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P Gruss

Publications and source records attributed to P Gruss.

At least 145 records · Page 8Linked to original sources
PubMed1995

A conserved enhancer of the human and murine Hoxa-7 gene specifies the anterior boundary of expression during embryonal development.

The murine homeobox-containing gene Hoxa-7 is expressed in restricted patterns during embryogenesis and plays an important role in the control of region-specific differentiation. Previous studies have shown that separate elements specify lineage restriction and expression boundaries of Hoxa-7. In particular 3.6 kb of 5' flanking sequences were sufficient to establish an anterior boundary of Hoxa-7 gene expression. To identify the minimal regulatory element specifying the anterior boundary of expression, transgenic mice were generated carrying chimeric constructs with deletions of 5' flanking sequences fused to a thymidine kinase minimal promoter/E. coli lacZ reporter construct. By deletion analysis, a 470 bp long control element (AX 470) located 1.6 kb upstream of the transcription start site was identified that directed expression of the beta-galactosidase protein in a pattern reflecting the anterior boundary of expression of the endogenous Hoxa-7 gene. This element was active in either orientation and conferred region-specific expression to unrelated promoters, thereby behaving like an enhancer element. In contrast, transgenic mice carrying further 5' and 3' deletions of the 470 bp long element did not exhibit an anterior boundary of Hoxa-7 expression. Based on these results the minimal control element (AX 470) specifying the anterior boundary of Hox expression was designated as Hoxa-7 enhancer. Furthermore, 3 kb of the human HOXA7 upstream region were sequenced and compared to its mouse homologue in order to identify conserved regions. Sequence comparison revealed motifs that were strongly conserved between both species. The human homologue of the mouse Hoxa-7 enhancer was 70% identical at the nucleotide level and was also capable of directing an anterior boundary in transgenic mice. Using transgenic lines a detailed analysis of the Hoxa-7 enhancer-directed expression during embryogenesis was performed. lacZ expression was first detected in the allantois at day 7.5 p.c. and in mesoderm and ectoderm at day 8.5 of gestation. Between gestational ages E8.5 to E12.5 beta-gal expression was observed in the somites, spinal cord, spinal ganglia and paraxial mesoderm as well as in mesenchymal layers of the kidney. A distinct anterior limit of expression was noted in transgenic lines at level C4 (neural tube) and C5 (spinal ganglia). Our deletion experiments defined a minimal enhancer element specifying the anterior boundary of Hox gene expression in early and late phases of development. Further studies aim at characterizing the trans-acting factors that mediate the spatial and temporal expression of Hox genes in the developing embryo.

Animals↗
PubMed1995

Targeted mutation of the murine goosecoid gene results in craniofacial defects and neonatal death.

The goosecoid gene encodes a homeodomain-containing protein that has been identified in a number of species and has been implicated in a variety of key developmental processes. Initially suggested to be involved in organizing the embryo during early development, goosecoid has since been demonstrated to be expressed during organogenesis-most notably in the head, the limbs and the ventrolateral body wall. To investigate the role of goosecoid in embryonic development, we have inactivated the gene by gene targeting to generate mice mutant for the goosecoid gene. Mice that are homozygous for the goosecoid mutation do not display a gastrulation phenotype and are born; however, they do not survive more than 24 hours. Analysis of the homozygotes revealed numerous developmental defects affecting those structures in which goosecoid is expressed during its second (late) phase of embryonic expression. Predominantly, these defects involve the lower mandible and its associated musculature including the tongue, the nasal cavity and the nasal pits, as well as the components of the inner ear (malleus, tympanic ring) and the external auditory meatus. Although the observed phenotype is in accordance with the late expression domains of goosecoid in wild-type embryos, we suggest that the lack of an earlier phenotype is the result of functional compensation by other genes.

Animals↗
PubMed1995

[Cerebral space-occupying lesion in thrombocytosis].

We report a case of venous thrombosis presented as ischemic intracranial expanding lesion localized in the parietal lobe of a young man with essential thrombocytosis. As surgical intervention became necessary we were able to investigate the morphological changes by histology and immunocytochemistry. The almost exclusive venous manifestation of thrombosis most probably initiated by elevated amounts of platelets suggests altered blood flow conditions as the predominant factor. Surgical interventions in such conditions are rare and might only proceed in space-occupying lesions with clinical symptoms. Treatment with inhibitors of platelet aggregation under strict control of the patient is the therapy of choice.

Adult↗
PubMed1995

PAX5 expression correlates with increasing malignancy in human astrocytomas.

