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W Gilbert

Publications and source records attributed to W Gilbert.

At least 73 records · Page 4Linked to original sources
PubMed1991

Neuronal differentiation and maturation in the mouse trigeminal sensory system, in vivo and in vitro.

We have isolated and characterized four monoclonal antibodies (mAbs B33, E1.9, B30, and B10) that recognize mouse trigeminal sensory neurons at specific times during development. These antibodies permit the study of neuronal differentiation, axon outgrowth, and neuronal maturation in the trigeminal sensory system. With B33, we can follow migrating neural crest and placode cells into the anlagen of the trigeminal ganglion. E1.9 immunoreactivity marks neuronal differentiation and appears in the central nervous system at embryonic day 8.5 (E8.5) and in the peripheral nervous system at E9, E1.9 and B30 show the axonal outgrowth of trigeminal sensory neurons and reveal the pioneering of the peripheral tracts by an early population of ganglionic neurons. At this stage, in the central nervous system, mesencephalic trigeminal neurons are also E1.9 and B30 positive as they migrate to their final location in the rostral metencephalon. B30 and B10 allow us to follow the maturation of these neurons. Also, in about 1% of the embryos, we identified mispositioned or misrouted trigeminal neurons. Furthermore, these biochemical markers facilitate the study of neuronal development in vitro. We find that, based on morphological and biochemical criteria, the maturation of trigeminal neurons in culture is target independent.

Animals↗
PubMed1991

The B30 ganglioside is a cell surface marker for neural crest-derived neurons in the developing mouse.

We have previously reported the isolation of a monoclonal antibody, mAb B30, that recognizes two minor gangliosides specifically expressed in a small subset of neurons in the developing mouse central nervous system (Stainier and Gilbert, 1989). B30 labels mesencephalic trigeminal neurons shortly after differentiation until about 2 weeks after birth. Postnatally, it also labels two specific monolayers of cerebellar neurons. In this study, we have characterized the B30 immunoreactivity in the developing peripheral nervous system of the mouse. We report that B30 is a marker for neural crest-derived neurons and have used it to follow the neuronal differentiation of neural crest cells in a serum-free chemically defined culture system. Within hours after plating, neural crest cells migrate away from the neural tube explant on a fibronectin or laminin substrate and by 24 hr, up to 15% of them have differentiated into morphologically identifable neurons. In vitro as in vivo, undifferentiated mouse neural crest cells express the GD3 ganglioside which is recognized by mAb B33, and neural crest-derived neurons can be labeled by mAbs B33, B30, and also E1.9, a specific neuronal cytoskeletal marker. We also show the unique biochemical specificity of mAb B30 and provide experimental evidence for the role of the B30 ganglioside in the cellular adhesion process.

Animals↗
PubMed1991

Zebrafish embryology and neural development.

The zebrafish is rapidly increasing in popularity with developmental biologists. Driving this interest are the elegant methods for in vivo observations and recovery of early developmental mutations. The past year has seen the introduction of additional methods for in vivo manipulation of identified cells and the application of these methods to mutant analysis.

Animals↗
PubMed1991

Spatial domains in the developing forebrain: developmental regulation of a restricted cell surface protein.

We have isolated a monoclonal antibody, mAb 52G9, that recognizes a 55-kDa cell surface protein restricted to the early embryonic rat forebrain and to placode-derived structures. In the central nervous system (CNS), 52G9 immunoreactivity appears at Embryonic Day 11 (E11) in the rostral-most area of the telencephalon. It then spreads to the neuroepithelium of the telencephalon and basal diencephalon. Most strikingly, it appears at E14 in a distinct zone at the caudal end of the ventral diencephalic neuroepithelium. This area is sharply defined by strong 52G9 immunoreactivity bounded by unlabeled neuroepithelium. The pattern revealed by 52G9 is the first biochemical demonstration of spatial domains in the forebrain at a time prior to neuronal differentiation. By E18, 52G9 immunoreactivity has progressively disappeared from the forebrain; the glomerular layer of the olfactory bulb is the only 52G9-positive area in the CNS. The olfactory, otic, and hypophyseal placodes, which can be identified as early as E10, are also 52G9 positive as are their derivatives, the sensory epithelial of the nasal passage and inner ear, and also Rathke's pouch. The distribution and regulation of the 52G9 protein suggests that this novel cell surface molecule may be involved in the formation of spatial domains in the developing forebrain.

