The proastroblasts gradually differentiate into astroblasts which have a similar morphology except that in addition to the singly distributed intermediate filaments they also contain intermediate filaments arranged into bundles of various sizes. The mature fibrous astrocytes have well-defined processes and distinct perikarya. They form from astroblasts in culture and also contain numerous bundles of intermediate filaments. The dibutyryl-cyclic AMP (dBcAMP)-induced astrocytes in culture in contrast are large stellate cells similar to reactive astrocytes found around sites of injury in the brain. On the basis of these and previous immunocytochemical studies of the formation and distribution of intermediate filaments in the cytoplasm of differentiating astrocytes, criteria are proposed for identification of different cells along the astrocyte lineage.

Animals↗

Evolutionary pattern of the H 3 haemagglutinin of equine influenza viruses: multiple evolutionary lineages and frozen replication.

The nucleotide and deduced amino acid sequences of the haemagglutinin genes coding for the HA 1 domain of H3N8 equine influenza viruses isolated over wide regions of the world were analyzed in detail to determine their evolutionary relationships. We have constructed a phylogenetic model tree by the neighbour-joining method using nucleotide sequences of 15 haemagglutinin genes, including those of five viruses determined in the present study. This gene tree revealed the existence of two major evolutionary pathways during a twenty five-year period between 1963 to 1988, and each pathway appeared to consist of two distinct lineages of haemagglutinin genes. Furthermore, our analysis of nucleotide sequences showed that two distinct lineages of equine H3N8 viruses were involved in an equine influenza outbreak during the period of December 1971-January 1972 in Japan. The number of nucleotide changes between strains was proportional to the length of time (in years) between their isolation except for three of the HA genes. However, there are three exceptional strains isolated in 1971, 1987, and 1988, respectively. The haemagglutinin gene in these strains showed a small number of nucleotide substitutions after they branched off around 1963, suggesting an example of frozen replication. Although the estimated rate (0.0094/site/year) of synonymous (silent) substitutions of the haemagglutinin gene of equine H3N8 viruses was nearly the same as that of human H 1 and H 3 haemagglutinin genes, the rate of nonsynonymous (amino-acid changing) substitutions of the former equine virus gene was estimated to be 0.00041/site/year--that is about 5 times lower than that estimated for the human H 3 haemagglutinin gene. The present study is the first demonstration that multiple evolutionary lineages of equine H3N8 influenza virus circulated since 1963.

Amino Acid Sequence↗

Age-related differences in the effect of in vivo administration of indomethacin on hemopoietic cell lineages of the spleen and bone marrow of mice.

During 21 days of indomethacin treatment, erythroid cells in the spleens of both young adult and older mice, and in the bone marrow of young adult mice, were increased significantly early in treatment, relative to age-matched control organs, and remained high throughout treatment. During drug exposure, the numbers of myeloid cells in young adult bone marrow, but not spleen, were reduced, but in older mice these cells were elevated in both organs. Lymphoid cells in the young adult and older mouse spleens decreased and increased, respectively, during treatment, but were unchanged and decreased, respectively, in the bone marrow of young adult and older mice. Monocyte-macrophage cells in the spleen were elevated but unchanged in the bone marrow of both age groups. During 14 days of indomethacin treatment of young adult mice, the proportions of precursor cells in DNA synthesis of only the splenic erythroid lineage were increased. Thus, the major hemopoietic lineages in both the bone marrow and spleen are affected by exposure to indomethacin in a time-dependent and age-dependent manner. For all lineages studied, those of the bone marrow were least disturbed and/or were first to recover, even during continued drug exposure.

Age Factors↗

Differences in pyrenoid morphology are correlated with differences in the rbcL genes of members of the Chloromonas lineage (volvocales, chlorophyceae).

