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Cell lineage analysis of the amphipod crustacean Parhyale hawaiensis reveals an early restriction of cell fates.

In the amphipod crustacean, Parhyale hawaiensis, the first few embryonic cleavages are total and generate a stereotypical arrangement of cells. In particular, at the eight-cell stage there are four macromeres and four micromeres, and each of these cells is uniquely identifiable. We describe our studies of the cell fate pattern of these eight blastomeres, and find that the eight clones resulting from these cells set up distinct cell lineages that differ in terms of proliferation, migration and cell fate. Remarkably, the cell fate of each blastomere is restricted to a single germ layer. The ectoderm originates from three of the macromeres, while the remaining macromere generates the visceral mesoderm. Two of the micromeres generate the somatic mesoderm, a third micromere generates the endoderm and the fourth micromere generates the germline. These findings demonstrate for the first time a total cleavage pattern in an arthropod which results in an invariant cell fate of the blastomeres, but notably, the cell lineage pattern of Parhyale reported shows no clear resemblance to those found in spiralians, nematodes or deuterostomes. Finally, the techniques we have developed for the analysis of Parhyale development suggest that this arthropod may be particularly useful for future functional analyses of crustacean development.

Animals↗

Two subpopulations of stem cells for T cell lineage.

An assay system for the stem cell that colonizes the thymus and differentiates into T cells was developed, and by using this assay system the existence of two subpopulations of stem cells for T cell lineage was clarified. Part-body-shielded and 900-R-irradiated C57BL/6 (H-2b, Thy-1.2) recipient mice, which do not require the transfer of pluripotent stem cells for their survival, were transferred with cells from B10 X Thy-1.1 (H-2b, Thy-1.1) donor mice. The reconstitution of the recipient's thymus lymphocytes was accomplished by stem cells in the donor cells and those spared in the shielded portion of the recipient that competitively colonize the thymus. Thus, the stem cell activity of donor cells can be evaluated by determining the proportion of donor-type (Thy-1.1+) cells in the recipient's thymus. Bone marrow cells were the most potent source of stem cells, the generation of donor-derived T cells being observed in two out of 14 recipients transferred with as few as 1.5 X 10(4) cells. The stem cell activity of spleen cells was estimated to be about 1% of that of bone marrow cells, and no activity was found in thymus cells. By contrast, when the stem cell activity was compared between spleen and bone marrow cells of whole-body-irradiated (800 R) C57BL/6 mice reconstituted with B10 X Thy-1.1 bone marrow cells by assaying in part-body-shielded and irradiated C57BL/6 mice, the activity of these two organs showed quite a different time course of development. Spleen cells showed a markedly high level of activity 7 days after the reconstitution, followed by a decline, whereas the activity of bone marrow cells was very low on day 7 and increased crosswise. The results strongly suggest that the stem cells for T cell lineage in the bone marrow comprise at least two subpopulations, spleen-seeking and bone marrow-seeking cells. Such patterns of compartmentalization of stem cells in the spleen and bone marrow of irradiated recipients completely conform to the general scheme of the relationship between restricted stem cells and less mature stem cells, including pluripotent stem cells, which became evident in other systems such as in the differentiation of spleen colony-forming cells or of stem cells for B cell lineage.

Animals↗

Growth of human T-cell lineage acute leukemia in severe combined immunodeficiency (SCID) mice and non-obese diabetic SCID mice.

