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PubMed2003

The horseradish peroxidase technique for cell lineage studies.

The identification of cell lineage for a given cell type of a particular tissue is an important step in understanding how this process contributes to histogenesis. The importance in understanding cell lineage has relevance for both theoretical and practical reasons. For example, delineating and identifying histogenic principals is required to advance stem cell research and tissue regeneration. To document cell lineage in a given experimental preparation, a number of techniques have been developed. This typically requires the injection of a tracer compound into a founder cell. As this cell produces progeny, the tracer is passed on to the daughter cells. By detecting the tracer in the progeny or daughter cells the investigator can determine which cells originated from the cell that was originally injected with the tracer. By using such an approach it is possible to trace the developmental path from the first cells of the embryo to the specialized cells making the tissue of the adult. A number of tracer compounds have been used with good results in lineage tracing. One of these tracer compounds is horseradish peroxidase (HRP). Several variations of the technique are available depending on what species are studied or what histological requirements are made by the study. A basic technique that can be adapted to individual needs is presented. Included in this protocol on lineage tracing are the procedures for injection, fixation, and the microscope evaluation of labelled cells in the Helobdella triseralis embryo. A brief discussion of the technique will note its advantages and disadvantages. Examples of labelled cell preparations are given to illustrate the technique.

Animals↗
PubMed1996

Lineage involvement by BCR/ABL in Ph+ lymphoblastic leukemias: chronic myelogenous leukemia presenting in lymphoid blast vs Ph+ acute lymphoblastic leukemia.

Chronic myelogenous leukemia (CML) can sometimes present in lymphoid blast phase (L-BP), and can be difficult to distinguish from Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). Some have suggested that the determination of cell lineages involved by the Ph chromosome may be used for distinguishing CML presenting in L-BP (presumably multilineage disease) from Ph+ ALL (presumably lymphoid-restricted), although others have suggested the term 'stem cell ALL' for the multilineage process. Because it has been difficult to perform lineage studies of the Ph chromosome, we investigated the use of fluorescence in situ hybridization (FISH) with probes for BCR (on chromosome 22) and ABL (on chromosome 9) to study lineage involvement in Ph+ lymphoblastic malignancies. We analyzed routine blood and marrow specimens from eight patients who presented with Ph+ lymphoblastic leukemia and found that FISH recognized the 9;22 translocation, distinguished between the two common molecular variants, and readily identified multilineage vs lymphoblast-restricted disease. In our series, four patients had multilineage and four had lymphoblast-restricted disease. Multilineage disease was associated with morphologic features of CML at diagnosis and/or reversion to chronic phase CML after treatment leading us to consider it as CML presenting in L-BP. Patients with lymphoid-restricted disease lacked such findings. The survival of three of our four patients with multilineage disease was prolonged, at 25, 28+, and 126+ months, and when data from our entire series are added to those of 18 previously reported cases that were studied for lineage involvement (reviewed in Leukemia 1993; 7: 147), the difference in overall survival between patients with multilineage and lymphoblast-restricted disease is significant (median overall survival of 47 months vs 8 months, respectively; P=0.013, log rank). Our findings illustrate that FISH analysis can be used to recognize lineage involvement in patients presenting with Ph+ lymphoblastic malignancies, and they provide further support to the notion that multilineage and lymphoblast-restricted disease are distinct clinically as well as biologically.

Adolescent↗
PubMed1997

Combination of osteoinductive bone proteins differentiates mesenchymal C3H/10T1/2 cells specifically to the cartilage lineage.

