Restricted Cell Populations Research Articles

Day/night fluctuations in melatonin content, arylalkylamine N-acetyltransferase activity and NAT mRNA expression in the CNS, peripheral tissues and hemolymph of the cockroach, Periplaneta americana

Melatonin content measured by a radioenzymatic assay in the brain of the American cockroach ( Periplaneta americana) showed a day/night fluctuation with higher levels at night under LD 12:12. The activity of arylalkylamine N-acetyltransferase (NAT) in brain was also higher at night and this pattern continued in constant darkness. The results suggest that the rhythmicity in melatonin content can be caused by NAT. Melatonin content in hemolymph showed an even greater day/night difference, more than 12 times that in brain under LD 12:12. Melatonin levels in retina were also higher at night while NAT activity was not significantly higher at night than at daytime. Using a probe designed from NAT cloned from testes we performed Northern blot analysis of total RNA, which revealed that the level of NAT mRNA was higher in midgut, ovary and female accessory glands than in fat body and brain. The level of transcript in midgut was higher at night, but the levels in ovary and female accessory reproductive gland showed the opposite pattern. We also used the antibody to whole Drosophila melanogaster aaNAT1 protein, seeking a homologous antigen in the cephalic ganglia. NAT-like antigen was detected in several restricted populations of cells in the brain that were partially co-localized with PER-like antigen. The results suggest that NAT exists in multiple forms in various tissues of the cockroach and that its functions and regulations can vary among tissues. The results in the brain led to the conclusion that NAT could be a clock-controlled gene functioning as an output regulator of the circadian clock.

Restricted expression of tumor necrosis factor-related apoptosis-inducing ligand receptor 4 in human peripheral blood lymphocytes

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumor but not normal cells, thus providing therapeutic possibilities for human cancers. However, it is not fully clear how widespread TRAIL receptors are, or how TRAIL signaling is modulated in normal cells. We characterized cell surface expression of TRAIL receptors in normal healthy donor peripheral blood and report that each of the TRAIL receptors are characteristically expressed on restricted cell populations. TRAIL-R1 is distinctively expressed on B-lymphocytes, TRAIL-R2 on monocytes, TRAIL-R3 on neutrophils and most impressively, CD8 + lymphocytes and NKT lymphocytes but not CD4 + lymphocytes express TRAIL-R4.

Gene therapy targeting in the central nervous system.

Targeted gene therapy aims at achieving the expression of therapeutic transgenes in specific and restricted cell populations, thus sparing all other cells of the unwanted effects of the gene product. This strategy is particularly appealing for therapy of the central nervous system (CNS), where many different cell types exist, and where the inappropriate expression of a molecule can produce grave consequences. To accomplish the objectives of targeted gene therapy, two different approaches have been developed. The first one consists in creating vectors that will deliver the transgene exclusively to the selected cells, that is manipulating the transductional capacities of the vector, and the second one is based on the transcriptional properties of the transgene, so that it will only be expressed in cells where the appropriate transcriptional machinery is present. Reaching the goals of targeted gene expression will greatly increase the specificity and safety of gene therapy, thus getting us closer to the fulfillment of the expectations generated by this new branch of molecular medicine.

Dynamic Analysis of Dorsal Closure in Drosophila: From Genetics to Cell Biology

Throughout development a series of epithelial bendings, sweepings, and fusions occur that collectively give shape to the embryo. These morphogenetic movements are driven by coordinated assembly and contraction of the actomyosin cytoskeleton in restricted populations of epithelial cells. One well-studied example of such a morphogenetic episode is dorsal closure in Drosophila embryogenesis. This process is tractable at a genetic level and has recently become the focus of live cell biology analysis because of the availability of flies expressing GFP-fusion proteins. This marriage of genetics and cell biology is very powerful and is allowing the dissection of fundamental signaling mechanisms that regulate the cytoskeletal reorganizations and contractions underlying coordinated tissue movements in the embryo.

Regulated gene expression in the chicken embryo by using replication-competent retroviral vectors.

Rous sarcoma virus (RSV)-derived retroviral vector could efficiently deliver the green fluorescent protein (GFP), which is driven by the internal cytomegalovirus enhancer/promoter, into restricted cell populations in the chicken embryo. RSV-derived vectors coupled with the tet regulatory elements also revealed doxycycline-dependent inducible GFP expression in the chicken embryo in ovo.

