Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos.

We show that a gene introduced into cells of mouse embryos by a retrovirus can serve as a heritable marker for the study of cell lineage in vivo. We constructed a defective recombinant retrovirus in which the Escherichia coli beta-galactosidase (lacZ) gene is inserted in the genome of a Muloney murine leukemia virus (M-MuLV). Expression of lacZ was detected with a histochemical stain that can be applied to cultured cells and embryonic tissue. Infection of cultured cells showed that lacZ has no detectable deleterious effects on cell viability or growth, that the enzyme is stably expressed in the progeny of infected cells for many generations in the absence of selective pressure, and that the virus can induce lacZ in a variety of cell types. Following injection of the virus into mid-gestation mouse embryos, clones of lacZ-positive cells were detected in skin, skull, meninges, brain, visceral yolk sac, and amnion. We identified the cell types comprising a series of lacZ-positive clones in the visceral yolk sac and skin to learn the lineage relationships of the labelled cells. In each tissue, we obtained evidence that several cell types have a pluripotential ancestor and that cell fate is progressively restricted as development proceeds.     

Reconstruction of cell lineage trees in mice

The cell lineage tree of a multicellular organism represents its history of cell divisions from the very first cell, the zygote. A new method for high-resolution reconstruction of parts of such cell lineage trees was recently developed based on phylogenetic analysis of somatic mutations accumulated during normal development of an organism. In this study we apply this method in mice to reconstruct the lineage trees of distinct cell types. We address for the first time basic questions in developmental biology of higher organisms, namely what is the correlation between the lineage relation among cells and their (1) function, (2) physical proximity and (3) anatomical proximity. We analyzed B-cells, kidney-, mesenchymal- and hematopoietic-stem cells, as well as satellite cells, which are adult skeletal muscle stem cells isolated from their niche on the muscle fibers (myofibers) from various skeletal muscles. Our results demonstrate that all analyzed cell types are intermingled in the lineage tree, indicating that none of these cell types are single exclusive clones. We also show a significant correlation between the physical proximity of satellite cells within muscles and their lineage. Furthermore, we show that satellite cells obtained from a single myofiber are significantly clustered in the lineage tree, reflecting their common developmental origin. Lineage analysis based on somatic mutations enables performing high resolution reconstruction of lineage trees in mice and humans, which can provide fundamental insights to many aspects of their development and tissue maintenance.     

Cell lineage in the developing retina of Drosophila.

Analysis of the cell lineage of the Drosophila retina is reported. Mitotic recombination within the white locus results in the formation of small red spots in white eyes; these are found under the dissecting microscope. The spot frequency is low (never more than $\text{1}\text{30}$ eyes) so that there can be no doubt that each spot is a single clone. Eyes bearing a clone are serially sectioned and all retinula and all pigment cells scored as white or white⁺. We describe the constitution of 101 clones and examine the disposition of the marked cells in the retinal lattice. The clones are apparently random combinations of the marked cell types—for example, two-celled clones containing one pigment and one retinula cell are frequently found. Our results appear to rule out fixed cell lineage as a determinative mechanism in ommatidial development.     

Making genomic surveillance deliver: A lineage classification and nomenclature system to inform rabies elimination

The availability of pathogen sequence data and use of genomic surveillance is rapidly increasing. Genomic tools and classification systems need updating to reflect this. Here, rabies virus is used as an example to showcase the potential value of updated genomic tools to enhance surveillance to better understand epidemiological dynamics and improve disease control. Previous studies have described the evolutionary history of rabies virus, however the resulting taxonomy lacks the definition necessary to identify incursions, lineage turnover and transmission routes at high resolution. Here we propose a lineage classification system based on the dynamic nomenclature used for SARS-CoV-2, defining a lineage by phylogenetic methods for tracking virus spread and comparing sequences across geographic areas. We demonstrate this system through application to the globally distributed Cosmopolitan clade of rabies virus, defining 96 total lineages within the clade, beyond the 22 previously reported. We further show how integration of this tool with a new rabies virus sequence data resource (RABV-GLUE) enables rapid application, for example, highlighting lineage dynamics relevant to control and elimination programmes, such as identifying importations and their sources, as well as areas of persistence and routes of virus movement, including transboundary incursions. This system and the tools developed should be useful for coordinating and targeting control programmes and monitoring progress as countries work towards eliminating dog-mediated rabies, as well as having potential for broader application to the surveillance of other viruses.     

