Expression of EphA5 during development of the olfactory nerve pathway in rat

The olfactory neuroepithelium is a highly plastic region of the nervous system that undergoes continual turnover of primary olfactory neurons throughout life. The mechanisms responsible for persistent growth and guidance of primary olfactory axons along the olfactory nerve are unknown. In the present study, we used antibodies against the Eph-related receptor, EphA5, to localise EphA5, and recombinant EDhA5-IgG fusion protein to localise its ligands. We found that although both EphA5 and its ligands were both expressed by primary olfactory neurons within the embryonic olfactory nerve pathway, there was no graded or complementary expression pattern. In contrast, the expression patterns altered postnatally such that primary olfactory neurons expressed the ligands, whereas the second-order olfactory neurons, the mitral cells, expressed EphA5. The role of EphA5 was analysed by blocking EphA5-ligand interactions in explant cultures of olfactory neuroepithelium using anti-EphA5 antibodies and recombinant EphA5. These perturbations reduced neurite outgrowth from explant cultures and suggest that intrafascicular axon repulsion may serve to limit adhesion and optimise conditions for axon growth. (C) 2000 Wiley-Liss, Inc.

Differential expression of hypoxia pathway genes in honey bee (Apis mellifera L.) caste development

Diphenism in social bees is essentially contingent on nutrient-induced cellular and systemic physiological responses resulting in divergent gene expression patterns. Analyses of juvenile hormone (JH) titers and functional genomics assays of the insulin-insulin-like signaling (IIS) pathway and its associated branch, target-of-rapamycin (TOR), revealed systemic responses underlying honey bee (Apis mellifera) caste development. Nevertheless, little attention has been paid to cellular metabolic responses. Following up earlier investigations showing major caste differences in oxidative metabolism and mitochondrial physiology, we herein identified honey bee homologs of hypoxia signaling factors, HIF alpha/Sima, HIF beta/Tango and PHD/Fatiga and we investigated their transcript levels throughout critical stages of larval development. Amsima, Amtango and Amfatiga showed correlated transcriptional activity, with two peaks of occurring in both queens and workers, the first one shortly after the last larval molt and the second during the cocoon-spinning phase. Transcript levels for the three genes were consistently higher in workers. As there is no evidence for major microenvironmental differences in oxygen levels within the brood nest area, this appears to be an inherent caste character. Quantitative PCR analyses on worker brain, ovary, and leg imaginal discs showed that these tissues differ in transcript levels. Being a highly conserved pathway and linked to IIS/TOR, the hypoxia gene expression pattern seen in honey bee larvae denotes that the hypoxia pathway has undergone a transformation, at least during larval development, from a response to environmental oxygen concentrations to an endogenous regulatory factor in the diphenic development of honey bee larvae. (C) 2010 Elsevier Ltd. All rights reserved.

Expression of fibroblast growth factor receptors during development and regression of the bovine corpus luteum

There is evidence that fibroblast growth factors (FGFs) are involved in the regulation of growth and regression of the corpus luteum (CL). However, the expression pattern of most FGF receptors (FGFRs) during CL lifespan is still unknown. The objective of the present study was to determine the pattern of expression of `B` and `C` splice variants of FGFRs in the bovine CL. Bovine CL were collected from an abattoir and classed as corpora hemorrhagica (Stage I), developing (Stage II), developed (Stage III) or regressed (Stage IV) CL. Expression of FGFR mRNA was measured by semiquantitative reverse transcription-polymerase chain reaction and FGFR protein was localised by immunohistochemistry. Expression of mRNA encoding the `B` and `C` spliced forms of FGFR1 and FGFR2 was readily detectable in the bovine CL and was accompanied by protein localisation. FGFR1C and FGFR2C mRNA expression did not vary throughout CL lifespan, whereas FGFR1B was upregulated in the developed (Stage III) CL. FGFR3B, FGFR3C and FGFR4 expression was inconsistent in the bovine CL. The present data indicate that FGFR1 and FGFR2 splice variants are the main receptors for FGF action in the bovine CL.