Cells containing immunoreactive estrogen receptor-α in the human basal forebrain

The distribution of estrogen receptor protein-α (ER-α)-containing cells in the human hypothalamus and adjacent regions was studied using a monoclonal antibody (H222) raised against ER-α derived from MCF-7 human breast cancer cells. Reaction product was found in restricted populations of neurons and astrocyte-like cells. Neurons immunoreactive for ER-α were diffusely distributed within the basal forebrain and preoptic area, infundibular region, central hypothalamus, basal ganglia and amygdala. Immunoreactive astrocyte-like cells were noted within specific brain regions, including the lamina terminalis and subependymal peri-third-ventricular region. These data are consistent with the location of estrogen receptors in the basal forebrain of other species and the known effects of estrogens on the cellular functions of both neurons and supporting elements within the human hypothalamus and basal forebrain.

D-Fenfluramine induces serotonin-mediated Fos expression in corticotropin-releasing factor and oxytocin neurons of the hypothalamus, and serotonin-independent Fos expression in enkephalin and neurotensin neurons of the amygdala

The neurotransmitters expressed by neurons activated by d-fenfluramine (5 mg/kg, i.p.) were identified in the hypothalamus, amygdala and bed nucleus of the stria terminalis. Induction of Fos immunoreactivity following d-fenfluramine injection was used as an index of neuronal activation. To test whether d-fenfluramine activated neurons by releasing serotonin from the serotonergic nerve terminals, rats were pretreated with fluoxetine (10 mg/kg, i.p.), a serotonin reuptake inhibitor that prevents the release of serotonin stimulated by d-fenfluramine, 12 h before d-fenfluramine injection. The approximate percentages of peptidergic neurons that contained Fos immunoreactivity after d-fenfluramine administration were 94% of corticotropin-releasing factor and 22% of oxytocin cells in the paraventricular nucleus of the hypothalamus, 6% of oxytocin cells in the supraoptic nucleus of the hypothalamus, 36% of enkephalin and 15% of neurotensin cells in the central amygdaloid nucleus, and 19% of enkephalin and 9% of neurotensin cells in the bed nucleus of the stria terminalis. Fluoxetine pretreatment blocked Fos expression in corticotropin-releasing factor- and oxytocin-expressing cells in the hypothalamus, but not in enkephalin- and neurotensin-expressing cells located in the bed nucleus of the stria terminalis and central amygdaloid nucleus. d-Fenfluramine did not induce Fos immunoreactivity in vasopressin-, thyrotropin-releasing hormone-, somatostatin- and tyrosine hydroxylase-containing cells in the hypothalamus, and corticotropin-releasing factor-expressing cells in the central amygdaloid nucleus and bed nucleus of the stria terminalis. These results show that d-fenfluramine stimulates corticotropin-releasing factor- and oxytocin-expressing cells in the hypothalamus via serotonin release. The enkephalin- and neurotensin-expressing cells in the amygdala are activated by d-fenfluramine via non-serotonergic mechanisms. Induction of Fos expression by d-fenfluramine in restricted populations of cells suggests a selective activation of neuronal circuitry that is likely to be involved in the appetite suppressant effects of d-fenfluramine.

Expression of zebrafish bHLH genes ngn1 and nrd defines distinct stages of neural differentiation.

Two zebrafish bHLH genes, neurogenin-related gene I (ngn1) and neuroD (nrd), have been isolated. ngn1 expression is initiated at the end of gastrulation in the neural plate and defines broad domains of cells that probably possess an ability to develop as neurons. This finding suggests that ngn1 may play a role during determination of cell fate in neuroblasts. ngn1 and pax-b are expressed in a mutually exclusive manner. nrd expression follows that of ngn1 in restricted populations of cells selected from ngn1-positive clusters of cells. The earliest nrd-positive cells in the brain and the trunk are a subset of the primary neurons. ngn1 is not expressed in the eye. Here, nrd transcription is activated at 25 hours postfertilization in the ventral retina. Expression of islet-1 occurs in nrd-positive cells after expression of nrd, and the expression of the two genes partially overlaps in time. These observations suggest that during eye development nrd expression may follow expression of some other neurodetermination gene(s). This supports the idea that expression of nrd is a necessary step leading toward overt neuronal differentiation.

