Heparin is a unique marker of progenitors in the glial cell lineage.

The oligodendrocyte-type-2 astrocyte progenitor cells (precursors of oligodendrocytes and type-2 astrocytes) are an excellent system in which to study differentiation as they can be manipulated in vitro. Maintenance of oligodendrocyte-type-2 astrocyte progenitor cells requires basic fibroblast growth factor, a growth factor whose action normally depends on a heparan sulfate coreceptor. Biochemical analysis revealed a most surprising result: that the oligodendrocyte-type-2 astrocyte progenitors did not synthesize heparan sulfate, the near ubiquitous N-sulfated cell surface polysaccharide, but the chemically related heparin in a form that was almost completely N- and O-sulfated. The heparin was detected in the pericellular fraction of the cells and the culture medium. In contrast the differentiated glial subpopulations (oligodendrocytes and type-2 astrocytes) synthesized typical heparan sulfate but with distinctive fine structural features for each cell type. Thus heparin is a unique differentiation marker in the glial lineage. Previously heparin has been found only in a subset of mature mast cells called the connective tissue mast cells. Its presence within the developing nervous system on a precise population of progenitors may confer specific and essential recognition properties on those cells in relation to binding soluble growth and/or differentiation factors and the extracellular matrix.     

Complexity of developmental control: analysis of embryonic cell lineage specification in Caenorhabditis elegans using pes-1 as an early marker.

In the early Caenorhabditis elegans embryo five somatic founder cells are born during the first cleavages. The first of these founder cells, named AB, gives rise to 389 of the 558 nuclei present in the hatching larva. Very few genes directly involved in the specification of the AB lineage have been identified so far. Here we describe a screen of a large collection of maternal-effect embryonic lethal mutations for their effect on the early expression of a pes-1::lacZ fusion gene. This fusion gene is expressed in a characteristic pattern in 14 of the 32 AB descendants present shortly after the initiation of gastrulation. Of the 37 mutations in 36 genes suspected to be required specifically during development, 12 alter the expression of the pes-1::lacZ marker construct. The gene expression pattern alterations are of four types: reduction of expression, variable expression, ectopic expression in addition to the normal pattern, and reduction of the normal pattern together with ectopic expression. We estimate that approximately 100 maternal functions are required to establish the pes-1 expression pattern in the early embryo.     

Expressing antisense P0 RNA in Schwann cells perturbs myelination.

Primary Schwann cells were infected in vitro with a recombinant retrovirus expressing a dominant selectable marker, neomycin phosphotransferase (conferring resistance to the drug G418), and antisense P0 RNA under the control of the human beta-actin promoter. A proportion of the G418-resistant cells failed to form myelin when cocultured with dorsal root ganglion neurons under conditions that promote Schwann cell differentiation. These cells expressed high levels of P0 antisense RNA. Among the impaired cells, the majority had segregated and ensheathed individual axon but had not differentiated further. They did not express P0 but did express myelin- associated glycoprotein and galactocerebroside. A minority of partially inhibited Schwann cells were also observed that elaborated thin myelin sheaths containing variable numbers of compacted and noncompacted lamellae. These data indicate that restricting the level of P0 expression inhibits spiralling of the Schwann cell membrane and subsequent compaction.     

Identification of a putative intestinal stem cell and early lineage marker; musashi-1

There are few reliable markers for adult stem cells and none for those of the intestinal epithelium. Previously, indirect experimental approaches have predicted stem cell position and numbers. The Musashi-1 (Msi-1) gene encodes an RNA binding protein associated with asymmetric divisions in neural progenitor cells. Two-day-old, adult, and 4.5 h, 1-, 2-, 4- and 12-day post-irradiation samples of BDF1 mouse small intestine, together with some samples of mouse colon were stained with a rat monoclonal antibody to Musashi-1 (14 H-1). Min ( + / - ) mice with small intestinal adenomas of varying sizes were also analysed. Samples of human small and large bowel were also studied but the antibody staining was weak. Musashi-1 expression was observed using immunohistochemistry in neonatal, adult, and regenerating crypts with a staining pattern consistent with the predicted number and distribution of early lineage cells including the functional stem cells in these situations. Early dysplastic crypts and adenomas were also strongly Musashi-1 positive. In situ hybridization studies showed similar expression patterns for the Musashi mRNA and real-time quantitative RT-PCR showed dramatically more Msi-1 mRNA expression in Min tumours compared with adjacent normal tissue. These observations suggest that Musashi-1 is a marker of stem and early lineage progenitor cells in murine intestinal tissue.     

