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PubMed2000

Modified intranuclear organization of regulatory factors in human acute leukemias: reversal after treatment.

Acute leukemias arise secondary to chromosomal aberrations that cause dysfunctions in gene regulation and regulatory factors. Significant differences in morphology between acute leukemic and nonleukemic hematopoietic cells are readily observed. How morphologic changes of the nuclei of acute leukemic cells relate to the underlying functional alterations of gene expression is minimally understood. Spatial modifications in the representation and/or organization of regulatory factors may be functionally linked to perturbations of gene expression in acute leukemic cells. Using in situ immunofluorescence microscopy, we addressed the interrelationships of modifications in nuclear morphology with the intranuclear distribution of leukemia-related regulatory factors (including ALL-1, PML, and AF-9) in cells from patients with acute leukemia. We compared the localization of leukemia-associated proteins with various factors involved in gene transcription and RNA processing (e.g., RNA polymerase II and SC-35). Our findings suggest that there are leukemia-associated aberrations in mechanisms that direct regulatory factors to sites within the nucleus. This misplacement of key cognate factors may contribute to perturbations in gene expression characteristic of leukemias.

Cell Nucleus↗
PubMed2001

Identification of novel elements which regulate the cell-type specificity of Dictyostelium 7E gene expression.

Previously, we have identified the Dictyostelium 7E gene promoter and shown that it is capable of driving expression in the same temporal and cAMP responsive manner as the endogenous gene during development. Furthermore, we have mapped the corresponding transcriptional regulatory sequences within the promoter. In the present study we used the lacZ reporter gene system to examine the role of 7E promoter elements in regulating cell-type specific expression during Dictyostelium morphogenesis. In situ detection of beta-galactosidase activity revealed that expression was induced within anterior prestalk cells at approximately 18 h of development. Subsequently, we found that promoter activity was independently regulated in subpopulations of prestalk cells. Element(s) upstream of position - 532 were necessary for expression in pstA cells while more proximal elements (located downstream of position - 426) were capable of directing expression in pstO cells. Deletion of a G-rich element ('GGT' box; 5'-GGT GAT GA-3') located between positions - 159 and - 152 resulted both in a loss of expression in pstA cells and aberrant expression in the prespore zone. Furthermore, the spatial organisation of reporter gene expression directed by this construct during culmination delineated a population of cells that have not been previously defined. These data suggest that the 7E gene is independently regulated in subpopulations of prestalk cells during development.

ATP-Binding Cassette Transporters↗
PubMed2002

Statistical analysis of multiplex brain gene expression images.

Analysis of variance (ANOVA) was employed to investigate 9,000 gene expression patterns from brains of both normal mice and mice with a pharmacological model of Parkinson's disease (PD). The data set was obtained using voxelation, a method that allows high-throughput acquisition of 3D gene expression patterns through analysis of spatially registered voxels (cubes). This method produces multiple volumetric maps of gene expression analogous to the images reconstructed in biomedical imaging systems. The ANOVA model was compared to the results from singular value decomposition (SVD) by using the first 42 singular vectors of the data matrix, a number equal to the rank of the ANOVA model. The ANOVA was also compared to the results from non-parametric statistics. Lastly, images were obtained for a subset of genes that emerged from the ANOVA as significant. The results suggest that ANOVA will be a valuable framework for insights into the large number of gene expression patterns obtained from voxelation.

Analysis of Variance↗
PubMed1994

Spatial and temporal expression patterns of FGF receptor genes type 1 and type 2 in the developing chick retina.