Glial-Restricted Precursors Are Derived from Multipotent Neuroepithelial Stem Cells

Neuroepithelial cells in the developing ventricular zone differentiate into neurons, astrocytes, and oligodendrocytes. It is not known, however, whether this differentiation occurs in a single step or is a pathway utilizing intermediate more restricted precursor cells. To characterize the generation of glial cells from multipotent stem cells we have cultured neuroepithelial (NEP) cells from E10.5 rat embryos. Cultured NEP cells do not express any glial differentiation markers when grown on fibronectin/laminin under nondifferentiation conditions. NEP cells, however, differentiate into A2B5 immunoreactive cells which can subsequently give rise to oligodendrocytes and astrocytes. Clonal analysis of NEP cells demonstrates that the A2B5 immunoreactive cells arise in clones that contain neurons and astrocytes, indicating that A2B5+cells arise from multipotent NEP precursor cells. A2B5+cells, maintained as undifferentiated cells over multiple passages, can subsequently give rise to both oligodendrocytes and astrocytes. A2B5+cells, however, do not generate neurons. Thus A2B5+cells represent a restricted progenitor cell population that differentiates from a multipotent NEP cell. Based on our results we propose that differentiation of the multipotential NEP cells to terminally differentiated glial cells occurs via intermediate restricted precursors.

Differential cellular distribution of glutathione – an endogenous antioxidant – in the guinea pig inner ear

Electronmicroscopic immunogold cytochemistry was applied to investigate the localization of glutathione (GSH) in the guinea pig inner ear. GSH immunoreactivity was preferentially distributed in basal cells and intermediate cells of the stria vascularis, and in fibrocytes and capillary endothelial cells in the spiral ligament and vestibular endorgans. The present findings suggest that the synthesis of GSH, a peptide known to protect against ototoxic compounds, depends on restricted cell populations in the inner ear.

Localization of natriuretic peptide-activated guanylate cyclase mRNAs in the rat brain.

Physiological actions of atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) are elaborated by membrane-bound natriuretic peptide receptors (NPRs). These receptors possess intracellular guanylate cyclase domains that mobilize cyclic guanosine monophosphate upon binding of peptide. Two distinct NPR subtypes have been described in brain: the NPR-A selectively binds ANP, whereas NPR-B exhibits high affinity for CNP. To define further the potential domains of ANP and CNP action in brain, the present study used in situ hybridization histochemistry to map NPR-A and NPR-B mRNA-expressing cell populations. Significant levels of neuronal NPR-A mRNA expression were observed only in the mitral cell layer of the olfactory bulb, medial habenula, subfornical organ, and area postrema. Expression of NPR-A mRNA was observed in forebrain white matter tracts, suggesting synthesis in glial cells. In contrast, NPR-B mRNA was widely expressed throughout the neuraxis. In the telencephalon, signal was abundant throughout limbic cortex and neocortex, olfactory bulb, hippocampus, and amygdala. Intense NPR-B mRNA hybridization was observed in preoptic-hypothalamic neuroendocrine circuits and in motor nuclei of cranial nerves. Intermediate expression of NPR-B mRNA was observed in brainstem nuclei controlling autonomic function. Labeling for NPR-B but not NPR-A mRNA was observed in pituicytes in the neural lobe of the pituitary and in scattered cells of the anterior pituitary. These results suggest that CNP is the primary biologically active natriuretic peptide in brain. In contrast with NPR-B, NPR-A appears to be expressed largely in restricted cell populations containing high levels of ANP and in circumventricular organs. These data implicate the NPR-A in autoregulation of ANP neurons and central registration of cardiac ANP release.

Immunohistochemical localization of Lyn (p56) protein in the adult rat brain.

