Oscillating expression of c-Hey2 in the presomitic mesoderm suggests that the segmentation clock may use combinatorial signaling through multiple interacting bHLH factors.

Vertebrate somitogenesis comprises the generation of a temporal periodicity, the establishment of anteroposterior compartment identity, and the translation of the temporal periodicity into the metameric pattern of somites. Molecular players at each of these steps are beginning to be identified. Especially, members of the Notch signaling cascade appear to be involved in setting up the somitogenesis clock and subsequent events. We had previously demonstrated specific expression of the mHey1 and mHey2 basic helix-loop-helix (bHLH) factors during somitogenesis. Here we show that perturbed Notch signaling in Dll1 and Notch1 knockout mutants affects this expression in the presomitic mesoderm (PSM) and the somites. In the caudal PSM, however, mHey2 expression is maintained and thus is likely to be independent of Notch signaling. Furthermore, we analysed the dynamic expression of the respective chicken c-Hey1 and c-Hey2 genes during somitogenesis. Not only is c-Hey2 rhythmically expressed across the chicken presomitic mesoderm like c-hairy1, but its transcription is similarly independent of de novo protein synthesis. In contrast, the dynamic expression of c-Hey1 is restricted to the anterior segmental plate. Both c-Hey genes are coexpressed with c-hairy1 in the posterior somite half. Further in vitro and in vivo interaction assays demonstrated direct homo- and heterodimerisation between these hairy-related bHLH proteins, suggesting a combinatorial action in both the generation of a temporal periodicity and the anterior-posterior somite compartmentalisation.

Molecular analysis of E-cadherin and cadherin-11 in Wilms' tumours.

Different studies of Wilms' tumours have demonstrated a loss of heterozygosity (LOH) of chromosome 16q ranging from 17 to 25%. In order to search for a potential tumour suppressor gene on 16q, we chose the calcium-dependent cell adhesion molecules E-cadherin and cadherin-11 as candidate genes, which are both located on the long arm of chromosome 16. E-cadherin is known to be expressed in epithelial structures, whereas cadherin-11 is supposed to be expressed in mesenchymal structures and developing epithelium, including renal tubules. For the present study, fresh frozen tissue from 30 Wilms' tumours and corresponding non-tumour tissues were analysed. Single nucleotide polymorphisms of the E-cadherin and cadherin-11 genes were chosen and analysed for allelic inactivation by polymerase chain reaction (PCR) amplification and sequence analysis. Loss of expression of one E-cadherin allele was seen in 10% (2/20) of the informative cases. Two out of 11 informative cases (18%) showed loss of expression of one cadherin-11 allele. No length alterations of either the E-cadherin or the cadherin-11 messenger RNAs were identified using reverse transcription PCR and agarose gel electrophoresis in tumour tissue. Sequencing of the entire E-cadherin coding region in seven cases showed the wild-type sequence. These data imply that E-cadherin and cadherin-11 are not likely to play typical tumour suppressor roles in Wilms' tumour. Interestingly, the E-cadherin immunohistochemistry showed a deviation from the normal reaction pattern in 50% of the cases, with 27% (8/30) showing an apical or cytoplasmic reaction and 23% (7/30) being completely negative. Northern blot analysis revealed that the overall expression of cadherin-11 is much stronger than that of E-cadherin. In several cases, the expression levels of the two genes were inversely correlated, suggesting the existence of a regulatory mechanism. Analysis of differential expression of the various cadherins and their subsequent signal transduction pathways might contribute to a better understanding of the complexity of Wilms' tumour formation.

Characterization of the human and mouse HEY1, HEY2, and HEYL genes: cloning, mapping, and mutation screening of a new bHLH gene family.

