Biomedical subjects
P Gruss
Publications and source records attributed to P Gruss.
Activation of the cytotactin promoter by the homeobox-containing gene Evx-1.
Cytotactin is a morphoregulatory molecule of the extracellular matrix affecting cell shape, division, and migration that appears in a characteristic and complex site-restricted pattern during embryogenesis. The promoter region of the gene that encodes chicken cytotactin contains a variety of potential regulatory sequences. These include putative binding sites for homeodomain proteins and a phorbol 12-O-tetradecanoate 13-acetate response element (TRE)/AP-1 element, a potential target for transcription factors thought to be involved in growth-factor signal transduction. To determine the effects of homeobox-containing genes on cytotactin promoter activity, we conducted a series of cotransfection experiments on NIH 3T3 cells using cytotactin promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs and plasmids driving the expression of mouse homeobox genes Evx-1 and Hox-1.3. cotransfection with Evx-1 stimulated cytotactin promoter activity whereas cotransfection in control experiments with Hox-1.3 had no effect. To localize the sequences required for Evx-1 activation, we tested a series of deletions in the cytotactin promoter. An 89-base-pair region containing a consensus TRE/AP-1 element was found to be required for activation. An oligonucleotide segment containing this TRE/AP-1 site was found to confer Evx-1 inducibility on a simian virus 40 minimal promoter; mutation of the TRE/AP-1 site abolished this activity. To explore the potential role of growth factors in cytotactin promoter activation, chicken embryo fibroblasts, which are known to synthesize cytotactin, were first transfected with cytotactin promoter constructs and cultured under minimal conditions in 1% fetal bovine serum. Although the cells exhibited only low levels of CAT activity under these conditions, cells exposed for 12 h to 10% (vol/vol) fetal bovine serum showed a marked increase in CAT activity. Cotransfection with Evx-1 and cytotactin promoter constructs of cells cultured in 1% fetal bovine serum was sufficient, however, to produce high levels of CAT activity. These findings are consistent with the hypothesis that Evx-1, a homeobox-containing gene, may activate the cytotactin promoter by a mechanism involving a growth-factor signal transduction pathway. More generally, the results support the hypothesis that the place-dependent expression of morphoregulatory molecules may depend upon local cues provided by homeobox genes and their encoded proteins.
Waardenburg's syndrome patients have mutations in the human homologue of the Pax-3 paired box gene.
Waardenburg's syndrome (WS) is an autosomal dominant combination of deafness and pigmentary disturbances, probably caused by defective function of the embryonic neural crest. We have mapped one gene for WS to the distal part of chromosome 2. On the basis of their homologous chromosomal location, their close linkage to an alkaline phosphatase gene, and their related phenotype, we suggested that WS and the mouse mutant Splotch might be homologous. Splotch is caused by mutation in the mouse Pax-3 gene. This gene is one of a family of eight Pax genes known in mice which are involved in regulating embryonic development; each contains a highly conserved transcription control sequence, the paired box. Here we show that some families with WS have mutations in the human homologue of Pax-3. Mutations in a related gene, Pax-6, which, like Pax-3, has both a paired box and a paired-type homeobox sequence, cause the Small-eye mutation in mice and aniridia in man. Thus mutations in the Pax genes are important causes of human developmental defects.
Development of the skeletal system.
The analysis of the development of the skeletal system has been greatly facilitated by the availability of a large number of mouse mutants with skeletal defects. Whereas for many of these mutants a description of the main phenotypic abnormalities is known, molecular insight into the ontogeny of the skeletal system is limited. One of the few skeletal mutants for which the molecular basis is known is undulated. These mice have a defect in the differentiation of the sclerotome and Pax-1, a mouse paired-box containing gene, has been identified as a candidate gene for this mutation. A molecular analysis of three independent undulated alleles revealed that in each case the Pax-1 gene is affected. One of the alleles could be classified as a null allele, in which the Pax-1 gene is deleted. A phenotypic analysis shows that Pax-1 is required for proper differentiation of intervertebral discs and vertebral bodies.
