Early fetal acquisition of the chromaffin and neuronal immunophenotype by human adrenal medullary cells. An immunohistological study using monoclonal antibodies to chromogranin A, synaptophysin, tyrosine hydroxylase, and neuronal cytoskeletal proteins.

The development of chromaffin and neuronal features in the adrenal medulla was studied in normal human fetuses with gestational ages (GAs) of 6-34 weeks. Monoclonal antibodies specific for chromogranin A, synaptophysin, and tyrosine hydroxylase; for different subunits and phosphoisoforms of neurofilament (NF) proteins; and for microtubule-associated proteins were applied. Morphologically, two major cell types could be distinguished, i.e., "large" cells with pale nuclei and ill-defined cytoplasm, which were present from 9 weeks GA on, and clusters of "small," primitive appearing cells, present from 14 weeks GA on. The large cells were immunoreactive for chromogranin A, synaptophysin, tyrosine hydroxylase, and NF proteins, similar to adult chromaffin cells. In contrast, small cells expressed NF proteins and tyrosine hydroxylase, but not chromogranin A or synaptophysin, more resembling ganglion cells in the adult adrenal medulla. At the latest developmental stages large cells were observed in the center of the clusters of "small" cells, which morphologically resembled immature ganglion cells and expressed NF proteins in their perikarya. These observations indicate that chromaffin and ganglion cells establish their immunophenotype early in embryogenesis. They suggest that "large" and "small" cells are progenitors of the chromaffin and the ganglion cells, respectively, of the mature adrenal medulla.

Comparison of V kappa gene family expression in adult and fetal B cells.

The functional B cell repertoires from adult and fetal mice were compared by examining V kappa gene family expression in individual cells. In addition, because little is known about the relative use of the various V kappa gene families in an immune response, adult B cells from several different strains of mice were analyzed. This was accomplished by stimulating B cells with the polyclonal activator, LPS. Activated cells were then analyzed for V kappa gene family expression at the single cell level by in situ hybridization using radiolabeled V kappa gene probes. It was found that all V kappa gene families tested were represented in the LPS-induced adult repertoire with V kappa 1, V kappa 4,5 and V kappa 19 being expressed to the largest degree in all strains tested. The LPS-induced adult V kappa gene family repertoire was then compared to the fetal repertoire and some differences were observed. In particular, a lower proportion of fetal B cells expressed V kappa 1 and a higher proportion of fetal B cells expressed V kappa 4,5 and V kappa 10. Importantly, compared with the adult response there was no evidence in the fetal response for an increased expression of V kappa 21, the family that maps closest to J kappa,C kappa. This is in contrast to what has been shown previously with H chain V region exons in which there was a clear preference for the VH gene families that mapped closest to DH.

A family of octamer-specific proteins present during mouse embryogenesis: evidence for germline-specific expression of an Oct factor.

We have analysed various adult organs and different developmental stages of mouse embryos for the presence of octamer-binding proteins. A variety of new octamer-binding proteins were identified in addition to the previously described Oct1 and Oct2. Oct1 is ubiquitously present in murine tissues, in agreement with cell culture data. Although Oct2 has been described as a B-cell-specific protein, similar complexes were also found with extracts from brain, kidney, embryo and sperm. In embryo and brain at least two other proteins, Oct3 and Oct7, are present. A new microextraction procedure allowed the detection of two maternally expressed octamer-binding proteins, Oct4 and Oct5. Both proteins are present in unfertilized oocytes and embryonic stem cells, the latter containing an additional protein, Oct6. Whereas Oct4 was not found in sperm or testis, it is expressed in male and female primordial germ cells. Therefore Oct4 expression is specific for the female germline at later stages of germ cell development. Our results indicate that a family of octamer-binding proteins is present during mouse development and is differentially expressed during early embryogenesis. Protease clipping experiments of Oct4 and Oct1 suggest that both proteins contain similar DNA-binding domains.

Possible recruitment of osteoblastic precursor cells from hypertrophic chondrocytes during initial osteogenesis in cartilaginous limbs of young rats.