Expression of a sarcoma proto-oncogene, c-lyn, was mapped in the adult rat brain using immunohistochemistry. Lyn protein was prevalent in restricted cell populations of the olfactory bulb and the basal forebrain which included nuclei of accumbens, fundal striatum, bed stria, ventral pallidum and central amygdala as well as deep entorhinal and pyriform cortices. Tightly packed Lyn-positive cells formed discrete multiple stripes crossing perpendicular to the rostral limb of the anterior commissure, and intense masses surrounding the caudal limb. In the thalamus, the habenula, anterodorsal nucleus and medial geniculate body, together with the paraventricular hypothalamic nuclei, had prominent reactive neuronal somata and dendrites in the neuropil. The lateral septal nucleus also had intense Lyn-positive neurons with overlapping dendritic fields. In addition, scattered neurons were evenly distributed throughout the striatum. The red, interpeduncular, auditory and trigeminal tract nuclei were intensely reactive. The cerebellar molecular layer was uniformly labeled except for a few isolated fiber bundles in the lowest part of this layer. The granule cells adjacent to the Purkinje cell layer appeared in reactive patches. In the spinal cord, the posteromarginal nucleus had intense labeling. The significance of this highly localized distribution pattern of Lyn protein may be related to connections forming functional compartments serving signal transduction within specific central nervous system circuitry.

Apoptosis in the developing tooth: association with an embryonic signaling center and suppression by EGF and FGF-4.

Apoptosis was localized in developing mouse teeth from initiation of morphogenesis to completion of cusp formation by using modified TUNEL method for serial sections and Nile Blue staining for whole mounts. Apoptosis was first detected at bud stage (E12-E13) in the central cells of the invaginating dental epithelium suggesting involvement of cell death in epithelial budding morphogenesis. During cusp development, apoptotic cells were located in the enamel knots, which are transient clusters of dental epithelial cells proposed to act as signaling centers directing the morphogenesis of tooth cusps. Apoptosis was also detected in other restricted epithelial cell populations including the dental lamina, ameloblasts, as well as stratum intermedium and stellate reticulum cells suggesting that the removal of these epithelial cells occurs by apoptosis. Apoptotic cells, presumably osteoclasts, were also located on the surfaces of the developing alveolar bone. When dissected E13 dental epithelium or mesenchyme were cultured in isolation, apoptotic cells were abundant throughout the tissues, whereas when cultured together, apoptosis was inhibited in both tissues close to their interface indicating that epithelial-mesenchymal tissue interactions prevent apoptosis. Epidermal growth factor (EGF) and fibroblast growth factor-4 (FGF-4) inhibited apoptosis in the dental mesenchyme when applied locally using agarose or heparin-coated acrylic beads, suggesting involvement of these or related growth factors in the prevention of apoptosis in dental tissues in vivo. The spatially and temporally restricted distribution patterns of apoptotic cells suggest multiple roles for programmed cell death in dental development. Of particular interest is the removal of the enamel knots by apoptosis which may terminate their tasks as regulators of the patterning of the tooth cusps. The apical ectodermal ridge (AER) of the limb bud has similar signaling characteristics as the enamel knot, and it also undergoes apoptosis. Hence, apoptosis may be a general mechanism for the silencing of embryonic signaling centers.

Selective immortalization of mammary epithelial cells by microinjection of SV40 DNA into human milk cells

Immortalization of mammary epithelial cells has been obtained by microinjection of SV40 DNA into epithelial cells from primary cultures of rabbit mammary glands. A modification of this technique, allowing selective immortalization of a specific cell type of mammary epithelial cells from human milk by microinjection of SV40 DNA, is described. It includes the selection of luminal epithelial cells from human milk cells after identification of these cells by antibodies directed against specific markers such as intermediate filaments and membrane antigens, and finally, the introduction of SV40 DNA into cell nuclei which will selectively immortalize this restricted cell population. This procedure can be used to derive permanent cell lines with a defined phenotype when only a few cells are available within a heterogeneous cell population.