Fibroblast growth factors are known to influence the growth and differentiation of cultured cells derived from the chick retina. The fibroblast growth factors can interact with a family of at least four closely related receptor kinases. To find a correlation between the presence of fibroblast growth factor receptors and eye development, the patterns of expression of transcripts encoding the type 1 (FGF-R1) and type 2 (FGF-R2) receptors in the developing chick retina have been studied. Northern blot analysis of RNA of the whole retina was used to observe that FGF-R1 transcripts are abundant at embryonic day 4 and then decrease until day 11. After this stage, the level of expression of FGF-R1 increases and its peak of expression at embryonic day 18 is concomitant with the detection of the opsin transcript. FGF-R2 transcript is also detected by Northern blots of RNA of the whole retina until embryonic day 6. However, the re-expression after embryonic day 11 of FGF-R2 could only be demonstrated by PCR studies. The same pattern of expression is observed with in situ hybridization of sagittal sections. The two genes are coexpressed in the pigmented epithelium and in the neural retina during embryonic development. The expression follows the retinal layering according to a gradient from the vitreous humor to the choroid. Quantification of the in situ hybridization signals demonstrates that the pattern of expression of both receptors diverges after embryonic day 6 between the pigmented epithelium and the neuronal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Temporal and spatial analysis of cartilage proteoglycan core protein gene expression during limb development by in situ hybridization.

As limb mesenchymal cells differentiate into chondrocytes they initiate the synthesis of a cartilage-specific sulfated proteoglycan, cartilage-characteristic type II collagen, and other cartilage-specific proteins. In the present study, in situ hybridization with a 32P-labeled cloned cDNA probe complementary to mRNA encoding the core protein of cartilage proteoglycan has been used to visualize and localize the accumulation of cartilage proteoglycan core protein mRNA sequences during development of the chick limb bud in vivo. When the probe was hybridized to sections through 7-day (stage 32) limbs, an intense hybridization signal was observed over the well-differentiated cartilage rudiments of the limb, while no signal above background was observed over nonchondrogenic tissues including muscle, loose connective tissue, and epidermis. At early stages of limb development, an accumulation of silver grains representing hybridizable core protein mRNA first became detectable in the proximal central core of the limb where the prechondrogenic condensation of mesenchymal cells that characterizes the onset of cartilage differentiation was occurring. In fact, the pattern of silver grain accumulation closely followed the pattern of mesenchymal cell condensation, and no hybridizable core protein mRNA sequences were detectable in the limb bud prior to condensation. Cartilage-characteristic type II collagen mRNA was colocalized with core protein mRNA in the condensing central core of the limb suggesting that the genes for these two major constituents of cartilage matrix are coordinately regulated at the onset of chondrogenesis. Furthermore, the appearance of hybridizable core protein mRNA was closely followed by the appearance of the protein for which it codes as detected by immunohistochemical staining with monospecific antibody. These observations support the hypothesis that at the initial stages of limb chondrogenesis core protein gene expression is controlled primarily at the transcriptional level.

Aggrecans↗
PubMed2004

Spatial and temporal expression pattern of a novel gene in the frog Xenopus laevis: correlations with adult intestinal epithelial differentiation during metamorphosis.

We report the cloning of a novel gene (ID14) and its expression pattern in tadpoles and adults of Xenopus laevis. ID14 encodes a 315-amino acid protein that has a signal peptide and a nidogen domain. Even though several genes have a nidogen domain, ID14 is not the homolog of any known gene. ID14 is a late thyroid hormone (TH)-regulated gene in the tadpole intestine, and its expression in the intestine does not begin until the climax of metamorphosis, correlating with adult intestinal epithelial differentiation. In contrast, ID14 is expressed in tadpole skin and tail and is not regulated by TH. In situ hybridization revealed that this putative extracellular matrix protein is expressed in the epithelia of the tadpole skin and tail and in the intestinal epithelium after metamorphosis. In the adult, ID14 is found predominantly in the intestine with weak expression in the stomach, lung, and testis. Its exclusive expression in the adult intestinal epithelial cells makes it a useful marker for developmental studies and may give insights into cell/cell interactions in intestinal metamorphosis and adult intestinal stem cell maintenance.

Amino Acid Sequence↗
PubMed2004

Spatial and temporal expression of Wnt and Dickkopf genes during murine lens development.