DIFFERENTIAL EXPRESSION OF GLUTARYL-CoA DEHYDROGENASE IN ADULT RAT CNS, PERIPHERAL TISSUES AND DURING EMBRYONIC DEVELOPMENT

Glutaryl-CoA dehydrogenase (GCDH, EC 1.3.99.7) deficiency, known as glutaric acidemia type I, is one of the more common organic acidurias. To investigate the role of this pathway in different organs we studied the tissue-specific expression pattern of rat Gcdh. The open reading frame cDNA of the rat Gcdh gene was cloned from rat brain mRNA by RT-PCR, allowing the synthesis of digoxigenin-labeled in situ hybridization (ISH) riboprobes. Gcdh mRNA expression was analyzed by ISH on cryosections of adult rat brain, kidney, liver, spleen and heart muscle, as well as on E15 and E18 rat embryos. Gcdh was found expressed in the whole rat brain, almost exclusively in neurons. Gcdh was absent from astrocytes but expressed in rare oligodendrocytes. Strong Gcdh expression was found in liver and spleen, where expression appears predominant to lymphatic nodules. In kidney, the highest Gcdh expression is found in the juxtamedullar cortex (but not in glomerula), and at lower levels in medulla. Heart muscle was negative. During embryonic development, Gcdh was found well expressed in liver, intestinal mucosa and skin, as well as at lower levels in CNS. Further studies are ongoing to provide evidence on the presence of the entire pathway in CNS in order to understand the mechanisms leading to neurotoxicity in glutaric aciduria. The high expression of Gcdh in kidney may explain why certain patients with residual enzyme activity are low excretors at the urine metabolite level.

Expression of dorsal-ventral genes during early development of Rhynchosciara americana embryos

The establishment of dorsal-ventral polarity in Drosophila is a complex process which involves the action of maternal and zygotically expressed genes. Interspecific differences in the expression pattern of some of these genes have been described in other species. Here we present the expression of dorsal-ventral genes during early embryogenesis in the lower dipteran Rhynchosciara americana. The expression of four genes, the ventralizing genes snail (sna) and twist (twi) and the dorsalizing genes decapentaplegic (dpp) and zerknüllt (zen), was investigated by whole-mount in situ hybridization. Sense and antisense mRNA were transcribed in vitro using UTP-digoxigenin and hybridized at 55°C with dechorionated fixed embryos. Staining was obtained with anti-digoxigenin alkaline phosphatase-conjugated antibody revealed with NBT-BCIP solution. The results showed that, in general, the spatial-temporal expression of R. americana dorsal-ventral genes is similar to that observed in Drosophila, where twi and sna are restricted to the ventral region, while dpp and zen are expressed in the dorsal side. The differences encountered were subtle and probably represent a particular aspect of dorsal-ventral axis determination in R. americana. In this lower dipteran sna is expressed slightly later than twi and dpp expression is expanded over the lateral ectoderm during cellular blastoderm stage. These data suggest that the establishment of dorsal-ventral polarity in R. americana embryos follows a program similar to that observed in Drosophila melanogaster.

Maternally expressed gene1 is a novel maize endosperm transfer cell-specific gene with a maternal parent-of-origin pattern of expression

Growth of the maize (Zea mays) endosperm is tightly regulated by maternal zygotic and sporophytic genes, some of which are subject to a parent-of-origin effect. We report here a novel gene, maternally expressed gene1 (meg1), which shows a maternal parent-of-origin expression pattern during early stages of endosperm development but biallelic expression at later stages. Interestingly, a stable reporter fusion containing the meg1 promoter exhibits a similar pattern of expression. meg1 is exclusively expressed in the basal transfer region of the endosperm. Further, we show that the putatively processed MEG1 protein is glycosylated and subsequently localized to the labyrinthine ingrowths of the transfer cell walls. Hence, the discovery of a parent-of-origin gene expressed solely in the basal transfer region opens the door to epigenetic mechanisms operating in the endosperm to regulate certain aspects of nutrient trafficking from the maternal tissue into the developing seed.

Expression of fibroblast growth factor receptors during development and regression of the bovine corpus luteum

There is evidence that fibroblast growth factors (FGFs) are involved in the regulation of growth and regression of the corpus luteum (CL). However, the expression pattern of most FGF receptors (FGFRs) during CL lifespan is still unknown. The objective of the present study was to determine the pattern of expression of 'B' and 'C' splice variants of FGFRs in the bovine CL. Bovine CL were collected from an abattoir and classed as corpora hemorrhagica (Stage I), developing (Stage II), developed (Stage III) or regressed (Stage IV) CL. Expression of FGFR mRNA was measured by semiquantitative reverse transcription-polymerase chain reaction and FGFR protein was localised by immunohistochemistry. Expression of mRNA encoding the 'B' and 'C' spliced forms of FGFR1 and FGFR2 was readily detectable in the bovine CL and was accompanied by protein localisation. FGFR1C and FGFR2C mRNA expression did not vary throughout CL lifespan, whereas FGFR1B was upregulated in the developed (Stage III) CL. FGFR3B, FGFR3C and FGFR4 expression was inconsistent in the bovine CL. The present data indicate that FGFR1 and FGFR2 splice variants are the main receptors for FGF action in the bovine CL.