Schwann cells: early lineage, regulation of proliferation and control of myelin formation.

This article reviews selected topics of particular relevance for understanding the process of Schwann cell development. It will discuss early commitment to the Schwann cell lineage and Schwann cell precursors, regulation of Schwann cell proliferation, and regulation of myelin formation.     

Pancreatic protein disulfide isomerase (XPDIp) is an early marker for the exocrine lineage of the developing pancreas in Xenopus laevis embryos

The pancreas develops from dorsal and ventral epithelial extensions at the foregut/midgut boundary in Xenopus embryos. Endocrine and exocrine specification is thought to occur from a pool of uniform precursor cells. While the genetic network controlling endocrine specification and differentiation has been the object of extensive investigations, the corresponding mechanism leading to the exocrine pancreas is much less understood. Here, we report on the identification and characterisation of a novel molecular marker for the early exocrine pancreas in Xenopus embryos. Xenopus pancreatic protein disulfide isomerase is expressed in both dorsal and ventral pancreatic buds. By whole mount in situ hybridization it is detected as early as stage 39 in the exocrine lineage of the developing pancreas; RT-PCR reveals onset of expression as early as stage 35/36.     

Schwann cell myelination requires timely and precise targeting of P(0) protein

This report investigated mechanisms responsible for failed Schwann cell myelination in mice that overexpress P(0) (P(0)(tg)), the major structural protein of PNS myelin. Quantitative ultrastructural immunocytochemistry established that P(0) protein was mistargeted to abaxonal, periaxonal, and mesaxon membranes in P(0)(tg) Schwann cells with arrested myelination. The extracellular leaflets of P(0)-containing mesaxon membranes were closely apposed with periodicities of compact myelin. The myelin-associated glycoprotein was appropriately sorted in the Golgi apparatus and targeted to periaxonal membranes. In adult mice, occasional Schwann cells myelinated axons possibly with the aid of endocytic removal of mistargeted P(0). These results indicate that P(0) gene multiplication causes P(0) mistargeting to mesaxon membranes, and through obligate P(0) homophilic adhesion, renders these dynamic membranes inert and halts myelination.     

In toto differentiation of human amniotic membrane towards the Schwann cell lineage

Human amniotic membrane (hAM) is a tissue containing cells with proven stem cell properties. In its decellularized form it has been successfully applied as nerve conduit biomaterial to improve peripheral nerve regeneration in injury models. We hypothesize that viable hAM without prior cell isolation can be differentiated towards the Schwann cell lineage to generate a possible alternative to commonly applied tissue engineering materials for nerve regeneration. For in vitro Schwann cell differentiation, biopsies of hAM of 8 mm diameter were incubated with a sequential order of neuronal induction and growth factors for 21 days and characterized for cellular viability and the typical glial markers glial fibrillary acidic protein (GFAP), S100β, p75 and neurotrophic tyrosine kinase receptor (NTRK) using immunohistology. The secretion of the neurotrophic factors brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) was quantified by ELISA. The hAM maintained high viability, especially under differentiation conditions (90.2 % ± 41.6 day 14; 80.0 % ± 44.5 day 21 compared to day 0). Both, BDNF and GDNF secretion was up-regulated upon differentiation. The fresh membrane stained positive for GFAP and p75 and NTRK, which was strongly increased after culture in differentiation conditions. Especially the epithelial layer within the membrane exhibited a change in morphology upon differentiation forming a multi-layered epithelium with intense accumulations of the marker proteins. However, S100β was expressed at equal levels and equal distribution in fresh and cultured hAM conditions. Viable hAM may be a promising alternative to present formulations used for peripheral nerve regeneration.     