Recent studies indicate a role for Wnt signalling in regulating lens cell differentiation (Stump et al., 2003). To further our understanding of this, we investigated the expression patterns of Wnts and Wnt signalling regulators, the Dickkopfs (Dkks), during murine lens development. In situ hybridisation showed that Wnt5a, Wnt5b, Wnt7a, Wnt7b, Wnt8a and Wnt8b genes are expressed throughout the early lens primordia. At embryonic day 14.5 (E14.5), Wnt5a, Wnt5b, Wnt7a, Wnt8a and Wnt8b are reduced in the primary fibres, whereas Wnt7b remains strongly expressed. This trend persists up to E15.5. At later embryonic stages, Wnt expression is predominantly localised to the epithelium and elongating cells at the lens equator. As fibre differentiation progresses, Wnt expression becomes undetectable in the cells of the lens cortex. The one exception is Wnt7b, which continues to be weakly expressed in cortical fibres. This pattern of expression continues through to early postnatal stages. However, by postnatal day 21 (P21), expression of all Wnts is distinctly weaker in the central lens epithelium compared with the equatorial region. This is most notable for Wnt5a, which is barely detectable in the central lens epithelium at P21. Dkk1, Dkk2 and Dkk3 have similar patterns of expression to each other and to the majority of the Wnts during lens development. This study shows that multiple Wnt and Dkk genes are expressed during lens development. Expression is predominantly in the epithelial compartment but is also associated, particularly in the case of Wnt7b, with early events in fibre differentiation.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Temporal and spatial expression of two isoforms of the Dutt1/Robo1 gene in mouse development.

The mammalian homologue of the Drosophila axonal guidance receptor roundabout is expressed in a wide range of tissues. Here we show that alternative splicing of the Dutt1/Robo1 gene results in two mRNA transcripts with different signal peptides, which are differentially expressed throughout mouse embryogenesis. Since mice with a targeted deletion in the Dutt1/Robo1 gene have abnormal lung pathology, immunohistochemistry was used to identify the cellular expression pattern of Dutt1/Robo1 during lung development. Dutt1/Robo1 expression was widespread and diffuse in the lung at embryonic day 17.5 but became increasingly localised to the bronchial epithelium in newborn and adult mice.

Alternative Splicing↗
PubMed2000

Spatial expression patterns of epidermal growth factor receptor gene transcripts in the postnatal mammalian cochlea.

Recent in vitro studies demonstrated that members of the epidermal growth factor (EGF) family are involved in hair cell replacement in the postnatal mammalian organ of Corti (OC) after ototoxic damage. This suggests a role for the EGF receptor (EGFR) in this process. We examined the expression of EGFR mRNA within the normal postnatal day 3 (P3) and adult rat cochlear epithelium by RT-PCR and examined its cellular localization with non-radioactive in situ hybridization in P3 and adult cochleae. RT-PCR demonstrated that EGFR mRNA is expressed in P3 and adult cochlear epithelium. In situ hybridization localized high levels of EGFR transcripts in the OC, spiral ganglion, Kölliker's organ and detectable levels in the supporting cells and the stria vascularis of P3 cochlea. In the adult cochlea, EGFR transcripts were detected only in the spiral ganglion. Our results support that the EGFR is implicated in the differentiation of several cochlear cell types and in the response of OC to ototoxic damage of the P3 rat. In the adult, it may participate in the maintenance of the mature neurons and its absence in the OC may contribute to the lack of regenerative responses in the adult cochlea.

Animals↗
PubMed1999

The genotype and epigenotype synergize to diversify the spatial pattern of expression of the imprinted H19 gene.

Little is known of how the genetic background effects the phenomenon of genomic imprinting. The H19 gene belongs to a cluster of imprinted genes on human chromosome 11. Here we show that the alternative splicing of a human H19 transcript is genotype-specific. Moreover, this variant transcript, which lacks exon 4, is either not found at all, is widely expressed or is confined to extra-villous cytotrophoblasts in first trimester placenta, depending on a combination of the genotype and the sex of the transmitting parent.

Alleles↗
PubMed1993

A repressor controls the timing and spatial localisation of stalk cell-specific gene expression in Dictyostelium.