The appearance of the bone phenotype during rat embryogenesis was studied by in situ hybridization using a cDNA clone to osteopontin. Radiolabeled sense and antisense RNA probes were prepared from the osteopontin cDNA by in vitro transcription. The probes were used to hybridize paraffin sections of the cartilaginous diaphysis from embryonic rats at day 17 of gestation. The hybridization pattern was analyzed by autoradiography. Hybridization with the antisense probe gave patterns of silver grain labeling, indicating the presence of osteopontin mRNA among the hypertrophic chondrocytes. No silver grains could be detected in the corresponding region following hybridization of consecutive sections with the sense probe, showing the specificity of the technique being used. Whether these results indicate that the osteopontin gene is transiently expressed by hypertrophic chondrocytes or that osteopontin is an early marker for osteoblastic precursor cells will have to be explored further.

Lineage, migration, and morphogenesis of longitudinal glia in the Drosophila CNS as revealed by a molecular lineage marker.

Previous studies described three different classes of glial cells in the developing CNS of the early Drosophila embryo that prefigure and ensheath the major CNS axon tracts. Among these are 6 longitudinal glial cells on each side of each segment that overlie the longitudinal axon tracts. Here we use transformant lines carrying a P element containing a 130 bp sequence from the fushi tarazu gene in front of the lacZ reporter gene to direct beta-galactosidase expression in the longitudinal glia. Using this molecular lineage marker, we show that 1 of the "neuroblasts" in each hemisegment is actually a glioblast, which divides once symmetrically, in contrast to the typical asymmetric neuroblast divisions, producing 2 glial cells, which migrate medially and divide to generate the 6 longitudinal glial cells. As with neuroblasts, mutations in Notch and other neurogenic genes lead to supernumerary glioblasts. The results indicate that the glioblast is similar to other neuroblasts; however, the positionally specified fate of this blast cell is to generate a specific lineage of glia rather than a specific family of neurons.

Interspersion of the VHQ52 and VH7183 gene families in the NFS/N mouse.

Deletion mapping analysis has shown that members of the VH7183 and VHQ52 gene families are interspersed in the NFS/N mouse. To obtain direct evidence that members of these gene families are physically linked, an NFS/N liver library was constructed and genomic clones were analyzed for hybridization to both VHQ52 and VH7183 gene probes. Four clones were identified which contained both VHQ52 and VH7183 hybridizable restriction fragments. Two clones containing rearranged VHQ52 genes were also found to hybridize with the VH7183 gene probe. Sequence analysis of three of the VH7183-containing restriction fragments indicate that all are pseudogenes which contain interruptions at either the 5' and/or 3' ends of the VH coding region. Given the D-proximal location of at least a portion of the VHQ52 gene family relative to VH7183 in NFS/N mice, and the known correlation between D proximity and the frequency of VH gene utilization, 22 NFS/N-derived pre-B cell lines were analyzed for VHQ52 gene utilization. More than 40% of the identified H chain (VHDJH) rearrangements in this survey used members of this gene family. Furthermore, analysis of poly(A)+ RNA from NFS/N fetal liver and adult spleen also indicates preferential utilization of VHQ52 family in fetal liver. Kinetic studies show, however, that there are no changes in relative utilization throughout fetal ontogeny. The implications of these findings for the expression and randomization of the VH repertoire are discussed.

Application of the ferrocyanide-reduced osmium method for mineralizing cartilage: further evidence for the enhancement of intracellular glycogen and visualization of matrix components.

The ferrocyanide-reduced osmium (FRO) fixation method was applied to neonatal mouse mandibular condylar cartilage for its processing for electron microscopy. The results were compared to those obtained by the conventional glutaraldehyde-osmium tetroxide fixation method. Three different stages in the life cycle of condylar cartilage cells were examined. FRO enabled the visualization of delicate fibrillar mesh in the matrix of all three zones of the cartilage, resulting in a dense appearance of the intercellular matrix. The classical stellate shape of matric granules seen in cartilage fixed with glutaraldehyde-osmium tetroxide was not observed in FRO-processed tissues. Chondrocytes that were FRO-processed almost entirely filled their lacunar space. In their pericellular area, fibrillar material and electron-dense aggregates could be demonstrated by the FRO method. As a conclusion of this study, it is recommended to supplement a conventional protocol with the FRO fixation method for routine and research purposes.

The mast cell-committed progenitor. I. Description of a cell capable of IL-3-independent proliferation and differentiation without contact with fibroblasts.