Many basic helix-loop-helix (bHLH) transcription factors are known as key regulators of embryonic development or differentiation in various species. We have isolated and characterized three new hairy-related bHLH transcription factor genes from mouse and human (hairy and Enhancer-of-split related with YRPW motif; HEY1, HEY2, and HEYL). All three HEY genes have a similar genomic structure with five exons. Together with a highly related Drosophila homologue, they form a new bHLH gene subfamily that is different from both hairy and the known vertebrate Hes and Her genes. While the overall structure with the bHLH domain, Orange domain, and WRPW motif is similar, the last motif is changed to KPYRPWG in Hey1/2 and absent in HeyL. This and other sequence features suggest Hey proteins to have unique functional properties. The genes were mapped by fluorescence in situ hybridization and RH mapping to the following human chromosomes: (HEY1) 8q21, (HEY2) 6q21, and (HEYL) 1p34.3. Based on expression patterns and map location, HEY genes are candidates for several human or mouse disease loci. However, initial screening of DNA from affected individuals for two human disorders and four mouse mutants did not reveal any diagnostic alterations in the coding regions.

Hey genes: a novel subfamily of hairy- and Enhancer of split related genes specifically expressed during mouse embryogenesis.

We have identified a novel subfamily of mammalian hairy/Enhancer of split (E(spl))-related basic helix-loop-helix (bHLH) genes together with a putative Drosophila homologue. While hairy/E(spl) proteins are characterized by an invariant proline residue in the basic domain and a carboxyterminal groucho-binding WRPW motif, our genes encode a carboxyterminal KPYRPWG sequence and were thus designated as Hey genes (Hairy/E(spl)-related with YRPW motif). Furthermore, they bear a unique C-terminal TE(I/V)GAF motif and the characteristic proline is changed in all Hey family members to glycine. RNA in situ hybridization analysis revealed specific expression of Hey1 during development of the nervous system, the somites, the heart and the craniofacial region. Hey2 is similarly expressed in the somites whereas it shows a complementary expression in the heart, the craniofacial region and the nervous system. The diversity of expression patterns implies unique functions in neurogenesis, somitogenesis and organogenesis.

Fjx1, the murine homologue of the Drosophila four-jointed gene, codes for a putative secreted protein expressed in restricted domains of the developing and adult brain.

The Drosophila gene four jointed (fj) codes for a secreted or cell surface protein important for growth and differentiation of legs and wings and for proper development of the eyes. Here we report the cloning of the mouse four-jointed gene (fjx1) and its pattern of expression in the brain during embryogenesis and in the adult. In the neural plate, fjx1 is expressed in the presumptive forebrain and midbrain, and in rhombomere 4, however a small rostral/medial area of the forebrain primordium is devoid of expression. Expression of fjx1 in the neural tube can be divided into three phases. (1) In the embryonic brain fjx1 is expressed in two patches of neuroepithelium: in the midbrain tectum and the telencephalic vesicles. (2) In fetal and early postnatal brain fjx1 is expressed mainly by the primordia of layered telencephalic structures: cortex (ventricular layer and cortical plate), olfactory bulb (subependymal layer and in the mitral cell layer). In addition expression is observed in the superior colliculus. (3) In the adult, fjx1 is expressed by neurones evenly distributed in the telencephalon (isocortex, striatum, hippocampus, olfactory bulb, piriform cortex), in the Purkinje cell layer of the cerebellum, and numerous medullary nuclei. In the embryo, strong expression can further be seen in the apical ectodermal ridge of fore- and hindlimbs and in the ectoderm of the branchial arches.

Allele loss in Wilms tumors of chromosome arms 11q, 16q, and 22q correlate with clinicopathological parameters.

An extended analysis for loss of heterozygosity (LOH) on eight chromosomes was conducted in a series of 82 Wilms tumors. Observed rates of allele loss were: 9.5% (1p), 5% (4q), 6% (6p), 3% (7p), 9.8% (11q), 28% (11p15), 13.4% (16q), 8.8% (18p), and 13.8% (22q). Known regions of frequent allele loss on chromosome arms 1p, 11p15, and 16q were analyzed with a series of markers, but their size could not be narrowed down to smaller intervals, making any positional cloning effort difficult. In contrast to most previous studies, several tumors exhibited allele loss for multiple chromosomes, suggesting an important role for genome instability in a subset of tumors. Comparison with clinical data revealed a possible prognostic significance, especially for LOH on chromosome arms 11q and 22q with high frequencies of anaplastic tumors, tumor recurrence, and fatal outcome. Similarly, LOH 16q was associated with anaplastic and recurrent tumors. These markers may be helpful in the future for selecting high-risk tumors for modified therapeutic regimens.

