Biomedical subjects

P Gruss

Publications and source records attributed to P Gruss.

At least 73 records · Page 4Linked to original sources

PubMed1999

Pax2/5 and Pax6 subdivide the early neural tube into three domains.

The nested expression patterns of the paired-box containing transcription factors Pax2/5 and Pax6 demarcate the midbrain and forebrain primordium at the neural plate stage. We demonstrate that, in Pax2/5 deficient mice, the mesencephalon/metencephalon primordium is completely missing, resulting in a fusion of the forebrain to the hindbrain. Morphologically, in the alar plate the deletion is characterized by the substitution of the tectum (dorsal midbrain) and cerebellum (dorsal metencephalon) by the caudal diencephalon and in the basal plate by the replacement of the midbrain tegmentum by the ventral metencephalon (pons). Molecularly, the loss of the tectum is demonstrated by an expanded expression of Pax6, (the molecular determinant of posterior commissure), and a rostral shift of the territory of expression of Gbx2 and Otp (markers for the pons), towards the caudal diencephalon. Our results suggest that an intact territory of expression of Pax2/5 in the neural plate, nested between the rostral and caudal territories of expression of Pax6, is necessary for defining the midbrain vesicle.

Animals↗

PubMed1999

Functional determinants for the tetracycline-dependent transactivator tTA in transgenic mouse embryos.

Tetracycline-dependent transgenes promise to be an important tool for investigating the time dependence of gene function during mouse development. The pivotal element of this approach is the recombinant tetracycline-dependent transactivator tTA. Using a modified gene trap approach we successfully generated mouse lines expressing tTA in a wide spread manner during embryogenesis. The transgenic model system which we established allowed us to depict transactivator and target gene expression patterns with high resolution by histochemical means. Our data provide evidence that with decreasing concentrations of tTA protein the state of chromatin acetylation becomes an increasingly important determinant for tTA function. The observation of tTA-dependent position effects on tetO-linked target genes suggests that transcription patterns can be encoded at the level of promoter preactivation.

Abdomen↗

PubMed1999

Six6 (Optx2) is a novel murine Six3-related homeobox gene that demarcates the presumptive pituitary/hypothalamic axis and the ventral optic stalk.

We report on the isolation of a murine homeobox-containing gene, Six6 (Optx2), that shows extended identity in its coding region with Six3, the only member of the mammalian Six gene family known to be expressed in the optic primordium. Phylogenetic analysis demonstrates that Six6 and Six3 belong to a separate group of homeobox-genes that are closely related to the recently identified Drosophila optix. Earliest Six6 expression was detected in the floor of the diencephalic portion of the primitive forebrain, a region predicted to give rise to the neurohypophysis and to the hypothalamus. Later on, Six6 mRNA was found in the primordial tissues giving rise to the mature pituitary: the Rathke's pouch and the infundibular recess. In the optic primordium, Six6 demarcates the presumptive ventral optic stalk and the ventral portion of the future neural retina. In the developing eye. Six6 expression was detected in the neural retina, the optic chiasma and optic stalk, but not in the lens. When compared to Six6, Six3 expression pattern was highly similar, but with a generally broader transcripts distribution in the brain and in the visual system. We finally show that Six6 does not require Pax6 for its expression in the optic primordium, suggesting that Six6 acts on a parallel and/or independent pathway with Pax6 in the genetic cascade governing early development of the eye.

Amino Acid Sequence↗

PubMed1999

Pancreas development and diabetes.

The past few years have seen an increase in interest about the molecular and genetic events regulating pancreas development. Transcription factors such as Pdx1, p48 and Nkx2.2 have been shown to be essential for the proper differentiation of exocrine and endocrine tissue; however, pancreas development also involves intricate interactions between the pancreatic epithelium and its surrounding mesenchyme. Signalling factors emanating from the notochord have been shown to repress Sonic hedgehog expression in the endoderm whereas signals originating from the pancreatic mesenchyme determine the proportion of exocrine to endocrine tissue. Understanding the molecular and genetic events underlying pancreas development also opens the door for devising new therapeutic strategies against pancreatic diseases such as diabetes and cancer.

Animals↗

PubMed1999

Role of Genes in Endoderm-derived Organs.