Rearrangements concerning chromosome 9p are a late event in the progression of human astrocytic tumors to their most malignant form. The expression of PAX5, which maps to chromosome 9p13, was studied in primary human brain tumors of astrocytic origin. Whereas murine Pax5 is not expressed in the forebrain at any stage, PAX5 expression was increased in a range of astrocytomas (WHO grades II-IV) which originated in the forebrain. Expression of PAX5 was limited to those cells which also expressed the oncogenes myc, fos, or jun singularly or in combination. The epidermal growth factor receptor was highly expressed in glioblastoma multiform tumors in areas which were also highly PAX5 positive. We conclude that the missappropriate expression of PAX5 may aid in promoting the progression of astrocytomal malignancy.

Adolescent↗
PubMed1994

Pax-3 contains domains for transcription activation and transcription inhibition.

Pax-3 is a member of the Pax family of transcription factors involved in transcriptional control events during embryonic development. Here we report a functional dissection of the Pax-3 protein and describe the protein domains which are responsible for different activities. A transcription inhibition activity is located in the first 90 N-terminal amino acids and includes part of the paired domain. Furthermore, the C terminus of Pax-3 is able to confer transcriptional activation of basal promoters. Pax-3 can utilize both transcription modulating functions and activates transcription over a narrow range of protein concentration in the presence of promoter elements containing functional binding sites.

Amino Acid Sequence↗
PubMed1994

Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter.

Promoters whose temporal activity can be directly manipulated in transgenic animals provide a tool for the study of gene functions in vivo. We have evaluated a tetracycline-responsive binary system for its ability to temporally control gene expression in transgenic mice. In this system, a tetracycline-controlled trans-activator protein (tTA), composed of the repressor of the tetracycline-resistance operon (tet from Escherichia coli transposon Tn10) and the activating domain of viral protein VP16 of herpes simplex virus, induces transcription from a minimal promoter (PhCMV*-1; see below) fused to seven tet operator sequences in the absence of tetracycline but not in its presence. Transgenic mice were generated that carried either a luciferase or a beta-galactosidase reporter gene under the control of PhCMV*-1 or a transgene containing the tTA coding sequence under the control of the human cytomegalovirus immediate early gene 1 (hCMV IE1) promoter/enhancer. Whereas little luciferase or beta-galactosidase activity was observed in tissues of mice carrying only the reporter genes, the presence of tTA in double-transgenic mice induced expression of the reporter genes up to several thousand-fold. This induction was abrogated to basal levels upon administration of tetracycline. These findings can be used, for example, to design dominant gain-of-function experiments in which temporal control of transgene expression is required.

Animals↗
PubMed1994

Pax-3-DNA interaction: flexibility in the DNA binding and induction of DNA conformational changes by paired domains.

The mouse Pax-3 gene encodes a protein that is a member of the Pax family of DNA binding proteins. Pax-3 contains two DNA binding domains: a paired domain (PD) and a paired type homeodomain (HD). Both domains are separated by 53 amino acids and interact synergistically with a sequence harboring an ATTA motif (binding to the HD) and a GTTCC site (binding to the PD) separated by 5 base pairs. Here we show that the interaction of Pax-3 with these two binding sites is independent of their angular orientation. In addition, the protein spacer region between the HD and the PD can be shortened without changing the spatial flexibility of the two DNA binding domains which interact with DNA. Furthermore, by using circular permutation analysis we determined that binding of Pax-3 to a DNA fragment containing a specific binding site causes conformational changes in the DNA, as indicated by the different mobilities of the Pax-3-DNA complexes. The ability to change the conformation of the DNA was found to be an intrinsic property of the Pax-3 PD and of all Pax proteins that we tested so far. These in vitro studies suggest that interaction of Pax proteins with their specific sequences in vivo may result in an altered DNA conformation.

Animals↗
PubMed1994

Molecular basis of splotch and Waardenburg Pax-3 mutations.

Pax genes control certain aspects of development, as mutations result in (semi)dominant defects apparent during embryogenesis. Pax-3 has been associated with the mouse mutant splotch (Sp) and the human Waardenburg syndrome type 1 (WS1). We have examined the molecular basis of splotch and WS1 by studying the effect of mutations on DNA binding, using a defined target sequence. Pax-3 contains two different types of functional DNA-binding domains, a paired domain and a homeodomain. Mutational analysis of Pax-3 reveals different modes of DNA binding depending on the presence of these domains. A segment of Pax-3 located between the two DNA-binding domains, including a conserved octapeptide, participates in protein homodimerization. Pax-3 mutations found in splotch alleles and WS1 individuals change DNA binding and, in the case of a protein product of the Sp allele, dimerization. These findings were taken as a basis to define the molecular nature of the mutants.

Animals↗
PubMed1994

Regulated expression of Brachyury(T), Nkx1.1 and Pax genes in embryoid bodies.