Gene expression in sea urchin development: Study of {\it LpS1\/} gene regulation and cloning and characterization of the {\it SpKrox}-{\it 1\/} gene

Cell differentiation are associated with activation of cell lineage-specific genes. The $LpS{\it 1}\beta$ gene of Lytechinus pictus is activated at the late cleavage stage. $LpS{\it 1}\beta$ transcripts accumulate exclusively in aboral ectoderm lineages. Previous studies demonstrated two G-string DNA-elements, proximal and distal G-strings, which bind to an ectoderm-enriched nuclear factor. In order to define the cis-elements which control positive expression of the $LpS{\it 1}\beta$ gene, the regulatory region from $-$108 to +17 bp of the $LpS{\it 1}\beta$ gene promoter was characterized. The ectoderm G-string factor binds to a G/C-rich region larger than the G-string itself and the binding of the G-string factor requires sequences immediately downstream from the G-string. These downstream sequences are essential for full promoter activity. In addition, only 108 bp of $LpS{\it 1}\beta\ 5\sp\prime$ flanking DNA drives $LpS{\it 1}\beta$ gene expression in aboral ectoderm/mesenchyme cells. Therefore, for positive control of $LpS{\it 1}\beta$ gene expression, two regions of 5$\sp\prime$ flanking DNA are required: region I from base pairs $-$762 to $-$511, and region II, which includes the G/C-rich element, from base pairs $-$108 to $-$61. A mesenchyme cell repressor element is located within region I.^ DNA-binding proteins play key roles in determination of cell differentiation. The zinc finger domain is a DNA-binding domain present in many transcription factors. Based on homologies in zinc fingers, a zinc finger-encoding gene, SpKrox-1, was cloned from S. purpuratus. The putative SpKrox-1 protein has all structural characteristics of a transcription factor: four zinc fingers for DNA binding; acidic domain for transactivation; basic domain for nuclear targeting; and leucine zipper for dimerization. SpKrox-1 RNA transcripts showed a transient expression pattern which correlates largely with early embryonic development. The spatial expression of SpKrox-1 mRNA was distributed throughout the gastrula and larva ectodermal wall. However, SpKrox-1 was not expressed in pigment cells. The SpKrox-1 gene is thus a marker of a subset of SMCs or ectoderm cells. The structural features, and the transient temporal and restricted spatial expression patterns suggest that SpKrox-1 plays a role in a specific developmental event. ^

Transcriptional and translational regulators of gene expression in germ cell development

Germ cell development is a highly coordinated process driven, in part, by regulatory mechanisms that control gene expression. Not only transcription, but also translation, is under regulatory control to direct proper germ cell development. In this dissertation, I have focused on two regulators of germ cell development. One is the homeobox protein RHOX10, which has the potential to be both a transcriptional and translational regulator in mouse male germ cell development. The other is the RNA-binding protein, Hermes, which functions as a translational regulator in Xenopus laevis female germ cell development. ^ Rhox10 is a member of reproductive homeobox gene X-(linked (Rhox) gene cluster, of which expression is developmentally regulated in developing mouse testes. To identify the cell types and developmental stages in which Rhox10 might function, I characterized its temporal and spatial expression pattern in mouse embryonic, neonatal, and adult tissues. Among other things, this analysis revealed that both the level and the subcellular localization of RHOX10 are regulated during germ cell development. To understand the role of Rhox10 in germ cell development, I generated transgenic mice expressing an artificial microRNA (miRNA) targeting Rhox10. While this artificial miRNA robustly downregulated RHOX10 protein expression in vitro, it did not significantly reduce RHOX10 expression in vivo. So I next elected to knockdown RHOX10 levels in spermatogonial stem cells (SSCs), which I found highly express both Rhox10 mRNA and RHOX10 protein. Using a recently developed in vitro culture system for SSCs combined with a short-hairpin RNA (shRNA) approach, I strongly depleted RHOX10 expression in SSCs. These RHOX10-depleted cells exhibited a defect in the ability to form stem cell clusters in vitro. Expression profiling analysis revealed many genes regulated by Rhox10, including many meiotic genes, which could be downstream of Rhox10 in a molecular pathway that controls SSC differentiation. ^ RNA recognition motif (RRM) containing protein, Hermes is localized in germ plasm, where dormant mRNAs are also located, of Xenopus oocytes, which implicates its role in translational regulator. To understand the function of Hermes in oocyte meiosis, I used a morpholino oligonucleotide (MO) based knockdown approach. Microinjection of Hermes MO into fully grown oocytes, which are arrested in meiotic prophase, caused acceleration of oocytes reentry into meiosis (i.e., maturation) upon progesterone induction. Using a candidate approach, I identified at least three targets of Hermes: Ringo/Spy, Xcat2, and Mos. Ringo/Spy and Mos are known to have functions in oocyte maturation, while Ringo/Spy, Xcat2 mRNA are localized in the germ plasm of oocytes, which drives germ cell specification after fertilization. This led me to propose that Hermes functions in both oocyte maturation and germ cell development through its ability to regulate 3 crucial target mRNAs. ^