The ecmA and ecmB genes of Dictyostelium encode related extracellular matrix proteins and both are induced by DIF, the stalk cell-specific morphogen. The ecmA gene is expressed throughout the prestalk region of the migrating slug but only later, at culmination, do the prestalk cells express the ecmB gene. Expression of the ecmB gene is induced at the entrance to the stalk tube and we have identified two, apparently redundant, promoter elements that control this process. They act as repressors, preventing transcription in the tip of the migrating slug and the apical papilla of the culminant. They have a semi-palindromic consensus sequence TTGnCAA, where n is in one case 2 and in the other 4 bp. Either element alone is able to repress ecmB promoter activity in prestalk cells. Introduction of a single repressor element into the promoter of the ecmA gene changes its expression pattern to resemble that of the ecmB gene. Mutant elements, where n is altered, cause repression during the slug stage but allow premature ecmB expression during culmination; suggesting that the effective strength of the inductive signal may increase during culmination. Inhibition of cAMP-dependent protein kinase (PKA) in prestalk cells blocks both stalk cell maturation and ecmB gene expression. We show that the block to gene expression correlates precisely with the presence of a functional repressor element and this is consistent with the notion that expression of the ecmB gene is controlled by a PKA-dependent release from transcriptional repression.

Animals↗
PubMed1987

Developmental and spatial patterns of expression of the mouse homeobox gene, Hox 2.1.

The Hox 2.1 gene forms part of a cluster of homeobox-containing genes on mouse chromosome 11. Analysis of Hox 2.1 cDNAs isolated from an 8 1/2-day p.c. mouse embryo library predicts that the gene encodes a 269 amino acid protein (Mr, 29,432). This deduced protein contains a homeobox 15 amino acids from the carboxy terminus and is very rich in serine and proline. A second partially conserved region present in several other genes containing homeoboxes, the hexapeptide Ile-Phe-Pro-Trp-Met-Arg, is located 12 amino acids upstream of the homeodomain and is encoded by a separate exon. Analysis of Hox 2.1 gene expression reveals a complex and tissue-specific series of RNA transcripts in a broad range of fetal tissues (lung, spinal cord, kidney, gut, spleen, liver and visceral yolk sac). Comparison of the temporal patterns of gene expression during development and in the adult suggests that Hox 2.1 is regulated independently in different tissues. Evidence is also presented that transcripts from other loci have extensive homology to the Hox 2.1 gene in sequences outside of the homeobox. In situ hybridization shows that Hox 2.1 transcripts are regionally localized in the spinal cord in an apparent anterior-posterior gradient extending from the hind brain. The distribution of RNA also displays a cell-type specificity in the lung, where mesodermal cells surrounding the branching epithelial cell layer accumulate high levels of Hox 2.1 transcripts.

Amino Acid Sequence↗
PubMed1997

Expression of actin genes in the arrow worm Paraspadella gotoi (Chaetognatha).

Arrow worms (the phylum Chaetognatha), one of the major marine planktonic animals, exhibit features characteristic to both deuterostomes and protostomes, and their ancestry therefore remains unknown. As the first step to elucidate the molecular bases of arrow worm phylogeny, physiology and embryology, we isolated cDNA clones for three different actin genes (PgAct1, PgAct2 and PgAct3) from the benthic species Paraspadella gotoi, and examined their expression patterns in adults and juveniles. The amino acid sequences of the three actins resembled each other, with identities ranging from 86% to 92%. However, the patterns of the spatial expression of the genes were independent. The PgAct1 gene might encode a cytoplasmic actin and was expressed in oogenic cells, spermatogenic cells, and cells in the ventral ganglion. The PgAct2 and PgAct3 genes encoded actins of divergent types. The former was expressed in well-developed muscle of the head (gnathic) region and trunk muscle cells, whereas the latter was expressed in muscle of the trunk and tail regions and oogenic cells. These results suggest that, similarly to other metazoans, the chaetognath contains multiple forms of actins, which are expressed in various manners in the adult and juvenile arrow worm.

Actins↗
PubMed2001

In vivo imaging of gene expression:.

With the ability to readily engineer genes, create knock-in and knock-out models of human disease, and replace and insert genes in clinical trials of gene therapy, it has become clear that imaging will play a critical role in these fields. Imaging is particularly helpful in recording temporal and spatial resolution of gene expression in vivo, determining vector distribution, and, ultimately, understanding endogenous gene expression during disease development. While endeavors are under way to image targets ranging from DNA to entire phenotypes in vivo, this short review focuses on in vivo imaging of gene expression with magnetic resonance and optical techniques.

Animals↗
PubMed1997

Spatial patterns of olfactory neurons expressing specific odor receptor genes in 48-hour-old embryos of zebrafish Danio rerio.