Primary leukemic cells from patients with acute lymphoblastic leukemia (ALL) can be injected intravenously into mice with severe combined immunodeficiency (SCID) to create a model of human leukemia. Leukemic cells disseminate to murine tissues in a clinicopathologic pattern similar to that seen in humans. Thus far, reports of engraftment of lymphoid leukemia in SCID mice have mainly been from patients with B-cell lineage ALL, for which engraftment occurs more frequently with cells from high-risk patients. There are few data on the engraftment of T-cell lineage ALL in SCID mice. Leukemic cells from 19 patients (16 adult and three pediatric) with T-cell lineage ALL were injected into SCID mice, with overt engraftment of 12 cases (63%). Engraftment of leukemia in SCID mice was associated with earlier death due to leukemia of the patient donors (P < .01, log-rank test). The recently developed non-obese diabetic (NOD)/SCID mouse may expand the uses of the SCID model. Cells from the seven patients with T-cell lineage ALL that failed to cause leukemia in SCID mice were injected into NOD/SCID mice. Overt leukemia engraftment was observed in all seven cases. Thus, growth of human T-cell lineage ALL cells in SCID mice was associated with a high-risk patient group. However, this association was not observed when NOD/SCID mice were used, suggesting that this model would no longer predict patients likely to die early of leukemia, but may provide a more realistic system for studying the biology and treatment of the disease.

Acute Disease↗

Preferential proliferation of immature B lineage cells in long-term stromal cell-dependent cultures with IL-4.

IL-4 influences the cellular composition of stromal cell dependent long term cultures. In bone marrow-derived long term lymphoid cultures initiated in presence of IL-4, the majority of cells exhibited a more immature phenotype than is usually seen in lymphoid cells grown in Whitlock-Witte type cultures. This immature cell population, lacking the B220 Ag, was purified by cell sorting. When transferred to mixed stromal layers used in conventional lymphoid long term cultures, these cells differentiated into B lineage cells that could be identified by expression of the B220 Ag and surface IgM. Abelson murine leukemia virus-transformed cell lines resulting from this immature cell population express a DJH rearrangement and contained RNA that hybridized with a VJ558 probe, suggesting transcription of germ-line V genes. A culture modification allowed selective proliferation of a non-transformed cell population with characteristics of very immature B lineage cells. The proliferation of these cells was supported by a homogeneous stromal cell line that was propagated with horse serum in presence of IL-4. The lymphoid cells proliferating under those culture conditions expressed the Ag detected by the BP-1 and 6C3 mAb and were Fc gamma RII and Ia-positive. However, more mature B cell markers were lacking. DNA analysis of these cell lines revealed JH rearrangement without evidence for deletion of any member of the DSP-2 family. These cell lines retained their immature phenotype after transfer to mixed stromal layers of Whitlock-Witte type. The mechanisms providing these unique culture conditions initiated by IL-4 in bone marrow stromal cells are discussed.

Abelson murine leukemia virus↗

SMAD1 signaling is critical for initial commitment of germ cell lineage from mouse epiblast.

Commitment of the germ cell lineage during embryogenesis depends on zygotic gene expression in mammals, but little is known about the signaling molecules required for germ cell formation. Here we show that the intracellular signaling molecule SMAD1, acting downstream of bone morphogenetic protein (BMP) receptors, is required for the commitment of germ cell lineage from epiblast in early mouse embryos. Smad1 homozygous mutant embryos (Smad1-/-) were generated by in-frame insertion of lacZ gene into an exon of the Smad1 gene. Most of the Smad1-/- embryos contained no primordial germ cells (PGCs) and had short allantois, while histological analysis and in situ hybridization for the mesoderm marker genes revealed that early mesoderm induction was normal in those embryos. Smad1 expression was observed in epiblast and in visceral endoderm during gastrulation, while only a few alkaline phosphatase-positive PGCs at 7.5 and 8.5 days post coitum (E7.5 and E8.5) expressed Smad1. Phosphorylated SMAD proteins were localized in the proximal region of epiblast at E6.0-6.5, where the progenitors of PGCs and of allantois reside. Single-cell reverse transcription-polymerase chain reaction analysis revealed that the expression of Smad1, -5 and -8 were sporadic and mutually independent in proximal epiblast cells. We also found that BMP4-induced differentiation of PGCs from epiblast in vitro was fully dependent on the existence of phosphorylated SMAD1. These results indicate that SMAD1 signaling possesses a critical and non-redundant function in the initial commitment of the germ cell lineage.

Alkaline Phosphatase↗

Establishment of a permanent rat brain-derived glial cell line as a source of purified oligodendrocyte-type 2 astrocyte lineage cell populations.