Fluorescent latex microparticles: A non-invasive short-term cell lineage marker suitable for use in the mouse early embryo

Summary We have examined the potential of fluorescent latex microparticles for use as a short term cell lineage marker in the mouse preimplantation embryo. Isolated blastomeres and intact embryos rapidly adsorb and subsequently endocytose the particles (0.2 m diameter) from a monodisperse suspension in normal medium, so that cytoplasmic endocytic organelles, but not the cytosol itself, becomes labelled. Latex fluorescence, either within intact embryos, disaggregated cells or thick resin sections, is stable during UV irradiation. The development of labelled embryos, both in terms of sequential morphological changes and their time of expression, was comparable to controls and resulted in blastocysts with normal cell numbers and capacity for tissue differentiation. Latex fluorescence is preserved within all the progeny of labelled blastomeres over several cell cycles (e.g. from 8-cell stage to 64-cell stage) and is not transmitted to unlabelled cells either by exocytosis or via midbodies. The particles are particularly suitable for labelling exclusively the entire population of outside cells in the intact embryo from the 16-cell stage onwards.     

The early life of a Schwann cell

Schwann cells are the major glial population of the vertebrate peripheral nervous system. In the adult, they build a protecting sheath around neuronal processes and myelinate large-caliber axons. Already early in development, Schwann cells and neurons establish close contacts. Later development and the maintenance of peripheral nerves are crucially dependent on the controlled bi-directional dialogue between these two cell types. Several major phases can be distinguished in the life of a Schwann cell: determination, differentiation, and potentially myelination. The aim of this review is to summarize the molecular and cellular characteristics of the first steps in the life of a Schwann cell, the development from a multipotent neural crest cell to a differentiated Schwann cell.     

Brain induces the expression of an early cell surface marker for blood-brain barrier-specific endothelium.

Capillaries derived from the perineural vascular plexus invade brain tissue early in embryonic development. Considerably later they differentiate into blood-brain barrier (BBB)-forming blood vessels. In the chick, the BBB as defined by impermeability for the protein horseradish peroxidase develops around embryonic day 13. We have previously found that brain endothelial cells start to express a number of proteins at around the same time, suggesting that these proteins play a role in BBB function. Here we describe a 74 kd protein defined by the monoclonal antibody HT7 that is expressed on the surface of chick embryonic blood cells and brain endothelial but on no other endothelial cells. This protein is not detectable on early embryonic brain endothelium, but is expressed by these cells on embryonic day 10. It is absent in choroid plexus endothelial cells which represent permeable fenestrated endothelial cells. The antigen is expressed on choroid plexus epithelium which is the site of the blood-cerebrospinal fluid barrier. Since it is also found in basolateral membranes of kidney tubules, it may be involved in specific carrier mechanisms. Embryonic mouse brain tissue transplanted on the chick chorio-allantoic membrane induces the expression of this antigen on endothelial cells derived from the chorio-allantois. Brain tissue can therefore induce in endothelial cells in vivo the expression of a molecule characteristic of brain endothelium.     

A non-canonical genetic code in an early diverging eukaryotic lineage.

The nearly invariant nature of the ''Universal Genetic Code'' attests to its early establishment in evolution and to the difficulty of altering it now, since so many molecules are required for, and depend upon, faithful translation. Nevertheless, variations on the universal code are known in a handful of genomes. We have found one such variant in diplomonads, an early-diverging eukaryotic lineage. Genes for alpha-tubulin, beta-tubulin and elongation factor 1 alpha (EF-1alpha) from two unclassified strains of Hexamitidae were found to contain TAA and TAG (TAR) triplets at positions suggesting a variant code in which TAR codes for glutamine. We found confirmation of this hypothesis by identifying genes encoding glutamine-tRNAs with CUA and UUA anticodons. The alpha-tubulin and EF-1alpha genes from two other diplomonads, Spironucleus muris and Hexamita inflata, were also sequenced and shown to contain no such non-canonical codons. However, tRNA genes with the anticodons UUA and CUA were found in H.inflata, suggesting that this diplomonad also uses these codons, albeit infrequently. The high GC content of these genomes and the presence of two isoaccepting tRNAs compound the difficulty of understanding how this variant code arose by strictly neutral means.     