During embryonic development, cartilage formation involves the condensation of mesenchymal stem cells and a series of maturation steps that ultimately results in the mineralized hypertrophic chondrocyte. The embryonic, murine, mesenchymal stem cell line, C3H/10T1/2, is pluripotent; exposure to azacytidine or to bone morphogenetic protein-2 or -4 results in low rates of differentiation to three mesengenic lineages. In contrast to previous studies, we report conditions for 10T1/2 differentiation specifically to the cartilage lineage and at high yields. These conditions include high cell density micromass cultures, a purified mixture of osteoinductive-proteins (BP; Intermedics Orthopedics, Denver, CO), a serum substitute, 50 micrograms/ml ascorbic acid, and 10 mM beta-glycerophosphate. The cartilagenous fate was confirmed by 1) histological detection of sulfated proteoglycans, 2) electron microscopic detection of proteoglycan and rounded cells separated by extracellular matrix containing short, disorganized collagen fibrils, 3) morphological detection of chondrocytes surrounded by a territorial matrix and encompassed within a distinct perichondrium, and 4) immunocytochemical detection of type II collagen and link protein. After 4 weeks in culture, mature although unmineralized cartilage was observed, as indicated by hypertrophic morphology, immunocytochemical detection of osteocalcin, and histological detection of lacunae. These conditions promote overt chondrogenesis for most of the treated cells and preclude lineage determination to the fat, muscle, and bone lineage, as assayed by electron microscopy and histomorphology. The faithful recapitulation of cartilage differentiation that we have established in vitro provides a versatile alternative to the use of chondrocyte and limb bud explant cultures. We propose this as a model system to study the factors that regulate commitment to the chondrogenic lineage, exclusion to related mesengenic pathways, and maturation during chondrogenesis.

Adipocytes↗
PubMed1997

Regulation of thymocyte lineage commitment by the level of classical protein kinase C activity.

Thymocyte-positive selection involves signaling through TCR and accessory molecules, and the signaling intensity appears to be critical for this event. The specific inhibitor of classical Ca2+-dependent protein kinase C (cPKC), Gö 6976, inhibited positive selection in fetal thymus organ culture, indicating that cPKC activation is essential for positive selection. The major protein kinase C isoforms in CD4+ CD8+ thymocytes are cPKC-alpha, cPKC-beta, and the novel Ca2+-independent protein kinase C, nPKC-epsilon. To analyze the effect of cPKC activation level on positive selection, we used thymocytes from TCR transgenic mice with nonselecting and RAG-2 -/- backgrounds as they were developmentally arrested at the CD4+ CD8+ stage without positive selection signals. These thymocytes survived and acquired CD4/CD8 lineage commitment in suspension culture upon transient stimulation with limited concentrations of the selective activator of cPKC-alpha and -beta, thymeleatoxin, and the calcium ionophore, ionomycin. However, neither 12-deoxyphorbol 13-phenylacetate 20-acetate, which selectively activates cPKC-beta, nor ingenol 3,20-dibenzoate, which selectively activates nPKC-epsilon, exerted such an effect. The thymeleatoxin/ionomycin concentrations corresponded to those that inhibit glucocorticoid-induced apoptosis in thymocytes and were lower than those that induce proliferation of mature T cells. The CD4 lineage commitment required a higher level of cPKC activity than the CD8 lineage commitment. CD8alpha or CD4 mRNA expression was down-regulated. Functional helper and killer T cells were induced from the CD4 and CD8 lineage-committed cells, respectively, by additional stimulation. These results suggest that thymocyte lineage commitment in positive selection is regulated by the level of cPKC-alpha activity or by the levels of cPKC-alpha and -beta activities.

Animals↗
PubMed1997

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↗
PubMed1999

The mucous neck cell in the human gastric corpus: a distinctive, functional cell lineage.