A permanent glial cell line (L3) has been established from mixed glial cultures obtained from neonatal rat forebrain by repetitive passaging and selection of the process-bearing cells growing on top of a flat cell monolayer. Continuous propagation of the process-bearing cells was supported by the flat cells, of presumed astroglial origin, which were present in negligible amounts following each passage but then grew and formed a basal, feeder layer. Throughout a culture period of over 2 years, the L3 cells have maintained a stable morphological and antigenic phenotype. In serum-containing culture medium, most of the process-bearing cells expressed at the same time features of immature oligodendrocytes (O4 positivity) and of astrocytes [glial fibrillary acidic protein (GFAP) positivity]. A smaller proportion of them was labeled by the monoclonal antibody LB1. LB1+ or O4+ cells were rarely GFAP-, and GFAP+ cells were rarely LB1- or O4-. GalC+ oligodendrocytes were seen only occasionally, but the proportion of these cells increased up to 30% upon culturing in chemically defined medium containing 0.5% fetal calf serum. The L3 process-bearing cells accumulated the neurotransmitter gamma-aminobutyric acid (GABA), expressed the proteoglycan chondroitin sulfate, and responded to the mitogenic action of platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). All these properties are characteristic of cells belonging to the O-2A (oligodendrocyte-type 2 astrocyte) cell lineage. The L3 flat cells were largely negative for the glial markers tested, but resembled type 1 astrocytes in their ability to support the growth of O-2A lineage cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

The embryonic cell lineage of the nematode Caenorhabditis elegans.

The embryonic cell lineage of Caenorhabditis elegans has been traced from zygote to newly hatched larva, with the result that the entire cell lineage of this organism is now known. During embryogenesis 671 cells are generated; in the hermaphrodite 113 of these (in the male 111) undergo programmed death and the remainder either differentiate terminally or become postembryonic blast cells. The embryonic lineage is highly invariant, as are the fates of the cells to which it gives rise. In spite of the fixed relationship between cell ancestry and cell fate, the correlation between them lacks much obvious pattern. Thus, although most neurons arise from the embryonic ectoderm, some are produced by the mesoderm and a few are sisters to muscles; again, lineal boundaries do not necessarily coincide with functional boundaries. Nevertheless, cell ablation experiments (as well as previous cell isolation experiments) demonstrate substantial cell autonomy in at least some sections of embryogenesis. We conclude that the cell lineage itself, complex as it is, plays an important role in determining cell fate. We discuss the origin of the repeat units (partial segments) in the body wall, the generation of the various orders of symmetry, the analysis of the lineage in terms of sublineages, and evolutionary implications.

Animals↗

Morphologic and morphometric study of the two main cell lineages involved in mycosis fungoides: the lymphoid cells and the Langerhans cells.

A comparative light and electron microscopic study was carried out on skin biopsies from 5 mycosis fungoides patients (stages II and III). In addition, certain zinc-iodide-osmium (ZIO) procedures that selectively reveal the Langerhans cells (LC) were also applied. Several morphometric analyses, such as relative frequency, cell size, nuclear:cell ratios and nuclear contour indexes, were performed. Two main cell lineages were found in the dermal infiltrate: the lymphoid cells, represented by lymphoblastlike cells (LB), lymphocytes (L), and mycosis fungoides cells (MFC), and the Langerhans cells. A pattern of cellular organization of the dermal infiltrate was recognized. The LC appeared as the "organizing" cell. The morphometric study indicated that all LB, L, and MFC have the chance, at the same time, to be in contact with a LC. The application of the ZIO procedures clearly indicated a "translocation" of the LC, since they were scarce or absent from the epidermis and numerous in the dermis. Evidence is presented indicating that differentiation of LB into L and malignant transformation of L into MFC are processes occurring predominantly in the dermis. It is postulated that in the chronic stages of mycosis fungoides, L-LC contacts occur mainly in the dermis and not in the epidermis and that the development of malignant clones may occur in the dermis.

