Observation of Roton Mode Population in a Dipolar Quantum Gas.

The concept of a roton, a special kind of elementary excitation, forming a minimum of energy at finite momentum, has been essential to understand the properties of superfluid 4He 1. In quantum liquids, rotons arise from the strong interparticle interactions, whose microscopic description remains debated 2. In the realm of highly-controllable quantum gases, a roton mode has been predicted to emerge due to magnetic dipole-dipole interactions despite of their weakly-interacting character 3. This prospect has raised considerable interest 4-12; yet roton modes in dipolar quantum gases have remained elusive to observations. Here we report experimental and theoretical studies of the momentum distribution in Bose-Einstein condensates of highly-magnetic erbium atoms, revealing the existence of the long-sought roton mode. Following an interaction quench, the roton mode manifests itself with the appearance of symmetric peaks at well-defined finite momentum. The roton momentum follows the predicted geometrical scaling with the inverse of the confinement length along the magnetisation axis. From the growth of the roton population, we probe the roton softening of the excitation spectrum in time and extract the corresponding imaginary roton gap. Our results provide a further step in the quest towards supersolidity in dipolar quantum gases 13.

Quantum Fluctuations in Quasi-One-Dimensional Dipolar Bose-Einstein Condensates.

Recent experiments have revealed that beyond-mean-field corrections are much more relevant in weakly interacting dipolar condensates than in their nondipolar counterparts. We show that in quasi-one-dimensional geometries quantum corrections in dipolar and nondipolar condensates are strikingly different due to the peculiar momentum dependence of the dipolar interactions. The energy correction of the condensate presents not only a modified density dependence, but it may even change from attractive to repulsive at a critical density due to the surprising role played by the transversal directions. The anomalous quantum correction translates into a strongly modified physics for quantum-stabilized droplets and dipolar solitons. Moreover, and for similar reasons, quantum corrections of three-body correlations, and hence of three-body losses, are strongly modified by the dipolar interactions. This intriguing physics can be readily probed in current experiments with magnetic atoms.

The apical ectodermal ridge, fibroblast growth factors (FGF-2 and FGF-4) and insulin-like growth factor I (IGF-I) control the migration of epidermal melanoblasts in chicken wing buds.

The role of the apical ectodermal ridge and of fibroblast growth factors FGF-2 and FGF-4 and of the insulin-like growth factor I (IGF-I) in the control of the migration of epidermal melanoblasts was investigated using quail-chicken chimeras. Wing buds of a strain of unpigmented chicken were microsurgically modified in several ways (ablation, displacement or implantation of additional apical ectodermal ridges, implantation of grafts devoid of apical ectodermal ridges, ectopic application of growth factors) and received grafts containing quail neural crest cells. The distribution of the epidermal melanoblasts which had differentiated from the quail grafts revealed that both the apical ectodermal ridge and the growth factors invariably caused the migration of epidermal melanoblasts towards them. This leads to the conclusion that the presence of the apical ectodermal ridge is the sufficient condition to direct the migration of epidermal melanoblasts within the avian embryonic wing bud. Furthermore, FGF-2 and IGF-I and to a lesser extent FGF-4 play a decisive role in directing the migration of epidermal melanoblasts within chicken wing buds and are likely to be involved in the molecular cascade by means of which the apical ectodermal ridge controls the migration of epidermal melanoblasts.

Signal enhancement at the electron microscopic level using Nanogold and gold-based autometallography.

Immunoelectron microscopy using ultrasmall gold markers is a very sensitive method to detect molecules at high resolution. In order to discriminate the gold particles in the electron microscope, enlargement of gold particles is necessary. So far, mostly silver ions were used for deposition onto the surface of gold grains. In our study, we tested the selective deposition of gold instead of silver ions to enlarge gold particles. This was performed following immunogold detection of DNA at the surface of ultrathin sections embedded in the acrylic resin LR White (postembedding approach). Morphometric analysis of the distribution of DNA in human spermatocytes revealed that the method offers very good specificity and sensitivity and therefore is a good alternative to the use of silver for signal enhancement. This technique was also applied to the detection of ribosomal genes in human testis at the electron microscopic level by in situ hybridization. Ribosomal genes were localized in peri- and intranucleolar chromatin as well as in the dense fibrillar component of nucleoli.