Animals↗
PubMed1991

The limited universe of exons.

The catalogue of mosaic proteins showing evidence of exon-shuffling continues to expand. The repeated use of exon modules suggests that current protein diversity could have been generated from a finite set of such exon modules, and that the size and character of this underlying exon universe can still be glimpsed in extant proteins.

Animals↗
PubMed1991

Murine memory B cells are multi-isotype expressors.

Flow cytometric analyses of the surface immunoglobulins of murine memory B cells revealed the existence of populations expressing multiple isotypes, including an IgM+/IgG+ population that could be stimulated in vitro with antigen to secrete both IgM and IgG. Female BALB/c mice were immunized with R-phycoerythrin (RPE), a fluorescent photosynthetic accessory protein from red algae. Pooled splenocytes from these mice at different stages of immunization were stained with RPE as well as with allophycocyanin- and fluorescein-conjugated anti-isotype antibodies and analysed on a two-laser FACS. RPE-binding cell sub-populations were defined and selectively sorted to verify their phenotype and to demonstrate that the various subpopulations (IgM+/IgG+, IgM+/IgG-, IgM-/IgG+) had different isotype-secretion patterns when challenged with RPE in vitro. These results re-affirm the notion that a transcriptional processing mechanism may be responsible for the simultaneous expression of multiple isotypes in memory cells.

Animals↗
PubMed1990

How big is the universe of exons?

If genes have been assembled from exon subunits, the frequency with which exons are reused leads to an estimate of the size of the underlying exon universe. An exon database was constructed from available protein sequences, and homologous exons were identified on the basis of amino acid identity; statistically significant matches were determined by Monte Carlo methods. It is estimated that only 1000 to 7000 exons were needed to construct all proteins.

Amino Acid Sequence↗
PubMed1990

A sodium-potassium switch in the formation of four-stranded G4-DNA.

Single-stranded complex guanine-rich DNA sequences from chromosomal telomeres and elsewhere can associate to form stable parallel four-stranded structures termed G4-DNA by a process that is anomalously dependent on the particular alkali metal cation that is present. The anomaly, which is not found in the formation of G4-DNA by oligonucleotides containing short, single runs of three or more guanines, is caused by potassium cations excessively stabilizing fold-back intermediate structures, or pathway by-products.

Animals↗
PubMed1990

Detection of mutations and DNA polymorphisms using whole genome Southern Cross hybridization.

We report a general method for the detection of restriction fragment length alterations associated with mutations or polymorphisms using whole genomic DNA rather than specific cloned DNA probes. We utilized a modified Southern Cross hybridization to display the hybridization pattern of all size-separated restriction fragments from wild-type Caenorhabditis elegans to all the corresponding fragments in a particular mutant strain and in a distinct C. elegans variety. In this analysis, almost all homologous restriction fragments are the same size in both strains and result in an intense diagonal of hybridization, whereas homologous fragments that differ in size between the two strains generate an off-diagonal spot. To attenuate the contribution of repeated sequences in the genome to spurious off-diagonal spots, restriction fragments from each genome were partially resected with a 3' or 5' exonuclease and not denatured, so that only the DNA sequences at the ends of these fragments could hybridize. Off-diagonal hybridization spots were detected at the expected locations when genomic DNA from wild-type was compared to an unc-54 mutant strain containing a 1.5 kb deletion or to a C. elegans variety that contains dispersed transposon insertions. We suggest that this modified Southern Cross hybridization technique could be used to identify restriction fragment length alterations associated with mutations or genome rearrangements in organisms with DNA complexities as large as 10(8) base pairs and, using rare-cutting enzymes and pulse-field gel electrophoresis, perhaps as large as mammalian genomes. This information could be used to clone fragments associated with such DNA alterations.

Animals↗
PubMed1990

Localization of an acetylcholine receptor intron to the nuclear membrane.

The first intron of the RNA for the acetylcholine receptor (AChR) alpha subunit shows a ringlike distribution around nuclei in multinucleated myotubes by in situ hybridization. This pattern is not observed for an actin intron or U1 RNA. Quantitation of the intron sequences reveals large variations in the amount of both the AChR and actin introns between nuclei within the same myotube, although all nuclei express equivalent amounts of U1 RNA. This differential RNA expression indicates that nuclei can individually control expression of messenger RNAs. The restricted distribution of the AChR intron RNA suggests a previously unknown step in RNA processing.

