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PubMed1988

In situ hybridization reveals differential spatial distribution of mRNAs for type I and type II collagen in the chick limb bud.

During limb development, type I collagen disappears from the region where cartilage develops and synthesis of type II collagen, which is characteristic of cartilage, begins. In situ hybridization using antisense RNA probes was used to investigate the spatial localization of type I and type II collagen mRNAs. The distribution of the mRNA for type II collagen corresponded well with the pattern of type II collagen synthesis, suggesting control at the level of transcription and mRNA accumulation. In contrast, the pattern of mRNA for type I collagen remained more or less uniform and did not correspond with the synthesis of the protein, suggesting control primarily at the level of translation or of RNA processing.

Animals↗
PubMed1990

A Drosophila growth factor homolog, decapentaplegic, regulates homeotic gene expression within and across germ layers during midgut morphogenesis.

The decapentaplegic (dpp) gene product, a member of the transforming growth factor-beta family, is required in Drosophila embryos for normal gastrulation and the establishment of dorsal-ventral polarity in the embryo. dpp is also expressed at specific positions in the visceral mesoderm along the developing midgut. We find that mutations that eliminate the visceral mesoderm expression of dpp lead to defects in midgut morphogenesis and alter the spatially localized expression of the homeotic genes Sex combs reduced (Scr), Ultrabithorax (Ubx), and Antennapedia (Antp) in the visceral mesoderm. The extracellular dpp protein migrates from the visceral mesoderm across the apposing endodermal cell layer in a region of the endoderm that expresses the homeotic gene labial (lab). Mesodermal expression of dpp is required for the expression of lab in these endodermal cells indicating that dpp mediates an inductive interaction between the two germ layers. We propose that extracellular dpp protein regulates gut morphogenesis, in part, by regulating homeotic gene expression in the visceral mesoderm and endoderm of the developing midgut.

Animals↗
PubMed1994

Dorsoventral axis formation in Drosophila depends on the correct dosage of the gene gurken.

The Drosophila gene gurken participates in a signaling process that occurs between the germ line and the somatic cells (follicle cells) of the ovary. This process is required for correct patterning of the dorsoventral axis of both the egg and the embryo. gurken produces a spatially localized transcript which encodes a TGF-alpha-like molecule (Neuman-Silberberg and Schupbach, Cell 75, 165-174, 1993). Mutations in gurken cause a ventralized phenotype in egg and embryo. To determine whether the gurken gene product plays an instructive role in dorsoventral patterning, we constructed females containing extra copies of a gurken transgene. Such females produce dorsalized eggs and embryos, which is expected if gurken acts as a limiting factor in the dorsoventral patterning process. In addition, the expression pattern of the gene rhomboid in the follicle cells is altered in ovaries of females containing extra copies of gurken. Our results indicate that changing gurken dosage in otherwise wild-type ovaries is sufficient to alter the number of somatic follicle cells directed to the dorsal fate. Therefore the gurken-torpedo signaling process plays an instructive role in oogenesis. It induces dorsal cell fates in the follicle cell epithelium and it controls the production of maternal components that will direct the embryonic dorsoventral pattern after fertilization.

Animals↗
PubMed1995

wingless inhibits morphogenetic furrow movement in the Drosophila eye disc.

Differentiation of the Drosophila eye imaginal disc is an asynchronous, repetitive process which proceeds across the disc from posterior to anterior. Its propagation correlates with the expression of decapentaplegic at the front of differentiation, in the morphogenetic furrow. Both differentiation and decapentaplegic expression are maintained by Hedgehog protein secreted by the differentiated cells posterior to the furrow. However, their initiation at the posterior margin occurs prior to hedgehog expression by an unknown mechanism. We show here that the wingless gene contributes to the correct spatial localization of initiation. Initiation of the morphogenetic furrow is restricted to the posterior margin by the presence of wingless at the lateral margins; removal of wingless allows lateral initiation. Ectopic expression of wingless at the posterior margin can also inhibit normal initiation. In addition, the presence of wingless in the center of the disc can prevent furrow progression. These effects of wingless are achieved without altering the expression of decapentaplegic.

Animals↗
PubMed1995

Target-induced neurogenesis in the leech CNS involves efferent projections to the target.