Intranuclear anchoring of repetitive DNA sequences: centromeres, telomeres, and ribosomal DNA.

Centromeres, telomeres, and ribosomal gene clusters consist of repetitive DNA sequences. To assess their contributions to the spatial organization of the interphase genome, their interactions with the nucleoskeleton were examined in quiescent and activated human lymphocytes. The nucleoskeletons were prepared using "physiological" conditions. The resulting structures were probed for specific DNA sequences of centromeres, telomeres, and ribosomal genes by in situ hybridization; the electroeluted DNA fractions were examined by blot hybridization. In both nonstimulated and stimulated lymphocytes, centromeric alpha-satellite repeats were almost exclusively found in the eluted fraction, while telomeric sequences remained attached to the nucleoskeleton. Ribosomal genes showed a transcription-dependent attachment pattern: in unstimulated lymphocytes, transcriptionally inactive ribosomal genes located outside the nucleolus were eluted completely. When comparing transcription unit and intergenic spacer, significantly more of the intergenic spacer was removed. In activated lymphocytes, considerable but similar amounts of both rDNA fragments were eluted. The results demonstrate that: (a) the various repetitive DNA sequences differ significantly in their intranuclear anchoring, (b) telomeric rather than centromeric DNA sequences form stable attachments to the nucleoskeleton, and (c) different attachment mechanisms might be responsible for the interaction of ribosomal genes with the nucleoskeleton.

Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice.

It is commonly accepted, that regenerative capacity of striated muscle is confined to skeletal muscle by activation of satellite cells that normally reside quiescent between the plasmalemma and the basement membrane of muscle fibers. Muscular dystrophies are characterized by repetitive cycles of de- and regeneration of skeletal muscle fibers and by the frequent involvement of the cardiac muscle. Since during the longstanding course of muscular dystrophies there is a permanent demand of myogenic progenitors we hypothesized that this may necessitate a recruitment of additional myogenic precursors from an undifferentiated, permanently renewed cell pool, such as bone marrow (BM) cells. To this end normal and dystrophic (mdx) female mice received bone marrow transplantation (BMT) from normal congenic male donor mice. After 70 days, histological sections of skeletal and cardiac muscle from BMT mice were probed for the donor-derived Y chromosomes. In normal BMT recipients, no Y chromosome-containing myonuclei were detected, either in skeletal or in cardiac muscle. However, in all samples from dystrophic mdx skeletal muscles Y chromosome-specific signals were detected within muscle fiber nuclei, which additionally were found to express the myoregulatory proteins myogenin and myf-5. Moreover, in the hearts of BMT-mdx mice single cardiomyocytes with donor derived nuclei were identified, indicating, that even cardiac muscle cells are able to regenerate by recruitment of circulating BM-derived progenitors. Our findings suggest that further characterization and identification of the BM cells capable of undergoing myogenic differentiation may have an outstanding impact on therapeutic strategies for diseases of skeletal and cardiac muscle.

Autosomal-dominant, prelingual, nonprogressive sensorineural hearing loss: localization of the gene (DFNA8) to chromosome 11q by linkage in an Austrian family.

A four-generation family suffering from an autosomal-dominant, congenital, nonprogressive, nonsyndromic hearing loss was found in a rural region of Austria. The hearing loss was moderate to severe, a pure tone audiogram showing a U-shaped form with maximum loss at 2, 000 Hz. An initial genome search led to a lod score of 3.01 with markers on chromosome 15. This locus was registered as DFNA8 in the HUGO data base. Further sampling of the family, however, yielded data that reduced the maximal lod score with chromosome 15 markers to 1.81. The genome search was restarted using an ABITM genotyper, which eventually detected several positive two-point lod scores with markers from the long arm of chromosome 11. The highest value was 3. 6, which was seen with the marker D11S934. Haplotype analysis excluded the gene from the chromosomal region proximal from D11S898 and distal to D11S1309. These results place the gene in the region of the hearing loss gene DFNA12. Recent evidence suggests that the somewhat different phenotypes found in these two families are due to two different mutations in the human alpha-tectorine gene (Verhoeven et al., 1998).

Arrangement of individual human ribosomal DNA fragments on stretched DNA fibers.