We have identified a late, committed stage in the differentiation of the mast cell progenitor just before granulation. Mast cell committed progenitors (MCCP) are nongranulated cells with a density of 1.060 to 1.070 g/ml which can be harvested from the mesenteric lymph node of mice infected with Nippostrongylus brasiliensis. Mast cell-committed progenitors are able to proliferate and differentiate in the absence of IL-3 or IL-4 when cultured on a monolayer of embryonic skin or 3T3 fibroblasts and can form colonies in methylcellulose supplemented with fibroblast conditioned medium. Fibroblast conditioned medium appears to contain a soluble MCCP proliferation factor that maintains biologic activity when heated to 56 degrees C for 45 min but is destroyed by incubation with either trypsin or chymotrypsin. It can be selectively precipitated with 60 to 70% saturated ammonium sulfate. The factor is not absorbed by immobilized antibodies to nerve growth factor. The MCCP proliferation activity of the factor could not be mimicked by IL-1, IL-2, IL-4, granulocyte-macrophage-CSF, granulocyte-CSF, macrophage-CSF, IFN-alpha/beta, IFN-gamma, nerve growth factor, epidermal growth factor, serum fibronectin, heparin, or a number of glycosaminoglycans. At high salt concentrations, the factor passes through a 50-kDa membrane and can be concentrated above a 5-kDa membrane. MCCP acquire a connective tissue phenotype when cultured on a fibroblast monolayer and a mucosal phenotype when cloned in the presence of conditioned medium from PWM-stimulated spleen cells. When cultured in the absence of IL-3 on a monolayer of embryonic skin or 3T3 fibroblasts, mast cell-committed progenitors produce mast cells which stain with berberine sulfate suggesting a connective tissue phenotype; however, the mast cells that develop when mast cell-committed progenitors are cultured in the presence of IL-3 or conditioned media from PWM-stimulated spleen cells do not stain with berberine sulfate. MCCP intercalate into monolayers of embryonic skin or 3T3 fibroblasts, but T cells are not able to associate with the monolayer and can be completely washed away. Attempts to enrich mast cell-committed progenitors by intercalation and elution from embryonic skin monolayers proved unsuccessful, but some enrichment of mast cell-committed progenitors could be achieved by discontinuous Percoll gradients. Thus, we have identified a way to obtain late-stage, mast cell-committed progenitors in an environment that is virtually uncontaminated with other hematopoietic progenitors.

PDGF receptors on cells of the oligodendrocyte-type-2 astrocyte (O-2A) cell lineage.

It has been shown previously that cultures of rat optic nerve contain three types of macroglial cells--oligodendrocytes and two types of astrocytes. Type-1 astrocytes develop from their own precursor cells beginning before birth, while oligodendrocytes and type-2 astrocytes develop postnatally from a common bipotential precursor called the O-2A progenitor cell. Proliferating O-2A progenitor cells give rise to postmitotic oligodendrocytes beginning around birth, and to type-2 astrocytes beginning in the second postnatal week. Studies in vitro have suggested that platelet-derived growth factor (PDGF), secreted by type-1 astrocytes, plays an important part in timing oligodendrocyte development: PDGF seems to keep O-2A progenitor cells proliferating until an intrinsic clock in the progenitor cells initiates the process leading to oligodendrocyte differentiation. The clock apparently determines when a progenitor cell becomes unresponsive to PDGF, at which point the cell stops dividing and, as a consequence, automatically differentiates into an oligodendrocyte. Here we have used radiolabelled PDGF to show that O-2A progenitor cells have PDGF receptors, suggesting that these cells respond directly to PDGF. The receptors resemble the type A PDGF receptor previously described on human fibroblasts and are initially retained when progenitor cells stop dividing and develop in vitro into oligodendrocytes. The latter finding indicates that receptor loss is not the reason that progenitor cells initially become mitotically unresponsive to PDGF.

Molecular cloning and characterization of a novel murine T cell surface antigen, YE1/48.