During a critical period in leech embryogenesis, the sex nerves that connect the 5th and 6th midbody ganglia (MG5 and MG6) to the primordium of the male sexual organ carry a spatially localized signal that induces the birth of several hundred neurons specific to these ganglia. We examined particular cellular elements (afferents, efferents, non-neuronal components) within these nerves as potential conveyors of the inductive signal. We show that axons of peripheral sensory neurons in the male genitalia travel along the sex nerves and into MG5 and MG6, but reach the CNS after the critical period has elapsed and cannot, therefore, be involved in the induction. Of the six sex nerves, four contain non-neuronal cells that span the entire distance between the male genitalia and the sex ganglia. However, when male genitalia were transplanted to ectopic locations close to MG6, induction occurred frequently but only in MG6, mediated by ectopic nerves that do not contain these cells. Thus, non-neuronal cells specific to the normal sex nerves are not necessary for induction. In addition, dye injections into the target during the critical period failed to reveal migrating cells in the sex nerves that could convey the inductive signal to the CNS. Finally, we show that 11 pairs of central neurons in each ganglion project to the male organ early during the critical period. In the adult, at least 3 additional pairs of neurons in MG6 also innervate this target. We conclude that the only components of the sex nerves that connect the sex ganglia to the target during the critical period that could be associated with induced central mitogenesis are the axons of central neurons that innervate the male genitalia.

Animals↗
PubMed1995

Regulation of the infraabdominal regions of the bithorax complex of Drosophila by gap genes.

The expression of the abdominal-A and Abdominal-B genes of the bithorax complex of Drosophila is controlled by cis-regulatory infraabdominal regions. The activation of these regions along the anteroposterior axis of the embryo determines where abdominal-A and Abdominal-B are transcribed. There is spatially restricted transcription of the infraabdominal regions (infraabdominal transcripts) that may reflect this specific activation. We show that the gap genes hunchback, Krüppel, tailless and knirps control abdominal-A and Abdominal-B expression early in development. The restriction of abdominal-A and Abdominal-B transcription is preceded by (and requires) the spatially localized activation of regulatory regions, which can be detected by the distribution of infraabdominal transcripts. The activation of these regions (except the infraabdominal-8 one) could require no specific gap gene. Instead, a general mechanism of activation, combined with repression by gap genes in the anteroposterior axis, seems to be responsible for delimiting infraabdominal active domains. The gradients of the hunchback and Krüppel products seem to be key elements in this restricted activation.

Animals↗
PubMed1998

In vivo clonal analyses reveal the properties of endogenous neural stem cell proliferation in the adult mammalian forebrain.

The adult mammalian forebrain contains a population of multipotential neural stem cells in the subependyma of the lateral ventricles whose progeny are the constitutively proliferating cells, which divide actively throughout life. The adult mammalian brain is ideal for examining the kinetics of the stem cells due to their strict spatial localization and the limited and discrete type of progeny generated (constitutively proliferating cells). Clonal lineage analyses 6 days after retrovirus infection revealed that under baseline conditions 60% of the constitutively proliferating cells undergo cell death, 25% migrate to the olfactory bulb and 15% remain confined to the lateral ventricle subependyma (where they reside for approximately 15 days). Analysis of single cell clones 31 days after retroviral infection revealed that the stem cell divides asymmetrically to self-renew and give rise to constitutively proliferating cells. Following repopulation of the depleted subependyma the average clone size is 2.8 times larger than control, yet the absolute number of cells migrating to the olfactory bulb is maintained and the stem cell retains its asymmetric mode of division. The number of neural stem cells in the adult forebrain 33 days after repopulation of the subependyma was estimated using bromodeoxyuridine labeling of subepenydmal cells. There were calculated to be 1200-1300 cells between the rostral corpus callosum and rostral anterior commissure; these data support a lineage model similar to those based on stem cell behavior in other tissue types.

Animals↗
PubMed1987

Fate map for the 32-cell stage of Xenopus laevis.

A complete fate map has been produced for the 32-cell stage of Xenopus laevis. Embryos with a regular cleavage pattern were selected and individual blastomeres were injected with the lineage label fluorescein-dextran-amine (FDA). The spatial location of the clones was deduced from three-dimensional (3D) reconstructions of later stages and the volume of each tissue colonized by labelled cells in each tissue was measured. The results from 107 cases were pooled to give a fate map which shows the fate of each blastomere in terms of tissue types, the composition of each tissue by blastomere, the location of each prospective region on the embryo and the fate of each blastomere in terms of spatial localization. Morphogenetic movements up to stage 10 (early gastrula) were assessed by carrying out a number of orthotopic grafts at blastula and gastrula stages using donor embryos uniformly labelled with FDA. Although there is a regular topographic projection from the 32-cell stage this varies a little between individuals because of variability of positions of cleavage planes and because of short-range cell mixing during gastrulation. The cell mixing means that the topographic projection fails for anteroposterior segments of the dorsal axial structures and it is not possible to include short segments of notochord or neural tube or individual somites on the pregastrulation fate map.