Actins↗
PubMed1990

DNA trapping electrophoresis.

Attempts to improve the size separation of single-stranded DNA in polyacrylamide gels by field-inversion gel electrophoresis (FIGE) have met with limited success. Here we show that attaching a neutral globular protein, streptavidin, to one end of a single-stranded DNA molecule profoundly alters the DNA mobility pattern and increases the band separation manyfold within a size range controlled by voltage and pulse cycle. In constant field, short modified fragments are only slightly retarded but long molecules are retarded dramatically above a 'threshold size' of 0.6 kilobases at 60 V per cm. At this voltage, molecules above a 1.2-kilobase 'cut-off' do not enter the gel. Both the threshold and the cut-off sizes decrease as the voltage increases. In FIGE, the longer the reverse pulse, the larger the modified fragments that enter the gel. We interpret these results as the trapping by the gel matrix of the protein attached to the DNA. The probability of release then depends on the balance between the electric field and thermal motion: the larger the DNA and the higher the voltage, the harder it is to release.

DNA↗
PubMed1990

Factors released by ciliary neurons and spinal cord explants induce acetylcholine receptor mRNA expression in cultured muscle cells.

The nuclei of cultured noninnervated muscle cells are heterogeneous with respect to production of mRNA for the nicotinic acetylcholine receptor (AChR). Some nuclei actively express AChR mRNA while others have a low level of activity or are inactive. To determine if innervation, or a factor released by neurons, influences nuclear expression of AChR mRNA, we examined mRNA at a single cell level via in situ hybridization and autoradiography with an alpha-subunit AChR genomic probe. Four days after plating, we co-cultured chicken primary muscle cells with spinal cord explants, ciliary neurons, or dorsal root ganglia (DRG) cells. In situ hybridization of the spinal-cord and muscle-cell co-cultures with the AChR alpha-subunit probe revealed a high density of silver grains on muscle cells, which were within two explant diameters of the spinal cord explant, and a graded decrease in silver grain density as the distance from the explant increased, as well as the appearance of a strikingly nonhomogenous distribution of active and inactive muscle cell nuclei. When ciliary neurons were uniformly distributed over the muscle cells, a high level of AChR mRNA was induced, but no gradients appeared. Neither an increased mRNA level nor a gradient was observed when DRG cells were co-cultured with muscle cells. When ciliary neurons are cultured within Costar permeable inserts, which prevent any contact between the neurons and the underlying muscle cells, AChR messenger RNA is still induced, showing that diffusible factors are responsible. Our results indicate that molecules released by cholinergic neurons regulate the expression of AChR mRNA in the myotubes and raise the possibility that AChR expression depends on both neuronal signals and on intracellular information from the muscle cell.

Animals↗
PubMed1990

Pioneer neurons in the mouse trigeminal sensory system.

Pioneer neurons establish preliminary nerve pathways that are followed by later-growing axons. The existence of pioneers and their importance is well documented in invertebrate systems. In mammals, early neuronal development has generally been difficult to study because of the size and complexity of the embryos, and the lack of adequate markers. Here we look at the time of earliest axonal outgrowth in the mouse embryo by using specific monoclonal antibodies to stain wholemount preparations. During the period of formation and closure of the neuropore beginning at embryonic day 8.5, we can follow the earliest trigeminal sensory neurons extending axons along stereotyped pathways. In the trigeminal ganglion, an early wave of neurogenesis gives rise to a small number of neurons whose axons pioneer the different trigeminal tracts in the periphery. After a brief pause (12 hr), these primary axons branch out to innervate individual targets. Emerging a day later, secondary fibers extend along the pioneers. By contrast, in the central nervous system, neurons of the mesencephalic trigeminal nucleus extend toward the rhombencephalon independently, ignoring preexisting fibers. These results show the existence of an early set of axonal tracts in the mouse peripheral nervous system that may be used for the guidance of later-differentiating neurons.

Animals↗
PubMed1990

Simultaneous visualization of neuronal protein and receptor mRNA.