Allele-Specific Expression of the MAOA Gene and X Chromosome Inactivation in In Vitro Produced Bovine Embryos

During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre-implantation bovine embryo development by characterizing the allele-specific expression pattern of the X chromosome-linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4-, 8- to 16-cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT-PCR-RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele-specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4-, 8-to 16-cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele-specific expression of an X-linked gene that is subject to XCI in in vitro bovine embryos from the 4-cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production. Mol. Reprod. Dev. MoL Reprod. Dev. 77: 615-621, 2010. (C) 2010 Wiley-Liss, Inc.

Increased expression of protease-activated receptor 1 (PAR-1) in human leukemias

Protease-activated receptor 1 (PAR-1) is a G-protein-coupled receptor that is overexpressed in solid tumors, being associated with several pro-tumoral responses including primary growth, invasion, metastasis and angiogenesis. Expression of PAR-1 in human leukemic cell lines is reported but the status of its expression in human leukemic patients is currently unknown. In this study we evaluated the expression pattern of PAR-1 in patients with the four main types of leukemia - chronic lymphocytic leukemia subtype B (B-CLL), acute lymphoblastic leukemia subtype B (B-ALL), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Flow cytometry analyses show that lymphocytes from B-CLL patients express this receptor at similar levels to healthy individuals. On the other hand, it was observed a significant increase in PAR-1 expression in B-ALL lymphocytes as compared to B-CLL and healthy donors. Flow cytometric and real-time PCR demonstrated a significant increase in PAR-1 expression in granulocytes from CML patients in blast phase (CML-BP) but not in chronic phase (CML-CP) as compared to healthy donors. Finally, a significant increase in PAR-1 expression has been also observed in blasts from AML (subtypes M4 and M5) patients, as compared to monocytes or granulocytes from healthy donors. We conclude that PAR-1 might play an important biological role in aggressive leukemias and might offer additional strategies for the development of new therapies. (C) 2010 Elsevier Inc. All rights reserved.

Expression of galectin-1 in the mouse olfactory system

Primary sensory olfactory axons arise from the olfactory neuroepithelium that lines the nasal cavity and then project via the olfactory nerve into the olfactory bulb. The P-galactoside binding lectin, galectin-1,and its laminin ligand have been implicated in the growth of these axons along this pathway. In galectin-1 null mutant mice, a subpopulation of primary sensory olfactory axons fails to reach its targets in the olfactory bulb. In the present study we examined the spatiotemporal expression pattern of galectin-1 in normal mice in order to understand its role in the development of the olfactory nerve pathway. At E15.5, when olfactory axons have already contacted the olfactory bulb, galectin-1 was expressed in the cartilage and mesenchyme surrounding the nasal cavity but was absent from the olfactory neuroepithelium, nerve and bulb. Between E16.5 and birth galectin-1 began to be expressed by olfactory nerve ensheathing cells in the lamina propria of the neuroepithelium and nerve fibre layer. Galectin-1 was neither expressed by primary sensory neurons in the olfactory neuroepithelium nor by their axons in the olfactory nerve. Laminin, a galectin-1 ligand, also exhibited a similar expression pattern in the embryonic olfactory nerve pathway. Our results reveal that galectin-1 is dynamically expressed by glial elements within the nerve fibre layer during a discrete period in the developing olfactory nerve pathway. Previous studies have reported galectin-1 acts as a substrate adhesion molecule by cross-linking primary sensory olfactory neurons to laminin. Thus, the coordinate expression of galectin-1 and laminin in the embryonic nerve fibre layer suggests that these molecules support the adhesion and fasciculation of axons en route to their glomerular targets.