Frasier syndrome is caused by defective alternative splicing of WT1 leading to an altered ratio of WT1 +/-KTS splice isoforms.

The Wilms' tumor gene WT1 plays a key role in genitourinary development and subsequent normal function. Homozygous mutations of WT1 can be found in approximately 15% of Wilms' tumors. Furthermore, somatic heterozygous loss of WT1 is known to lead to cryptorchidism and hypospadias in males. A much more severe phenotype is seen in patients with Denys-Drash syndrome which results from heterozygous dominant-negative mutations of the gene. Characteristic features are mesangial sclerosis with early kidney failure, varying degrees of gonadal dysgenesis and high risk of Wilms' tumors. Here we show that a related disease, Frasier syndrome, characterized by focal glomerular sclerosis, delayed kidney failure and complete gonadal dysgenesis, is probably caused by specific intronic point mutations of WT1 that preferentially affect a CpG dinucleotide. Disruption of alternative splicing at the exon 9 splice donor site prevents synthesis of the usually more abundant WT1 +KTS isoform from the mutant allele. In contrast to Denys-Drash syndrome, no mutant protein is produced. The splice mutation leads to an imbalance of WT1 isoforms in vivo , as detected by RT-PCR on streak gonadal tissue. Thus, WT1 isoforms must have quite different functions, and the pathology of Frasier syndrome suggests that especially gonadal development may be particularly sensitive to imbalance or relative underrepresentation of the WT1 +KTS isoform.

An integrated YAC clone contig for the WAGR region on human chromosome 11p13-p14.1.

The WAGR syndrome (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) deletion region on chromosome 11p13 has been extensively characterized by deletion analysis and long-range restriction mapping. A dense probe set is available for this genomic region, which harbors a number of disease gene loci, some of which still are not cloned. The identification of candidates for these genes would be greatly facilitated by a complete gene map for this chromosomal segment. As an initial step toward this goal, we have isolated the entire region in 58 overlapping YAC clones. The contig spanning 8 Mb from RAG1 to KCNA4 has been assembled by STS and probe content mapping for 76 loci with an average spacing of about 100 kb. A subset of clones has been analyzed by PFG analysis to position these within the known physical map. Common microsatellite markers permit an alignment of the YAC contig with the genetic and radiation hybrid maps of chromosome 11. Ten known genes, some with much more refined map positions, are placed in the contig. The severalfold coverage of 11p13-p14.1 provides a reliable resource for the future development of a complete gene map of this region.

CpG island clones for chromosome 11p--a resource for mapping and gene identification.

A NotI end fragment library has been constructed for human Chromosome (Chr) 11p. Seventy-two clones were mapped to chromosomal subregions by use of somatic cell hybrids. The clones detect 44 different CpG islands, and we have isolated cosmid contigs for 36 of them. Extrapolation from the known 11p13 NotI restriction map suggests that every second CpG island from 11p containing a Not site is already represented in the clone collection. By sequence analysis all of the 11p13 clones exhibit typical features of CpG islands, and cross-species hybridization has been detected with at least one fragment in most cases. The cosmids serve as valuable linking clones for long-range restriction mapping. They also provide excellent starting material for transcript isolation procedures to identify genes on chromosome 11p associated with developmental anomalies and various tumor types. Several transcribed sequences have already been isolated with some of these clones.

Elements of a general theory of joints. 6. General kinematical structure of mandibular movements.

Movements of the mandible are recorded in vivo by a measuring system (MT 1602) that takes all 6 degrees of freedom of a rigid body into account. Class-I-patients were asked to move their mandible in the sagittal-vertical plane. The evaluation of the measurements yields an almost plane mandibular movement that only uses 2 degrees of freedom although a general plane movement normally possesses 3 degrees and although the human temporomandibular joint (TMJ) has a certain space of motion. This quantitative reduction of the degrees of freedom by one is produced by a neuro-muscularly guided dimeric link chain that cannot directly be related to anatomical landmarks. The diverse types of mandibular motion of a sound patient differ in the constant ratio of the angular velocities around the 2 axes of the dimeric link chain. Therefore, the paths of the individual mandibular points are epicycloids or hypocycloids. Patients with disorders of the TMJ and the neuromuscular feedback system do no longer show this constancy of the angular velocities' ratio. Besides that, we theoretically derive and empirically prove the fact that common axiographs do not record the "path of the hinge axis" of the TMJ, on principle. In this context we discuss some--in dentistry and anatomy widespread--fundamental misconceptions of the rigid body's kinematics.