We describe a combined immunocytochemistry/in situ hybridization technique which allows for the simultaneous localization of protein and mRNA in a single cell. We have carried out these studies either on non-innervated skeletal myotubes or on myotubes which we have innervated with spinal cord explants or ciliary neurons. Our methods allowed us to detect acetylcholine receptor gene mRNA sequences which are expressed in low abundance within the cells and to determine the intracellular and intranuclear domains where these sequences are concentrated, as well as to identify neurons and their processes. The isotopic detection of RNA in combination with fluorescence microscopy produces high-resolution double-label images, with little background and good preservation of morphology, providing a powerful tool for detection of gene expression and protein content at the single-cell level.

Animals↗
PubMed1989

One-sided polymerase chain reaction: the amplification of cDNA.

We report a rapid technique, based on the polymerase chain reaction (PCR), for the direct targeting, enhancement, and sequencing of previously uncharacterized cDNAs. This method is not limited to previously sequenced transcripts, since it requires only two adjacent or partially overlapping specific primers from only one side of the region to be amplified. These primers can be located anywhere within the message. The specific primers are used in conjunction with nonspecific primers targeted either to the poly(A)+ region of the message or to an enzymatically synthesized d(A) tail. Pairwise combinations of specific and general primers allow for the amplification of regions both 3' and 5' to the point of entry into the message. The amplified PCR products can be cloned, sequenced directly by genomic sequencing, or labeled for sequencing by amplifying with a radioactive primer. We illustrate the power of this approach by deriving the cDNA sequences for the skeletal muscle alpha-tropomyosins of European common frog (Rana temporaria) and zebrafish (Brachydanio rerio) using only 300 ng of a total poly(A)+ preparation. In these examples, we gained initial entry into the tropomyosin messages by using heterologous primers (to conserved regions) derived from the rat skeletal muscle alpha-tropomyosin sequence. The frog and zebrafish sequences are used in an analysis of tropomyosin evolution across the vertebrate phylogenetic spectrum. The results underscore the conservative nature of the tropomyosin molecule and support the notion of a constrained heptapeptide unit as the fundamental structural motif of tropomyosin.

Animals↗
PubMed1989

Direct genomic sequencing of bacterial DNA: the pyruvate kinase I gene of Escherichia coli.

The genomic sequencing procedure is applied to the direct sequencing of uncharacterized regions of bacterial DNA by a "multiplex walking" approach. Samples of bulk Escherichia coli DNA are cut with various restriction enzymes, subjected to chemical sequencing degradations, run in a sequencing gel, and transferred to nylon membranes. When a labeled oligomer is hybridized to a membrane, a sequence ladder appears wherever the probe lies near a restriction cut. New probes, based on sequence that lies beyond other restriction sites, are then synthesized, and the membranes are reprobed to reveal new sequence. Repeated cycles of oligomer probe synthesis and subsequent reprobing permit rapid sequence walking along the genome. This oligomer walking technique was used to sequence the pyruvate kinase (EC 2.7.1.40) gene in E. coli without resorting to cloning or to library construction. The sequenced region was amplified by the polymerase chain reaction and subsequently transcribed and translated using both in vivo and in vitro systems, and the resultant gene product characterized to show that the gene encodes the type I isoform of pyruvate kinase.

Base Sequence↗
PubMed1989

Differential expression of acetylcholine receptor mRNA in nuclei of cultured muscle cells.

Muscle cells in vitro and in vivo are multinucleated and express acetylcholine receptors (AcChoRs). On innervated cells, the AcChoRs form clusters which lie under the nerve terminals. However, noninnervated cells in culture also express clusters of AcChoR. Both in vivo and in vitro the AcChoR clusters appear to be associated with clusters of nuclei. We have used in situ hybridization to determine whether all the nuclei in cultured chicken embryo myotubes are equally active in expressing the AcChoR alpha subunit message. Cells were hybridized with 35S-labeled probes that contained either both an exon and an intron region or only exon sequences. Control cultures were hybridized with a labeled actin DNA probe or poly(U). The hybrids were detected by emulsion autoradiography; simultaneously, the nuclei were visualized with bisbenzamide. Cells hybridized with the intron/exon probe showed a striking preferential silver grain localization in and around some of the myotube nuclei, whereas those hybridized with the exon probe gave a rather homogeneous grain distribution in the cytoplasm. These results show that myotube nuclei possess differential activation capacities for the expression of AcChoR alpha subunit mRNA and that this difference is due to differential rates of transcription.

Animals↗