Animals↗
PubMed1997

Binding of urokinase to plasminogen activator inhibitor type-1 mediates cell adhesion and spreading.

Urokinase plasminogen activator and its receptor are both found at the surface of the cell membrane in many cell types. The plasminogen activator inhibitor type-1 (PAI-1) is often associated with the extracellular matrix. The spatial localization of these three molecules could account for their involvement in cell adhesion and/or migration. We have shown previously that the urokinase receptor mediates mechanical force transmission across the cell surface to the cytoskeleton. Here we investigated whether immobilized plasminogen activator inhibitor type 1 (PAI-1) could regulate cell spreading and cytoskeleton reorganization. Serum deprived human myogenic cells were plated in serum free medium onto bacteriologic dishes precoated with different extracellular matrix ligands (fibronectin, vitronectin, or type 1 collagen) or PAI-1 at increasing concentrations. The number of adherent cells and their projected area were quantitated after 3 hours of plating. PAI-1 promoted cell adhesion and spreading in a dose dependent manner. Addition of antibodies to PAI-1 inhibited the adhesion on PAI-1 coated dishes in a dose dependent way. The PAI-1 mediated cell adhesion required the presence of urokinase at the cell surface. Removal of the glycosylphosphatidylinositol (GPI)-linked proteins abolished cell adhesion on PAI-1 dish, suggesting its dependence on the presence of the urokinase receptor, a GPI-linked receptor. Furthermore, addition of antibodies against alpha v beta3 integrin completely inhibited cell adhesion on PAI-1, suggesting that alpha v beta3 might be the transmembrane molecule that physically connects the complex of PAI-1, urokinase, and urokinase receptor to the cytoskeleton. Visualization of spread cells stained for filamentous actin with confocal microscopy showed a dose-dependent increase of filopodia on PAI-1 coated dishes and cytoskeletal reorganization, suggesting a migratory profile. These data indicate that PAI-1 plays a direct role in dynamic cell adhesion particularly at the leading edge, where increased levels of urokinase plasminogen activator (uPA) and its receptor (uPAR) are localized in migrating cells. Immobilized PAI-1 could therefore serve to bridge the cell surface with the extracellular matrix via the formation of a multimolecular complex that includes alpha v beta3 integrins in myogenic cells.

Antibodies↗
PubMed1999

Globular domains of agrin are functional units that collaborate to induce acetylcholine receptor clustering.

Agrin, an extracellular matrix protein involved in neuromuscular junction formation, directs clustering of postsynaptic molecules, including acetylcholine receptors (AChRs). This activity resides entirely in the C-terminal portion of the protein, which consists of three laminin-like globular domains (G-domains: G1, G2 and G3) and four EGF-like repeats. Additionally, alternate mRNA splicing yields G-domain variants G2(0,4) with 0- or 4-amino-acid inserts, and G3(0, 8,11,19) with 0-, 8-, 11- or 19-amino-acid inserts. In order to better understand the contributions of individual domains and alternate splicing to agrin activity, single G-domains and covalently linked pairs of G-domains were expressed as soluble proteins and their AChR clustering activity measured on cultured C2 myotubes. These analyses reveal the following: (1) While only G3(8) exhibits detectable activity by itself, all G-domains studied (G1, G2(0), G2(4), G3(0) and G3(8)) enhance G3(8) activity when physically linked to G3(8). This effect is most pronounced when G2(4) is linked to G3(8) and is independent of the order of the G-domains. (2) The deletion of EGF-like repeats enhances activity. (3) Increasing the physical separation between linked G1 and G3(8) domains produces a significant increase in activity; similar alterations to linked G2 and G3(8) domains are without effect. (4) Clusters induced by two concatenated G3(8) domains are significantly smaller than all other agrin forms studied. These data suggest that agrin G-domains are the functional units which interact independently of their specific organization to yield AChR clustering. G-domain synergism resulting in biological output could be due to physical interactions between G-domains or, alternatively, independent interactions of G-domains with cell surface receptors which require spatially localized coactivation for optimal signal transduction.

Agrin↗
PubMed1985

New approaches to the study of cellulose biosynthesis.