Analysis of the myogenic lineage in chick embryos. IV. Effects of conditioned medium

Immunocytochemical analysis of small myogenic clones was used to compare the effects of fresh medium (FM) and conditioned medium (CM) on muscle differentiation. In order to compare the same population of cells, clones were initiated in FM and then switched to either new FM or to CM. Clones were fixed at 12-hour intervals up to 76 hours, then assayed for the presence of post-mitotic myoblasts by immunoperoxidase staining for muscle myosin heavy chain (MHC) or M-creatine kinase (MCK). In both media, myogenic cells occurred predominantly in homogeneous positive clones (all cells (MHC +/MCK +) which contained 2" cells. At 76 hours, the percentages of 1-, 2-, and 4-cell positive clones did not differ statistically in the two conditions; however, the percentages of 8- and 16-cell positive clones were significantly reduced in CM, and the percentages of small negative clones were concomitantly increased. We conclude from these data that CM affects myogenesis by slowing progression through a predetermined lineage rather than by changing the number of mitoses an individual cell will undergo before terminally differentiating. These results further support the idea that progress through the myogenic lineage is mediated by cell divisions.     

Neurogenin 3 and the enteroendocrine cell lineage in the adult mouse small intestinal epithelium

It is thought that small intestinal epithelial stem cell progeny, via Notch signaling, yield a Hes1-expressing columnar lineage progenitor and an Atoh1 (also known as Math1)-expressing common progenitor for all granulocytic lineages including enteroendocrine cells, one of the body's largest populations of endocrine cells. Because Neurogenin 3 (Neurog3) null mice lack enteroendocrine cells, Neurog3-expressing progenitors derived from the common granulocytic progenitor are thought to produce the enteroendocrine lineage, although more recent work indicates that Neurog3+ progenitors also contribute to non-enteroendocrine lineages. We aimed to test this model and better characterize the progenitors leading from the stem cells to the enteroendocrine lineage. We investigated clones derived from enteroendocrine precursors and found no evidence of a common granulocytic progenitor that routinely yields all granulocytic lineages. Rather, enteroendocrine cells are derived from a short-lived bipotential progenitor whose offspring, probably via Notch signaling, yield a Neurog3+ cell committed to the enteroendocrine lineage and a progenitor committed to the columnar lineage. The Neurog3+ cell population is heterogeneous; only about 1/3 are slowly cycling progenitors, the rest are postmitotic cells in early stages of enteroendocrine differentiation. No evidence was found that Neurog3+ cells contribute to non-enteroendocrine lineages. Revised lineage models for the small intestinal epithelium are introduced.     

Virulence strategies of the dominant USA300 lineage of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA)

Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious threat to worldwide health. Historically, MRSA clones have strictly been associated with hospital settings, and most hospital-associated MRSA (HA-MRSA) disease resulted from a limited number of virulent clones. Recently, MRSA has spread into the community causing disease in otherwise healthy people with no discernible contact with healthcare environments. These community-associated MRSA clones (CA-MRSA) are phylogenetically distinct from traditional HA-MRSA clones, and CA-MRSA strains seem to exhibit hypervirulence and more efficient host : host transmission. Consequently, CA-MRSA clones belonging to the USA300 lineage have become dominant sources of MRSA infections in North America. The rise of this successful USA300 lineage represents an important step in the evolution of emerging pathogens and a great deal of effort has been exerted to understand how these clones evolved. Here, we review much of the recent literature aimed at illuminating the source of USA300 success and broadly categorize these findings into three main categories: newly acquired virulence genes, altered expression of common virulence determinants and alterations in protein sequence that increase fitness. We argue that none of these evolutionary events alone account for the success of USA300, but rather their combination may be responsible for the rise and spread of CA-MRSA.     

Analysis of neural crest cell lineage and migration.

In this review, we describe the results of recent experiments designed to investigate various aspects of neural crest cell lineage and migration. We have analyzed the lineage of individual premigratory neural crest cells by injecting a fluorescent lineage tracer dye, lysinated fluorescein dextran, into cells within the dorsal neural tube. Individual clones contained cells that were located in very diverse sites consistent with their being sensory neurons, prepigment cells, Schwann cells, adrenergic cells, and neural tube cells. These results suggest that some neural crest cells in the trunk and cranial regions are multipotent prior to their emigration from the neural tube. The environment through which neural crest cells move influences both the pattern and direction of their migration. We have shown that the sclerotomal portion of the somites are responsible for the rostrocaudal pattern of trunk neural crest cell movement, whereas the neural tube appears to govern the dorsoventral position of neural crest-derived ganglia. In addition, the notochord inhibits the movement of neural crest cells. In order to understand necessary cell-matrix interactions in neural crest migration, we have performed perturbation experiments, in which antibodies directed against cell surface or extracellular matrix molecules were introduced along neural crest pathways. We find that integrins, fibronectin, laminin, and tenascin all play some role in cranial neural crest emigration. Thus, multiple factors may be involved in controlling neural crest cell migration, and different factors may be important for migration in different regions of the embryo.     

Envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1 West Nile virus strains

The introduction of West Nile virus (WNV) into North America has been associated with relatively high rates of neurological disease and death in humans, birds, horses, and some other animals. Previous studies identified strains in both genetic lineage 1 and genetic lineage 2, including North American isolates of lineage 1, that were highly virulent in a mouse neuroinvasion model, while other strains were avirulent or significantly attenuated (D. W. C. Beasley, L. Li, M. T. Suderman, and A. D. T. Barrett, Virology 296:17-23, 2002). To begin to elucidate the basis for these differences, we compared a highly virulent New York 1999 (NY99) isolate with a related Old World lineage 1 strain, An4766 (ETH76a), which is attenuated for mouse neuroinvasion. Genomic sequencing of ETH76a revealed a relatively small number of nucleotide (5.1%) and amino acid (0.6%) differences compared with NY99. These differences were located throughout the genome and included five amino acid differences in the envelope protein gene. Substitution of premembrane and envelope genes of ETH76a into a NY99 infectious clone backbone yielded a virus with altered in vitro growth characteristics and a mouse virulence phenotype comparable to ETH76a. Further site-specific mutagenesis studies revealed that the altered phenotype was primarily mediated via loss of envelope protein glycosylation and that this was associated with altered stability of the virion at mildly acidic pH. Therefore, the enhanced virulence of North American WNV strains compared with other Old World lineage 1 strains is at least partly mediated by envelope protein glycosylation.     

Cell lineage and segmentation in the leech

Segments in the leech arise by the proliferation of longitudinally arrayed bandlets of blast cells derived from ten identifiable embryonic stem cells, two M, two N, four O/P and two Q teloblasts. In each bandlet, older blast cells lie ahead of those born later. By using microinjected cell lineage tracers it was shown previously that the teloblasts give rise to characteristic cell patterns made up of segmentally iterated complements of progeny designated as M, N, O, P and Q kinship groups. When a teloblast is injected after it has begun generating blast cells, a boundary is observed later in development between anterior, unlabelled progeny of blast cells produced before injection and posterior, labelled progeny of blast cells produced after injection. We have examined such boundaries in detail to establish the precise relationship between blast cell clones and segments, with the following conclusions: (i) in the M, O and P cell lines, one blast cell generates one segmental complement of progeny, but serially homologous blast clones intermix so that no segment boundaries can be defined based on primary blast cell clones; (ii) in the N and Q cell lines, two blast cells are required to generate a complete segmental complement of progeny; (iii) in the process of forming the germinal plate, cells derived from the N and Q teloblasts move past those derived from the M and O/P teloblasts, so that consegmental blast cell clones do not come into register until well after the establishment of segmentally iterated units within each bandlet.     

Propagation of human parvovirus B19 in primary culture of erythroid lineage cells derived from fetal liver.

Erythroid lineage cells derived from fetal liver were demonstrated to be target cells for human parvovirus B19 infection. B19 virus antigen-positive serum was inoculated into primary cultures containing erythroid lineage cells enriched from fetal liver. The B19 virus antigen was detected on about 5% of cells in the culture by immunofluorescence staining, and the stained cells were identified as erythroid lineage cells by double staining with anti-B19 virus-positive serum and anti-erythroid lineage monoclonal antibody. The immunofluorescence staining study also revealed that the B19 virus antigen localized in the nucleus and the periphery of cytoplasm. We also detected B19 virus DNA, which was generated by replication in the infected cells, not only in the cells but also in the culture supernatants, in which the amount of B19 DNA increased depending on the period of culture, indicating that the cells infected with B19 virus produced B19 virus and released it into the medium. The ability of B19 virus released into the medium to infect fetal erythroid lineage cells was demonstrated quantitatively. Because of the absence of any cytopathic effect of B19 virus during culture periods of at least 15 days, this culture system should be useful in the study of B19 virus replication and in vitro generation of B19 virus. In addition, the present study may contribute to a better understanding of the pathogenesis of hydrops fetalis, which is probably associated with B19 virus infection during pregnancy.     