We studied the arrangement of individual human ribosomal (r)DNA repeats by direct visualization of rDNA sequences. We used high resolution fluorescence in situ hybridization on preparations of DNA fibers released from interphase nuclei of HeLa cells. Probes from both the transcription unit and the intergenic spacer were used, and lengths of signals and of the gaps in between were measured and compared to molecular data. We could visualize the repetitive arrangement of individual rDNA sequences at the single gene level. No inversions or deletions were detected. The intergenic spacer was found to be shorter than expected, indicating a length polymorphism.

Mutations in the human alpha-tectorin gene cause autosomal dominant non-syndromic hearing impairment.

The tectorial membrane is an extracellular matrix of the inner ear that contacts the stereocilia bundles of specialized sensory hair cells. Sound induces movement of these hair cells relative to the tectorial membrane, deflects the stereocilia, and leads to fluctuations in hair-cell membrane potential, transducing sound into electrical signals. Alpha-tectorin is one of the major non-collagenous components of the tectorial membrane. Recently, the gene encoding mouse alpha-tectorin (Tecta) was mapped to a region of mouse chromosome 9, which shows evolutionary conservation with human chromosome 11q (ref. 3), where linkage was found in two families, one Belgian (DFNA12; ref. 4) and the other, Austrian (DFNA8; unpublished data), with autosomal dominant non-syndromic hearing impairment. We determined the complete sequence and the intron-exon structure of the human TECTA gene. In both families, mutation analysis revealed missense mutations which replace conserved amino-acid residues within the zona pellucida domain of TECTA. These findings indicate that mutations in TECTA are responsible for hearing impairment in these families, and implicate a new type of protein in the pathogenesis of hearing impairment.

Ribosomal gene transcription is organized in foci within nucleolar components.

We investigated how only three morphologically distinguished nucleolar components can integrate the many necessary tasks in ribosome biogenesis. For the mapping of ribosomal (r)DNA transcription loci, we combined non-autoradiographic in situ transcription assays with the immunological analysis of the ultrastructural distribution of transcription-associated proteins, i.e., polymerase I, the human polymerase I-specific upstream binding factor, and topoisomerase I. Furthermore, we visualized the nascent transcripts simultaneously with the rDNA in the nucleoli. All tested transcription proteins were found in both the fibrillar center and the dense fibrillar component (DF) of nucleoli in human cells. In the DF the nascent transcripts, detected by bromouridine incorporation, were found colocalized with the transcription proteins only within circumscribed regions. We did not observe colocalization of rDNA with nascent transcripts within the fibrillar centers, which corroborates the view that transcription proteins in this component are rather inactive. Our results suggests that only a minor portion of the DF is involved in transcriptional activity. Transcription appears to be confined to small foci, which exist close to or associated with the DF. Our results are in favor of the view that the DF has different functions which are localized in subcompartments of the DF.

Signal amplification at the ultrastructural level using biotinylated tyramides and immunogold detection.

The tyramide amplification technique has recently been developed for signal enhancement in enzyme-linked immunosorbent assays and western blots. This method relies on using labelled tyramides as substrates for peroxidase, resulting in an immobilization of the labelled tyramide residues (tyramide reaction). We succeeded in establishing reliable protocols for the use of the tyramide reaction at the electron microscopic (EM) level. As model systems we chose the visualization of DNA in late spermatocytes, of actin in skeletal muscle, and the visualization of an rDNA probe after DNA-DNA in situ hybridization. We observed a significant increase in signal density after performing the tyramide reaction at the EM level. The tyramide amplification technique at the ultrastructural level therefore appears to be a useful tool to detect even a few epitopes present at the surface of a section as shown after in situ hybridization. It offers advantages over other amplification systems, such as the peroxidase-mediated deposition of diaminobenzidine, because of an increased spatial resolution, whereas specificity and sensitivity are comparable to the conventional immunogold detection method.

Dystrophin expression in heterozygous mdx/+ mice indicates imprinting of X chromosome inactivation by parent-of-origin-, tissue-, strain- and position-dependent factors.