There is considerable debate about whether the mucous neck cell (MNC) in the mucosa of the gastric corpus is merely a transit cell population, intermediate between gastric stem cells and the differentiated zymogenic (chief or peptic) cell lineages, or has distinct functions of its own. To cast light on these possibilities, the secretory phenotype of the MNC has been examined. Archival gastric body samples from non-ulcer dyspepsia biopsies and gastrectomies performed for peptic ulcer disease were stained with antibodies to the trefoil peptides TFF1/pS2 and TFF2/SP, pancreatic secretory trypsin inhibitor (PSTI), epidermal growth factor (EGF) and its receptor (EGFR), and to the MUC1 gene product--HMFG2. Human MNCs express PSTI, TFF1/pS2, TFF2/SP, and EGF proteins, while rat MNCs express TFF2/SP; the mucin contained in the MNCs is diastase/periodic acid Schiff (D/PAS)-positive and stains with human milk fat globulin (HMFG2). The canaliculi but not the cytoplasm of adjacent parietal cells were also decorated focally by D/PAS, by HMFG2, and by antibodies to TFF2/SP and TFF1/pS2. These findings favour the hypothesis that MNCs have a defined phenotype and are thus a separate and distinct cell lineage, secreting a number of luminally-active peptides which protect the gastric mucosa, and in particular the adjacent parietal cells, from the effects of secreted gastric acid. Moreover, a considerable degree of similarity in secretory profile is noted between MNCs and the so-called 'reparative lineages' in the gut--the ulcer-associated cell lineage (UACL) and hyperplastic polyp epithelium. If, on the other hand, the MNCs are indeed a transit population differentiating into zymogenic or peptic cells, then it is clear that having differentiated into one secretory phenotype producing a range of peptides, the MNC then proceeds to differentiate into a cell with a totally different secretory phenotype, a phenomenon unique in gastrointestinal cell lineage relationships.

Animals↗
PubMed1999

Lineage-related susceptibility of human hemopoietic cell lines to apoptosis.

Apoptosis plays a fundamental role in shaping normal hematopoiesis. We have investigated the relationship existing between susceptibility to apoptosis and lineage commitment in hemopoietic cells. The presence and degree of apoptosis were investigated in myeloid (HL-60 and K562), T (Jurkat and MOLT-4), and B (CESS and Raji) lymphoid cell lines by using a variety of techniques-transmission electron and light microscopy, flow cytometry and DNA gel electrophoresis. The major achievement of this study is that hematopoietic cells respond to different chemical (staurosporin, tiazofurin, camptothecin) and physical (hyperthermia or hypothermia) stimuli by apoptosis in a lineage-related way. Moreover, with respect to the methods used to detect apoptosis, a strong correlation was observed between the presence of the hypodiploid peak determined by flow cytometry and the DNA laddering evaluated by gel electrophoresis, but both techniques failed to demonstrate the presence of apoptosis in some cases. We conclude that cells of different hematopoietic lineages mostly show a lineage-related behaviour in their apoptotic response to different stimuli, suggesting that the lineage commitment and the stage of differentiation can confer different sensitivities to specific apoptotic stimuli. Moreover, morphological techniques still represent the most reliable approach to detect apoptosis in hemopoietic cells.

Apoptosis↗
PubMed1990

Cell lineage markers in human pancreatic cancer.

The normal pancreas consists of three major cell types or lineages that share a common embryologic origin from pluripotent endodermal precursors. The type of cell that undergoes neoplastic transformation to form a pancreatic carcinoma is controversial and may influence the phenotype and biologic behavior of the tumor. In this study, immunohistologic techniques were used to determine the cell lineage differentiation expressed in 29 primary exocrine pancreatic adenocarcinomas, five metastatic exocrine pancreatic adenocarcinomas, and five islet cell neoplasma. Specimens of normal pancreas and chronic pancreatitis were used for comparison. The cell lineage markers consisted of monoclonal and polyclonal antibodies against trypsin and lipase (acinar cells); secretory component, carbonic anhydrase II, and pancreatic cancer mucin SPan-1 (ductal cells); and chromogranin-A and somatostatin (islet cells). The expression of carcinoembryonic antigen (CEA) and lysozyme were also determined. This collection of markers allowed the differentiation between acinar, ductal, and islet cells of normal pancreas and chronic pancreatitis specimens. The expression of cell lineage markers in islet cell tumors was homogeneous and restricted to chromogranin-A. In contrast, the expression of these markers in primary and metastatic exocrine pancreatic adenocarcinomas was variable. Reactivity with monoclonal anti-CEA was absent in normal pancreas, and was present in 83% of chronic pancreatitis specimens as well as 90% of exocrine pancreatic adenocarcinomas. In addition, lysozyme reactivity was absent in normal pancreas; however, lysozyme was expressed in one case of chronic pancreatitis, 17 cases of primary carcinoma, and three cases of metastatic carcinoma. These findings support the concept that the original transformed cell type in many pancreatic exocrine carcinomas resemble endodermal "stem cells" that retain the capability of differentiation along more than one cell lineage pathway.