The murine even-skipped-like gene Evx-2 is closely linked to the Hox-4 complex, but is transcribed in the opposite direction.
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The gene for the POU domain transcription factor Oct-6 maps to the distal end of mouse chromosome 4.
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Mini-Oct and Oct-2c: two novel, functionally diverse murine Oct-2 gene products are differentially expressed in the CNS.
We report that two novel alternatively spliced products of the murine Oct-2 gene encode Mini-Oct (Oct-2d), a protein consisting of almost only the POU domain, and Oct-2c, a protein lacking the last 12 amino acids of Oct-2a. Ectopic expression in HeLa cells shows that Oct-2c is a transactivator, whereas Mini-Oct fails to transactivate if the octamer motif is in a promoter position next to TATA box. Mini-Oct can repress the transcriptional signal generated by endogenous octamer factors in F9 cells. It seems that Mini-Oct has the potential to serve as a transcriptional modulator for genes regulated by different octamer-binding factors. In situ hybridization reveals that Mini-Oct expression follows the general pattern of other known Oct-2 transcripts. However, it is absent from the Purkinje cell layer in the cerebellum of adult mice, and strong expression is observed in the developing nasal neuroepithelium and primary spermatids. Differential expression patterns of the Oct-2 transcripts with different transactivation/repression capacities of the encoded proteins may have a specific role in gene expression in the developing nervous system and in adult brain.
Pax-5 is expressed at the midbrain-hindbrain boundary during mouse development.
The murine paired-box-containing gene 5, Pax-5, is highly homologous to two other Pax genes, Pax-2 and Pax-8. The expression pattern of Pax-5 during mouse embryogenesis was examined by in situ RNA hybridization and compared to those of Pax-2 and Pax-8. Beginning at day 9.5 postcoitum (p.c.), Pax-5 was expressed in the developing brain, predominantly at the midbrain-hindbrain boundary, and in the neural tube. While the neural tube expression pattern overlapped completely with Pax-2 and Pax-8, the expression pattern in the brain was only partially overlapping. Unlike Pax-2 and Pax-8, Pax-5 was not expressed in the developing excretory system, thyroid, eye or ear. Our data suggest that Pax-5 has a role in the development of the central nervous system.
Pax1, a member of the paired box-containing class of developmental control genes, is mapped to human chromosome 20p11.2 by in situ hybridization (ISH and FISH).
Pax-1, a member of a murine multigene family, belongs to the paired box-containing class of developmental control genes first identified in Drosophila. The Pax-1 gene encodes a sequence-specific DNA-binding protein with transcriptional activating properties and has been found to be mutated in the autosomal recessive mutation undulated (un) on mouse chromosome 2 with vertebral anomalies along the entire rostrocaudal axis. By radioactive in situ hybridization (ISH) using a fragment from the murine Pax-1 paired box that is almost identical to the respective sequences from the cognate human gene HuP48 and fluorescence in situ hybridization (FISH) using a complete mouse Pax-1 cDNA, we have assigned the human homologue of murine Pax-1, the PAX1 locus, to chromosome 20p. The map position of PAX1 after FISH (FL-pter value of 0.34 +/- 0.04) corresponds to band p11.2. These results confirm the exceptional homology between human chromosome 20 and the distal segment of mouse chromosome 2, extending from bands F to G, and add PAX1 to the group of genes on 20p like PTPA, PRNP, SCG1, BMP2A, which are located in proximity on both chromosomes.
[Follow-up of spondylodiscitis following intervertebral disk operation--on the etiology, therapy and prevention].