Pax genes, which encode a family of transcription factors, are essentially required for the formation of several tissues from all germ layers in the mammalian embryo. Specifically, in organogenesis, they are involved in triggering early events of cell differentiation. The differentiation of endoderm-derived endocrine pancreas is mediated through Pax4 and Pax6. In the thyroid gland, Pax8 is essential for the formation of thyroxine-producing follicular cells, also of endodermal origin. The analysis of loss-of-function mutants revealed a common function of Pax genes in organogenesis.

Journal Article↗

PubMed1999

Prox1 function is crucial for mouse lens-fibre elongation.

Although insights have emerged regarding genes controlling the early stages of eye formation, little is known about lens-fibre differentiation and elongation. The expression pattern of the Prox1 homeobox gene suggests it has a role in a variety of embryonic tissues, including lens. To analyse the requirement for Prox1 during mammalian development, we inactivated the locus in mice. Homozygous Prox1-null mice die at mid-gestation from multiple developmental defects; here we describe the specific effect on lens development. Prox1 inactivation causes abnormal cellular proliferation, downregulated expression of the cell-cycle inhibitors Cdkn1b (also known as p27KIP1) and Cdkn1c (also known as p57KIP2), misexpression of E-cadherin and inappropriate apoptosis. Consequently, mutant lens cells fail to polarize and elongate properly, resulting in a hollow lens. Our data provide evidence that the progression of terminal fibre differentiation and elongation is dependent on Prox1 activity during lens development.

Animals↗

PubMed1999

The role of the notochord for epaxial myotome formation in the mouse.

The vertebrate somite is the source of all trunk skeletal muscles. Myogenesis in avian embryos is thought to depend on signals from notochord and neural tube for the epaxial muscles, and signals from lateral mesoderm and surface ectoderm for the hypaxial muscles. However, this hypothesis has to be tested because in mouse mutants lacking a notochord the presence of a fused myotome beneath the neural tube has been reported. We have compared the expression pattern of myogenic markers and markers for the hypaxial muscle precursors in the mutants Brachyury curtailed, truncate, Danforth's short tail and Pintail. In regions lacking notochord and sclerotome, we found small, ventrally located domains of Myf5 and MyoD expression, concomitant with ventrally expanded Pax3 signals and upregulated expression of the hypaxial marker Lbx1, suggesting that only the hypaxial program is active. We therefore hypothesise that in mammals, as in birds, the formation of the epaxial musculature depends on the presence of notochord derived signals.

Animals↗

PubMed1998

Efficient gene trap screening for novel developmental genes using IRES beta geo vector and in vitro preselection.

We have used different gene trap vectors and in vitro preselection of embryonic stem (ES) cells for a large scale screening of insertional mutations in developmentally regulated genes. A gene trap vector was constructed, which contains an internal ribosome entry site (IRES) upstream from a betageo selectable-reporter fusion gene. Analysis of 801 independent integrations revealed that the IRESbetageo vector allows for a global enrichment of about 15 folds in the number of detectable gene trap events when compared with a conventional betageo vector. Characterization of in vitro and in vivo lacZ expression suggested that this IRES-based vector is able to capture a wide range of genes expressed in a variety of tissues and developmental stages, and it can also allow trapping of genes expressed at very low levels in ES cells. A preselection protocol was devised, where gene-trapped ES cells were grown in the presence of specific growth/differentiation factors such as follistatin, nerve growth factor, and retinoic acid. Several gene trap integrations were found to be either activated or repressed by one of these factors. Characterization of lacZ expression during embryogenesis showed a strong enrichment of restricted patterns in vivo after ES cell preselection. These results suggest that a combination of IRESbetageo vector and in vitro preselection is more effective for the capture and mutation of a large number of developmental genes.

Animals↗

PubMed1998

Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development.

The cytosolic protein APAF1, human homolog of C. elegans CED-4, participates in the CASPASE 9 (CASP9)-dependent activation of CASP3 in the general apoptotic pathway. We have generated by gene trap a null allele of the murine Apaf1. Homozygous mutants die at embryonic day 16.5. Their phenotype includes severe craniofacial malformations, brain overgrowth, persistence of the interdigital webs, and dramatic alterations of the lens and retina. Homozygous embryonic fibroblasts exhibit reduced response to various apoptotic stimuli. In situ immunodetection shows that the absence of Apaf1 protein prevents the activation of Casp3 in vivo. In agreement with the reported function of CED-4 in C. elegans, this phenotype can be correlated with a defect of apoptosis. Our findings suggest that Apaf1 is essential for Casp3 activation in embryonic brain and is a key regulator of developmental programmed cell death in mammals.