Allelic exclusion at the TCR delta locus and commitment to gamma delta lineage: different modalities apply to distinct human gamma delta subsets

Expression of a beta-chain, as a pre-TCR, in T cell precursors prevents further rearrangements on the alternate beta allele through a strict allelic exclusion process and enables precursors to undergo differentiation. However, whether allelic exclusion applies to the TCR delta locus is unknown and the role of the gamma delta TCR in gamma delta lineage commitment is still unclear. Through the analysis of the rearrangement status of the TCR gamma, delta, and beta loci in human gamma delta T cell clones, expressing either the TCR V delta 1 or V delta 2 variable regions, we show that the rate of partial rearrangements at the delta locus is consistent with an allelic exclusion process. The overrepresentation of clones with two functional TCR gamma chains indicates that a gamma delta TCR selection process is required for the commitment of T cell precursors to the gamma delta lineage. Finally, while complete TCR beta rearrangements were observed in several V delta 2 T cell clones, these were seldom found in V delta 1 cells. This suggests a competitive alpha beta/gamma delta lineage commitment in the former subset and a precommitment to the gamma delta lineage in the latter. We propose that these distinct behaviors are related to the developmental stage at which rearrangements occur, as suggested by the patterns of accessibility to recombination sites that characterize the V delta 1 and V delta 2 subsets.     

Spontaneous substitutions in the vicinity of the V3 analog affect cell tropism and pathogenicity of simian immunodeficiency virus.

Simian immunodeficiency virus (SIV) exists within tissues of infected macaques as a mixture of diverse genotypes. The goal of this study was to investigate the biologic significance of this variation in terms of cellular tropism and pathogenicity. PCR was used to amplify and clone 3'-half genomes from the spleen of an immunodeficiency SIV-infected pig-tailed macaque (Macaca nemestrina). Eight infectious clones were generated by ligation of respective 3' clones into a related SIVsm 5' clone, and virus stocks were generated by transient transfection. Four of these viruses were infectious for macaque peripheral blood mononuclear cells (PBMC) or monocyte-derived macrophages (MDM). Three viruses with distinct tropism for macaque PBMC or MDM were tested for in vivo infectivity and pathogenicity. The ability of these three viruses to infect PBMC and macrophages correlated with differences in infectivity and pathogenicity. Thus, a virus that was infectious for both PBMC and MDM was highly infectious for macaques and induced AIDS in half of the inoculated animals. In contrast, virus that was less infectious for PBMC and not infectious for MDM induced only transient viremia. Finally, a virus that was not infectious for either primary cell type did not infect macaques. Chimeric clones exchanging portions of the envelope gene of the 62A and smH4 molecular clones and a series of point mutants were used to map the determinant of tropism to a 60-amino-acid region of gp120 encompassing the V3 analog of SIV. Naturally occurring mutations within this region were critical for determining tropism and, as a result, pathogenicity of these SIVsm clones.     

Engrailed expression in the anterior lineage compartment of the developing wing blade of Drosophila.

The developing wing of Drosophila melanogaster was examined at larval and pupal stages of development to determine whether the anterior-posterior lineage boundary, as identified by lineage restrictions, was congruent with the boundaries defined by the expression of posterior-specific (engrailed, invected), and anterior-specific (cubitus interruptus-D) genes. The lineage boundary was identified by marking mitotic recombinant clones, using an enhancer trap line with ubiquitous beta-gal expression in imaginal tissues; clones of +/+ cells were identified by their lack of beta-gal expression. Domains of gene expression were localized using antibodies and gene specific lacZ constructs. Surprisingly, it was found that engrailed expression extended a small distance into the anterior lineage compartment of the wing blade, as identified with anti-en/inv mAb, anti-en polyclonal antiserum, or an en-promoter-lacZ insert, ryxho25. This anterior expression was not present in early third instar discs, but appeared during subsequent larval and pupal development. In contrast, the expression of cubitus interruptus-D, as identified using the ci-Dplac insert, appeared to be limited to the anterior lineage compartment. Thus, en expression is not limited to cells from the posterior lineage compartment, and en and ci-D activities can overlap in a region just anterior to the lineage compartment boundary in the developing wing. The lineage boundary could also be identified by a line of aligned cells in the prospective wing blade region of wandering third instar discs. A decapentaplegic-lacZ construct was expressed in a stripe several cells anterior to the lineage boundary, and did not define or overlap into the posterior lineage compartment.     

Muscle fiber pattern is independent of cell lineage in postnatal rodent development.

Muscle fibers specialized for fast or slow contraction are arrayed in characteristic patterns within developing limbs. Clones of myoblasts analyzed in vitro express fast and slow myosin isoforms typical of the muscle from which they derive. As a result, it has been suggested that distinct myoblast lineages generate and maintain muscle fiber pattern. We tested this hypothesis in vivo by using a retrovirus to label myoblasts genetically so that the fate of individual clones could be monitored. Both myoblast clones labeled in muscle in situ and clones labeled in tissue culture and then injected into various muscles contribute progeny to all fiber types encountered. Thus, extrinsic signals override the intrinsic commitment of myoblast nuclei to particular programs of gene expression. We conclude that in postnatal development, pattern is not dictated by myoblast lineage.     