Inactivation of one X chromosome (X inactivation) in female mammals results in dosage compensation of X-chromosomally encoded genes between sexes. In the embryo proper of most mammals X inactivation is thought to occur at random with respect to the parental origin of the X chromosome. We determined on the cellular level the expression of the X-chromosomally encoded protein dystrophin in skeletal and cardiac muscle of female mice heterozygous for a null mutation of the dystrophin gene (mdx/+). In all muscles investigated (cardiac, anterior venter of digastric muscle, biceps brachii and tibialis anterior muscle) we found a mosaic expression of dystrophin-expressing versus non-expressing cells and determined their proportion with respect to the parental origin of the X chromosome. In all groups of mdx/+ mice the level and pattern of dystrophin expression were found to be dependent on the parental origin of the mdx mutation. Additionally, the extent of dystrophin expression was clearly dependent on the mouse strains (C57BL/10 and BALB/c) used to produce heterozygous mdx/+ mice. Variable differences and patterns of dystrophin expression in skeletal versus cardiac muscle were found that were strictly dependent on the parental source of the mdx mutation and the strain used to breed mdx/+ mice. Moreover, dystrophin expression was found to be different between the right side and the left side of the body in individual muscles, and this difference was clearly dependent on the parental origin of the X chromosome. Our data provide evidence that in the mouse embryo proper there is a non-random distribution of cells showing inactivation of the paternal versus the maternal X chromosome in skeletal and cardiac muscle, indicating a non-random X-inactivation. Besides gametic imprinting, strain-, tissue and position-dependent factors also appear to bias X inactivation.

Test tube systems with cutting/recombination operations.

We introduce test tube systems based on operations that are closely related to the splicing operation, i.e. we consider the operations of cutting a string at a specific site into two pieces with marking them at the cut ends and of recombining two strings with specifically marked endings. Whereas in the splicing of two strings these strings are cut at specific sites and the cut pieces are recombined immediately in a crosswise way, in CR(cutting/recombination)-schemes cutting can happen independently from recombining the cut pieces. Test tube systems based on these operations of cutting and recombination turn out to have maximal generative power even if only very restricted types of input filters for the test tubes are used for the redistribution of the contents of the test tubes after a period of cuttings and recombinations in the test tubes.

The transcription unit of ribosomal genes is attached to the nuclear skeleton.

The relationship between various loci of the ribosomal gene repeat and the nucleoskeleton was examined in agarose-embedded HeLa cells. The accessibility of intranucleolar structures to molecular probes was improved by dispersing the granular component of nucleoli, and unattached DNA was removed from permeabilized nuclei under "physiological" conditions by enzymatic digestion and subsequent electroelution. The cells were then hybridized in situ with various human rDNA probes for the transcription unit or for the intergenic spacer. A strong signal was detected with probes for the transcription unit but no signal was seen with probes for the intergenic spacer. These results show that only the transcription unit is strongly attached to the nucle(ol)ar skeleton and imply that rDNA is probably attached to the skeleton primarily via RNA polymerase complexes rather than via sequence-specific attachment sites. Nucleolar fibrillar centers, embedded into the nucle(ol)ar skeleton, provide structural support for these attachments.

Redistribution of ribosomal DNA after blocking of transcription induced by actinomycin D.

We report on the effect of different doses and times of incubation of the cytostatic drug actinomycin D (AMD) on nucleolar morphology, rRNA gene transcription and rDNA gene localization using in situ hybridization and the immunocytochemical detection of the human upstream binding factor (UBF) at the electron microscopic level in HeLa cells. Low doses of AMD (0.001 micrograms/ml, 30 min) selectively block rRNA gene transcription but alter neither nucleolar morphology nor the localization of rDNA with respect to the nucleolar components. Treatment with high doses of AMD (0.05 micrograms/ml, 1 h) resulted in a retraction of the rDNA out of the nucleolus in addition to the well-known blocking of rDNA transcription, total nuclear transcription and nucleolar segregation. Under these conditions accumulations of rDNA were found in patches of chromatin at the nucleolar periphery. We conclude that the blocking of rRNA gene transcription and the changes in nucleolar morphology, both induced by AMD at different doses, are independent phenomena.

Spatial distribution of sex chromosomes and ribosomal genes: a study on human lymphocytes and testicular cells.

