Biomedical subjects

D ten Berge

Publications and source records attributed to D ten Berge.

6 recordsLinked to original sources

PubMed2001

Automated topographical cell proliferation analysis.

BACKGROUND: Cell proliferation is often studied using the incorporation of bromodeoxyuridine (BrdU). Immunohistochemical staining is then used to detect BrdU in the nucleus. To circumvent the observer bias and labor-intensive nature of manually counting BrdU-labeled nuclei, an automated topographical cell proliferation analysis method is developed. METHODS: Sections stained with fluorescein-labeled anti-BrdU and counterstained with To-Pro-3 are scanned using confocal laser scanning microscopy (CLSM). For every point in the image, the nucleus density of BrdU-labeled nuclei and the total nucleus density of the neighborhood of that point are calculated from the BrdU and the To-Pro-3 signal, respectively. The ratio of these densities gives an indication of the amount of cell proliferation at that point. The automated measure is validated by comparing it with the ratio of BrdU-stained nuclei to the total number of nuclei obtained from a manual count. RESULTS: A positive correlation is found between the automated measure and the ratios calculated from the manual counting (r = 0.86, P < 0.001). Calculating the topographical cell proliferation using the automated method is faster and does not suffer from interobserver variability. CONCLUSIONS: Automated topographical cell proliferation analysis is a fast method to objectively find differences in cell proliferation within a tissue. This can be visualized by a topographical map that corresponds to the tissue under study.

Animals↗

PubMed2001

Prx1 and Prx2 are upstream regulators of sonic hedgehog and control cell proliferation during mandibular arch morphogenesis.

The aristaless-related homeobox genes Prx1 and Prx2 are required for correct skeletogenesis in many structures. Mice that lack both Prx1 and Prx2 functions display reduction or absence of skeletal elements in the skull, face, limbs and vertebral column. A striking phenotype is found in the lower jaw, which shows loss of midline structures, and the presence of a single, medially located incisor. We investigated development of the mandibular arch of Prx1(-/-)Prx2(-/-) mutants to obtain insight into the molecular basis of the lower jaw abnormalities. We observed in mutant embryos a local decrease in proliferation of mandibular arch mesenchyme in a medial area. Interestingly, in the oral epithelium adjacent to this mesenchyme, sonic hedgehog (Shh) expression was strongly reduced, indicative of a function for Prx genes in indirect regulation of SHH: Wild-type embryos that were exposed to the hedgehog-pathway inhibitor, jervine, partially phenocopied the lower jaw defects of Prx1(-/-)Prx2(-/-) mutants. In addition, this treatment led to loss of the mandibular incisors. We present a model that describes how loss of Shh expression in Prx1(-/-)Prx2(-/-) mutants leads to abnormal morphogenesis of the mandibular arch.

Animals↗

PubMed1999

Expression of retinoic acid 4-hydroxylase (CYP26) during mouse and Xenopus laevis embryogenesis.

Retinoids are important signal molecules during vertebrate embryonic development and their synthesis as well as catabolism should therefore be strictly regulated. The retinoic acid (RA) 4-hydroxylase, belonging to the cytochrome P450 family CYP26, is an enzyme catalyzing the 4-hydroxylation of RA, thereby regulating RA homeostasis. Here we describe the temporal and spatial expression patterns of mouse (mCYP26) and Xenopus laevis (xCYP26) homologues. In mouse, expression is detected in uterine crypt, around differentiating cartilage, several regions of the head, regions of the pharynx, the neural retina, and several regions of the trunk. In Xenopus, Northern blot analysis shows presence of xCYP26 transcripts before the MBT and an increased expression level during gastrulation. Whole-mount in situ hybridization shows a specific expression pattern arising at onset of gastrulation, with a ring around the blastopore. By mid gastrulation there is an anterior and a posterior expression domain, each of which gets more complex later in development. There are some important similarities and differences in expression pattern between Xenopus and mouse.

Animals↗

PubMed1998

Mouse Alx3: an aristaless-like homeobox gene expressed during embryogenesis in ectomesenchyme and lateral plate mesoderm.