Biomarkers↗
PubMed1992

Commitment to the T cell receptor-alpha beta or -gamma delta lineages can occur just prior to the onset of CD4 and CD8 expression among immature thymocytes.

Two types of T lymphocytes, distinguishable by their surface expression of either the gamma delta or the alpha beta T cell receptor (TcR) for antigen, populate the periphery in the adult. In addition, immature precursors of both T cell types can be found in the thymus. While it is generally accepted that these two cell types represent distinct lineages, it is not known at which developmental stage these lineages diverge. The most mature thymocyte precursor population not yet expressing T lineage-specific surface markers (i.e. CD3, CD4, and CD8) is known to be capable of generating TcR-alpha beta T cells, and has been thought to be preprogrammed into the TcR-alpha beta lineage at an earlier developmental stage. We now show that this late-stage precursor is capable of giving rise to cells of both the TcR-alpha beta and -gamma delta lineages, both in vitro after intrathymic transplantation, and in vitro in simple culture medium or medium with cytokines. Thus it appears that the divergence of TcR-alpha beta and -gamma delta cells can occur at a relatively late stage of intrathymic development, just prior to the onset of CD4 and CD8 expression in most cells.

Animals↗
PubMed1995

Intrathymic T cell receptor (TcR) targeting in mice lacking CD4 or major histocompatibility complex (MHC) class II: rescue of CD4 T cell lineage without co-engagement of TcR/CD4 by MHC class II.

A critical step during intrathymic T cell development, termed positive selection, is associated with rescue of short-lived, immature thymocytes from programmed cell death, T cell lineage commitment, and induction of lineage-specific differentiation programs. T cell receptor (TcR)-major histocompatibility complex (MHC) interactions during positive selection can be closely mimicked by targeting TcR on immature thymocytes to cortical epithelial cells in situ via hybrid antibodies. Here, we show that antibody-mediated TcR signaling in mice deficient for CD4 or MHC class II expression induces polyclonal differentiation of the CD4 T cell lineage. Following a single TcR signal pulse in situ, a temporal sequence of phenotype changes can be discerned: CD69 up-regulation (< 1 day), CD8 down-regulation, TcR up-regulation (1-1.5 days) and down-regulation of the heat-stable antigen (1.5-2 days). Differentiation of phenotypically and functionally mature CD4 T cells in situ is attained within 3 days. Rescue of CD4 lineage T cells in the absence of TcR/CD4 co-engagement by MHC class II in this experimental system supports the stochastic/selective model of T cell lineage commitment.

Animals↗
PubMed1991

Tracing glial cell lineages in the mammalian forebrain.

Astrocytes and oligodendrocytes emerge in late gestational and early post-natal development in the mammalian CNS. The nature, and number, of progenitors for each glial type is a central question. This review will focus upon several unresolved issues relating to glial cell lineages and describe new methods to try to illuminate these issues further: 1) How can developmental patterns by which immature neuroectodermal cells give rise to classes of neurons and glia be understood in the context of lineage? 2) What are the lineage relationships among the various cell classes, how many glial lineages are there in the developing CNS, and how can recent methods of clonal analysis using stable markers be used to clarify lineage patterns? 3) Do patterns of gliogenesis vary in different regions of the CNS? 4) How do patterns of gliogenesis observed in vitro relate to those in vivo?

Animals↗
PubMed1991

Division of astroblasts and oligodendroblasts in postnatal rodent brain: evidence for separate astrocyte and oligodendrocyte lineages.