Alleles↗

PubMed1998

Genes expressed after retinoic acid-mediated differentiation of embryoid bodies are likely to be expressed during embryo development.

In order to test if retinoic acid-mediated differentiation of embryoid bodies can be used as an in vitro preselection method for ES cell lines generated by gene trap, we correlated gene expression after in vitro differentiation and in 11.5-day embryos. Fifty-two genes captured by gene trap and expressed in undifferentiated embryonic stem cells were analyzed. Most genes expressed after differentiation in vitro were also expressed during embryo development. In order to correlate the expression patterns in vitro and in vivo, the in vitro expression in the center and in the periphery of the embryoid body outgrowths was observed. This allowed us to distinguish, according to in vitro expression, not expressed genes from those expressed widely in 11.5-day embryos. Consequently, with this parameter we increased the probability to obtain the restricted expression patterns in vivo. This study demonstrates the potential of the differentiation procedure in combination with the gene trap to select in vitro for genes expressed during embryo development.

Animals↗

PubMed1998

Xiro3 encodes a Xenopus homolog of the Drosophila Iroquois genes and functions in neural specification.

We have identified in Xenopus and in the mouse two highly related genes, Xiro3 and Irx3 respectively, that encode a Drosophila Iroquois-related homeobox transcription factor. Xiro3 in Xenopus and Irx3 in the mouse are expressed early in the prospective neural plate in a subset of neural precursor cells. In Xenopus, injection of Xiro3 mRNA expands the neural tube and induces ectopic neural tissue in the epidermis, based on the ectopic expression of early neural markers such as Xsox3. In contrast, the differentiation of the early forming primary neurons, as revealed by the expression of the neuronal marker N-tubulin, is prevented by Xiro3 expression. Activation of Xiro3 expression itself requires the combination of a neural inducing (noggin) and a posteriorizing signal (basic fibroblast growth factor). These results suggest that Xiro3 activation constitutes one of the earliest steps in the development of the neural plate and that it functions in the specification of a neural precursor state.

Amino Acid Sequence↗

PubMed1998

Efficiency assessment of the gene trap approach.

The trapping of genes in murine embryonic stem (ES) cells offers three features in one experimental approach: 1) analysis of the expression patterns of unknown genes by using a simple staining method, 2) rapid cloning of unknown genes, and 3) generation of mutant mouse lines. We performed a gene trap screen aimed at the discovery of new genes regulating embryonic development. We have processed 209 gene trap events for expression patterns in chimeric murine embryos. Randomly tested, beta-galactosidase-positive ES cell clones resulted in vivo in 35% gene trap events showing no beta-galactosidase activity, 39% gene trap events with ubiquitous beta-galactosidase activity, and 26% gene trap events showing beta-galactosidase activity restricted to specific cell types or organs. In vitro preselection reduced gene trap events with ubiquitous beta-galactosidase activity to 10% and increased the gene trap events with restricted beta-galactosidase activity to 64%, making the screening procedure for genes expressed in a restricted manner 2.5-fold more efficient. In five of the seven gene trap insertions into genes in which the expression pattern during embryogenesis was known, the beta-galactosidase marker gene reproduced faithfully the expression pattern of the trapped gene. 5'-Rapid amplification of cDNA ends (5'-RACE) of 28 gene trap events revealed 19 novel mouse genes, 8 known mouse genes, and 1 random transsplicing event. Twelve of the 25 mouse lines that crossed to homozygosity showed overt abnormalities. The genomic structure was investigated in four of these gene trap events, which caused obvious abnormalities. In all four cases, the splice-acceptor gene trap construct was inserted into an exon. One of the 13 gene trap events that did not result in overt abnormalities was examined for the presence of wild-type mRNA. Homozygous animals were found to produce normal levels of wild-type mRNA. Evidently, gene trapping does not always provide all three of the features mentioned above. In this paper, we discuss the efficiency of gene trapping and ways in which some problems may be overcome.