Mouse Alx3 is a homeobox gene that is related to the Drosophila aristaless gene and to a group of vertebrate genes including Prx1, Prx2, Cart1, and Alx4. The protein encoded contains a diverged variant of a conserved peptide sequence present near the carboxyl terminus of at least 15 different paired-class-homeodomain proteins. Alx3 is expressed in mouse embryos from 8 days of gestation onward in a characteristic pattern, predominantly in neural crest-derived mesenchyme and in lateral plate mesoderm. We detected prominent expression in frontonasal head mesenchyme and in the first and second pharyngeal arches and some of their derivatives. High expression was also seen in the tail and in many derivatives of the lateral plate mesoderm including the limbs, the body wall, and the genital tubercle. aristaless-related genes like Alx3, Cart1, and Prx2 are expressed in overlapping proximodistal patterns in the pharyngeal arches. Similar, but more lateral patterns have been described for the Distal-less-related (Dlx) genes. Intriguingly, expression and to some extent function of aristaless and Distal-less in Drosophila also have overlapping as well as complementary aspects. Alx3 was localized to chromosome 3, near the droopy-ear (de) mutation.

Amino Acid Sequence↗

PubMed1998

Prx1 and Prx2 in skeletogenesis: roles in the craniofacial region, inner ear and limbs.

Prx1 and Prx2 are closely related paired-class homeobox genes that are expressed in very similar patterns predominantly in mesenchyme. Prx1 loss-of-function mutants show skeletal defects in skull, limbs and vertebral column (Martin, J. F., Bradley, A. and Olson, E. N. (1995) Genes Dev. 9, 1237-1249). We report here that mice in which Prx2 is inactivated by a lacZ insertion had no skeletal defects, whereas Prx1/Prx2 double mutants showed many novel abnormalities in addition to an aggravation of the Prx1 single mutant phenotype. We found defects in external, middle and inner ear, reduction or loss of skull bones, a reduced and sometimes cleft mandible, and limb abnormalities including postaxial polydactyly and bent zeugopods. A single, or no incisor was present in the lower jaw, and ectopic expression of Fgf8 and Pax9 was found medially in the mandibular arch. A novel method to detect &bgr ;-galactosidase activity in hydroxyethylmethacrylate sections allowed detailed analysis of Prx2 expression in affected structures. Our results suggest a role for Prx genes in mediating epitheliomesenchymal interactions in inner ear and lower jaw. In addition, Prx1 and Prx2 are involved in interactions between perichondrium and chondrocytes that regulate their proliferation or differentiation in the bones of the zeugopods.

Amino Acid Sequence↗

PubMed1996

Mode of insertion of the signal sequence of a bacterial precursor protein into phospholipid bilayers as revealed by cysteine-based site-directed spectroscopy.

The interactions between a bacterial precursor protein and phospholipids in bilayer-based model membrane systems is addressed in this study. The precursor-lipid interactions were assessed from the side of the lipid phase by fluorescence and electron spin resonance spectroscopy, using the precursor of the Escherichia coli outer membrane protein PhoE. The role of the signal sequence, as part of the precursor, in this interaction was investigated by using cysteine-based site-directed spectroscopy. For this purpose, purified cysteine-containing mutants of prePhoE, which were made by site-directed mutagenesis of the signal sequence part and of the mature part, and defined lipids were used. The location of the fluorescently labeled cysteine residues was established by resonance energy transfer and quenching experiments and those of the corresponding spin-labeled cysteine residues by paramagnetic relaxation enhancement. It was demonstrated that precursor-phospholipid interactions exist in model membrane systems and also that these interactions were dependent on the presence of anionic phospholipids and resulted in a deep insertion of (parts of) the precursor into the lipid bilayer. Furthermore, the results with the cysteine mutations in the signal sequence of the precursor indicate that both termini of the signal sequence are located near or at the membrane surface, with only the fluorescence of the labeled cysteines in the signal sequence part being protected against aqueous quenchers. The results demonstrate that, when part of the intact precursor, the signal sequence experiences similar lipid-protein interactions as do isolated signal peptides. They also indicate that the signal sequence inserts entirely as a looped structure into the membrane. In addition, the data also indicate that the mature part of the precursor has an affinity for the membrane.

Amino Acid Sequence↗