Cadherin-11 Expressed in Association with Mesenchymal Morphogenesis in the Head, Somite, and Limb Bud of Early Mouse Embryos

Cadherin-11 (cad-11) is a novel member of the cadherin family of cell adhesion molecules, having recently been identified by means of the polymerase chain reaction. To study the function and expression of this molecule, we cloned mouse cad-11 cDNA. Transfection of L cells with cDNA led them to acquire a typical cadherin-dependent cell-cell adhesiveness, and the L cells expressing cad-11 did not coaggregate with L cells expressing E-, P-, N-, or R-cadherin when they were mixed, indicating that this novel cadherin has a homophilic binding specificity, as found for other cadherins. To determine the developmental expression pattern of this molecule, we performed in situ hybridization analysis on early mouse embryos. Cad-11 first appeared in mesodermal layers only in the head and tail regions at the mid-to-late primitive streak stages. In the head, this appearance was followed by strong expression in mesenchymal tissues including bronchial arches. In the trunk, the paraxial mesoderm initially did not express cad-11. However, as the somites formed, they expressed cad-11, and this expression was strictly correlated with their initial condensation and segregation from the presomitic mesoderm. The cad-11 expression in the somites was eventually restricted to sclerotome cells. As the limb buds developed, cad-11 appeared in the distal portion of the limb mesenchyme, and, at later stages, its expression was most evident at the peripheral mesenchyme. Cad-11 was thus expressed by restricted populations of mesenchymal cells in early embryos, although it was also expressed in parts of the neural tube, such as the optic vesicle and dorsal midline, and in part of the otic vesicle. As a step to investigate the role of cad-11 in mesenchymal cell adhesion, we dissociated the limb bud mesenchyme into single cells, pelleted them, and cultured them as aggregates. In these cultures, cad-11-positive cells clearly sorted out of the negative cell population, suggesting that cad-11 might be involved in selective association of mesenchymal cells. For comparison, we studied the expression of N-cadherin and found that the expressions of these two cadherins were differential, and complementary in some tissues. These results suggest that cad-11 is involved in specific associations of subsets of mesenchymal cells and also of some neural cells during early embryogenesis.

Current Excitement about the Glutamate Receptor Family

Glutamatergic synaptic transmission provides the major excitatory drive to neurons within the mammalian central nervous system, and the consequent activation of glutamate re ceptors participates in a variety of physiological and pathophysiological processes. There are several classes of ligand-gated glutamate receptors that express characteristic elec trophysiological properties. Glutamate receptors are multimeric proteins encoded by a large family of glutamate receptor genes, and individual genes are expressed by restricted populations of cells. One source of the functional diversity of glutamate receptors is the assembly of subunits into hetero-oligomeric complexes. An additional source of diversity comes from splice variants of individual subunits and editing of the messenger RNA se quence of some subunits at critical sites. Glutamate receptor subunit gene expression is dynamically regulated during development and in response to physiological and patho logical stimuli. Particular combinations of glutamate receptor subunits may express elec trophysiological properties that promote neuronal plasticity, whereas other combinations may have neurotoxic effects. The linkage of particular glutamate receptors to specific neuronal functions may provide an avenue for therapeutic intervention. The Neuroscientist 1:60-63, 1995

Distribution of β‐endorphin‐like‐immunoreactive structures in the chicken and quail brain as demonstrated with a new homologous antibody directed against a synthetic peptide

A polyclonal rabbit antibody was raised against a synthetic peptide fragment located at the C-terminal end of turkey beta-endorphin (beta-END) and used to analyze the distribution of beta-END-immunoreactive-like structures in the quail and chicken brain. Three major groups of immunopositive cells were detected in the preoptic area-hypothalamus complex. A thin layer of immunopositive cells was parallel and adjacent to the ventral edge of the brain in the preoptic and anterior hypothalamic region, a more numerous group of immunoreactive perikarya was located along the walls of the third ventricle in these same regions, and, finally, a few scattered cells were found in a more lateral position on both the internal and external sides of the tip of the fasciculus prosencephali lateralis. The periventricular cell population extended in the caudal direction until the posterior hypothalamus. Labelled fibers were always associated with these immunoreactive perikarya, and they were also found in the adjacent hypothalamic regions. A dense innervation of the median eminence was also detected. These data are compared with previous studies in mammals and birds that had identified more restricted populations of immunoreactive cells and the possible sources of the observed discrepancy are discussed. The functional significance of the present data is also briefly analyzed.

Specification and segmentation of the paraxial mesoderm.