Aged↗

Flow karyology of serially cultured Chinese hamster cell lineages.

Flow karyology of serially cultured Chinese hamster cell lineages has been observed to be influenced by the degree of cellular heterogeneity in culture. The minimum coefficient of variation (CV) and debris fraction obtainable vary as a cell lineage evolves from a primary cell culture to an established cell line. The cell lineages pass through a stage of decreased cellular heterogeneity from which flow karyotypes can be obtained with lower CV and debris fraction. The influence of cellular heterogeneity on flow karyology is observed with constant preparative protocol and constant instrument performance, and can be an additional source of variability.

Animals↗

NIH gerontology research support: policy evaluation of in vitro cell lineage studies.

Populations of cultured human diploid cells, used extensively in research on aging, are not well understood in terms of kinetics and mitotic activity of constituent cells. NICHD has supported, by contract, cell lineage studies to elucidate such cell characteristics contributing to the cell population degeneration and death. The policy of NICHD (NIA) encouragment of such studies has been evaluated. A need for cell lineage and related data from cultured-cell populations is recognized, and research necessary to achieve these data will be encouraged. Grant applications to this end are sought.

Cell Line↗

Chromosomal excision of TCRdelta chain genes is dispensable for alphabeta T cell lineage commitment.

TCRbeta, delta and gamma chain genes are assembled and expressed in double-negative thymocytes prior to alphabeta or gammadelta T cell lineage commitment. Thus, cells committed to the alphabeta T cell lineage can possess completely assembled TCRdelta and/or TCRgamma chain genes. However, these genes are not expressed. TCRgamma chain gene expression may be silenced through the activity of a cis-acting silencer element. In the TCRalpha/delta locus, the TCRdelta genes lie between the Valpha and Jalpha gene segments, which rearrange by deletion. Moreover, Valpha to Jalpha rearrangements occur on both alleles in essentially all developing alphabeta T cells. Consequently, both TCRdelta chain genes are excised from the chromosome and placed on extrachromosomal circles in mature alphabeta T cells. It has been proposed that this excision process is important for silencing TCRdelta gene expression and permitting alphabeta T cell lineage commitment. A gene-targeting Cre-loxP strategy was used to invert a 75-kb region of the TCRalpha/delta locus encompassing all the Jalpha gene segments, generating the TCRalpha/delta(I) allele. Initial Valpha to Jalpha rearrangements on the TCRalpha/delta(I) allele occur by inversion, resulting in chromosomal retention of TCRdelta chain genes. These TCRdelta chain genes can be productively rearranged and are expressed at levels similar to TCRdelta chain genes in gammadelta T cells. However, alphabeta T cell development appears unperturbed in TCRalpha/delta(I/I) mice. Thus, excision of TCRdelta genes from the chromosome per se is not required for commitment of developing lymphocytes to the alphabeta T cell lineage.

Alleles↗

Violation of cell lineage restriction compartments in the chick hindbrain.

Previous cell lineage studies indicate that the repeated neuromeres of the chick hindbrain, the rhombomeres, are cell lineage restriction compartments. We have extended these results and tested if the restrictions are absolute. Two different cell marking techniques were used to label cells shortly after rhombomeres form (stage 9+ to 13) so that the resultant clones could be followed up to stage 25. Either small groups of cells were labelled with the lipophilic dye DiI or single cells were injected intracellularly with fluorescent dextran. The majority of the descendants labelled by either technique were restricted to within a single rhombomere. However, in a small but reproducible proportion of the cases (greater than 5%), the clones expanded across a rhombomere boundary. Neither the stage of injection, the stage of analysis, the dorsoventral position, nor the rhombomere identity correlated with the boundary crossing. Judging from the morphology of the cells, both neurons and non-neuronal cells were able to expand over a boundary. These results demonstrate that the rhombomere boundaries represent cell lineage restriction barriers which are not impenetrable in normal development.

Animals↗

Apoptosis in rodent peri-implantation embryos: differential susceptibility of inner cell mass and trophectoderm cell lineages--a review.