Operations on lumbar disc prolapses are the most frequent operations in German neurosurgery divisions. After such operations, spondylodiscitis is a dreaded complication which is sometimes difficult to diagnose. Treatment of spondylodiscitis is always protracted and a burden for doctors and patients. Hence, it appears worthwhile to present a further report on discitis and spondylodiscitis, infections of the intervertebral space and the surrounding tissues after disk operations. Various clinical pictures are described: septic progress forms with neurological disorders and the necessity of open wound treatment as well as clinical pictures without septic signs with good recovery after immobilisation and antibiotic treatment. In two cases, CT-guided puncture of purulent suppuration with identification of the causative organisms and specific antibiotic treatment was possible. All patients had a relatively good result: pareses and/or bladder/rectal disorders disappeared completely in every case. The causes of discitis are discussed. The condition arises when nosocomial microorganisms, or very frequently even harmless skin bacteria, enter the wound. A large number of operations are carried out under pressure of time and under hectic conditions, as well as in a confined space in operation theatres which are too warm; these factors increase the susceptibility to infection. However, the resistance of the patient to infection is also weakened after longterm prior antiinflammatory treatment and a stay in hospital before the operation. Besides appropria to treatment of the infection (immobilisation, wound treatment, antibiotic therapy), psychological management of the patient is an important component of therapy.(ABSTRACT TRUNCATED AT 250 WORDS)
Sequence and expression pattern of pax-6 are highly conserved between zebrafish and mice.
Despite obvious differences in the patterns of early embryonic development, vertebrates share a number of developmental mechanisms and control genes, suggesting that they use similar genetic programs at some stages of development. To examine this idea, we isolated and characterized one such gene, pax-6, a member of the pax gene family, from the zebrafish Brachydanio rerio and determined the evolutionary conservation in the structure and expression of this gene by comparison to its homolog in mice. We found two alternatively spliced forms of the zebrafish pax-6 message. Sequence and expression pattern of the zebrafish pax-6 gene are remarkably similar to its murine homolog. pax-6 expression begins during early neurulation. A stripe of cells in the neuroectoderm, including the prospective diencephalon and a part of the telencephalon, expresses pax-6 as well as the hindbrain and the ventral spinal cord extending from the level of the first rhombomere to the posterior end of the CNS. During later development more limited regions of the brain including the eye, the olfactory bulb and the pituitary gland express pax-6. Cells at the midbrain-hindbrain junction express eng genes and are separated from the neighboring pax-6 regions by several cells that express neither gene, indicating a complex subdivision of this region. pax-6 expression appears during processes when cell-to-cell signalling is thought to be important, for example during induction of the eye and regionalization of the spinal cord and brain, suggesting that it may be one component mediating the response to inductive interactions.
PAX8, a human paired box gene: isolation and expression in developing thyroid, kidney and Wilms' tumors.
Recent evidence indicates a crucial role for paired box genes in mouse and human embryogenesis. The murine Pax8 gene encodes a sequence-specific transcription factor and is expressed in the developing secretory system as well as in the developing and adult thyroid. This restricted expression pattern suggested involvement of the Pax8 gene in the morphogenesis of the above organs and prompted us to investigate the PAX8 gene in humans. In this report, we describe the isolation and characterization of PAX8 cDNAs from a human adult kidney cDNA library. An open reading frame of 450 amino acids contains the 128 amino acid paired domain at its amino-terminal end. The predicted human and mouse Pax8 proteins show 97.8% conservation and are identical in their paired domains. Two independent cDNA clones reveal differential splicing of the PAX8 transcripts resulting in the removal of a 63 amino acid serine-rich region from the carboxy end of the predicted Pax8 protein. The truncated Pax8 protein becomes more similar to the predicted murine Pax2 protein, that is also expressed during kidney development and lacks the serine rich region. RNAse protection analysis shows the presence of both PAX8 transcripts in human thyroid, kidney and five Wilms' tumors. No truncated Pax8 transcripts could be detected in mouse kidney. In situ hybridization to sections of human embryonic and fetal kidney showed expression of PAX8 in condensed mesenchyme, comma-shaped and S-shaped bodies. In contrast, PAX2 expression was present mainly in the very early stages of differentiation, in the induced, condensing mesenchyme. This restricted expression pattern suggests a specific role for both genes during glomeruli maturation.(ABSTRACT TRUNCATED AT 250 WORDS)
Pax genes, mutants and molecular function.