[Arrangement of neuromuscular movement cycles of free mandibular movements following Le Fort I-operations]. / Anordnung neuro-muskulärer Bewegungszyklen freier Unterkeiferbewegungen nach Le-Fort-I-Operationen.

In orthognathic surgery the Le Fort I osteotomy changes the structure of motion of the mandible. This structure and its changes could reliably and quantitatively be described if it was evaluated by a projection of the mandibular movement upon a couple's movement in a gearing system. The comparison of Le Fort I cases with orthodontically treated class-I-cases shows significant differences.

[Structural arrangement of mandibular neuromuscular guided drive systems]. / Anordnungsstrukturen der mandibulären, neuro-muskulär gesteuerten Getriebesysteme.

The free movements of mandibular, oral apertures can be related to the couples' movements of neuromuscular throttle cranks which reveal a common specific property: a double dead position of the mandible (couple). As the neuromuscular system uses the same cyclic path of a well-defined mandibular point for the opening and the closing process of a specific mandibular movement, the mandible can follow the same or two different trajectories although the positive drive works on. The different movements of oral aperture are related to the geometrical positions of the cranks at the fixed plane. Geometrical properties and measures of the gear systems of eleven class-I-patients are reported and discussed.

[Posterior guidance of the mandible as a neuromuscular assigned dimeric joint chain]. / Die posteriore Führung der Mandibula als neuro-muskulär gegebene dimere Gelenkkette.

Measurements of the mandible's movements which take its six degrees of freedom into account show that the conventional concept of a condylar hinge axis does not hold: on principle the structure of motion of the condylar hinge axis is not different from that of the incisal edge. The spaces of movement which are related to the condylar hinge axis and the incisal edge are factually enforced by a hinge axis of the neuromuscular system. This neuromuscular hinge axis cannot directly be related to anatomical structures although in the sagittal-vertical plane it reduces the number of the degrees of freedom from three to two.

[The general even mandibular movements as couple movements in neuromuscular guided mechanism]. / Die allgemeinen ebenen Mandibulabewegungen als Koppelbewegungen in neuro-muskulär gesteuerten Getriebesystemen.

The manifold mandibular movements of oral aperture can be modelled by movements of couples in neuromuscular gear systems. These systems consist of the dimeric link chain of the neuromuscular hinge axis (rocking arm) and a neuromuscularly enforced cyclic trajectory of a well-defined point of the mandible. The neuromuscular hinge axis is the common constant of all gear systems whereas the position of the cyclic trajectory at the fixed plane (maxilla) is closely related to the specific path of the entire rigid body mandible. The presented theory is inferred by measurements of the mandible's movement that take all six degrees of freedom into account.

[Comparative studies of measuring methods for the six-dimensional recording of spatial mandibular movements]. / Vergleichende Untersuchung von Messmethoden zur Aufzeichnung der räumlichen Mandibulabewegung.

A catalog of criteria has been developed for testing and judging methods to record extensive movements of the mandible. The aim is to find a method that functions in all six dimensions (translation and rotation) and precludes system errors. Two methods that seem to be in principle able to fulfil the criteria have been tested with calibration sets. The catalog, the results of the survey and developments to improve these methods are presented.

The biomechanical relation between incisal and condylar guidance in man.

The arrangement, form and movement of incisal and condylar guidance allow representation of the stomatognathic system by a gearing with positive drive and closed linkage. Because of problems of stability, this gearing has to be a throttle crank. Therefore, the stomatognathic elements have to fulfill the condition of Grashoff. The analysis of 14 Class I patient data confirms this condition and yields that their stomatognathic systems seem to be determined by only two parameters. The properties of these systems are largely independent of the jaw's growth if the momentary growth takes place in the direction of the initial inclination of hinge-axis movement out of centric relation.