Animals↗

PubMed1998

Gene trap expression and mutational analysis for genes involved in the development of the mammalian nervous system.

We have used a large-scale gene trap approach for the isolation and mutation of genes that might play roles in the developing nervous system. After in vitro integration of two different gene trap vectors (pGT1.8geo: Skarnes et al. [1995] Proc. Natl. Acad. Sci. USA 92:6592-6596; IRES beta geo: Chowdhury et al. [1997] Nucleic Acids Res. 25:1531-1536) in mouse embryonic stem (ES) cell lines, we created 64 transgenic mouse lines. The expression analysis of the reporter gene during embryogenesis of heterozygous embryos revealed 47 lines with a variety of patterns. Around one-third (36%) of these gene trap lines showed spatiotemporal expression that was either restricted predominantly in the developing nervous system (11 lines; 17%) or widespread but with very high levels of expression in the nervous tissue (12 lines; 19%). In most cases, a correlation was found between the in vitro and the in vivo patterns of the reporter gene expression. Thus far, preliminary mutant analysis of 16 gene trap lines with potentially interesting expression patterns in the developing nervous system showed that mice homozygous for eight (50%) insertions were lethal, whereas the homozygous mice from five gene trap lines (31%) showed a lower than expected Mendelian ratio of live homozygous animals. Analysis of beta-galactosidase reporter gene expression during embryogenesis has shown that four transgenic lines are useful lacZ in situ markers for specific regions of the developing nervous system. Here, we discuss some in vivo and in vitro selection criteria that may increase the number of the trapped genes potentially involved in the control of neural development and some future strategies to improve further the efficiency of the gene trap approach.

Animals↗

PubMed1998

Distribution of a murine protein tyrosine phosphatase BL-beta-galactosidase fusion protein suggests a role in neurite outgrowth.

We have generated a gene trap insertion into the protein tyrosine phosphatase-BL (PTP-BL) locus, which produces a fusion of the N-terminal half of PTP-BL with beta-galactosidase. During development, beta-galactosidase activity was seen in all epithelial cells: strong staining was observed in the stomach and kidney epithelium, the ependymal layer of the central nervous system, and the surface ectoderm. Particularly prominent beta-galactosidase activity was seen in the peripheral nervous system, which correlated with neurite outgrowth. In epithelial cells, staining was seen in the apical portion of the cells. In nerves, beta-galactosidase activity was associated with growth cones as well as with Schwann cells. This suggests that the amino-terminal portion of PTP-BL contains sequences sufficient to target the fusion protein to specific subcellular compartments. In situ hybridization with a PTP-BL probe demonstrated that all tissues in which beta-galactosidase activity was seen were genuine sites of expression of the PTP-BL gene, although differences in the stability of the PTP-BL protein and the PTP-BL-beta-galactosidase fusion protein may exist. The distribution of beta-galactosidase activity in the peripheral nervous system, together with the structure of the wild-type protein, suggests that this phosphatase may have a role in regulation of the cytoskeleton during the development of the peripheral nervous system.

Animals↗

PubMed1998

Compensation for a gene trap mutation in the murine microtubule-associated protein 4 locus by alternative polyadenylation and alternative splicing.

One of the features expected of the gene trap approach is the functional mutation of a gene, allowing its loss-of-function phenotype analysis. We have mutated the murine microtubule-associated protein 4 (MAP-4) locus by inserting a splice-acceptor gene trap construct. Because the MAP-4 gene has been cloned, sufficient information is available to investigate the efficiency of the gene trap insertion in disrupting the protein-coding region. The fusion mRNA contains the first 905 bases of the MAP-4 mRNA and is expected to code for a truncated, nonfunctional MAP-4 protein missing, among others, the microtubule-binding domain. Activity of the lacZ marker gene of the gene trap construct was observed in all tissues throughout development and in all cells examined in adult animals. However, some cells and tissues showed higher levels of activity than others: for example, blood vessel endothelium, heart, aspects of the developing nervous system, surface ectoderm of embryonic day 11.5 embryos, and the ependymal layer and blood vessel endothelium in adult brain. MAP-4 binds to microtubules and is thought to modulate their stability. It is expressed differentially in different tissues as 5.5-kb, 6.5-kb, 8-kb, 9-kb, and 10-kb mRNA species from a single copy gene in mice. Northern hybridization with a 5', MAP-4-specific probe revealed a 3.3-kb splice variant, which has not been described previously, that was expressed as the most abundant MAP-4 mRNA species in the brain but not in other tissues. Mice homozygous for the reported gene trap insertion in the MAP-4 locus (MAP-4gt/gt) are viable and appear to be phenotypically normal. They exhibited normal levels of all MAP-4 mRNA species in brain and kidney, showing that the simian virus 40-polyadenylation signal of the gene trap construct was ignored and also showing compensation for the gene trap insertion by splicing around the gene trap construct.