Embryonic stem cells (ES) can be exploited to analyze in vitro mechanisms of cellular differentiation. We have utilized ES-derived embryoid body formation in an attempt to study cell types resulting from in vitro differentiation. To this end, a variety of molecular markers, preferably those which have been associated with regulatory events during mouse embryogenesis, was employed. Specifically, Brachyury (T), Pax-3 and Pax-6 genes as well as Nkx-1.1 were used. We could demonstrate that the expression of these genes in vitro was regulated by growth factors such as activin A or bFGF. Implications of these findings and the possible applications for identifying new genes are discussed.

Animals↗
PubMed1994

Pax: genes for mice and men.

The murine Pax family consists of nine genes containing a highly conserved sequence, the paired box. The expression of these genes is temporally and spatially restricted during development. Evidence gathered indicates that Pax genes are involved in the regionalization of the nervous system and in important inductive events leading to the formation of various organs. The demonstration that mutations in Pax-1, Pax-3 and Pax-6 are linked with various murine mutants (undulated, splotch and small eye) and human diseases (Waardenburg syndrome and aniridia) confirms the importance of these genes as essential morphoregulators. Recent observations also indicate that inappropriate expression of these genes can lead to the appearance of cancer.

Animals↗
PubMed1994

Differential induction of Pax genes by NGF and BDNF in cerebellar primary cultures.

The Pax genes encode sequence-specific DNA binding transcription factors that are expressed in embryonic development of the nervous system. Primary neuronal cell cultures derived from the cerebellar cortex of embryonic day 14, newborn and 7-d old mice, were used to investigate the cell-type specific expression patterns of three members of the murine paired box containing gene family (Pax gene family), in vitro. Cell types which express Pax-2, Pax-3, and Pax-6 RNA in primary cultures correspond to those found in regions of the cerebellum which show RNA signals in sections of the developing mouse brain. To find mechanisms regulating Pax gene expression during cerebellar development, the differential regulation of Pax-2, Pax-3, and Pax-6 by NGF and BDNF, two structurally related neurotrophins, was studied in such primary cultures. Pax-2 and Pax-6 RNA increased slightly by 1 h and remained elevated throughout a 24-h treatment with BDNF and NGF. Pax-3 RNA was not detected in newborn cultures, but underwent a rapid (1 h) and transient (2 h) induction upon treatment with either BDNF or NGF. No response was seen with EGF or FGF. Cycloheximide treatment amplified Pax-3 induction and prolonged the signal. Thus, Pax-3 induction resembles that of the immediate-early gene c-fos, which transduces growth factor signals during the development of particular neuronal/glial cell types. The changes in Pax expression were inductive rather than trophic.

Animals↗
PubMed1994

Identification and promoter activity of DNase I hypersensitive sites in the region of the Hox-1.3 gene.

The Hox genes encode transcriptional regulatory proteins that play a critical role in rostrocaudal specification in the developing embryo. The genes lie in four clusters in the mouse and human genome and are arranged such that a colinear relation exists between a gene's position in the cluster and the time of activation of the gene's expression. We have analyzed the Hox-1.3 region within the Hox-1 gene cluster for DNase I hypersensitive sites to identify putative regulatory sequences. Fragments identified in this way were then analyzed for transcriptional activity using gene transfer experiments in embryonal carcinoma (EC) cells. Three DNase I hypersensitive sites were identified, one of which includes the Hox-1.3 promoter and another, located 550 bp upstream, which enhances the Hox-1.3 promoter activity. The third occurs in the intron and may represent a Hox binding site. Significantly, the DNase I hypersensitive site pattern of this region of the Hox-1 cluster is not altered when F9 stem cells are differentiated with retinoic acid, suggesting that sequential activation of Hox genes by retinoic acid is not due to a sequential opening of the chromatin structure in the Hox gene region.

Animals↗
PubMed1994

Characterization of Pax-6 and Hoxa-1 binding to the promoter region of the neural cell adhesion molecule L1.

The neural cell adhesion molecule L1, a member of the immunoglobulin superfamily, mediates cell interactions in the developing and regenerating nervous system of mammals and is also detectable in the immune system and in the epithelia of intestine, skin, lung, and kidney. This diverse pattern of expression begs the question as to the regulatory mechanisms underlying transcription of the L1 gene. We demonstrate here that the paired domain and homeodomain containing Pax-6 protein binds to three different sites in the promoter region of the L1 gene. The promoter proximal binding site is also recognized by Hoxa-1 and lies approximately 60 bp upstream from the transcription start site only few base pairs upstream of a putative binding site for the TFII-I transcription initiation factor. On the basis of this sequence, we have characterized the binding of Pax-6 and explored two modes of its DNA binding activities.