YE1/48 is a murine cell surface disulphide-linked dimeric Ag consisting of two 45,000-50,000 Mr subunits. It is expressed on some T lymphoma lines at high levels but its expression on normal lymphocytes is very low. The functional significance of this Ag is currently unknown. We have now cloned a cDNA encoding the YE1/48. Sequence analysis revealed that it encodes a Type II membrane protein of 262 amino acids (30,500 MW), with 44 amino acids in the N-terminal cytoplasmic domain, 22 amino acids in the transmembrane domain and 196 amino acids in the C-terminal extracellular domain. There are three potential N-linked glycosylation sites in the extracellular domain all of which are probably used in the mature protein. No significant homology can be identified with other known protein sequences in the data base or with human CD28(T44), a human T cell activation antigen consisting of two 44,000 Mr subunits. The protein sequence includes in its extracellular domain the arginine-glycine-aspartic acid tripeptide, a potential cell-adhesive binding site, and a sequence similar to the consensus domain of any metal-binding proteins. However, whether these sequences are functional is unknown. Genomic Southern analysis of C57BL/6, BALB/c and C3H mice has demonstrated a restriction fragment length polymorphism. The analysis has also strongly suggested the existence of some other genes with sequences highly homologous to the YE1/48 gene. The YE1/48 gene appears to be expressed at very low levels in a wide range of lymphoid cells with no restriction to their differentiation stages. Interestingly, YE1/48 expression appears to be induced in pre-B cells after transformation by Abelson virus, suggesting an association of YE1/48 expression with the transformation of T and pre-B Cells.

Bu-1 antigen expression as a marker for B cell precursors in chicken embryos.

Genetically polymorphic cell surface antigen, Bu-1, is expressed on B cells as well as on a subset of macrophages. Bu-1+ cells are also present in embryonic spleen and bone marrow, and these could represent prebursal precursors for B cells and Bu-1+ macrophages. To test the repopulation capacity of these cells we sorted 14-day embryonic spleen cells from Bu-1a-homozygous donors into Bu-1a+ and Bu-1a- fractions and transferred them into age-matched irradiated Bu-1b-homozygous recipients. Four to six weeks after hatching, the recipients were analyzed for Bu-1 chimerism. The results demonstrate that B cell precursors are exclusively present in the Bu-1+ population of 14-day embryonic spleen, whereas the Bu-1+ macrophage subpopulation can be repopulated by either the Bu-1+ or the Bu-1- fraction of these embryonic cells. Bone marrow cells from young chickens could also repopulate the Bu-1+ macrophage subset but not the B cell compartment, thus confirming previous data that postnatal bone marrow does not contain B cell precursors. These results demonstrate that all B cell precursors in the 14-day embryonic spleen carry the Bu-1 antigen, and suggest that there is no lineage relationship between the Bu-1+ cells and macrophages.

MLR3 molecule is an activation antigen shared by human B, T lymphocytes and T cell precursors.

MLR3 molecule is a membrane glycoprotein (mol. mass range 28-34 kDa) present on activated, but not resting human peripheral T cells, B cells and thymocytes. Its kinetics of appearance on the cell surface (3 h after the addition of the inductive signal to the cells) suggests that it is an early activation antigen. The proliferative response of cultured T and B lymphocytes and thymocytes to different activation signals is inhibited by the addition of MLR3 monoclonal antibody. Moreover the antibody in combination with non-mitogenic doses of phorbol myristate acetate leads to proliferation of thymocytes and resting B and T lymphocytes. In the latter, synthesis of interleukin 2 is also induced. Biochemical analysis of MLR3 antigen indicates that it is a phosphorylated protein with N-linked sugar moieties. Together these data suggest a role for MLR3 antigen in the signal transduction process during activation, both for mature lymphocytes and for T cell precursors.

Expression of CSF-1-receptor-related proteins in muscular stem cells.

We previously reported that consistent levels of c-fms-related transcripts are expressed during the growth of rat myogenic cells as well as in all neoplastic myoblasts. The present study extends this observation to mouse myogenic cells and demonstrates that a tyrosine-kinase-associated gp170, very similar or identical to the receptor for the macrophage stimulating factor CSF-1, is synthesized in myoblasts via a short-lived precursor of 115-116 kD and an immature gp130. These gene products are eliminated during the myogenic process, suggesting their role in the proliferation of muscular cells.

Restriction fragment analysis of V preB and lambda 5 within the genus Mus.