Examples of a variety of approaches for studying the mechanism and regulation of cellulose biosynthesis are presented. Attempts to demonstrate conclusively a cellulose synthase activity using membrane preparations derived from higher plants have not been successful; the predominant UDP-glucose: beta-glucan-beta-glucosyltransferase detected in these preparations is a beta-(1----3)-glucan synthase that is dependent upon Ca2+ and a beta-glucobiose, such as laminaribiose or cellobiose, for activity. Xyloglucan glucosyltransferase activity is detected in all plant preparations examined and, in cotton fibres, the activity of this enzyme correlates well with the level of xyloglucan found in the fibre. Low activity for a Mg2+-dependent beta-(1----4)-glucan synthase is found in extracts from soybeans and mung beans, but not cotton fibres; this enzyme could represent either a dissociated form of xyloglucan glucosyltransferase or a partially latent form of cellulose synthase. In vitro translation of RNA from developing cotton fibres shows notable increases in the level of several relatively abundant translatable mRNAs associated with the time of onset of secondary-wall cellulose synthesis. In order to determine whether these apparent changes in gene expression represent enhanced expression of specific genes required for cellulose synthesis, several strategies are being developed for identification of specific polypeptides required for this process. One strategy involves the successful development of a technique for detection of glucan synthase activity in acrylamide gels, a technique that should prove useful for characterization of the polypeptide composition of such enzymes. We have also synthesized a photoaffinity analogue of 2,6-dichlorobenzonitrile (DCB), a potent and specific inhibitor of cellulose synthesis. The analogue is also an effective inhibitor in vivo, and upon ultraviolet irradiation of extracts in the presence of the radioactive analogue, we observe a relatively specific labelling of a polypeptide of 18 X 10(3)Mr. Finally, we have studied the spatial regulation and structural requirements for cellulose synthesis in internode cells of the alga Chara corallina. Cellulose deposition in vivo shows spatial localization, which correlates with acid and base bands along the cell. Using internodes perfused with solutions containing UDP-[14C]glucose and subsequently ligated, we were able to demonstrate synthesis of a highly insoluble cell-wall-localized glucan, thus offering hope that Chara can be developed as another useful system for studying the mechanism and regulation of cellulose synthesis.

Cell Membrane↗
PubMed1989

Three-dimensional analysis of the organization of human chromosome domains in human and human-hamster hybrid interphase nuclei.

This report describes the intranuclear organization of chromosomes in human-hamster hybrid nuclei and in human cell nuclei. The target chromosomes were stained using in situ hybridization with biotinylated, chromosome-specific DNA probes. Bound probe was detected with fluorescein-avidin. Hybridizations were performed to fixed nuclei in aqueous suspension in order to preserve their three-dimensional morphology. Total nuclear DNA was stained with DAPI. Three-dimensional information about the organization of DNA and probe within the nucleus was obtained by optical sectioning. The human chromosomes in human-hamster hybrid nuclei were found to be confined to 'domains' that were maintained during the cell cycle. Different spatial localization patterns of the human chromosomes were seen in interphase nuclei of two different hybrid cell lines. The positions of chromosome-specific repetitive sequences in human fibroblast interphase nuclei were also studied using probes for the telomeric region of chromosome 1p (1p36), the centromeric region of chromosome 9 (9q12) and the long arm of the Y chromosome (Yq12). These studies showed that the two 1p telomeric loci are located near the nuclear surface. The chromosome 9 centromeric loci are similarly located. Simultaneous hybridization of the chromosome 1 telomeric probe (target size approximately 200 kb; b, base) and the Y-specific probe (target size greater than 2Mb), demonstrate that the binding sites of the two probes can be distinguished in the same nucleus on the basis of domain size.

Animals↗
PubMed2002

Comment on "Excitation with a focused, pulsed optical beam in scattering media: diffraction effects".

To incorporate the wave properties of light, it was recently proposed to modify the Monte Carlo-based photon transport model to a semi-quantum-mechanical representation by considering each ray as a photon wave packet [Appl. Opt. 39, 5244 (2000)]. It is not clear from the paper whether each photon wave packet is considered representative of an entire plane wave or if each is spatially localized. However, for each interpretation we identify problems with the approach suggested for combining the wave-packet contributions. These include violation of the principle of conservation of energy and the use of a scattering phase function that is incompatible with the suggested way of calculating the intensity values. These issues render the approach impractical.

Journal Article↗
PubMed1997

Orientation tuning of the spatial-frequency-tuned mechanisms of the red-green channel.