Somite formation in the mouse embryo begins with the recruitment of mesenchymal cells into the paraxial mesoderm. Cells destined for the paraxial mesoderm are recruited from a progenitor population found first in the embryonic ectoderm and later in the primitive streak and the tail bud. Experimental evidence suggests that the allocation of precursor cells to different mesodermal lineages may be related to the site at which the cells ingress through the primitive streak. An increasing number of genes, such as those encoding growth factor and transcription factors, are now known to be expressed in the primitive streak. It is not known whether the specification of mesodermal cell fate has any relationship with the activity of genes that are expressed in the restricted cell populations of the primitive streak. Somitomeres, which are spherical clusters of mesenchymal cells in the presomitic mesoderm, presage the segmentation of somites in the paraxial mesoderm. The somitomeric organization denotes a pre-pattern of segmentation that defines the physical boundary and the bilateral symmetry of the mesodermal segments in the body axis. The establishment of new somitomeres seems to require the interaction of a resident cell population in the presomitic mesoderm and the incoming primitive streak cells. Cell mixing, which occurs in the somitomeres prior to somite segmentation, poses problems in understanding the developmental role of the somitomere and the real significance of the partitioning of the node-derived and primitive streak-derived cells in the mesodermal segments. In the presomitic mesoderm, the expression of some genes that encode transcription factors, growth factors or tyrosine kinase receptor, and the localization of certain cell adhesion molecules are closely associated with distinct morphogenetic events, such as cell clustering in the presomitic mesoderm and the formation of epithelial somites. There is, however, very little direct relationship between the spatial pattern of gene expression and the somitomeric organization in the presomitic mesoderm. Results of somite transplantation experiments suggest that both the segmental address and the morphogenetic characteristics of the somite may be determined during somite segmentation. Regional identity of the paraxial mesodermal segment is conferred by the expression of a combination of Hox genes in the sclerotome and probably other lineage-specific genes that are subject to imprinting. Superimposed on the global metameric pattern, two orthogonal polarities of cell differentiation are endowed in each mesodermal segment. The rostro-caudal polarity is established prior to somite segmentation. This polarity is later manifested by the subdivision of the sclerotome and the alliance of the neural crest cells and motor axons with the rostral half-somite.(ABSTRACT TRUNCATED AT 400 WORDS)

The sequence of 5′ flanking DNA from the rat preprotachykinin gene; analysis of putative transcription factor binding sites

The rat preprotachykinin A (rPPT-A) gene is expressed in restricted populations of neurons and endocrine cells, including a subset of dorsal root ganglion (DRG) neurons. As part of a study to investigate the DNA sequences responsible for tissue-specific expression of the gene, we have determined the sequence of the 5′ flanking DNA to 3350 bp upstream of the transcription start site. The sequenced region encodes a number of putative transcription factor binding sites which may play important roles in the regulation of rPPT-A gene transcription.

Comparison of molecular and cytogenetic methods in the evaluation of engraftment following allogeneic bone marrow transplantation

Cytogenetic evaluation of patients after bone marrow transplantation (BMT) has provided a standard method of documentation of hematopoietic engraftment. More recently, recombinant DNA technology has also been applied to determine engraftment status. In order to establish the relative utility of these methods in clinical practice we have directly compared the data from cytogenetic and recombinant DNA methods, evaluating engraftment status in 68 BMT recipients. Patients were evaluated pre-transplant, 30, 60, 90, and 180 days after BMT, and yearly thereafter for 1) the presence or absence of the Y chromosome in sex-mismatched allogeneic transplant recipients, 2) the presence or absence of the Philadelphia chromosome [t(9;22)] in patients transplanted for chronic myelogenous leukemia (CML), 3) restriction fragment length polymorphism (RFLP) profiles, and/or 4) clonal rearrangement of the bcr gene. Cytogenetic examination of unstimulated bone marrow and recombinant DNA tests of nucleated peripheral blood or bone marrow cells produce qualitatively similar data in the identification of patient and donor cells and/or normal and tumor cells. Differences in the results obtained by the two analytic methods were most often due to the restricted cell populations evaluable by cytogenetic studies of PHA-stimulated peripheral blood specimens. DNA analyses could frequently be applied at earlier intervals after transplantation and, in cases of graft rejection, when cell counts were low. Although recombinant DNA methods required fewer cells and demonstrated greater sensitivity in detection of minor cell populations in the majority of instances, the cytogenetic evaluation may complement the DNA studies and allow detection of additional chromosomal anomalies.