DNA from a panel of inbred strains of mice and colony bred mice, isolated from different geographical locations, was hybridized to mouse V preB and lambda 5 probes under stringent conditions, indicating sequence similarities greater than 80%. The probe for lambda 5 detects one gene and the probe for V preB detects two genes (V preB1 and V preB2) in the inbred strains of mice examined under the stringency used. No restriction endonuclease fragment length polymorphisms (RFLP) were detected with the V preB and lambda 5 DNA probes among the inbred strains of mice using Bam HI and Hind III. Very few RFLP were detected among Mus musculus subspecies, and the intensity of the hybridization did not differ significantly with either DNA probe. The number of RFLP increased slightly when different species and subgenera were examined, and the intensity of the hybridization signal began to decrease in samples from the different subgenera, suggesting a slight decrease in sequence similarity for both V preB genes with increased time of divergence. Fewer RFLP were detected with the lambda 5 DNA probe. DNA from 11 different Mus species representing 4 subgenera, genetically isolated from laboratory mice for approximately 1-12 million years, continued to hybridize under high stringency conditions using both DNA probes. A comigrating lambda 5 and V preB restriction endonuclease fragment was detected in most of the samples examined, suggesting the close physical linkage of V preB1 and lambda 5 is maintained within the genus Mus. These results suggest that V preB1, V preB2 and lambda 5 have been present for over 12 million years.

[C3 and IgG Fc receptors of granulocytes in human pathology]. / Receptores para C3 y para Fc de IgG del sector granulocítico en patología humana.

Blood neutrophils and their bone marrow cell progenitors have membrane receptors for C3 and for the Fc portion of IgG. To test possible changes in the expression of those receptors associated to diseases we studied: a) blood of 31 and bone marrow of 9 normal individuals; b) blasts of 29 patients with acute myeloblastic leukemia; c) bone marrow of 8 patients with severe bacterial infections. The receptors were evaluated by rosette techniques: Saccharomyces C3 and sheep erythrocyte specific IgG antibody. Dried droplet stained smears were used to count rosettes of cells at different stages of maturation. The studies disclosed the following: a) these receptors are detected in progressively increasing percentage of cells throughout the differentiation steps of the granulocytic series; receptor for C3 is depicted as starting in promyelocytes, and receptor for Fc in myelocytes; the percentage of bone marrow neutrophils expressing these receptors is lower than that of blood neutrophils; b) in acute myeloblastic leukemia, blasts frequently express receptors normally found at more mature levels of differentiation which is an expression of nucleocytoplasmic asynchronism; there is good correlation between the FAB classification and the expression of these receptors; c) in severe bacterial infections, the receptors are found at earlier stages and in a higher proportion of cells at early maturation steps, marking a shift to the left in the expression of receptors.

Receptores para C3 y para Fc de IgG del sector granulocitico en patologia humana / Receptors for C3 and IgG Fc receptors of the granulocytic sector in human pathologies

Los neutrófilos y sus progenitores medulares tienen receptores para C3 y para Fc de IgG. Con el objeto de estudiar las modificaciones de la expersión de estos receptores en patología se estudiaron: a) sangre de 31 y médula ósea de 9 individuos normales; b) blastos de 29 pacientes de leucemia mieloblástica aguda; c) médula ósea de 8 pacientes con infecciones bacterianas severas. Se trabajó con técnica de rosetas de Saccharomyces-C3 glóbulos rojos de carnero sensibilizados con IgG. Los resultados demuestran que: a) los receptores aparecen gradual y progresivamente en las etapas de diferenciación granulocítica; el receptor de C3 se evidencia en promielocitos y el de Fc en mielocitos; el porcentaje de neutrófilos de médula ósea que expresan estos receptores es inferior al de neutrófilos de la sangre; b) en leucemia mielobástica aguda, a pesar de los signos morfológicos de inmadurez, los blastos expresan frecuentemente receptores que normalmente aparecen en estadios más diferenciados, mostrando otra evidencia de asincronismo núcleocitoplasmático; se observa buena concordancia entre la expresión de receptores y la clasificación FAB; c) en infecciones bacterianas severas aparece desviación izquierda de la expresión de los receptores en la línea de diferenciación de la progenie granulocítica (AU)

Receptores para C3 y para Fc de IgG del sector granulocitico en patologia humana / Receptors for C3 and IgG Fc receptors of the granulocytic sector in human pathologies