Effect of vaccine use in the evolution of Mexican lineage H5N2 avian influenza virus

An outbreak of avian influenza (AI) caused by a low-pathogenic H5N2 type A influenza virus began in Mexico in 1993 and several highly pathogenic strains of the virus emerged in 1994-1995. The highly pathogenic virus has not been reported since 1996, but the low-pathogenic virus remains endemic in Mexico and has spread to two adjacent countries, Guatemala and El Salvador. Measures implemented to control the outbreak and eradicate the virus in Mexico have included a widespread vaccination program in effect since 1995. Because this is the first case of long-term use of AI vaccines in poultry, the Mexican lineage virus presented us with a unique opportunity to examine the evolution of type A influenza virus circulating in poultry populations where there was elevated herd immunity due to maternal and active immunity. We analyzed the coding sequence of the HA1 subunit and the NS gene of 52 Mexican lineage viruses that were isolated between 1993 and 2002. Phylogenetic analysis indicated the presence of multiple sublineages of Mexican lineage isolates at the time vaccine was introduced. Further, most of the viruses isolated after the introduction of vaccine belonged to sublineages separate from the vaccine's sublineage. Serologic analysis using hemagglutination inhibition and virus neutralization tests showed major antigenic differences among isolates belonging to the different sublineages. Vaccine protection studies further confirmed the in vitro serologic results indicating that commercial vaccine was not able to prevent virus shedding when chickens were challenged with antigenically different isolates. These findings indicate that multilineage antigenic drift, which has not been observed in AI virus, is occurring in the Mexican lineage AI viruses and the persistence of the virus in the field is likely aided by its large antigenic difference from the vaccine strain.     

Postembryonic lineages of the Drosophila brain: II. Identification of lineage projection patterns based on MARCM clones

The Drosophila central brain is largely composed of lineages, units of sibling neurons derived from a single progenitor cell or neuroblast. During the early embryonic period, neuroblasts generate the primary neurons that constitute the larval brain. Neuroblasts reactivate in the larva, adding to their lineages a large number of secondary neurons which, according to previous studies in which selected lineages were labeled by stably expressed markers, differentiate during metamorphosis, sending terminal axonal and dendritic branches into defined volumes of the brain neuropil. We call the overall projection pattern of neurons forming a given lineage the “projection envelope” of that lineage. By inducing MARCM clones at the early larval stage, we labeled the secondary progeny of each neuroblast. For the supraesophageal ganglion excluding mushroom body (the part of the brain investigated in the present work) we obtained 81 different types of clones. Based on the trajectory of their secondary axon tracts (described in the accompanying paper, Lovick et al., 2013), we assigned these clones to specific lineages defined in the larva. Since a labeled clone reveals all aspects (cell bodies, axon tracts, terminal arborization) of a lineage, we were able to describe projection envelopes for all secondary lineages of the supraesophageal ganglion. This work provides a framework by which the secondary neurons (forming the vast majority of adult brain neurons) can be assigned to genetically and developmentally defined groups. It also represents a step towards the goal to establish, for each lineage, the link between its mature anatomical and functional phenotype, and the genetic make-up of the neuroblast it descends from.     

Cell lineage in the rat cerebral cortex: a study using retroviral-mediated gene transfer

We have used a retroviral vector that codes for the bacterial enzyme beta-galactosidase to study cell lineage in the rat cerebral cortex. This vector has been used to label progenitor cells in the cerebral cortices of rat embryos during the period of neurogenesis. When these embryos are allowed to develop to adulthood, the clones of cells derived from the marked progenitor cells can be identified histochemically. In this way, we can ask what are the lineage relationships between different neural cell types. From these studies, we conclude that there are two distinct types of progenitor cells in the developing cortex. One generates only grey matter astrocytes, whereas the second gives rise to neurones - both pyramidal and nonpyramidal - and to another class of cells that we have tentatively identified as glial cells of the white matter. We have also been able to address the question of how neurones are dispersed in the cortex during histogenesis. It had been previously hypothesized that clonally related neurones migrated radially to form columns in the mature cortex. However, we find that clones of neurones do not form radial columns; rather, they tend to occupy the same or neighbouring cortical laminae and to be spread over several hundreds of micrometers of cortex in the horizontal dimension. This spread occurs in both mediolateral and rostrocaudal directions.