The paired domain is a conserved DNA binding motif which was first found in Drosophila segmentation gene products. This paired domain is encoded by a well conserved, paired box DNA sequence, also detected in the genomes of other species. The mouse paired box-containing genes are referred to as Pax genes and are expressed in a distinct spatiotemporal pattern during embryogenesis. Pax proteins are able to bind to specific DNA sequences and modulate transcriptional activity. Interestingly, three different Pax genes have already been shown to correspond to some mouse and human mutants, emphasizing their role as developmental control genes.
[Adolf Butenandt lecture 1992. Molecular mechanisms of mouse embryogenesis].
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Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid.
Exposure of murine embryos to teratogenic doses of retinoic acid (RA) induced homeotic transformations of vertebrae. Posterior transformations occurred along the complete body axis after RA administration on day 7 of gestation and were accompanied by anterior shifts of Hox gene expression domains in embryos. Anterior transformations of vertebrae in the caudal half of the vertebral column were induced on day 8.5. We suggest that the identity of a vertebral segment is specified by a combination of functionally active Hox genes, a "Hox code." In this concept the sequential activation of Hox genes defines sequentially more posterior axial levels, while mesodermal cells leave the primitive streak. Exogenous RA interferes with the normal establishment of Hox codes and thus with axial specification.
The molecular basis of the undulated/Pax-1 mutation.
The murine paired box gene Pax-1 has been associated with the mouse developmental mutant undulated (un), which exhibits malformations in the vertebral column. In un mice, a point mutation leading to a Gly-Ser exchange in a conserved part of the paired domain of Pax-1 is present. Here we show that Pax-1 encodes a DNA-binding protein with transcriptional activating properties. The DNA-binding specificity of the Pax-1 protein has been extensively analyzed in gel shift assays, and in conjunction with binding interference experiments, a DNA-binding core motif was defined. Comparison of the DNA-binding properties of wild-type and un Pax-1 proteins demonstrates that the Gly-Ser replacement at position 15 within the paired domain dramatically decreases the DNA-binding affinity of the un Pax-1 protein and alters its DNA-binding specificity. These results decipher the molecular basis of the un mutation.
A nexus between Oct-4 and E1A: implications for gene regulation in embryonic stem cells.
Oct-4 is a transcription factor expressed in the pluripotent progenitor cells of the early mouse embryo. Additional factors are required for the distal activation of genes in differentiated cells containing ectopically expressed Oct-4. Here we show that Oct-4 and E1A are sufficient for distance-independent activation of the basal transcription machinery. The ratio of Oct-4 to E1A is critical for transcriptional activation, because higher levels of either factor are less efficient. Activation depends on a transactivation domain linked to the POU domain of Oct-4 and also on the conserved domain 3 of the 289RE1A protein. This domain is required for binding to the C-terminal part of Oct-4 including the POU domain. Our results indicate that E1A can serve as a bridging factor between Oct-4 and the basal initiation complex, and we postulate that an E1A-like factor acts as a cellular bridging factor of Oct-4 in pluripotent cells.
Pax-3, a novel murine DNA binding protein expressed during early neurogenesis.
We describe the isolation and characterization of Pax-3, a novel murine paired box gene expressed exclusively during embryogenesis. Pax-3 encodes a 479 amino acid protein with an Mr of 56 kd containing both a paired domain and a paired-type homeodomain. The Pax-3 protein is a DNA binding protein that specifically recognizes the e5 sequence present upstream of the Drosophila even-skipped gene. Pax-3 transcripts are first detected in 8.5 day mouse embryos where they are restricted to the dorsal part of the neuroepithelium and to the adjacent segmented dermomyotome. During early neurogenesis, Pax-3 expression is limited to mitotic cells in the ventricular zone of the developing spinal cord and to distinct regions in the hindbrain, midbrain and diencephalon. In 10-12 day embryos, expression of Pax-3 is also seen in neural crest cells of the developing spinal ganglia, the craniofacial mesectoderm and in limb mesenchyme of 10 and 11 day embryos.