Olfactory neurons have a complex phenotype characterized by their expression of a specific odor receptor (OR) gene and their targeting of an equally specific locus in the olfactory bulb. In the adult fish, olfactory neurons expressing specific ORs are broadly distributed in the epithelium, intermingling with neurons expressing other OR phenotypes. This distributed adult pattern has led to the suggestion that olfactory neuron phenotype is determined by a stochastic process, independent of external positional cues. However, when the fish olfactory system is established during embryogenesis it is simple in its organization, with few olfactory neurons and an olfactory epithelium that has not yet folded into the adult morphology. It is possible that positional cues might act in the embryo to establish an initial population and pattern of olfactory neuron phenotypes and that subsequent morphogenesis and neuronal addition lead to the randomized distribution of neurons. To test this possibility, we examined the spatial patterns of olfactory neurons expressing specific OR genes in 48 h embryos, a time of relative simplicity in the developing olfactory epithelium. Three-dimensional plots of neuron distributions were made, and comparison of OR expression patterns were made between right and left epithelia, between individual animals and between different OR genes. The patterns of OR gene expression were not conserved in these comparison. Mathematical analysis of 21 epithelia for the degree of order in the distribution of olfactory neurons argued strongly that the neurons expressing given ORs are randomly distributed in the 48 h embryos. These results are consistent with those observed from adult tissue and support models suggesting that extrinsic positional cues do not have a major role in specifying olfactory neuron phenotypes.

Animals↗
PubMed2002

Improved expression by cytomegalovirus promoter/enhancer and behavior of vascular endothelial growth factor gene after myocardial injection of naked DNA.

Direct injection of the vascular endothelial growth factor (VEGF) gene plasmid DNA into the myocardium was shown to induce development of new blood vessels to increase the circulation in the heart of patients with coronary artery diseases. However, such angiogenic gene therapy (via naked DNA) was limited by low level of gene expression. Furthermore, the temporal and spatial characteristics of VEGF gene transfer in the heart are not known. In this study, we demonstrated that a plasmid vector, containing the human cytomegalovirus immediate early (HCMV IE) promoter and enhancer, induces greater expression of gene in the rat heart monitored by gene fused to the chloramphenicol acetyl transferase (CAT) reporter, than four different viral and cellular promoters. Interestingly, expression of VEGF121 protein showed an earlier peak, a shorter duration, and a wider distribution than that of CAT only. Therefore, a plasmid vector with an HCMV IE promoter/enhancer provides clear advantages over other previously developed plasmids. Furthermore, expression profile of VEGF121 gene may provide useful information in the design of angiogenic gene therapy in the heart.

Animals↗
PubMed2006

In situ hybridization of whole-mount embryos.

The ability to visualize the expression of a gene in both time and space is an essential tool of developmental biology. Here, we detail a robust method for in situ hybridization of RNA probes to whole pieces of fixed tissue. This method has been optimized for reliable and sensitive visualization of the spatial patterns of gene expression in mouse embryo tissue.

Animals↗
PubMed1997

Meis2, a novel mouse Pbx-related homeobox gene induced by retinoic acid during differentiation of P19 embryonal carcinoma cells.

We report the cDNA cloning, partial genomic organization, and expression pattern of Stra10, a novel retinoic acid-inducible gene in P19 embryonal carcinoma cells. Four murine cDNA isoforms have been isolated, which are likely to result from alternative splicing. The predicted protein sequences exhibit approximately 85% identity with the Pbx-related Meis1 homeobox gene products, which are involved in myeloid leukemia in BXH-2 mice, and one of the Stra10 isoforms corresponds to the recently published Meis2 sequence (Nakamura et al. [1996] Oncogene 13:2235-2242). The Meis2 homeodomain is identical to that of Meis1, and is most closely related to those of the Pbx/TGIF homeobox gene products. By in situ hybridization analysis, we show that the Meis2 gene displays spatially restricted expression patterns in the developing nervous system, limbs, face, and in various viscera. In adult mice, Meis2 is mainly expressed in the brain and female genital tract, with a different distribution of the alternative splice forms in these organs.

Amino Acid Sequence↗