Inner cell mass (ICM) and trophectoderm cell lineages diverge early in cleavage in response to a complex combination of cellular and molecular determinative events. The resulting differences in metabolic requirements, cell positioning and micro-environments are considered as some of the major causes underlying the differential sensitivity of ICM and trophectoderm cell lines to embryotoxic agents. In most instances, ICM cells appear less resistant to disruption than trophectoderm cells, and past observations suggest that over-stimulation of apoptosis is probably one of the mechanisms leading to selective ICM depletion at the blastocyst stage. Disproportionate deficiency in this lineage below a certain threshold level may then prevent the ICM core from providing sufficient prefetal stem cells during gastrulation and from sending regulatory signals to the trophectoderm, leading to compromised post-implantation development. The aim of this review article is to discuss the above observations and to show the value of the impact of hyperglycaemia on blastocyst metabolism and development as an exciting model for further studies.

Animals↗

Differentiation in culture of murine primitive lymphohematopoietic progenitors toward T-cell lineage.

Earlier, we described a stromal cell-free two-step clonal culture system in which murine primitive lymphohematopoietic progenitors produce myeloid and B-lymphoid lineage cells. In the same culture T-cell potential of the progenitors was maintained. We now report that, in addition to myeloid and B-lymphoid cells, putative T-cell progenitors are also produced in culture. Lineage-negative (Lin-) Ly-6A/E+ c-kit+ bone marrow cells from 5-fluorouracil-treated mice were cultured in methylcellulose in the presence of SF (Steel factor), interleukin (IL)-11, and IL-7, and the resulting primary colonies were picked and pooled. When injected into severe combined immune deficiency (scid) mice, the pooled cells reconstituted the T-cell compartment of the scid mice earlier than freshly prepared primitive marrow cells. This reconstitution activity of the pooled primary colony cells was enriched in the Ly-6A/E+ and FcgammaRII/III-/low cell fractions. Reverse transcriptase-polymerase chain reaction (RT-PCR) and DNA-PCR analyses showed that some of the primary colony cells are differentiated sufficiently to express messenger RNA (mRNA) of T-cell receptor (TCR) beta-chain and pre-TCR alpha (pTalpha) and, although not frequently, to perform Dbeta-Jbeta rearrangement of the TCR gene. Micromanipulation studies confirmed the clonal origin of myeloid lineage cells and the cells positive for the T-cell-specific transcripts and D-J rearrangement of TCR beta-chain. These results suggested that, in the presence of SF, IL-11, and IL-7, primitive lymphohematopoietic progenitors differentiate toward T-cell lineage in addition to myeloid and B-cell lineages.

Animals↗

Sertoli cell-specific expression of rat androgen-binding protein in transgenic mice: effects on somatic cell lineages.

The Sertoli cells of many species produce an androgen binding protein (ABP) which carries testicular androgens to the epididymis and is thought to play a role in sperm maturation. In the present report we analyzed the morphological modifications present in Leydig, Sertoli, and peritubular cells of the testis of young adult male mice transgenic for ABP gene, which overproduce ABP in testis. By in situ hybridization we demonstrated that ABP is specifically produced by Sertoli cells. Using light and electron microscopy, we detected scattered alterations of the seminiferous tubule cells which include cell degeneration and vacuolization. Leydig and Sertoli cells present morphological signs of hyperfunctioning compensatory mechanisms which include increased amounts of lipid droplets probably due to the existence of a stimulated steroid synthesis that in turn could be a consequence of the decreased unbound testosterone and/or a direct paracrine effect of ABP. Peritubular cells also present numerous signs of hyperstimulation.

Androgen-Binding Protein↗

Mixed chimerism in the B cell lineage is a rapid and sensitive indicator of minimal residual disease in bone marrow transplant recipients with pre-B cell acute lymphoblastic leukemia.