Los neutrófilos y sus progenitores medulares tienen receptores para C3 y para Fc de IgG. Con el objeto de estudiar las modificaciones de la expersión de estos receptores en patología se estudiaron: a) sangre de 31 y médula ósea de 9 individuos normales; b) blastos de 29 pacientes de leucemia mieloblástica aguda; c) médula ósea de 8 pacientes con infecciones bacterianas severas. Se trabajó con técnica de rosetas de Saccharomyces-C3 glóbulos rojos de carnero sensibilizados con IgG. Los resultados demuestran que: a) los receptores aparecen gradual y progresivamente en las etapas de diferenciación granulocítica; el receptor de C3 se evidencia en promielocitos y el de Fc en mielocitos; el porcentaje de neutrófilos de médula ósea que expresan estos receptores es inferior al de neutrófilos de la sangre; b) en leucemia mielobástica aguda, a pesar de los signos morfológicos de inmadurez, los blastos expresan frecuentemente receptores que normalmente aparecen en estadios más diferenciados, mostrando otra evidencia de asincronismo núcleocitoplasmático; se observa buena concordancia entre la expresión de receptores y la clasificación FAB; c) en infecciones bacterianas severas aparece desviación izquierda de la expresión de los receptores en la línea de diferenciación de la progenie granulocítica

Normal thymic cortical epithelial cells developmentally regulate the expression of a B-lineage transformation-associated antigen.

Nonlymphoid, stromal cells in the mouse thymus are believed to be important in T cell maturation and have been proposed to play a central role in the acquisition of major histocompatibility complex (MHC) restriction and self-tolerance by maturing thymocytes. Both cortical and medullary epithelial cells in the thymus express high levels of class II (A) major histocompatibility antigens (MHC Ags). We show here that a specific subset of these A+ epithelial cells express a transformation-associated antigen (6C3Ag) found previously on the surfaces of Abelson murine leukemia virus-transformed pre-B cells and on those bone marrow-derived stromal cell clones which support normal and preneoplastic pre-B cell proliferation. Among solid lymphoid organs, only the thymus contains 6C3Ag+ cells and within the thymus, this antigen is found exclusively on A+ epithelial cells in cortical regions. It is striking that the expression of the 6C3Ag on thymic epithelium is developmentally regulated, suggesting a role for this lymphostromal antigen in the maturation of the thymic microenvironment.

Purification of murine hemopoietic stem cells and committed progenitors by fluorescence activated cell sorting using wheat germ agglutinin and monoclonal antibodies.

Two procedures to purify and separate pluripotent hemopoietic stem cells (PHSC) and committed progenitor cells from mouse bone marrow cells, and a three colour stem cell staining procedure are described. Visser et al., (J. Exp. Med. 59, 1576-1590, 1984) described the purification of PHSC by metrizamide density gradient centrifugation followed by wheat germ agglutinin-FITC (WGA-FITC) and light scatter sorting using a fluorescence activated cell sorter (FACS). The light density, WGA-positive, high forward (FLS) and low perpendicular light scatter (PLS) blast cells, after removal of the lectin from the sorted cells by the competing sugar, are restained with biotinylated anti-H-2K plus avidin-FITC. The H-2K positive cells proved to be pure pluripotent stem cells. Bauman et al. (J. Cell. Physiol. 128, 133-142, 1986) started by sorting the 6% most positive fluorescent cells with low PLS and high FLS from WGA-FITC stained normal bone marrow. After lectin removal the sorted cells are restained with anti-GM-1.2. Sorted, GM-1.2 negative cells are almost exclusively PHSC plus committed colony forming cells. A three colour staining procedure was designed to measure stem cells in bone marrow samples by flow cytometry. Cells are simultaneously stained with anti-H-2K-biotin plus avidin-phycoerythrin, a rat monoclonal antibody detecting a cell surface antigen plus goat anti rat Ig-FITC and 1-butyryl-pyrene-WGA. Analysis and sorting on the two laser Rijswijk Experimental Light-Activated Cell Sorter (RELACS II) using selected windows indicated that these windows contained high frequencies of PHSC. This multicolour analysis and sorting therefore is equivalent to the multistep sorting procedures.