Significant orthogonal masking for color stimuli [Invest. Ophthalmol. Visual Sci. 34, 782 (No. 405-54) (1993)] but not for achromatic stimuli [J. Opt. Soc. Am. A 1, 226 (1984)] under sustained presentation led us to investigate the orientation tuning of the spatial-frequency- (SF-) tuned color mechanisms. The Red-Green channel was isolated from the achromatic channel by the minimum flicker technique and from the Yellow-Blue channel by the hue cancellation technique. Contrast sensitivity functions, threshold elevation versus mask orientation curves (measured by orientation masking), and threshold elevation versus mask contrast curves were measured by the method of constant stimuli and a two-interval forced-choice technique on two normal observers. Test targets were spatially localized (D6), vertical color patterns, and masks were sinusoidal color patterns oriented 15 degrees-90 degrees from the vertical in 15 degrees steps and had the same SF's as those of test patterns. Mask contrasts were varied between 1.2% and 60%. The orientation tuning curves of the SF-tuned color mechanisms were extracted by obtaining the best fit to contrast sensitivity and threshold elevation data simultaneously at a given SF with use of the masking model. Results show that threshold elevations depend on test SF, mask SF, mask orientation, and mask contrast. Half-bandwidths at half-height (with respect to 15 degrees from the vertical) of threshold elevation versus mask orientation curves range from 90 degrees to 29 degrees depending on SF's. The slopes of threshold elevation versus mask contrast curves range from 0.76 to 0.29 on octave-octave coordinates depending on SF's. Orientation half-bandwidths at half-height of orientation tuning curves of the SF-tuned color mechanisms (1) range from 79 degrees to 28 degrees and (2) average from 68 degrees to 30 degrees for SF's 0.063-8 cycles per degree (cpd). Data suggest that the orientation tuning curves of the SF-tuned chromatic mechanisms are broader (except at 2 cpd) than those of the achromatic mechanisms (orientation half-bandwidths: 32 degrees-15 degrees for 0.5-11.3 cpd [J. Opt. Soc. Am. A 1, 226 (1984)]); moreover, the orientation bandwidths are SF dependent.

Algorithms↗
PubMed1998

Spatial-frequency tuning of sustained nonoriented units of the red-green channel.

The existence of nonoriented cells but not of sustained orthogonal masking for achromatic stimuli led to an investigation of the spatial-frequency (SF) tuning of sustained nonoriented color units. For this purpose the Red-Green channel was isolated by the minimum flicker and hue cancellation techniques. Chromatic contrast sensitivity functions (CSF's), threshold elevation (TE) curves, and contrast nonlinearities (TE-versus-mask-contrast curves) were measured with spatially localized vertical color tests and sinusoidal orthogonal color masks by the method of constant stimuli under Gaussian temporal presentation. Results show that (1) color CSF's are a low-pass function of SF, whereas TE curves are a bandpass function of mask SF, and (2) a minimum of six SF-tuned color mechanisms (one low pass and five bandpass functions of SF, with peak SF's of 0.13, 0.5, 2, 4, and 8 cycles per degree and bandwidths of 3.9, 4.4, 2.9, 2.1, 1.1, and 1.2 octaves), similar to oblique-masking color mechanisms, are extracted by the multiple-mechanisms model. These data imply that (1) most of the SF tuning of the broadly oriented color units is already present in the circularly symmetric units, and (2) the latter may be an input to the former.

Color↗
PubMed2001

Computational modeling of non-Fourier motion: further evidence for a single luminance-based mechanism.

It is generally assumed that the perception of non-Fourier motion requires the operation of some nonlinearity before motion analysis. We apply a computational model of biological motion processing to a class of non-Fourier motion stimuli designed to investigate nonlinearity in human visual processing. The model correctly detects direction of motion in these non-Fourier stimuli without recourse to any preprocessing nonlinearity. This demonstrates that the non-Fourier motion in some non-Fourier stimuli is directly available to luminance-based motion mechanisms operating on measurements of local spatial and temporal gradients.

Fourier Analysis↗
PubMed2001

Bessel pulse beams and focus wave modes.

Free-space propagation of ultrashort pulses is investigated. Space-time couplings are reduced for a particular form of beams that is termed a pulse beam, or a type 3 pulsed beam. General conditions for the formation of pulse beams in the paraxial approximation are presented. The free-space propagation of spatially localized ultrashort laser pulses is investigated. This treatment is based on a particular pulsed form of the well-known Bessel beam, which is termed a Bessel pulse beam. The connections with focus wave modes and X waves are discussed.

Journal Article↗
PubMed2003

Dispersion compensation in whispering-gallery modes.

We show that manipulation by a spatial profile of the refractive index of a circularly symmetric dielectric cavity results in a novel way of fine tuning frequency separations as well as spatial localizations of high-Q whispering-gallery modes excited in the cavity. The method permits dispersion compensation in the modes (spectrum equalization), diminishes the quality-factor limitation by surface roughness and contamination, and allows critical coupling to ultra-high-Q modes without maintaining an air gap with evanescent couplers.

Journal Article↗