What precursor cells are the source of the macroglia generated during postnatal development? In order to answer this question, we studied the expression of glial specific antigens in proliferating neuroglia in postnatal rodent brain and optic nerve. Immunocytochemistry using antibodies to oligodendrocyte (OL) specific markers (sulfatide and galactocerebroside) and an astrocyte (AS) specific marker (glial fibrillary acidic protein) was combined with thymidine autoradiography. During the first week of postnatal development when most ASs are being generated, one third to one half of the proliferating cells in the optic system are positive for glial fibrillary acidic protein after a 1 h injection of thymidine (Skoff, Dev. Biol., 139:149-168, 1990). During the second postnatal week when OLs are being generated, 30 to 100% of the proliferating cells in presumptive white matter tracts are sulfatide positive and at least 10% are galactocerebroside positive. This finding demonstrates that ASs and OLs divide during postnatal development. These results confirm previous electron microscopic autoradiographic studies showing that the vast majority of proliferating cells in postnatal rat optic nerve have the morphologic characteristics of differentiating ASs or OLs (Skoff, J. Comp. Neurol., 169:291-312, 1976). Since proliferating ASs (astroblasts) and OLs (oligodendroblasts) constitute the majority of the dividing cells at the time that ASs and OLs are being generated, these glioblasts must be the major source for the macroglia generated postnatally. The findings strongly suggest that separate lineages exist for ASs and OLs during postnatal development. There is no compelling in vivo evidence for a bipotential progenitor cell that generates the majority of OLs and certain ASs in postnatal rodent brain. There may, of course, be distinct lineages for the subtypes of ASs and possibly even for subtypes of OLs. We review the concepts of commitment and plasticity and apply these terms to glial differentiation. In situ, the presence of oligodendroblasts and astroblasts demonstrates the COMMITMENT of proliferating cells to a specific glial lineage during normal development. Culture conditions may provide an environment that permits proliferating glial cells to vacillate in their selection of a specific lineage. This situation demonstrates developmental PLASTICITY and the ability of glia to adapt to an altered environment. Whether committed glial cells in situ can be induced to switch their lineage when normal CNS conditions are altered is an intriguing question that remains to be answered.

Adaptation, Physiological↗
PubMed1991

Diversification of glial lineages: a novel method to clone brain cells in vitro on nitrocellulose substratum.

We have developed a novel in vitro method to analyze the diversification of glial cells during development. The primary advantage of the approach is that glial lineages are formed in discrete clones on a nitrocellulose substratum where the relationship of the progeny is strictly defined. This method facilitates the comparison of a large complement of astrocyte and oligodendrocyte lineages under controlled conditions. Clones were formed by plating a brain dissociate on nitrocellulose at very low density (5,000-40,000 cells/154 mm2). However, growth depended on diffusible factors produced by brain cells growing under the nitrocellulose support at high density (feeder layer). The cloning efficiency of cells from mouse forebrain (P0) was 1-3%. This means we can detect 100,000 to 300,000 clonal progenitors in the dissociate (10(7) cells per forebrain) using the clonal culture technique. Cell phenotypes were determined by immunocytochemical staining with anti-glial fibrillary acidic protein (GFAP) to label astrocytes and anti-galactocerebroside (GC) and anti-myelin basic protein (MBP) to label oligodendrocytes. There was a remarkable diversity of glia represented in different lineages. The number of astrocyte clones was greater than the number of oligodendrocyte clones but combined their total was 90%. Clone sizes were distributed over a wide range, which indicated that growth rates varied. Clones appeared compact or dispersed but astrocyte clones exhibited three different morphologies-fibroblast-like, stellate, and elongated. Oligodendrocytes had different morphologies distinct from astrocytes. Although there were different glial lineages the cells in most clones were homogeneous, indicating the progeny had the same fate. However, a small number of the clones, approximately 2%, were heterogeneous and contained both astrocytes and oligodendrocytes. The application of this technique to glial lineages demonstrates that intrinsic factors have a role in determining cell fate since different clones formed under the same external conditions. Finally, these results are consistent with the existence of multiple glial progenitors or the continued presence of multipotential progenitors at the time of birth.

