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At least 1,099 records · Page 61Linked to original sources
PubMed2005

Applications of mRNA injections for analyzing cell lineage and asymmetric cell divisions during segmentation in the leech Helobdella robusta.

Synthetic mRNAs can be injected to achieve transient gene expression even for 'non-model' organisms in which genetic approaches are not feasible. Here, we have used this technique to express proteins that can serve as lineage tracers or reporters of cellular events in embryos of the glossiphoniid leech Helobdella robusta (phylum Annelida). As representatives of the proposed super-phylum Lophotrochozoa, glossiphoniid leeches are of interest for developmental and evolutionary comparisons. Their embryos are suitable for microinjection, but no genetic approaches are currently available. We have injected segmentation stem cells (teloblasts) with mRNAs encoding nuclear localized green fluorescent protein (nGFP) and its spectral variants, and have used tandem injections of nGFP mRNA followed by antisense morpholino oligomer (AS MO), to label single blast cell clones. These techniques permit high resolution cell lineage tracing in living embryos. We have applied them to the primary neurogenic (N) lineage, in which alternate segmental founder cells (nf and ns blast cells) contribute distinct sets of progeny to the segmental ganglia. The nf and ns blast cell clones exhibit strikingly different cell division patterns: the increase in cell number within the nf clone is roughly linear, while that in the ns clone is almost exponential. To analyze spindle dynamics in the asymmetric divisions of individual blast cells, we have injected teloblasts with mRNA encoding a tau::GFP fusion protein. Our results show that the asymmetric divisions of n blast cells result from a posterior shift of both the spindle within the cell and the midbody within the mitotic spindle, with differential regulation of these processes between nf and ns.

Animals↗
PubMed2005

Lineage restriction maintains a stable organizer cell population at the zebrafish midbrain-hindbrain boundary.

The vertebrate hindbrain is subdivided into segments, termed neuromeres, that are units of gene expression, cell differentiation and behavior. A key property of such segments is that cells show a restricted ability to mix across segment borders -- termed lineage restriction. In order to address segmentation in the midbrain-hindbrain boundary (mhb) region, we have analyzed single cell behavior in the living embryo by acquiring time-lapse movies of the developing mhb region in a transgenic zebrafish line. We traced the movement of hundreds of nuclei, and by matching their position with the expression of a midbrain marker, we demonstrate that midbrain and hindbrain cells arise from two distinct cell populations. Single cell labeling and analysis of the distribution of their progeny shows that lineage restriction is probably established during late gastrulation stages. Our findings suggest that segmentation as an organizing principle in early brain development can be extended to the mhb region. We argue that lineage restriction serves to constrain the position of the mhb organizer cell population.

Animals↗
PubMed1996

An indelible lineage marker for Xenopus using a mutated green fluorescent protein.

We describe the use of a DNA construct (named GFP.RN3) encoding green fluorescent protein as a lineage marker for Xenopus embryos. This offers the following advantages over other lineage markers so far used in Xenopus. When injected as synthetic mRNA, its protein emits intense fluorescence in living embryos. It is non-toxic, and the fluorescence does not bleach when viewed under 480 nm light. It is surprisingly stable, being strongly visible up to the feeding tadpole stage (5 days), and in some tissues for several weeks after mRNA injection. We also describe a construct that encodes a blue fluorescent protein. We exemplify the use of this GFP.RN3 construct for marking the lineage of individual blastomeres at the 32- to 64-cell stage, and as a marker for single transplanted blastula cells. Both procedures have revealed that the descendants of one embryonic cell can contribute single muscle cells to nearly all segmental myotomes rather than predominantly to any one myotome. An independent aim of our work has been to follow the fate of cells in which an early regulatory gene has been temporarily overexpressed. For this purpose, we co-injected GFP.RN3 mRNA and mRNA for the early Xenopus gene Eomes, and found that a high concentration of Eomes results in ectopic muscle gene activation in only the injected cells. This marker may therefore be of general value in providing long term identification of those cells in which an early gene with ephemeral expression has been overexpressed.