Serologic identification of early members of the T cell lineage

The SC-1 antigen, identified by thymus-absorbed rabbit anti-mouse brain antiserum, is present on multipotential stem cells. Its presence on cells of the T lineage has been examined by immunofluorescence, and we have demonstrated that it is also a marker for thymic development. Although it is present on the majority of fetal and neonatal thymocytes, its expression declines rapidly and, as early as 1 mo after birth, adult levels are reached. In normal animals, these do not change during adult life, even in mice destined to develop a T cell leukemia. SC-1 does reappear transiently on cells in the regenerating thymus of sublethally irradiated mice. Although the antigen is not associated with preleukemic changes in the thymus, it is expressed on some cells of all spontaneous T lymphomas, and it is uniformly present on tissue culture lines of T cell lymphomas.     

Stomatal patterning in Tradescantia: an evaluation of the cell lineage theory.

The cell lineage theory, which explains stomatal patterning in monocot leaves as a consequence of orderly divisions, was studied in Tradescantia. Data were collected to test the theory at three levels of organization: the individual stoma; stomata distributed in one dimension, in linear fashion along cell files; and stomata apportioned in two dimensions, across the length and breadth of the leaf. In an attempt to watch the patterning process through regeneration, stomata in all visible stages of development were laser ablated. The results showed that the formation of stomatal initials was highly regular, and measurements of stomatal frequency and spacing showed that pattern was determined near the basal meristem when the stomatal initials arose. Following the origin of initials, the pattern was not readjusted by division of epidermal cells. Stomatal initials were not committed when first present and a small percentage of them arrested. The arrested cells, unlike stomata, were consistently positioned in cell files midway between a developed pair of stomata. At the one-dimensional level of pattern, stomata in longitudinal files were separated by a variable number of epidermal cells and the frequency of these separations was not random. The sequential spacing of stomata also was not random, and stomata separated by single epidermal cells were grouped into more short and long series than expected by chance. The stomatal pattern across the width of the leaf resulted from cell files free of stomata which alternated with cell files containing stomata, but not with a recurring periodicity. Files lacking stomata were found only over longitudinal vascular bundles. Laser ablations of developing stomata did not disrupt the pattern in nearby cells or result in stomatal regeneration. We conclude that the cell lineage theory explains pattern as an individual stomatal initial arises from its immediate precursor and satisfactorily accounts for the minimum spacing of stomata in a cell file, i.e., stoma-epidermal cell-stoma. However, the theory does not explain the collective stomatal pattern along the cell files, at the one-dimensional level of patterning. Nor does the theory account for the for the two-dimensional distribution of stomata in which regions devoid of stomata alternate with regions enriched with stomata, but not in a highly regular nor haphazard manner. We suggest that the grouping of epidermal cells and stomata separated by single epidermal cells in cell files may result from cell lineages at a specific position in the cell cycle as they traverse the zone where stomatal initials form.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

SSEA-1 is a specific marker for the spinal sensory neuron lineage in the quail embryo and in neural crest cell cultures

Investigation of the early phases of the development of primary sensory neurons has been limited to cells obtained from sensory ganglia. Due to the lack of an early, lineage-specific marker for sensory neuroblasts, it has not been possible to use the neural crest, which gives rise to all spinal and some cranial primary sensory neurons, as a source of precursor cells. In the present study, we show that in neural crest derivatives of the quail embryo, the stage-specific embryonic antigen-1 (SSEA-1) is expressed specifically by developing sensory neuroblasts. The monoclonal antibodies anti-SSEA-1 and AC4 were used to characterize sensory neuron development in vivo and in neural crest cell cultures. In the rat and mouse, both antibodies recognize the same carbohydrate sequence [galactose beta 1-4(fucose alpha 1-3)N-acetylglucosamine] which characterizes SSEA-1. In the quail embryo, this epitope is a marker with several attractive characteristics. Among neural crest derivatives, it is specific for the sensory lineage and is expressed by all detectable sensory neuroblasts at all spinal axial levels. In addition, the carbohydrate sequence appears early and persists throughout development. Expression of SSEA-1 was also studied in neural crest cell cultures, in which two populations of sensory neuroblasts were observed. One population differentiated before or shortly after explanation into culture; these cells did not emigrate from the neural tube. A second population appeared in older cultures. Forming the leading edge of the emigrating neural crest cells, they became SSEA-1+ 3 days after the nonmigrating SSEA-1+ cells. Double staining experiments revealed no obvious differences between the two populations with regard to morphology, neurofilament expression, and neurotransmitter content.