Animals↗
PubMed1994

Identification of pro-thymocytes in murine fetal blood: T lineage commitment can precede thymus colonization.

Phenotype and commitment of thymus-colonizing precursors are unknown. Here we report the identification of T lineage-committed precursors (designated prothymocytes) in murine fetal blood at day 15.5 of development. Fetal blood pro-thymocytes are Thy-1+c-kit(low)CD3- in contrast to fetal blood-derived pluripotent hematopoietic progenitors which are Thy-1-c-kit+. Upon transfer into the thymus, fetal blood pro-thymocytes generate a single wave of CD4+CD8+ thymocytes and subsequently mature TCR alpha beta+ peripheral T cells. However, fetal blood pro-thymocytes lack multipotent progenitor potential since they fail to reconstitute B lymphocytes and myeloid and erythroid lineages. In contrast, T and B lymphocytes as well as myeloid and erythroid lineages are reconstituted from fetal blood-derived pluripotent progenitors. Pro-thymocytes are equally present in peripheral blood of athymic fetal mice, suggesting that this novel precursor population is T lineage-committed prior to thymus colonization and represents the earliest T lineage precursor identified.

Animals↗
PubMed2003

Genetic variability among serotype G6 human rotaviruses: identification of a novel lineage isolated in Hungary.

Rotavirus serotype G6 has been demonstrated to be a rare cause of gastroenteritis in man. To date, only a few well characterized strains have been described from Italy, Australia, and the United States. Nucleotide sequencing of G6 VP7 genes shows that these strains belong to two distinct G6 lineages, one for strains of serotype P11[14],G6 (PA169-like strains) and one for strains of serotype P3[9],G6 (PA151-like strains). In this study, we sequenced the VP7 genes and VP8* gene fragments of human rotavirus G6 strains detected in Hungary. Phylogenetic analysis demonstrated that the VP7 genes of Hungarian G6 strains fell into three lineages, represented by a single PA169-like strain, three PA151-like strains, and two novel G6 strains, respectively. The amino acid sequence identity of VP7 was 97.2-100% within each lineage and 92-93.9% between any two lineages. The sequence analysis of VP8* revealed that the single PA169-like Hungarian G6 strain belonged to genotype P[14] and was phylogenetically closely related to P11[14],G6 strains characterized previously. In contrast, the VP8* of PA151-like Hungarian G6 strains clustered in accordance with their VP7 genes representing genetically distinguishable variants of genotype P[9]. This finding raises the possibility that Hungarian genotype P[9],G6 strains might have been generated through independent reassortment events. Serotype G6-specific primers for each human G6 lineage were also developed. The use of these primers in reverse-transcription polymerase chain reaction genotyping may help determine the epidemiological role of G6 strains in humans.

Amino Acid Sequence↗
PubMed1995

CD34+ cell expansion and expression of lineage markers during liquid culture of human progenitor cells.

A 96-well-based suspension culture system for human hematopoietic progenitor cells has been developed to monitor the commitment and differentiation of CD34+ cells to specific lineages and the maintenance and expansion of CD34+ cells in vitro. The expression of maturation and lineage markers on the cells in culture was measured by enzyme-linked immunosorbent assay (ELISA). CD34+ cells were isolated from umbilical cord blood and fetal liver (90% purity) and were grown in liquid culture in 96-well plates for 10 days. The cells were then fixed with a glutaraldehyde-paraformaldehyde mixture, attaching the cells firmly to the plastic. An ELISA was performed, using appropriate primary antibodies directed against cell surface markers. The expression of four different lineage markers was measured: CD14 (monocyte), CD15 (neutrophil), platelet glycoprotein (GP) IIb/IIIa (CD41a, megakaryocyte) and glycophorin A (erythroid). The two-growth factor combination of interleukin 3 (IL-3) and stem cell factor (SCF) stimulated expression of CD14, CD15 and GP IIb/IIIa. Lineage-restricted growth factors such as erythropoietin (EPO), in combination with SCF, stimulated expression of glycophorin A. The three-factor combination of IL-3, SCF and EPO stimulated expression of all four lineage markers. Other multiple growth factor combinations all stimulated myeloid and megakaryocyte growth, as measured by ELISA and flow cytometry, but erythroid growth was present only when EPO was included in the growth factor mixture. In serum-free medium or plasma-containing medium, CD14 expression was markedly reduced, whereas glycophorin A expression was greatly elevated in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗
PubMed1993