Animals↗
PubMed1997

Smoothened-mediated Hedgehog signalling is required for the maintenance of the anterior-posterior lineage restriction in the developing wing of Drosophila.

It is thought that the posterior expression of the 'selector' genes engrailed and invected control the subdivision of the growing wing imaginal disc of Drosophila into anterior and posterior lineage compartments. At present, the cellular mechanisms by which separate lineage compartments are maintained are not known. Most models have assumed that the presence or absence of selector gene expression autonomously drives the expression of compartment-specific adhesion or recognition molecules that inhibit intermixing between compartments. However, our present understanding of Hedgehog signalling from posterior to anterior cells raises some interesting alternative models based on a cell's response to signalling. We show here that anterior cells that lack smoothened, and thus the ability to receive the Hedgehog signal, no longer obey a lineage restriction in the normal position of the anterior-posterior boundary. Rather these clones extend into anatomically posterior territory, without any changes in engrailed/invected gene expression. We have also examined clones lacking both en and inv; these too show complex behaviors near the normal site of the compartment boundary, and do not always cross entirely into anatomically anterior territory. Our results suggest that compartmentalization is a complex process involving intercompartmental signalling; models based on changes in affinity or growth will be discussed.

Animals↗
PubMed1999

Restriction of developmental potential during divergence of the enteric and sympathetic neuronal lineages.

In the peripheral nervous system, enteric and sympathetic neurons develop from multipotent neural crest cells. While local environmental signals in the gut and in the region of the sympathetic ganglia play a role in the choice of cell fate, little is known about the mechanisms that underlie restriction to specific neuronal phenotypes. We investigated the divergence and restriction of the enteric and sympathetic neuronal lineages using immuno-isolated neural crest-derived cells from the gut and sympathetic ganglia. Analysis of neuronal and lineage-specific mRNAs and proteins indicated that neural crest-derived cells from the gut and sympathetic ganglia had initiated neuronal differentiation and phenotypic divergence by E14.5 in the rat. We investigated the developmental potential of these cells using expression of tyrosine hydroxylase as a marker for a sympathetic phenotype. Tyrosine hydroxylase expression was examined in neurons that developed from sympathetic and enteric neuroblasts under the following culture conditions: culture alone; coculture with gut monolayers to promote enteric differentiation; or coculture with dorsal aorta monolayers to promote noradrenergic differentiation. Both enteric and sympathetic neuroblasts displayed developmental plasticity at E14.5. Sympathetic neuroblasts downregulated tyrosine hydroxylase in response to signals from the gut environment and enteric neuroblasts increased expression of tyrosine hydroxylase when grown on dorsal aorta or in the absence of other cell types. Tracking of individual sympathetic cells displaying a neuronal morphology at the time of plating indicated that neuroblasts retained phenotypic plasticity even after initial neuronal differentiation had occurred. By E19.5 both enteric and sympathetic neuroblasts had undergone a significant loss of their developmental potential, with most neuroblasts retaining their lineage-specific phenotype in all environments tested. Together our data indicate that the developmental potential of enteric and sympathetic neuroblasts becomes restricted over time and that this restriction takes place not as a consequence of initial neuronal differentiation but during the period of neuronal maturation. Further, we have characterized a default pathway of adrenergic differentiation in the enteric nervous system and have defined a transient requirement for gut-derived factors in the maintenance of the enteric neuronal phenotype.

Animals↗
PubMed1999

Development of the cardiac conduction system involves recruitment within a multipotent cardiomyogenic lineage.