The location of the sex chromosomes in relation to the rRNA genes in the nuclei of human lymphocytes and testicular cells was examined. Sex chromosomes were found to be located closer to ribosomal genes than would be expected assuming a random arrangement of these chromosomes with respect to rRNA genes. This proximity could be observed irrespective of the transcriptional activity of ribosomal genes indicating that the chromosomal material and not transcriptional activity is responsible for the intranuclear order of these chromosomes.

The RNA polymerase I transcription factor UBF and rDNA are located at the same major sites in both interphase and mitotic pig embryonic kidney (PK) cells.

Indirect immunolabeling with anti-UBF antibodies, in situ hybridization with an rDNA probe, and confocal scanning laser microscopy were used to study nucleolar organizer regions (NORs) during the cell cycle in pig embryonic kidney (PK) cells. The chromosomal distribution of the polymerase I transcription factor UBF and rDNA was compared with the number of silver-stained NORs (Ag-NORs) present and nucleolar size. It was shown, both at interphase and mitosis, that the majority of UBF and rDNA signals were located at the same foci and that the amounts of UBF and rDNA at any given site were in a striking positive correlation. At mitosis, only the NORs were labeled; at interphase, the signals for both UBF and rDNA were arranged in necklace-like structures around the nucleoli. No chromosomal NORs without Ag-proteins or UBF were present, indicating that all NORs in PK cells are active at interphase. It was concluded that (1) UBF and rDNA co-localize throughout the cell cycle in PK cells; (2) their association with mitotic NORs is determined by the number of rDNA repeats, rather than by any differential ability of NORs to recruit the transcription factor; and (3) the amount of UBF can be correlated with the size and activity of the nucleoli at interphase.

The metameric pattern of the head mesoderm--does it exist?

It is a long-standing question whether the paraxial head mesoderm of vertebrate embryos is segmentally organized into somites like the trunk or not. On the one hand, no somites are seen in the anterior head mesoderm in vertebrate embryos, on the other hand, such a segmental pattern has been described under the name of somitomeres. In order to investigate the patterning of mesodermal cells in the head of avian embryos we performed scanning electron microscopy, computer assisted reconstructions of the head mesoderm and density analyses of head mesoderm cells. We observed regional differences within the head mesoderm of avian embryos, but we could not see a consistent somitomeric pattern in the head mesoderm. In sum, we consider that the avian head mesoderm is not arranged in a metameric pattern.

Emergence of myogenic and endothelial cell lineages in avian embryos.

The roles of cell cycles and of cell-cell interactions in the emergence of myogenic and endothelial cell lineages were studied in avian embryos using the quail-chicken marker system. Quail embryos were treated with drugs preventing either DNA replication or the movement of cells. Portions of drug-treated or untreated quail blastoderms were grafted into chicken wing buds. After an incubation for an additional 4 to 10 days, the embryos were analyzed for the presence of quail muscle or quail endothelial cells by the Feulgen reaction and by immunostaining. Both cell lineages differ in the time of their commitment as well as in the conditions necessary for their emergence. Muscle cells did not differentiate from unincubated blastoderms nor did they develop from drug-treated blastoderms. These results corroborate that the commitment of myogenic cells occurs during gastrulation and indicate that this commitment requires both DNA replication and cellular movements allowing cell-cell and/or cell-matrix interactions. Endothelial cells, on the contrary, developed both from drug-treated and from unincubated blastoderms, indicating that their commitment occurs before and independent of gastrulation and does not require DNA replication during gastrulation.

Site of transcription of ribosomal RNA and intranucleolar structure in HeLa cells.

Sites of transcription of ribosomal RNA in HeLa cells were visualized by electron microscopy. Cells were either incubated with Br-uridine, or permeabilized and then incubated with BrUTP, before sites containing Br-RNA were immunolabeled with gold particles. Short incubations ensured that most incorporated analogue remained at synthetic sites. Fibrillar centres were unlabelled except at their periphery; label was concentrated over certain regions of the surrounding dense fibrillar component. These results suggest that the dense fibrillar component is the site of rRNA transcription. After dispersing the granular component and the dense fibrillar component by a hypotonic treatment, removal of most chromatin and preparation of resinless sections, fibrillar centres remained fixed to a nucleoskeleton. These structural and functional features are incorporated into a model for rRNA transcription.