Genetic analysis of developmental mechanisms in hydra. XXI. Enhancement of regeneration in a regeneration-deficient mutant strain by the elimination of the interstitial cell lineage.

The interstitial cell lineage, including interstitial stem cells, nerve cells, and nematocytes, was eliminated from a regeneration-deficient mutant strain (reg-16) of Hydra magnipapillata. The resultant interstitial cell lineage-free (or "epithelial") reg-16 animals showed a marked enhancement in the ability to regenerate head structures. The epithelial reg-16 polyps regenerated nearly the same number of tentacles as was originally present within 8 days after head removal, while interstitial cell lineage-containing (or "complete") reg-16 polyps restored less than one-third of their original tentacle number under the same conditions. Lateral tissue transplantation was used to examine the head activation and inhibition potentials. The gradients of the two potentials along the body axis of intact epithelial 105 (a wild-type strain) and intact epithelial reg-16 polyps were nearly identical to the gradients in their complete counterparts. The changes of the two potentials occurring after head removal in the epithelial 105 animals were also similar to those in the complete 105 animals. However, the postdecapitation changes in the epithelial reg-16 polyps were different from those in complete reg-16 polyps. The changes in the epithelial reg-16 animals were similar to those observed in wild-type hydra while those of complete reg-16 polyps were highly abnormal. These observations suggest that the phenotypic expression of the genetic defect present in the reg-16 mutant strain is attenuated when the interstitial cell lineage is eliminated from its tissue. The role of the interstitial cell lineage in head regeneration and the nature of the defect present in the reg-16 strain are discussed based on the observations made in this and previous related studies.

Animals↗
PubMed1996

A contact-dependent animal-to-vegetal signal biases neural lineages during Xenopus cleavage stages.

The central nervous system (CNS) of Xenopus is derived from three of four tiers of blastomeres of the 32-cell embryo, and each blastomere in these tiers produces a characteristic number of primary spinal neurons. The C-tier blastomeres constitute the boundary between those that contribute to the CNS (A-, B-, and C-tiers) and those that do not (D-tier). To test whether the neural lineages descended from the C-tier are established by intrinsic information or by cell-cell interactions, single B-tier blastomeres were deleted and the lineage of their C-tier neighbors mapped. The contributions of C-tier blastomeres to subdivisions of the CNS and to specific spinal neurons were significantly reduced. Contributions of these blastomeres to other tissues were mostly normal, indicating that those C-tier progeny that no longer contribute to CNS are distributed in small numbers throughout the rest of the clone. To test whether the changes in neural lineages after B-tier deletions were the result of the C-tier blastomere changing position, contacting new neighbors, or losing contact with inductive B-tier neighbors, intact embryos were transiently dissociated within their vitelline membranes at different time points prior to the midblastula transition. This treatment disrupted cell-cell contact, but not gap junction-mediated dye coupling or the positions of neighboring cells. C-tier CNS lineages were reduced as after deletion of the B-tier neighbor, suggesting that the neural fate of C-tier cells depends upon specific B-tier interactions. To determine whether these interactions occurred specifically between B-tier and C-tier neighbors, barriers were inserted transiently between individual B/C pairs; similar reductions in C-tier CNS lineages were observed. These data demonstrate that an animal-to-vegetal, contact-dependent signal passes from B-tier to C-tier blastomeres and is required for the normal C-tier contribution to the CNS. This cell-cell interaction occurs many hours before the onset of zygotic transcription or neural induction and may bias the field of cells that can respond to neural induction.

Animals↗