The cardiac pacemaking and conduction system sets and maintains the rhythmic pumping action of the heart. Previously, we have shown that peripheral cells of the conduction network in chick (periarterial Purkinje fibers) are selected within a cardiomyogenic lineage and that this recruitment occurs as a result of paracrine cues from coronary arteries. At present, the cellular derivation of other elements of this specialized system (e.g. the nodes and bundles of the central conduction system) are controversial, with some proposing that the evidence supports a neurogenic and others a myogenic origin for these tissues. While such ontological questions remain, it is unlikely that progress can be made on the molecular mechanisms governing patterning and induction of the central conduction system. Here, we have undertaken lineage-tracing strategies based on the distinct properties of replication-incompetent adenoviral and retroviral lacZ-expressing constructs. Using these complementary approaches, it is shown that cells constituting both peripheral and central conduction tissues originate from cardiomyogenic progenitors present in the looped, tubular heart with no detectable contribution by migratory neuroectoderm-derived populations. Moreover, clonal analyses of retrovirally infected cells incorporated within any part of the conduction system suggest that such cells share closer lineage relationships with nearby contractive myocytes than with other, more distal elements of the conduction system. Differentiation birthdating by label dilution using [(3)H]thymidine also demonstrates the occurrence of ongoing myocyte conscription to conductive specialization and provides a time course for this active and localized selection process in different parts of the system. Together, these data suggest that the cardiac conduction system does not develop by outgrowth from a prespecified pool of 'primary' myogenic progenitors. Rather, its assembly and elaboration occur via processes that include progressive and localized recruitment of multipotent cardiomyogenic cells to the developing network of specialized cardiac tissues.

Adenoviridae↗
PubMed2000

Mechanism of glia-neuron cell-fate switch in the Drosophila thoracic neuroblast 6-4 lineage.

During development of the Drosophila central nervous system, neuroblast 6-4 in the thoracic segment (NB6-4T) divides asymmetrically into a medially located glial precursor cell and a laterally located neuronal precursor cell. In this study, to understand the molecular basis for this glia-neuron cell-fate decision, we examined the effects of some known mutations on the NB6-4T lineage. First, we found that prospero (pros) mutations led to a loss of expression of Glial cells missing, which is essential to trigger glial differentiation, in the NB6-4T lineage. In wild-type embryos, Pros protein was localized at the medial cell cortex of dividing NB6-4T and segregated to the nucleus of the glial precursor cell. miranda and inscuteable mutations altered the behavior of Pros, resulting in failure to correctly switch the glial and neuronal fates. Our results suggested that NB6-4T used the same molecular machinery in the asymmetric cell division as other neuroblasts in cell divisions producing ganglion mother cells. Furthermore, we showed that outside the NB6-4T lineage most glial cells appeared independently of Pros.

Animals↗
PubMed2001

Specification of ectodermal teloblast lineages in embryos of the oligochaete annelid Tubifex: involvement of novel cell-cell interactions.

In embryos of clitellate annelids (i.e. oligochaetes and leeches), four ectodermal teloblasts (ectoteloblasts N, O, P and Q) are generated on either side through a stereotyped sequence of cell divisions of a proteloblast, NOPQ. The four ectoteloblasts assume distinct fates and produce bandlets of smaller progeny cells, which join together to form an ectodermal germ band. The pattern of the germ band, with respect to the ventrodorsal order of the bandlets, has been highly preserved in clitellate annelids. We show that specification of ectoteloblast lineages in the oligochaete annelid Tubifex involves cell interaction networks distinct from those in leeches. Cell ablation experiments have shown that fates of teloblasts N, P and Q in Tubifex embryos are determined rigidly as early as their birth. In contrast, the O teloblast and its progeny are initially pluripotent and their fate becomes restricted to the O fate through an inductive signal emanating from the P lineage. In the absence of this signal, the O lineage assumes the P fate. These results differ significantly from those obtained in embryos of the leech Helobdella, suggesting the diversity of patterning mechanisms that give rise to germ bands with similar morphological pattern.

Animals↗
PubMed2002

Xcl1 causes delayed oblique periclinal cell divisions in developing maize leaves, leading to cellular differentiation by lineage instead of position.