Base Sequence↗
PubMed1994

Functional analysis of mouse Hoxa-7 in Saccharomyces cerevisiae: sequences outside the homeodomain base contact zone influence binding and activation.

The murine developmental control gene product, Hoxa-7, was shown to function as a DNA-binding transactivator in Saccharomyces cerevisiae. The importance of the ATTA core, the preference for antp class flanking nucleotides, the importance of Asn-51 of the homeodomain (HD), and the synergism of multiple binding sites all reflect properties that have previously been described for HOM or Hox proteins in tissue culture systems. A comparison of contact positions among genes of paralog groups and classes of mammalian HDs points to a lack of diversity in positions that make base contact, suggesting that besides the combination of HD amino acid-base pair contacts, another means of recognizing differences between targets must exist if Hox genes select different targets. The HD of antennapedia is identical to the Hoxa-7 HD. The interaction of Hoxa-7 with the exact sequence used in the nuclear magnetic resonance three-dimensional structural analysis on the antennapedia HD was studied. Hoxa-7 binding and transactivation was influenced by sequences outside of the known base contact zone of this site. We conclude that Hoxa-7 protein has a second means to interact with DNA or/and that the sequences flanking the base contact zone influence HD interactions by distorting DNA within the contact zone (base or backbone). This result is discussed in terms of DNA flexure and two modes of transcription used in S. cerevisiae.

Animals↗
PubMed1994

Pax-3 is required for the development of limb muscles: a possible role for the migration of dermomyotomal muscle progenitor cells.

Limb muscles in vertebrates originate from dermomyotomal cells, which during early development migrate from the ventrolateral region of somites into the limb buds. These progenitor cells do not express any muscle-specific marker genes or myogenic transcription factors until they reach their destination in the limbs. Here, we demonstrate by in situ hybridization that myogenic cells in somites and a population of presumably migratory muscle precursor cells in somatopleural tissue as well as myoblasts in the developing limbs express Pax-3. Significantly, in homozygous splotch mutant mice, which synthesize altered Pax-3 mRNA but make no normal protein, no cells positive for Pax-3 transcripts can be detected in the region of migrating limb muscle precursors or in the limb itself. In contrast, myotomal precursor cells and axial skeletal muscles contain Pax-3 transcripts also in the mutant. Interestingly, these animals fail to develop limb musculature as demonstrated by the lack of hybridization with various probes for myogenic transcription factors (Myf-5, myogenin, MyoD) but make apparently normal axial muscles. These observations suggest that Pax-3 is necessary for the formation of limb muscles, affecting either the generation of myogenic precursors in the somitic dermomyotome or the migration of these cells into the limb field.

Animals↗
PubMed1994

Pax genes in development.

The Pax gene family consists of nine members encoding nuclear transcription factors. Their temporally and spatially restricted expression pattern during embryogenesis suggests that they may play a key role during embryogenesis. Direct evidence for the important role of the Pax genes during embryonic development has been demonstrated by the correlation of mouse developmental mutants and human syndromes with mutations in some Pax genes. To date three Pax genes have been shown to be mutated in undulated, Splotch and small eye, respectively. In man, Pax-3 is mutated in the Waardenburg syndrome, while in aniridia Pax-6 is mutated.

Animals↗
PubMed1994

Roles of Pax-genes in developing and adult brain as suggested by expression patterns.

We have examined the transcript distribution of six members of the murine paired box-containing gene family (Pax-gene family) in midgestation embryo and adult brain using in situ hybridization analysis. The expression domains of several Pax-genes in the embryo brain were found to correspond with anatomical boundaries that coincide with neuromere landmarks and therefore respect former neuromere territories in the forebrain. The results are consistent with the concept of brain segmentation and suggest a role for Pax-genes in the brain regionalization. In the adult brain the expression of Pax-genes was observed in discreet areas, with a caudal to rostral restriction in the number of the expressed genes. In general the distribution of transcripts along the anterior-posterior axis was similar to that found in midgestation embryo brain, suggesting a role for Pax-genes in the commitment of the precursor cells to different neuronal cell fates and in the maintenance of specific brain cell subtypes. In the cerebellar cortex, the granular cell layer was found to express high levels of the Pax-6 gene, while putative Bergmann glia and cells surrounding the Purkinje cells contained Pax-3 transcripts. The main adult brain structures that expressed distinct Pax-mRNAs were the periglomerular and granular cell layer of olfactory bulb, nuclei of the septum, amygdala, and isthmus, which suggests a role for the Pax-gene family in the specification of the subcortical domains of the evolutionary old limbic system.

Aging↗