Expression of galectin-1 in the olfactory nerve pathway of rat

The olfactory neuroepithelium is characterised by the mosaic distribution of primary olfactory neurons that express different odorant receptors and cell surface glycoconjugates. Carbohydrates are believed to form a glycocode that mediates sorting out and fasciculation of primary olfactory axons through interactions with carbohydrate-binding proteins such as galectin-1. In the present study, we describe in detail the expression pattern of galectin-1 in the developing and adult rat olfactory system. We demonstrate that galectin-1 is expressed by olfactory ensheathing cells both in olfactory nerve and within the nerve fibre layer of the olfactory bulb of the embryonic and adult rat. In the adult rat, galectin-1 was preferentially expressed by olfactory ensheathing cells in the nerve fibre layer of the ventromedial and lateral surfaces of the olfactory bulb. Galectin-1 was also expressed by subsets of periglomerular cells and granule cells, particularly in the ventromedial region of the olfactory bulb. In adult rat, the galectin-1 ligand, N-acetyl-lactosamine, was expressed by primary olfactory axons that terminated in glomeruli present in the ventromedial and lateral olfactory bulb. These results suggest that expression of galectin-1 may provide a mechanism for the sorting of subpopulations of axons in the nerve fibre layer of the olfactory bulb during development as well as play a role in the postnatal maintenance of specific glomerular connections. (C) 1999 Elsevier Science B.V. All rights reserved.

Molecular profiling of isolated histological components of Wilms tumor implicates a common role for the Wnt signaling pathway in kidney and tumor development

Wilms tumor (WT), a tumor composed of three histological components - blastema (BL), epithelia and stroma - is considered an appropriate model system to study the biological relationship between differentiation and tumorigenesis. To investigate molecular associations between nephrogenesis and WT, the gene expression pattern of individual cellular components was analyzed, using a customized platform containing 4,608 genes. WT gene expression patterns were compared to genes regulated during kidney differentiation. BL had a closer gene expression pattern to the earliest stage of normal renal development. The BL gene expression pattern was compared to that of fetal kidney (FK) and also between FK and mature kidney, identifying 25 common de-regulated genes supposedly involved in the earliest events of WT onset. Quantitative RT-PCR was performed, confirming the difference in expression levels for 13 of 16 genes (81.2%) in the initial set and 8 of 13 (61.5%) in an independent set of samples. An overrepresentation of genes belonging to the Wnt signaling pathway was identified, namely PLCG2, ROCK2 and adenomatous polyposis coli (APC). Activation of the Wnt pathway was confirmed in WT, using APC at protein level and PLCG2 at mRNA and protein level. APC showed positive nuclear immunostaining for an independent set of WT samples, similarly to the FK in week 11. Lack of PLCG2 expression was confirmed in WT and in FK until week 18. Taken together, these results provided molecular evidence of the recapitulation of the embryonic kidney by WT as well as involvement of the Wnt pathway in the earliest events of WT onset. Copyright (C) 2008 S. Karger AG, Basel.

Influence of the interaction between nodal fibroblast and breast cancer cells on gene expression

Our aim was to evaluate the interaction between breast cancer cells and nodal fibroblasts, by means of their gene expression profile. Fibroblast primary cultures were established from negative and positive lymph nodes from breast cancer patients and a similar gene expression pattern was identified, following cell culture. Fibroblasts and breast cancer cells (MDA-MB231, MDA-MB435, and MCF7) were cultured alone or co-cultured separated by a porous membrane (which allows passage of soluble factors) for comparison. Each breast cancer lineage exerted a particular effect on fibroblasts viability and transcriptional profile. However, fibroblasts from positive and negative nodes had a parallel transcriptional behavior when co-cultured with a specific breast cancer cell line. The effects of nodal fibroblasts on breast cancer cells were also investigated. MDA MB-231 cells viability and migration were enhanced by the presence of fibroblasts and accordingly, MDA-MB435 and MCF7 cells viability followed a similar pattern. MDA-MB231 gene expression profile, as evaluated by cDNA microarray, was influenced by the fibroblasts presence, and HNMT, COMT, FN3K, and SOD2 were confirmed downregulated in MDA-MB231 co-cultured cells with fibroblasts from both negative and positive nodes, in a new series of RT-PCR assays. In summary, transcriptional changes induced in breast cancer cells by fibroblasts from positive as well as negative nodes are very much alike in a specific lineage. However, fibroblasts effects are distinct in each one of the breast cancer lineages, suggesting that the inter-relationships between stromal and malignant cells are dependent on the intrinsic subtype of the tumor.