Alternative Splicing↗

PubMed1998

Bodenin: a novel murine gene expressed in restricted areas of the brain.

A gene trap screen designed to isolate novel mouse genes involved in nervous system development was performed. Here, we describe the isolation and characterization of a novel gene, bodenin, which is expressed in restricted areas of the brain. beta-Galactosidase marker gene activity was detected in the embryo at the start of organogenesis (embryonic day 8.5; E8.5). Staining gradually became stronger until E12.5, when embryos exhibited widespread expression. In brains of newborn and adult mice, beta-galactosidase staining was confined predominantly to forebrain structures. Transcriptional activity was also observed in kidney, liver, lung, heart, skeletal muscle, and testes. Part of the trapped gene was isolated by 5'-rapid amplification of cDNA ends (5'-RACE). Isolation and sequencing of the complete cDNA revealed an unknown gene encoding a 200 amino acid protein. Comparison with published sequences showed 94% amino acid identity to a human integrin cytoplasmic domain-associated protein. Mice homozygous for the mutation were viable and did not exhibit any obvious abnormal phenotype. However, concealed phenotypic abnormalities cannot be excluded. The lack of readily visible abnormalities may also be due to functional compensation or to the production of low levels of wild-type protein in mice homozygous for the gene trap vector insertion. Nevertheless, the restricted expression of bodenin in the brain of newborn and adult mice suggests a role for this novel gene in the developing and mature central nervous system.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Gene trap insertion into a novel gene expressed during mouse limb development.

Gene trapping is a useful method to identify new genes involved in development. Here we describe the spatiotemporal expression of a gene identified in a gene-trap screen. This gene is first expressed at 9.5 days postcoitum (E9.5) in the forelimbs and in the branchial arches region. At E11.5, expression was detected in the stomach, genital bud, and pharyngeal epithelium. At later stages, expression includes the hair follicles, whereas the expression in the stomach and pharynx disappears. We performed 5'-rapid amplification of cDNA ends (RACE) to amplify and clone a partial cDNA of the endogenous sequence fused to the lacZ reporter gene. The sequence did not reveal any similarity to known sequences and was named paddy. The expression pattern suggests multiple roles during limb development. The early phase of expression, for instance, correlates with anteroposterior (A/P) regionalization. In contrast to other molecules involved in A/P polarization, paddy expression fades away distally as the bud elongates. This suggests that expression of paddy in late stages does not depend on apical ectodermal ridge (AER) and zone of polarizing activity (ZPA) signaling and is probably involved in posterior determination in more proximal regions of the limb.

Animals↗

PubMed1998

Efficient poly A trap approach allows the capture of genes specifically active in differentiated embryonic stem cells and in mouse embryos.

Special vectors have been constructed that allow the trapping of genes in mouse embryonic stem (ES) cells. These vectors generally contain the neomycin phosphotransferase (neo) gene for selection and the beta-galactosidase (beta-gal) gene as a marker. Promoterless vectors can be used to identify genes that are active in undifferentiated ES cells. To also have access to genes that are inactive in totipotent ES cells, we constructed a polyadenylation (poly A) trap vector in which the expression of a poly A less neo gene is driven by a constitutive promoter, whereas the expression of beta-gal depends on the trapped sequences. We demonstrate here that this vector integrates with a high frequency into transcription units and that it traps genes with very different expression patterns in vitro and in vivo. The vector integrates efficiently into transcription units that are inactive in undifferentiated ES cells and which can be activated through in vitro differentiation. Furthermore, in vivo expression patterns demonstrate that this vector integrates into genes that exhibit a highly specific temporal and spatial expression pattern during embryogenesis.

Animals↗