Differentiation of plant cells is regulated by position-dependent mechanisms rather than lineage. The maize Extra cell layers1 (Xcl1) mutation causes oblique, periclinal divisions to occur in the protoderm layer. These protodermal periclinal divisions occur at the expense of normal anticlinal divisions and cause the production of extra cell layers with epidermal characteristics, indicating that cells are differentiating according to lineage instead of position. Mutant kernels have several aleurone layers instead of one, indicating that Xcl1 alters cell division orientation in cells that divide predominantly in the anticlinal plane. Dosage analysis of Xcl1 reveals that the mutant phenotype is caused by overproduction of a normal gene product. This allows cells that have already received differentiation signals to continue to divide in aberrant planes and suggests that the timing of cell division determines differentiation. Cells that divide early and in the absence of differentiation signals use positional information, while cells that divide late after perceiving differentiation signals use lineage information instead of position.

Cell Differentiation↗
PubMed2001

Lineage allocation during early embryogenesis. Mapping of the neural primordia and application to the analysis of mouse mutants.

The methods outlined in this chapter discuss a range of techniques that have been employed for lineage analysis studies of the neural primordia from the onset of gastrulation and during neurulation. As the mouse has been extensively mapped, lineage analysis during normal morphogenesis is well understood. Attention is now focused on the tissue interactions that are essential for gastrulation and neurulation to proceed normally. The key to understanding these tissue interactions lies in the study of mutant embryos where abnormal development of specific tissue types affects the processes of gastrulation and neurulation. Lineage analysis and tissue potency experiments on particular mutant embryos will provide insight into these essential tissue interactions. As the first step toward undertaking such analysis of the neural derivatives, we have outlined the mutant strains available and detailed a protocol for the introgression of the lacZ transgene onto the mutant background.

Animals↗
PubMed2005

A new clonal assay system for lymphoid and myeloid lineages.

It has long been unclear how the pluripotent hematopoietic stem cell is restricted to the major lineage progenitors including the progenitors for myeloid, T- and B-cells. This is the result of the absence of a methodology capable of determining the developmental potential of individual progenitors to generate these major lineage cells. We have established such an assay system, termed the multilineage progenitor assay, as a modification of the fetal thymic organ culture system. By examining cells from murine fetal tissues with this assay, we have succeeded in elucidating the process of lineage restrictions in early hematopoiesis.

Animals↗
PubMed2006

Methods for analyzing microRNA expression and function during hematopoietic lineage differentiation.

MicroRNAs (miRNAs), an abundant class of approx 22-nucleotide (nt) small RNAs that control gene expression at the posttranscriptional level, may play important roles during normal hematopoiesis and leukemogenesis. This chapter focuses on the methods and strategies for dissecting miRNA function during hematopoietic lineage differentiation. We describe a modified miRNA cloning method and expression analysis approach for determining miRNA expression during hematopoietic lineage differentiation. We illustrate a retroviral vector and a general strategy for the ectopic expression of miRNAs in hemato-poietic stem/progenitor cells. We discuss in vitro and in vivo functional assays that can be used to examine the roles of miRNAs during hematopoietic lineage differentiation. The methods and principles described here should also be applicable to study the roles of miRNAs in the differentiation and function of nonhematopoietic cell types.

Animals↗
PubMed2006

Evolution of hypervariable microsatellites in apomictic polyploid lineages of Ranunculus carpaticola: directional bias at dinucleotide loci.