One of the major problems after allogeneic bone marrow transplantation (BMT) is a high frequency of leukemia relapse. We have prospectively studied the presence of donor- and recipient-derived chimeric cells in bone marrow recipients with pre-B cell acute lymphoblastic leukemia (pre-B-ALL). The chimeric status of BMT recipients was compared to minimal residual disease (MRD) detection by analysis of immunoglobulin heavy chain (IgH) and T cell receptor (TcR) genes. Post-transplant blood and bone marrow samples from 12 patients with pre-B-ALL were studied. Five patients showed mixed chimerism (MC) in the CD19-positive cell fraction. Four of them have relapsed to date. The remaining patient with MC in the B cell lineage was also MRD positive in the same samples. All seven patients with donor chimerism in the B cell fraction remain in clinical remission (P = 0.01). In samples from all five patients having MC in the B cell lineage, the patient-specific IgH or TcR rearrangement was also detected. In three of four patients who relapsed, MC in the B cell lineage was seen more than 2.5 months prior to morphologically verified relapse. The results of this comparison suggest that routinely performed MC analysis of the affected cell lineage may facilitate post-BMT monitoring and rapid therapeutic decisions in transplanted patients with pre-B-ALL.

Adolescent↗

Soluble interleukin-6 (IL-6) receptor/IL-6 fusion protein enhances in vitro differentiation of purified rat oligodendroglial lineage cells.

We investigated the effects of a chimeric protein (IL6RIL6 chimera) containing interleukin-6 (IL-6) fused to its soluble receptor (sIL-6R) on the proliferation and/or differentiation of rat oligodendrocyte progenitor cells (OPCs) and on oligodendrocyte survival. Exposure of OPCs to IL6RIL6 chimera for 48 h induced a dose-dependent decrease of bromodeoxyuridine (BrdU) incorporation. IL6RIL6 chimera treatment for 48 h also strongly increased the reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) by mitochondrial enzymes and enhanced oligodendrocyte staining with a mitochondrial fluorescent dye. A strong, dose-dependent increase in the number and length of processes immunostained for early (galactocerebroside) or late (myelin basic protein) oligodendrocyte differentiation markers was revealed after OPC treatment with IL6RIL6 chimera for 2-7 days, respectively. Moreover, treatment with IL6RIL6 chimera improved oligodendrocyte survival. The chimera-induced increase of oligodendrocyte arborization was mimicked, although with lower efficacy, by ciliary neurotrophic factor (CNTF) but not by IL-6 and was reduced in the presence of a gp130 soluble peptide which is able to inhibit the gp130-mediated signals of the IL-6/sIL-6R complex. Oligodendrocyte treatment with IL6RIL6 chimera for 30 min induced both signal transducer and the activator of transcription-1 (STAT-1) and STAT-3 phosphorylation and nuclear translocation. We conclude that, by interacting with membrane gp130 and possibly by activating Janus kinase/STAT pathways, IL6RIL6 chimera induces OPCs to differentiate into mature oligodendrocytes, promotes their survival, and could deserve investigation as a therapeutic strategy for enhancing remyelination.

Animals↗

Cell lineage analysis of maize bundle sheath and mesophyll cells.

Maize leaves are divided into repeated longitudinal units consisting of vascular tissue, bundle sheath (BS), and mesophyll (M) cells. We have carried out a cell lineage analysis of these cell types using six spontaneous striping mutants of maize. We show that certain cell division patterns are preferentially utilized, but not required, to form the characteristic arrangement of cell types. Our data suggest that early in development a central cell layer is formed, most frequently by periclinal divisions in the adaxial subepidermal layer of the leaf primordium. Lateral and intermediate veins are initiated in this central layer, most often by divisions which contribute daughter cells to both the procambium and the ground meristem. These divisions generate "half vein" units which comprise half of the bundle sheath cells around a vein and a single adjacent M cell. We show that intermediate veins are multiclonal both in this transverse direction and along their lengths. BS cells are more closely related to M cells in the middle layer of the leaf than to those in the upper and lower subepidermal layers. An examination of sector boundaries has shown that photosynthetic differentiation in M cells is affected by the phenotype of neighboring BS cells.

Cell Differentiation↗