Chloromonas is distinguished from Chlamydomonas primarily by the absence of pyrenoids, which are structures that are present in the chloroplasts of most algae and are composed primarily of the CO2-fixing enzyme Rubisco. In this study we compared sequences of the rbcL (Rubisco large subunit-encoding) genes of pyrenoid-less Chloromonas species with those of closely related pyrenoid-containing Chlamydomonas species in the "Chloromonas lineage" and with those of 45 other green algae. We found that the proteins encoded by the rbcL genes had a much higher level of amino acid substitution in members of the Chloromonas lineage than they did in other algae. This kind of elevated substitution rate was not observed, however, in the deduced proteins encoded by two other chloroplast genes that we analyzed: atpB and psaB. The rates of synonymous and nonsynonymous nucleotide substitutions in the rbcL genes indicate that the rapid evolution of these genes in members of the Chloromonas lineage is not due to relaxed selection (as it presumably is in parasitic land plants). A phylogenetic tree based on rbcL nucleotide sequences nested two Chlamydomonas species as a "pyrenoid-regained" clade within a monophyletic Chloromonas "pyrenoid-lost" clade. Character-state optimization with this tree suggested that the loss and the regain of pyrenoids were accompanied by eight synapomorphic amino acid replacements in the Rubisco large subunit, four of which are positioned in the region involved in its dimerization. However, both the atpB and the psaB sequence data gave robust support for a rather different set of phylogenetic relationships in which neither the "pyrenoid-lost" nor the "pyrenoid-regained" clade was resolved. The appearance of such clades in the rbcL-based tree may be an artifact of convergent evolutionary changes that have occurred in a region of the large subunit that determines whether Rubisco molecules will aggregate to form a visible pyrenoid.

Amino Acid Substitution↗

Molecular evolution of a plastid tandem repeat locus in an orchid lineage.

The molecular evolution of a chloroplast minisatellite locus in the Anacamptis palustris (Orchidaceae) lineage and haplotype variation in two Italian A. palustris populations were investigated. A phylogenetic analyses of the chloroplast tRNA(LEU) intron, where the minisatellite locus is located, revealed that a deletion in the ancestor of the A. palustris lineage led to the formation of two noncontiguous, complementary sequence motifs. We propose a model to explain the initial formation of the minisatellite repeat motif, starting with the two noncontiguous, complementary sequence motifs. A survey of minisatellite variation in four species of the A. palustris lineage revealed several haplotypes that differed not only in repeat number, but also in repeat organization. A haplotype network suggests that three different minisatellite loci evolved independently at the same position in the tRNA(LEU) intron. A secondary structure model revealed that the A. palustris minisatellite repeat forms a stem region of the tRNA(LEU) intron, which allows its notable expansion without negatively affecting splicing. Minisatellite variation was high in the two examined A. palustris populations where 20 haplotypes were detected, whereas no length variation was detected in a neighboring poly (A) microsatellite locus. We estimated a chloroplast minisatellite mutation rate of 3.2 x 10(-3) mutations per generation. Southern blot analyses did not find evidence for chloroplast heteroplasmy. Based on the analysis of the largest known, extant A. palustris population, a stepwise mutation model (SMM) was inferred.

Evolution, Molecular↗

Explosive lineage-specific expansion of the orphan nuclear receptor HNF4 in nematodes.

The nuclear receptor superfamily expanded in at least two episodes: one early in metazoan evolution, the second within the vertebrate lineage. An exception to this pattern is the genome of the nematode Caenorhabditis elegans, which encodes more than 270 nuclear receptors, most of them highly divergent. We generated 128 cDNA sequences for 76 C. elegans nuclear receptors, confirming that these are active genes. Among these numerous receptors are 13 orthologues of nuclear receptors found in arthropods and/or vertebrates. We show that the supplementary nuclear receptors (supnrs) originated from an explosive burst of duplications of a unique orphan receptor, HNF4. This origin has specific implications for the role of ligand binding in the function and evolution of the nematode supplementary nuclear receptors. Moreover, the supplementary nuclear receptors include a group of very rapidly evolving genes found primarily on chromosome V. We propose a model of lineage-specific duplications from a chromosome on which duplication and substitution rates are highly increased. Our results provide a framework to study nuclear receptors in nematodes, as well as to consider the functional and evolutionary consequences of lineage-specific duplications.

Animals↗