Microsatellites are widely used in genetic and evolutionary analyses, but their own evolution is far from simple. The mechanisms maintaining the mutational patterns of simple repeats and the typical stable allele-frequency distributions are still poorly understood. Asexual lineages may provide particularly informative models for the indirect study of microsatellite evolution, because their genomes act as complete linkage groups, with mutations being the only source of genetic variation. Here, we study the direction of accumulated dinucleotide microsatellite mutations in wild asexual lineages of hexaploid Ranunculus carpaticola. Whereas the overall number of contractions is not significantly different from that of expansions, the within-locus frequency of contractions, but not of expansions, significantly increases with allele length. Moreover, within-locus polymorphism is positively correlated with allele length, but this relationship is due solely to the influence of contraction mutations. Such asymmetries may explain length constraints generally observed with microsatellites and are consistent with stable, bell-shaped allele-frequency distributions. Although apomictic and allohexaploid, the R. carpaticola lineages show mutational patterns resembling the trends observed in a broad range of organisms, including sexuals and diploids, suggesting that, even if not of germline origin, the mutations in these apomicts may be the consequence of similar mechanisms.

Cell Lineage↗
PubMed2002

Age-related alterations in the lymphohematopoietic and B-lineage precursor populations in NZB mice.

Significant disturbances in B lineage populations of New Zealand Black (NZB) mice have been reported, both with respect to their phenotypes as well as to their function. Notably, there is a profound age-dependent decrease in B-cell precursors in this strain of lupus prone mice. In efforts to characterize the impact of this disturbance in disease, we performed an intensive phenotypic and B-cell population analysis in young and old NZB mice. Our results revealed that there was a significant age-dependent decrease in B cell precursors at all levels of the B-cell-lineage developmental pathway. Analysis of the proliferative capacity of these cell populations showed a comparative decrease in cycling activity in the B-cell-lineage populations of old NZB mice. Furthermore, these cell subsets were much more susceptible to spontaneous apoptosis when compared with similar populations from age-matched BALB/c or young NZB mice. Since the frequency of cells that express the interleukin-7 receptor (IL-7R) declines as NZB mice age, we hypothesize that impairment of IL-7R signal transduction pathways could contribute to severe perturbations of B-cell function in aged NZB mice.

Aging↗
PubMed2004

Directing stem cell differentiation into the chondrogenic lineage in vitro.

A major area in regenerative medicine is the application of stem cells in cartilage tissue engineering and reconstructive surgery. This requires well-defined and efficient protocols for directing the differentiation of stem cells into the chondrogenic lineage, followed by their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages upon transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying chondrogenesis and cartilaginous tissue biology. The development of pharmacokinetic and cytotoxicity/genotoxicity screening tests for cartilage-related biomaterials and drugs could also utilize protocols developed for the chondrogenic differentiation of stem cells. Hence, this review critically examines the various strategies that could be used to direct the differentiation of stem cells into the chondrogenic lineage in vitro.

Animals↗
PubMed2005

Crucial roles of mesodermal cell lineages in a murine embryonic stem cell-derived in vitro liver organogenesis system.

Recent studies in the field of regenerative medicine have exploited the pluripotency of embryonic stem (ES) cells to generate a variety of cell lineages. However, the target has always been only a single lineage, which was isolated from other differentiated cell populations. In the present study, we selected sublines with a high capability for differentiation to contracting cardiomyocytes and also produced germ-line chimeric mice from a parent ES line. We also succeed in establishing embryoid bodies prepared from the ES cells that differentiated into not only hepatocytes but also at least two mesodermal lineages: cardiomyocytes that supported liver development and endothelial cells corresponding to sinusoids. This allowed the development of an in vitro system using murine ES cells that approximated the events of liver development in vivo. The expression of albumin was significantly higher in cardiomyocytes that had arisen in differentiated ES cells than in those that had not. Our in vitro system for liver organogenesis consists of a blood/sinusoid vascular-like network and hepatocyte layers and shows higher levels of hepatic function, such as albumin production and ammonia degradation, than hepatic cell lines and primary cultures of murine adult hepatocytes. This innovative system will lead to the development of second-generation regenerative medicine techniques using ES cells and is expected to be useful for the development of bioartificial liver systems and drug-metabolism assays.

Ammonia↗
PubMed2001

Immortalization of equine trophoblast cell lines of chorionic girdle cell lineage by simian virus-40 large T antigen.

Immortalized cell lines have many potential experimental applications including the analysis of molecular mechanisms underlying cell-specific gene expression. We have utilized a recombinant retrovirus encoding the simian virus-40 (SV-40) large T antigen to construct several immortalized cell lines of equine chorionic girdle cell lineage - the progenitor cells that differentiate into the equine chorionic gonadotropin (eCG) producing endometrial cups. Morphologically, the immortalized cell lines appear similar to normal chorionic girdle cells. Derivation of the immortalized cell lines from a chorionic girdle cell lineage was verified by immunological detection of cell-surface antigens specific to equine invasive trophoblasts. The cell lines differed, however, from mature chorionic girdle cells or endometrial cup cells in that they did not produce eCG and did express MHC class I molecules. Thus, these cell lines appear to have been arrested at a stage of development prior to final differentiation into endometrial cup cells. It was also determined that some of these cell lines as well as endometrial cups express the estrogen receptor-related receptor beta gene, but not the glial cell missing gene (GCMa) both of which are expressed in the murine and human placenta. Among these cell lines, three (eCG 50.5, 100.6 and 500.1) express eCG alpha mRNA. Since regulation of eCG alpha subunit gene is largely unknown, we investigated the signal transduction pathways regulating the eCG alpha subunit gene. Both activators of protein kinase A (PKA) and protein kinase C (PKC) induced the expression of eCG alpha subunit expression 3.2 (P<0.05)- and 1.9 (P<0.05)-fold respectively, in the eCG 500.1 cell line. However, activation of these pathways failed to induce eCG beta subunit expression. In conclusion, lines of equine trophoblast cells have been immortalized that display markers characteristic of those with the equine chorionic girdle and endometrial cup cell lineage. A subset of these cells expresses the eCG alpha subunit gene which is responsive to activators of the PKA and PKC signal transduction pathways.

Analysis of Variance↗
PubMed2004

Flow cytometric analyses on lineage-specific cell surface antigens of rat bone marrow to seek potential myelotoxic biomarkers: status after repeated dose of 5-fluorouracil.

Flow cytometry (FCM) analysis of CD45, CD45R, CD71 and CD90 expression on Crj:CD(SD)IGS rat bone marrow cells was done after 5-fluorouracil (5-FU) administration to examine whether these lineage-specific cell surface antigens could be myelotoxic biomarkers. The expression of CD45 (CD45Low and CD45High: differing in expression intensity), CD45R, CD71 and CD90 on bone marrow cells coincided with previous reports. After repeated administration of 5-FU at 50 mg/kg/day for 1-5 days, a time-dependent decrease in cells expressing CD45Low, CD71 and CD90 was observed, whereas a decrease in the CD45High expressing cells was not observed. Furthermore, the decrease was dose-dependent in CD45Low, CD71 and CD90 expressing cells after administration of 5-FU between 2 and 50 mg/kg/day for 4 days. After 4-day repeated dose of 5-FU at 50 mg/kg/day followed by a recovery period, the change in number of CD45Low, CD45R, CD71 and CD90 cells to the bottom and in recovery showed different kinetics. In contrast, the change in number of CD45High cells was minimal, and relatively stable after 5-FU administration. The results suggest that CD45, CD45R and CD90 could each be potential myelotoxic biomarkers for a total proportion of common leukocytes including T- and B-lymphocytes, for a total proportion of B-lymphocytes, and for a total proportion of T-lymphocytes plus immature B-lymphocytes and common progenitor cells, respectively. CD71 could be a single myelotoxic biomarker for erythroid cells. Further study is required for isolation of each of the myelo-lymphocytic lineages. However, the present study showed that FCM analysis could be available to assess the lineage or differentiation stage-specific response, such as the different extent and time-course or the kinetics (the time to reach the bottom and to recover to the normal level) of myelotoxic effect in rat bone marrow.

Animals↗